DEVELOPMENTAL BIOLOGY OF THE SEA URCHIN XXII
APRIL 23 – 27, 2014
MARINE BIOLOGICAL LABORATORY
WOODS HOLE, MA
The sea urchin featured on the front cover is Coelopleurus maculatus, Family Arbaciidae.
It was collected by fisherman's net from 100m depth in the entrance area of Tokyo Bay and
given to Dr. Masato Kiyomoto, Director of the Tateyama Marine Laboratory.
DEVELOPMENTAL BIOLOGY OF THE SEA URCHIN XXII
APRIL 23 – 27, 2014
MARINE BIOLOGICAL LABORATORY
WOODS HOLE, MA
Oganizers:
Dave McClay
Eric Davidson
David Burgess
Julia Morales
The Organizers would like also to acknowledge the generous support
of the following sponsors:
The Society for Developmental Biology
lumencor
TABLE OF CONTENTS
Program
2-6
Poster Presenters
7-8
Abstracts. Talk Sessions
9 – 36
Abstracts, Posters
37 - 51
Participants List
52
PROGRAM
Sea Urchin XXII
Wednesday April 23 A
Dinner 6:00-7:30 pm
Evening Opening Plenary Session: Complex Spatial patterning following
gastrulation
7:30-9:00 PM Lillie Auditorium
Chair, Dave McClay
7:30 Isabelle Peter: Use of regulatory complexity in gut organogenesis
8:00 Dave McClay: Spatial regulatory states in the ectoderm.
8:30 Thierry Lepage: A maternal determinant of dorsal-ventral axis in Paracentrotus
lividus.
Posters/Mixer 9:00-11:00 pm
Meigs Room
Thursday April 24
Breakfast 7:00-8:30 am
Plenary Session II: Genomic Logic and Cell Specification
8:30-12:00 Lillie Auditorium
Chair, Robert Burke
8:30 Eric Davidson: Sea urchin GRN model
9:00 Patrick Lemaire: Similar embryos with divergent genomes: the ascidian
paradigm
9:30 Robert Burke: Neurogenesis
Coffee Break 10:00-10:30 Lillie Auditorium
10:30 Steve Small: Transcriptional networks and gradients that control body plan
formation in Drosophila
11:00 Len Zon: Programs Regulating Stem Cell Self Renewal and Migration
11:30 Chuck Ettensohn: Morphogenesis of the Embryonic Skeleton
Lunch 12:00-1:30 pm
Plenary session III: Information at the bottom of the egg
1:30-3:00 PM Lillie Auditorium Chair, Julia Morales
1:30 Athula Wikramanayake: Deconstructing the Animal-Vegetal Axis
2:00 Amro Hamdoun: Membrane transport activity changes necessary for small
micromere motility and pigment cell formation.
2:30 Zak Swartz: Post transcriptional mechanisms for germ line segregation in
echinoderms
Coffee Break 3:00-3:30 pm Lillie Auditorium
Concurrent Session A: Gametes, Fertilization & Cleavage
3:30-5:45 PM Speck Auditorium
Chair, Brad Shuster
3:30 Dominic Poccia: Quantification of Exocytosis Kinetics by DIC Image Analysis of
Cortical Lawns
3:50 Vanessa Zazueta, Hedgehog signaling functions during sea star development
4:10 Nathalie Oulhen, Nanos protein enrichment in the small micromeres
4:30 Nadine Peyrieras: Sea urchins as models for the construction of prototypic
embryonic development
4:50 Joe Campanale: ATP-binding cassette transporters mediate small micromere
migration and left/right coelomic pouch segregation in Strongylocentrotus pupuratus
5:10 Henson, John: Inhibition of the Arp2/3 Complex in Sea Urchin Coelomocytes
Induces a Lamellipodial to Filopodial Shape Change and Alters the Cell Spreading
Process
Concurrent Session B: Specification toward specific cell types
3:30-5:45 PM Lillie Auditorium
Chair, Chuck Ettensohn,
3:30 Julius Barsi: General approach for in vivo recovery of cell-type specific effector
gene sets
3:50 Ryan Range Wnt signaling modulators establish an anterior signaling center that
patterns the anterior neuroectoderm territory in the sea urchin embryo
4:10 Jeni Croce: A Comprehensive Survey of Wnt and Frizzled Expression in the Sea
Urchin Paracentrotus lividus
4:30 Oliver Krupke: Pigment cell migration: The role of Eph-Ephrin signaling
4:50 Megan Martik: Mechanisms of Small Micromere Homing
5:10 Catherine Schrankel: Transcriptional control of immune cell development in the
purple sea urchin embryo
Dinner 5:45-7:15 pm
Plenary Session IV: New model systems for deuterostome development
7:30-9:00 pm Lillie Auditorium
Chair, Veronica Hinman
7:30 Dan Medeiros: Insights from amphioxus and lamprey into the evolution of
vertebrate head skeleton development
8:00 Paola Oliveri: Amphiura filiformis: an emerging model system to study
skeletogenesis during development and regeneration
8:30 Chris Lowe: The role of Nodal signaling in axis formation of the hemichordate
Saccoglossus kowalevskii
Posters and Mixer 9:00-11:00 pm
Meigs Room
Friday April 25
Breakfast 7:00-8:30 am
Plenary Sesson V. Specification
8:30am-12pm Lillie Auditorium
Chair, Ina Arnone
8:30 Andy Ransick: Cis-reg logic of Ese and Prox genes underlying output in
blastocoelar mesoderm founders
9:00 Yi-Hsien Su: Asymmetrical stabilization of hypoxia inducible factor α during sea
urchin embryogenesis
9:30 Enhu Li - Gene Regulatory Network Governing 2-Dimensional Expression
Patterns in the Sea Urchin Ectoderm
Coffee Break 10-10:30 pm
Lillie Auditorium
10:30 Miao Cui: A global survey of Wnt signaling in the early development of purple
sea urchin embryos
11:00 Carmen Andrikou - A gene regulatory network orchestrating myogenesis in the
sea urchin embryo
11:30 Cyndi Bradham: New Genes in Dorsal-Ventral Skeletal Patterning
Lunch 12:00-1:30 pm
Plenary Session VI: Motile components important for early divisions
1:30-3:00 PM Lillie Auditorium
Chair, Gary Wessel
1:30 Brad Shuster: Dynamics and regulation in the early embryo
2:00 Alex McDougall: Three cell behaviors involved in shaping the ascidian gastrula :
cell cycle duration, unequal cleavage, and oriented cell division.
2:30 David Burgess: Membrane domains regulating cleavage
Coffee Break and Group Photo 3:00-3:30 pm Lillie Auditorium
Concurrent Session C: Embryonic molecular biology
3:30-5:45 PM Speck Auditorium
Chair, Jia Song
3:30 Jia Song: Functional analysis of microRNAs in development.
3:50 Smadar Ben-Tabou de-Leon: The ectoderm-mesoderm connection and the
upstream regulation of VEGF and VEGFR.
4:10 Michael Piacentino: Alk4/5/7 Activity is Required for Animal Skeletal Patterning.
4:30 Dolores (Loli) Molina Jiménez: Segregation of pigment and blastocoelar cells
depends on the interplay between the lineage specific transcription factors ESE and
GCM
4:50 Zheng Wei: SoxC functions in neural precursor cells in sea urchin embryo
neurogenesis.
5:10 Vincenzo Cavalieri: Suppression of nodal expression in prospective dorsal cells
of the early sea urchin embryo by the Hbox12 homeodomain regulator.
Concurrent Session D: Evolutionary Mechanism
3:30-5:45 PM Lillie Auditorium
Chair, Yi-Hsien Su
3:30 Dede Lyons: Evolution of ectoderm-mesoderm communication during skeletal
patterning in echinoid larva
3:50 Feng Gao Experimental approach to divergence in test organization between
euechinoid and cidaroid sea urchins
4:10 Margherita Perillo: Evolution of pancreatic cell types
4:30 Jon Valencia: Regulatory gene use within the gastrulating sea urchin
4:50 Eric Erkenbrack: Delta-Notch signaling and HesC mediate the spatial
confinement of the skeletogenic-specific regulatory gene alx1 to micromeredescendants in Eucidaris tribuloides
5:10 Alys Cheatle: Transcription factor evolution.
Dinner 5:45-7:15 pm
Plenary Session VII: Intracellular mechanisms that drive early development
7:30-9:00 Lillie Auditorium
Chair, David Burgess
7:30 Gary Wessel: Something awesome about echinoderms
8:00 Michael Whitaker: Activation of DNA and protein synthesis at fertilization in the
sea urchin.
8:30 Julia Morales: Translatome analysis following fertilization
Posters and Mixer 9:00-11:00 pm
Meigs Room
Saturday April 26
Breakfast 7:00-8:30
A
Plenary Session VIII: Evolution
8:30-12:00 Lillie Auditorium
Chair, Greg Wray
8:30 Greg Wray: Intraspecific variation in GRNs
9:00 Jeff Thompson: A prelude to the present: Paleontological perspectives on 450
million years of echinoid evolution
9:30 Ina Arnone: Pattern and process during gut morphogenesis: an evolutionary
perspective
Coffee Break 10:00-10:30 am
Lillie Auditorium
10:30 Jonathan Rast: Differentiation of immunocytes and the emergence of the larval
immune system
11:00 Veronica Hinman: A gene regulatory network for neurogenenesis in
development and regeneration
11:30 Stephan Schneider: Spiralian embryogenesis at your fingertips: the quest for
an early annelid GRN
Lunch 12:00-1:30 pm
Plenary Session IX: Differentiation of cell types
1:30-3:00 Lillie Auditorium
Chair, Jonathan Rast
1:30 Mamiko Yajima – Developmental plasticity: A broad utilization of germ line
molecules in multipotent cells of the sea urchin
2:00 ShunsukeYaguchi: Maintenance of the anterior neuroectoderm of the sea urchin
embryo
2:30 Courtney Smith: Coelomocytes and the Sea Urchin Immune System
Coffee Break 3:00-3:30 pm Lillie Auditorium
Concurrent Session E: Embryo transcriptomes and Genomics
3:30-5:00 PM Lillie Auditorium
Chair: Andy Cameron
3:30 Jennifer Wygoda: Transcriptome analysis of direct vs indirect development
3:50 David Dylus: De-novo developmental transcriptome of the brittle star Amphiura
filiformis
4:10 Andy Cameron: Sequencing Echinoderm Genomes
4:30 Sarah Tulin: Genome wide identification of regulatory elements
4:50 Jongmin Nam: Experimental measurement of embryonic regulatory states
Concurrent Session F. Cytoskeleton and signaling.
3:30-5:00 PM Speck Auditorium
Chair, Cyndi Bradham
3:30 Kathleen Moorhouse, cytoskeleton and polarity
3:50 Tara Fresques: Germ cell associated gene expression is regulated by Nodal
signaling in the sea star P. miniata.
4:10 Bob Morris: Hedgehog and cilia
4:30 Andrea Bodnar Tissue homeostasis, regeneration and negligible senescence:
insight from the sea urchin
4:50 Meike Stumpp: Deuterostome origins of gastric pH regulation
Business Meeting: Lillie Auditorium 5:15 - 5:30 pm
POSTER SESSION/Mixer: 5:30-7:00 pm Swope Hall
BANQUET : 7:00 – 8:30 pm
Sunday April 27
Breakfast 7:00-8:30
Check-Out 10:00 am
- Pick up Bag Lunch
Poster Presenters
71 The small GTPase Arf6 is essential for the development of the sea urchin larval gut
Ahiakonu, Priscilla; Stepicheva, Nadezda; Dumas, Megan; Song, Jia L.
72 1-MA signaling and Hox cluster in the crown-of-thorns Acanthaster planci starfish.
Baughman, Ken; Satoh, Nori; Shoguchi, Eiichi
73 A transcriptomic strategy for identifying novel mediators of the sea urchin larval immune
response
Buckley, Katherine M.; Ho, Eric; Rast, Jonathan
74 Expression of the karyopherin-alpha family of nuclear transport proteins in Lytechinus
variegatus.
Byrum, Christine; Smith, Jason; Easterling, Marietta; Bridges, M. Catherine
75 Transcriptome Analysis of Late Gastrula Pigment Cells
Calestani, Cristina; Barsi, Julius C; Tu, Qian; Ortiz, Antonio; Buckley, Kate M; Stearnes, Ariel; Rast,
Jonathan P; Davidson, Eric H
76 mTOR regulation of polysomal recruitment at fertilization
Chassé, Héloïse; Boulben, Sandrine; Cormier, Patrick; Morales, Julia
77 SLC and Notch2 regulate dorsal-ventral PMC positioning and skeletal patterning
Chung, Oliver; Piacentino, Michael; Hewitt, Finnegan; Patel, Vijeta; Ferrell, Patrick; Chaves, James; Li,
Christy; Hameeduddin, Hajerah; Poutska, Albert; Bradham, Cynthia
78 Mesoderm and Micromere Development in Eucidaris tribuloides
Coots, Ashley D.; Covington, Rae Ann; Lung, Kara; Wood, Maureen; Sweet, Hyla
79 Expression of embryonic skeletal development genes during adult arm regeneration of the
brittle star Amphiura filiformis
Czarkwiani, Anna; Dylus, David; Oliveri, Paola
80 Polarity of small micromeres and its impact on localization of plasma membrane proteins
Espinoza, Jose A.; Campanale, Joseph; Gokirmak, Tufan; Hamdoun, Amro
81 Ophioplocus esmarki Embryo and Larvae Swimming Behavior
Freyn, Alec W.; Coots, Ashley; Sweet, Hyla
82 Identifying and measuring voltage gradients in normal and perturbed embryos
Hadyniak, Sarah E.; Schatzberg, Daphne; Lawton, Matthew L.; Bishop, Jacob; Beane, Wendy; Levin,
Michael; Bradham, Cynthia
83 Semi-dry sea urchin experiment using preserved egg and sperm supply
Kiyomoto, Masato
84 Development of the nervous system in a sea cucumber, Apostichopus japonicus: shift from
bilateral to pentaradial symmetry
Kondo, Mariko; Nagai, Akiko; Kikuchi, Mani; Omori, Akihito; Akasaka, Koji
85 Expression of germ cell markers in hemichordate Ptychodera flava: implication to the
embryonic origin of PGCs in Ambulacraria
Lin, Ching-Yi; Yu, Jr-Kai; Su, Yi-Hsien
86 BioTapestry: Interacting With the Network Directly in the Web Browser
Longabaugh, William; Paquette, Suzanne; Leinonen, Kalle
87 How pluteus arms were evolved?
Morino, Yoshiaki; Koga, Hiroyuki; Wada, Hiroshi
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88 5-LOX is Required for Skeletal Patterning in Sea Urchin Embryos
Murray, Ian S.; Patel, Vijeta; Li, Christy; Yu, Annie; Hameeduddin, Hajerah; Hewitt, Finnegan; Poustka,
Albert; Bradham, Cynthia
89 Histological and molecular biological analysis on the adult nervous system of the feather star
Oxycomanthus japonicus
Omori, Akihito; Kurokawa, Daisuke; Akasaka, Koji
90 Circadian clock in the S. purpuratus larva: a diverged time-keeping mechanism that drives 24h
rhythmicity?
Petrone, Libero; Lerner, Avigdor; Oliveri, Paola
91 Appearance of Order Level Traits in the Triassic Echinoid Fossil Record
Petsios, Elizabeth; Thompson, Jeffery; Bottjer, David
92 Concanamycin A perturbs dorsal-ventral specification in sea urchin embryos
Reidy, Patrick; Bishop, Jacob; Schatzberg, Daphne; Zushin, Peter; Ross, Erik; Carney, Tamara;
Bradham, Cynthia
93 LvBMP5-8 is required for normal skeletal patterning but not dorsal-ventral specification in the
sea urchin embryo
Ramachandran, Janani; Chung, Oliver; Piacentino, Michael; Reyna, Arlene; Yu, Jia; Hameeduddin,
Hajerah; Poustka, Albert; Bradham, Cynthia
94 H+/K+ antiport activity is required for PMC differentiation and skeletogenesis
Schatzberg, Daphne; Lawton, Matthew; Hadyniak, Sarah; Bishop, Jacob; Ross, Erik; Carney, Tamara;
Beane, Wendy; Levin, Michael; Bradham, Cynthia
95 Genome-wide analysis of the skeletogenic gene regulatory network of sea urchins Shashikant,
Tanvi; Rafiq, Kiran; Ettensohn, Charles
96 Expression of the ATP-Binding Cassette transporter Sp-ABCC5a in pigment cells is required for
sea urchin gastrulation
Shipp, Lauren E.; Hill, Rose; Moy, Gary; Gokirmak, Tufan; Hamdoun, Amro
97 microRNA-31 Regulates Skeletogenesis of the Sea Urchin Embryo
Stepicheva, Nadezda; Song, Jia
98 miR-124 Regulation of the Delta/ Notch signaling pathway
Suarez, Santiago N.; Song, Jia
99 Spatial Regulation of Gene Expression in the Skeletogenic Mesenchyme by Extrinsic Cues Sun,
Zhongling; Ettensohn, Charles
100 A probabilistic modeling of the cell lineage highlights interindividual variability in
Paracentrotus lividus early development
villoutreix, paul; Rizzi, Barbara; Delile, Julien; Duloquin, Louise; Faure, Emmanuel; Savy, Thierry;
Bourgine, Paul; Peyriéras, Nadine
101 The embryonic transcriptome for Lytechinus variegatus
Zuch, Daniel; Hogan, J.D.; Keenan, Jessica; Luo, Lingqi; Saji, Akhil; Sundermeyer, Mary Ann;
Piacentino, Michael; Schatzberg, Daphne; Azzizi, Elham; Zhang, Shile; Heilbut, Adrien; Poustka, Albert;
Bradham, Cynthia
Page 8 of 51
Talk Abstracts
1 Use of regulatory complexity in gut organogenesis
Peter, Isabelle (Cal-Tech); Davidson, Eric; Valencia, Jonathan
So far no gene regulatory network has been solved for any animal organogenesis process, the reason
being both the enormous regulatory complexity underlying the formation of most organs and the
challenges of experimental network analysis. The advanced state of GRN analysis in sea urchin
embryos thus provides a unique opportunity to approach such large scale GRNs. We are particularly
interested in solving the genomic program for gut development, because of its importance to animal
development and evolution. GRN analysis for a process as complex as the formation of a
compartmentalized gut however requires strategies different from the analysis of smaller pre-gastrular
GRNs. To assess the dimensions of the gut GRN, we have analyzed the spatial expression of a majority
of regulatory genes encoded in the sea urchin genome and expressed during the time from beginning of
gastrulation up to 72h. The results show that more than 190 regulatory genes are expressed in the
developing gut. The function of this so far unknown GRN is to specify within the anterior and posterior
endoderm at least 25 distinct regulatory domains which pattern the mature gut at 72h. Thus even to form
the relatively simple larval gut in sea urchin embryos requires a large part of the regulatory system
encoded in the genome.
2 Spatial regulatory states in the ectoderm.
McClay, David R. (Duke University)
Two new territories of transcription factor expression have been identified in the ectoderm of the sea
urchin embryo Lytechinus variegatus. The first region is a band of cells about 4 cells wide immediately
adjacent to the endoderm. Wnt5 from the endoderm induces the border ectoderm via short range
signaling. Nodal and BMP signaling then subdivides this band of ectoderm into dorsal and ventral zones.
Perturbation experiments establish that the border ectoderm releases signals necessary for skeletal
patterning. Especially important is a group of cells at the intersection of the border ectoderm and the
dorsal-ventral margin. VEGF and FGF are produced at that intersection causing the ventrolateral cluster
of PMCs to form immediately beneath, and causing initiation of skeletogenesis. The second territory of
specialized gene expression is initiated by expression of ectodermal SoxC in a group of cells
immediately above the ventral border ectoderm. That group of cells migrates to the ciliary band and
differentiates as neurons. Perturbation of SoxC eliminates all neurons selectively, including the neurons
originating in the ventral ectoderm. The ventral ectodermal SoxC pre-neurons also express
achaete-scute and later express a series of known neural markers. This territory of pre-neural cell origin
is distinct from the apical ectoderm where other neurons are known to originate. A large number of
perturbation studies, guided in part by the ectodermal gene regulatory network model, establish SoxC as
an early transcription factor in pre-neural cells.
3 A maternal determinant of the dorsal-ventral axis in Paracentrotus lividus?
Lepage, Thierry (CNRS); Haillot, Emmanuel (Institute of Biology Valrose)
Classical studies showed that, unlike the animal-vegetal axis, the dorsal-ventral axis of the sea urchin
embryo is not pre-established rigidly in the egg. Consistent with this idea, specification of the
dorsal-ventral axis critically relies on cell interactions and on the zygotic expression of nodal. Nodal
expression starts around the 32-60-cell stage and is first very broad, before being restricted to the
Page 9 of 51
ventral ectoderm. It has been proposed that Redox gradients generated by asymmetrically distributed
mitochondria, and possibly acting through p38, may provide the initial spatial cue that initiates nodal
expression or alternatively, that Redox gradients may be required for the spatial restriction of nodal.
However, manipulating redox gradients has only modest effects on the orientation of the D/V axis and
how the polarized expression of nodal is established is not well understood. We discovered a maternal
factor that we named Panda (Paracentrotus Anti Nodal Dorsalizing Activity) that directs the orientation of
the D/V axis by controlling the spatial expression of nodal. panda transcripts are broadly distributed, with
a slight gradient present in a fraction of the embryos. Embryos injected with morpholino oligonucleotides
targeting maternal panda transcripts failed to restrict nodal expression. Misexpression of panda oriented
the dorsal-ventral axis in nearly 100% of the injected embryos. Finally, rescue experiments
demonstrated that the activity of Panda is required locally in the early embryo. These findings identify
Panda as a maternal factor that is both required for the spatial restriction of nodal and sufficient to orient
the dorsal-ventral axis when misexpressed. Panda therefore fulfils some of the requirements for a
maternal determinant of the D/V axis. These results suggest that, although the dorsal-ventral axis of the
sea urchin embryo is not rigidly fixed, this axis may be prefigured in the egg in the form of a broad
gradient of this maternally deposited factor.
4 Sea urchin GRN model
Davidson, Eric (CalTech)
The sea urchin embryo GRN up to gastrulation has now been expanded to include all but the apical
neurogenic domain and the mesenchyme blastula stage oral/aboral mesoderm domains. Thus it
becomes possible to consider the form of a global, genomically encoded network that encompasses the
majority of a developing organism. I briefly discuss both the recurrent overall characteristics of the global
developmental program explicit in this GRN, and the interesting special features of certain of its
subregions.
5 Developmental systems drift: making similar embryos with divergent genomes.
Lemaire, Patrick (CRBM, UMR5237, CNRS/University Montpellier; Institut de Biologie
Computationnelle, Montpellier, France)
The relationships between genotype and phenotype during evolution are complex and poorly
understood. Surprisingly dissimilar genotypes and developmental programmes can translate into very
similar phenotypes. This may explain how some species can remain morphologically similar for long
periods of time in spite of extensive genome divergence. It also highlights that morphological similarity
does not necessarily reflects molecular homology, a phenomenon that should be taken into
consideration when extrapolating to Man results obtained with mammalian model organisms. Ascidian
embryos constitute a remarkable system to study morphological stasis. Slow evolution of their
stereotyped embryonic morphologies, based on invariant cell lineages, allows comparison of the same
developmental processes across hundreds of millions of years. Extreme genome intra-specific
polymorphism and inter-specific divergence suggests an astounding level of plasticity in the underlying
developmental pathways. During the talk, I will present the computational tools that we are developing in
collaboration with the Virtual PLant research team (Montpellier) to quantify embryonic morphologies and
their variability from light-sheet microcopy recordings of live embryos. I will also give a preliminary
assessment of genome and transcriptome divergence within and between ascidian genera. I will
conclude by presenting cis-regulatory mechanisms that explain, in part, how divergent genomes can
support morphological invariance.
Page 10 of 51
6 Embryonic Neurogenesis
Burke, Robert D. (University of Victoria)
Urchins appear to share many of the common features of neurogenesis of metazoan embryos. The
morphological aspects of the differentiation phase of neurogenesis in urchin embryos are documented,
however, our understanding of neural specification and the regulation of proneural networks is
fragmentary. This is in part due to a lack of early markers for neural progenitors. Sox B2 is expressed
broadly in oral ectoderm and ciliary band and some, but not all Sox B2 expressing cells become
neurons. Six3 is necessary for neural specification, but expression is transitory and it does not serve to
identify neural progenitors. In blastulae, Sox C is expressed in vegetal mesendoderm cells of the animal
pole domain, and during gastrulation it appears in cells scattered throughout the oral ectoderm. SoxC
has several of the features of a marker for neural progenitors. Delta-Notch signaling is a common
feature of metazoan neurogenesis that produces committed progenitors and it appears to be a critical
phase of neurogenesis in urchin embryos. We are attempting to determine the critical functions of these
neurogenic proteins to develop a model that articulates the gene regulatory networks with the cellular
context in which neural development occurs. Urchins are a facile, but powerful model with the potential
of revealing many shared and derived features of deuterostome neurogenesis.
7 Gradients and networks that pattern the Drosophila embryo
Small, Stephen (New York University); Chen, Hongtao (New York University); Ochoa-Espinosa,
Amanda (New York University); Xu, Zhe (New York University); Cao, Jinshuai (New York University)
The early Drosophila embryo develops as a syncytium of nuclei, and spatial patterning of the embryo is
controlled by a well-characterized network of transcription factors. Here we focus on the patterning
activities of the Bicoid transcription factor, which is distributed in a long-range gradient along the anterior
posterior axis. Previous studies suggest that Bcd functions as a “morphogen”, which establishes multiple
gene expression boundaries by concentration-dependent activation mechanisms. We have used in vivo
gradient manipulations and an extensive analysis of Bcd-dependent enhancers to critically test this
hypothesis. Our results strongly suggest that most target genes do not respond to specific concentration
thresholds within the gradient. Rather, target gene boundaries are positioned primarily by repressive
gradients that antagonize Bcd-dependent activation. Thus, embryo organization is more accurately
described as an emergent property of the whole network of interacting genes.
8 Pathways Regulating Stem Cell Induction, Self-Renewal and Engraftment
Zon, Leonard (HHMI/Boston Children's Hospital)
Hematopoietic stem cell transplantation involves the homing of stem cells to the marrow, an active
process of engraftment, and the self-renewal of the blood stem cells. We have been using the zebrafish
as a model to study the molecular biology of this process. By imaging RUNX1 GFP+ cells arriving in the
next site of hematopoiesis (the caudal hematopoietic territory), engraftment can be visualized. This
process involves an attachment phase and then an extravasation to the abluminal side of the endothelial
cells. The endothelial cells cuddle the hematopoietic stem cell and the stem cells have the ability to be
maintained in a quiescent fashion or to divide symmetrically or asymmetrically. Using chemical screens,
we have found small molecules that can enhance engraftment or suppress engraftment. We also have
developed a new technique of culturing zebrafish blastomeres and examining tissue differentiation. Our
initial work led to a chemical screen that demonstrated that activation of the FGF, wnt, and cAMP
pathways stimulate muscle development. A cocktail of chemicals found in our zebrafish culture assays
was sufficient to reprogram human iPS cells to skeletal muscle. The system has now been used to
reprogram neural crest cells and blood stem cells. Our studies have found fundamental aspects of stem
cell biology that may be therapeutically useful for patients.
Page 11 of 51
9 Chuck Ettensohn
10 Athula Wikramanayaka
11 Amro Hamdoun
12 Evolution of post transcriptional mechanisms for germ line segregation in echinoderms
Swartz, S. Zachary (Brown University Molec Biology, Cell Biology & Biochemistry); Reich, Adrian
(Brown University); Oulhen, Nathalie (Brown University); Raz, Tal (Helicos Biosciences); Milos, Patrice
(Helicos Biosciences); Campanale, Joseph (UCSD Scripps); Hamdoun, Amro (UCSD Scripps); Wessel,
Gary (Brown University)
A critical event in animal development is the specification of primordial germ cells (PGCs), which
become the stem cells that create sperm and eggs. Germ line segregation can be categorized within a
continuum of inherited and inductive mechanisms. The inherited mode, typified by organisms such as
the fruit fly Drosophila, involves spatial localization of maternally supplied molecular determinants,
collectively called a germ plasm. The embryonic cells that inherit this material are thus directed toward
germ line fate. Animals such as the mouse do not employ a germ plasm, but rather rely upon inductive
signaling between tissue layers to specify germ cells. We find that the PGCs of the sea urchin
Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of
inherited mRNAs. Enrichment of several germ line determinants in the PGCs requires the RNA binding
protein Nanos to deplete the transcript encoding CNOT6, a deadenylase, in the PGCs, thereby creating
a stable environment for RNA. Alteration of CNOT6 levels in the PGCs and somatic cells results in their
failure to selectively retain Seawi transcripts and Vasa protein. We propose a "time-capsule" model of
germ line determination and continuity of a cryptic germ plasm in the sea urchin, which has classically
been thought of as an inductive embryo. Comparisons with the pencil urchin, sea star, and sea
cucumber suggest Nanos depletion of CNOT6 is a conserved mechanism within echinoderms. In
addition to this primary inherited mechanism, the sea urchin embryo is capable of compensating for the
loss of its germ line under certain conditions. We identify z426, a maternally supplied putative RNA
binding protein, as a potential mediator for this process.
13 Quantification of Exocytosis Kinetics by DIC Image Analysis of Cortical Lawns
Poccia, Dominic L. (Amherst College)
Cortical lawns prepared from sea urchin eggs (Vacquier in Dev Biol 43:62–74, 1975) have served for
many years as a robust in vitro system for study of regulated exocytosis and membrane fusion events.
Lawns have been imaged by various microscopy techniques and quantification of exocytosis kinetics
has been achieved primarily with specialized light scattering techniques and flow cells. We present
simple differential interference contrast image analysis procedures for quantifying the kinetics and extent
of exocytosis in cortical lawns using an open vessel that allows rapid solvent equilibration and
modification. These preparations maintain biologically relevant architecture of the original cortices, allow
for cytological and immunocytochemical analyses, and permit quantification of variation within and
between lawns. This simple technique should allow investigations into the contributions of proteins and
phospholipids of endoplasmic reticulum, granule and plasma membranes in regulation of exocytosis.
14 Hedgehog signaling functions during sea star development
Zazueta-Novoa, Vanesa (Brown University); Wessel, Gary (Brown University)
The Hedgehog pathway controls a wide variety of developmental processes. One such role for Hh
signaling is in the development of the gut during embryonic development. Hedgehog (Hh) is a secreted
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protein that is enzymatically modified to make it active and it binds to its receptor, Patched (Ptch). In
absence of Hh, Ptch acts as an inhibitor, binding and blocking Smoothened (Smo) to activate the
pathway. When Hh is present, it binds to Ptch, releasing the inhibition of Smo. Smo antagonizes
kinesin-related protein Costal 2 (Co2) activity, preventing the transcription factor cubitus interruptus (Ci
in Drosophila or Gli in vertebrates) from cleavage. Then, full-length Ci protein proceeds to the nucleus
where it activates transcription. In sea urchin, Hh expression profiles showed that Hh transcripts are
enriched in the endoderm, suggesting that Hedgehog signal is produced by the endodermal tissue of the
archenteron and function in formation of the mesodermal tissues. By addition of cyclopamine, sea urchin
embryos revealed several defects, but the most relevant was the disruption of coelomic pouch
morphogenesis. In cyclopamine-treated embryos, coelomic pouch cell progenitors at the tip of the
archenteron did not split in two groups and relocate laterally to form the coelomic pouches as they do in
normal conditions. Instead these cells remained atop the archenteron and formed one pouch-like
structure. We studied the Hh signaling pathway in sea star and we identified two Hh sequences, two
Ptch sequences and one Smo sequence using a de novo transcriptome database. We analyzed their
expression profiles and the effect on early development using cyclopamine. At lower concentrations,
cyclopamine incubation of the embryos caused a general delay in the development of the larva and at
higher concentrations, it caused defects in gut formation or death of the larvae as it was reported
previously in sea urchin. However, defects in archenteron and gut patterning are being addressed.
15 Nanos protein is specifically retained in the small micromeres.
Oulhen, Nathalie (Brown University); Wessel, Gary (Brown University)
Nanos is a translational regulator required for the survival and maintenance of primordial germ cells
during embryogenesis. It is usually expressed uniquely in germ cells, and is often "toxic" if active
elsewhere in the embryo. Three nanos homologs are present in the genome of the sea urchin
Strongylocentrotus purpuratus (Sp),each nanos mRNA accumulates specifically in the small micromeres
(SMM), the lineage that contributes to the germline. Sp nanos2 3’UTR is sufficient for RNA retention and
protein accumulation selectively in the SMM lineage. We found that an additional mechanism regulates
Sp nanos2 expression. Sp nanos2 ORF also leads to a selective reporter enrichment in the small
micromeres, independently of the 3’UTR. Our results suggest that post translational modifications such
as sumoylation, ubiquitination, or acetylation do not affect nanos expression. Moreover, mutations in
nanos zinc finger domains do not affect the protein stability in the SMM. We previously showed that
nanos expression is controlled by a combination of selective RNA retention and translational control
mechanisms, we are now adding an additional level of regulation affecting its protein stability.
16 Sea urchins as models for the construction of prototypic embryonic development
Peyrieras, Nadine (CNRS); Fabreges, Dimitri (CNRS); Barbara, Rizzi (CNRS); Villoutreix, Paul (CNRS);
Delile, Julien (CNRS); Savy, Thierry (CNRS); Duloquin, Louise (CNRS); Suret, Pierre (Lille University);
Doursat, René (Drexel Unviversity); Bourgine, Paul (CNRS)
On the way to a better understanding of embryonic development, we aim at systematizing a quantitative
and modeling approach of developing organisms based on live imaging and automated image
processing. Sea urchins should be ideal models for a proof of concept, thanks to their egg accessibility,
small size, rapid embryonic development and relative transparency. Most importantly, several species
have been extensively explored at the cellular, genetic and molecular level, and an impressive amount
of data is available to be assimilated in integrative models. Immobilization of the embryos to image them
in toto by state of the art 2-photon laser scanning or selective plane imaging microscopy, remains
however a major issue. Our current model explored the space of phenotypic features in Paracentrotus
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lividus at blastula stages. A small cohort of individuals was used to propose probabilistic models for cell
behavior and their integration in a spatial multi agent model was developed to assess biomechanical
constraints shaping the blastula. The comparative study of Paracentrotus lividus and Sphaerechinus
granularis aims at quantifying intra- and inter-specific variability. S. granularis has the advantage to
undergo primary mesenchymal cells ingression prior swimming. It thus appears as an interesting model
for the quantitative approach of the variation components in the twin paradigm. The development of
twins obtained by the classical separation of 2-cell stage blastomeres challenges our understanding of
normal development. Again, the need for cell lineage analysis points to the usefulness of a quantitative
automated approach based on live imaging. We expect that the community will synergize in exploiting
an open source database of digital specimens and make it grow by using the BioEmergences
webservice for 3D+time data processing.
17 ATP-binding cassette transporters mediate small micromere migration and left/right coelomic
pouch segregation in Strongylocentrotus pupuratus
Campanale, Joseph P. (Scripps Institution of Oceanography); Espinoza, Jose (Scripps Institution of
Oceanography); Gokirmak, Tufan (Scripps Institution of Oceanography); Hamdoun, Amro (Scripps
Institution of Oceanography)
One function of ATP-binding cassette (ABC) transporters is to establish morphogenetic gradients in
embryos. The small micromeres (Smics) undergo a 65% reduction in ABC-transporter activity at
formation, presumably to concentrate morphogenetic signals necessary for segregation between the two
coelomic pouches (CPs). In controls, an average of five Smics are found in the left coelomic pouch (CP),
while embryos grown in the presence of ABC-transporter inhibitors have random left/right Smic
distributions. To test how ABC transporters control segregation, we fused Sp-ABCB1a to Sp-nanos
UTRs and selectively overexpressed it in Smics. In these embryos a dose-dependent increase in the
number of Smics in the left CP was observed, while targeted expression of membrane markers and null
mutants of ABCB1a did not alter Smic distribution. These results suggest that the expression of ABCB1a
in Smics disrupts their left/right migration, possibly by dampening the detection of chemoattractants. We
recently showed that Smics transition from non-motile epithelial cells to motile quasi-mesenchymal cells
during gastrulation. Smics in gastrulae are embedded in the tip of the archenteron, but remain motile
and develop cortical blebs and dynamic filopodia that contact ectoderm. Smics in prism larvae move in
the plane of the blastoderm towards the coelomic pouches. Using this information, we are currently
investigating whether ABC-transporter activity disrupts Smic migration by altering bleb and filopodia
dynamics. Preliminary results indicate Smics expressing ABCB1a in vitro extend smaller blebs and fewer
filopodia than Smics expressing non-transporter membrane proteins. Collectively, our findings suggest
that ABC-transporter mediated secretion of morphogens controls Smic migration leading to their left/right
patterning. We speculate that Smic left/right patterning is linked to ABC-transporter mediated Smic
migration dynamics.
18 Inhibition of the Arp2/3 Complex in Sea Urchin Coelomocytes Induces a Lamellipodial to
Filopodial Shape Change and Alters the Cell Spreading Process
Henson, John (Dickinson College); Goldson, Brandon (Dickinson College); Patterson, Rebecca
(Dickinson College); Shen, Eileen (Dickinson College); Brown, Briana (Dickinson College); Medrano,
Angela (Dickinson College)
Our previous studies on sea urchin coelomocytes have indicated that Arp2/3 complex-facilitated actin
filament polymerization is crucial for structuring the dendritic actin filament network characteristic of the
peripheral lamellipodial region, for generating the pushing force component of actin-based centripetal
flow, and for closing cytoplasmic wounds. In the present study live cell, fluorescence, and 3D structured
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illumination super-resolution light microscopy, as well as critical-point-dry and rotary shadow
transmission electron microscopy were used to study the impact of treating coelomocytes in suspension
with the specific inhibitor of the Arp2/3 complex, CK666. In adherent cells treated with CK666 the
dendritic network of actin filaments present in the lamellipodium is replaced by an array of transversely
oriented arcs of elongate actin filaments. In contrast, when coelomocytes are treated in suspension with
CK666 they undergo a radical morphological transformation from a lamellipodial to highly filopodial form.
In addition, when these filopodial cells are allowed to settle onto a glass substrate their spreading
process proceeds via a novel structural reorganization of actin involving the presence of concentric
circle patterns of elongate actin filaments in regions of filopodial spreading. Comparison of these cells
with control cells in which spreading takes place in the absence of CK666 indicates that the rate of the
overall spreading process is also significantly slowed by the presence of the inhibitor. These results
demonstrate that Arp2/3 complex-facilitated actin polymerization is essential for the maintenance of the
lamellipodial form of coelomocytes in suspension and for the normal spreading of these cells on a
substrate. They also indicate that cells retain the ability to spread even in the absence of the machinery
necessary to generate the dendritic array of branched actin filaments present in lamellipodia.
19 General approach for in vivo recovery of cell type specific effector gene sets
Barsi, Julius C. (Caltech); Tu, Qiang (Caltech); Davidson, Eric (Caltech)
Differentially expressed, cell type specific effector gene sets hold the key to multiple important problems
in biology, from theoretical aspects of developmental gene regulatory networks (GRNs) to various
practical applications. Although individual cell types of interest have been recovered by various methods
and analyzed, systematic recovery of multiple cell type specific gene sets from whole developing
organisms has remained problematical. My presentation will delineate general methodology aimed at
obtaining this biological information and is applicable to all model organisms for which transgenic tools
are available. It has been devised using the sea urchin embryo, material of choice because of the
large-scale GRNs already solved for this model system. The method utilizes the regulatory states
expressed by given cells of the embryo to define cell type, and includes a Fluorescence Activated Cell
Sorting (FACS) procedure that results in no perturbation of transcript representation. I have extensively
validated the method by spatial and qualitative analyses of the transcriptome expressed in isolated
embryonic skeletogenic cells and as a consequence, generated a prototypical cell type specific
transcriptome database: http://www.spbase.org:3838/cellspecific/
20 An anterior signaling center patterns and sizes the anterior neuroectoderm territory of the sea
urchin embryo
Range, Ryan (Mississippi State University); Angerer, Robert (NIDCR, National Institutes of Health);
Angerer, Lynne (NIDCR, National Institutes of Health)
Anterior signaling centers are essential to specify and pattern the early anterior neuroectoderm (ANE) in
several deuterostome embryos. In the sea urchin embryo, the early ANE is restricted to the anterior end
of the late blastula-stage embryo where it separates into inner and outer regulatory domains expressing
the cardinal ANE transcriptional regulators, FoxQ2 and Six3, respectively. This patterning process is
driven by FoxQ2, which is required to eliminate expression of six3 from the inner domain. FoxQ2 also
activates the expression of two secreted Wnt regulators, sFrp1/5 and Dkk3, the activities of which define
the correct sizes of the inner and outer ANE territories. Furthermore, the levels of sFrp1/5 and Dkk3 are
rigidly maintained via auto-repressive and cross-repressive interactions with Wnt signaling components
and ANE transcription factors. Our data support a model in which Six3 and FoxQ2 establish an anterior
patterning center that ensures correct ANE patterning and border positions. Comparisons of functional
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and expression studies in sea urchins, hemichordates and vertebrates show striking similarities in
deuterostome ANE regulatory states and the molecular mechanisms that position and define its borders.
These data provide strong support for the idea that the sea urchin embryo uses an ancient anterior
patterning system that was present in the common ambulacrarian/chordate ancestor.
21 A Comprehensive Survey of wnt and frizzled Expression in the Sea Urchin Paracentrotus
lividus
Croce, Jenifer (CNRS); Robert, Nicolas (UPMC); Lhomond, Guy (CNRS); Schubert, Michael (CNRS)
WNT signaling is, in all multicellular animals, an essential intercellular communication pathway that is
critical for shaping the embryo. At the molecular level, WNT signals can be transmitted by several
transduction cascades, all activated commonly by the binding of WNT ligands to receptors of the
FRIZZLED family. The first step in assessing the biological functions of WNT signaling during
embryogenesis is thus the establishment of the spatiotemporal expression profiles of wnt and frizzled
genes during development. To this end, using qPCR, Northern blot and in situ hybridization assays, we
have thus determined the comprehensive expression patterns of all eleven wnt and four frizzled genes
present in the genome of the sea urchin Paracentrotus lividus throughout its embryogenesis. Our
findings indicate that the expression of these wnt ligands and frizzled receptors is highly dynamic in both
time and space. We further establish that all wnt genes are chiefly transcribed in the vegetal hemisphere
of the embryo, whereas expression of the frizzled genes is distributed more widely across the embryonic
territories. Thus, in P. lividus, WNT ligands might act both as short- and long-range signaling molecules
that may operate in all cell lineages and tissues to control various developmental processes throughout
embryogenesis.
22 Pigment cell migration: The role of Eph-Ephrin signaling
Krupke, Oliver (University of Victoria)
Studies on mesenchyme specification in the sea urchin have produced one of the most clearly defined
gene regulatory networks to date and there is an ongoing effort to characterise effectors that relay this
genetic information to produce morphological changes and cell movements that further define cell fate.
Following specification and epithelial-mesenchyme transition, mesenchyme cells ingress into the embryo
and migrate to specific embryonic regions where they differentiate to skeletonogenic or non-skeletogenic
fates. Pigment cells are specialized, echinochrome-containing, non-skeletogenic secondary
mesenchyme with apparent roles in innate immunity. Using targeted gene knockdown, direct inhibition of
Eph kinase and ectopic expression of Ephrin, we show Eph-Ephrin signaling is necessary and sufficient
for pigment cell migration, directing them from within the blastocoel to positions adjacent the aboral
basement membrane where they extend processes into the overlying ectoderm. Using
immunofluorescence, we identify a putative mesenchyme-epithelial transition in pigment cells following
contact with the basal lamina and pigment cells exhibit a number of features characteristic of an MET
event. Combined our results highlight a developing model for MET wherein the upstream and
downstream components can be visualized in an easily manipulated embryo.
23 Mechanisms of Small Micromere Homing
Martik, Megan L. (Duke University); McClay, David R. (Duke University)
The small micromeres arise at the vegetal pole from an unequal 5 th cleavage, and their progeny are
specified to become the primordial germ cells. Throughout gastrulation, small micromeres actively
extend filopodia and lamellipodia at the tip of the gut though they continue to remain part of the
invaginating epithelium and express LvG-cadherin. Once gastrulation nears completion, the tip of the gut
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undergoes basement membrane remodeling that facilitates the small micromeres’
epithelial-mesenchymal transition (EMT) and migration over the archenteron to the posterior halves of
the forming coelomic pouches. We show that the small micromeres reach the coelomic pouches via a
directed homing mechanism. Ectopically placed small micromeres are able to find their way home to the
coelomic pouches from any starting position in the embryo. Spatially and temporally misplaced 16-cell
stage micromeres also home upon insertion into the blastocoel of a late gastrula host. The small
micromeres home directly to the non-skeletogenic mesoderm (NSM) lineage of the forming coelomic
pouch. Small micromeres are directed to the NSM via evolutionarily conserved signaling and
chemoattractant mechanisms. When the NSM is not specified, the small micromeres are unable to
home. Transcription factor knockdowns within the NSM lineage shed light on the regulatory
underpinnings of homing mechanisms. By using the robust homing behavior of these cells, we
uncovered a transcriptional circuitry responsible, in part, for directed homing mechanisms of germ cells.
Current aims are to connect the underlying transcriptional regulation of the signaling with the
chemoattractant mechanisms by which primordial germ cells undergo such a dramatic feat of finding
their way home.
24 Transcriptional control of immune cell development in the purple sea urchin embryo
Schrankel, Catherine S. (University of Toronto)
Immune response in the purple sea urchin larva is mediated by granular pigment cells and several
derivatives of the blastocoelar cells. Blastocoelar immunocytes develop from an oral patch of NSM cells
in the 24-hr embryo that co-express orthologs of the vertebrate hematopoietic stem cell transcription
factors Gata1/2/3, Scl/Tal-2/Lyl-1 and Erg/Fli-1. Upon down-regulation of these genes, immunocyte
precursors initiate epithelial-to-mesenchymal transition (EMT), undergo limited cell division and begin to
express subsets of immune differentiation markers. When SpGata123 is perturbed, precursors fail to
migrate and terminal gene expression is greatly reduced. SpGata123 itself appears to provide negative
feedback into this system (as does Gata1 in vertebrate stem cells). Thus in the sea urchin embryo, the
co-expression of SpGata123, Scl, Erg/Fli-1 may affect a transient multipotent state that switches towards
immune differentiation upon the down-regulation. Targets of SpGata123 in immunoctye precursors may
include EMT genes and upstream regulators of terminal gene batteries. Class I bHLH transcription
factors also play a role in this system, including SpE-protein, the single homolog of vertebrate
E2A/HEB/ITF2 that is the relevant binding partner of Scl, and SpId, a negative regulator of class I BHLH
activity. We have identified an alternative start in SpE-protein, also found in vertebrate HEB and ITF2.
This is the first such domain to be characterized in an invertebrate. SpEalt is expressed specifically and
dynamically in subsets of the developing NSM. As sea urchins are evolutionarily situated at the base of
the deuterostomes, the regulatory links between SpGata123 down-regulation and E-protein activity to
programs of immunocyte EMT and differentiation may be conserved in a minimal gene network that is
ancient to deuterostome immune cell development.
25 Insights from amphioxus and lamprey into the evolution of vertebrate head skeleton
development
Medeiros, Daniel (University of Colorado, Boulder)
A defining feature of vertebrates (craniates) is a pronounced head supported and protected by a robust,
cellular endoskeleton. In the first vertebrates, this skeleton was likely built of collagenous cellular
cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless
and cartilaginous fish. In the head, most cellular cartilage is derived from the neural crest, a migratory
cell population that arises from the edges of the central nervous system (CNS). Because collagenous
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cellular cartilage and neural crest cells (NCC) have never been described in invertebrates, the
appearance of NCC-derived cellular cartilage is considered a turning point in vertebrate evolution.
Using new methods for the continuous lab culture of the cephalochordate Branchiostoma floridae, we
find that a skeletal tissue displaying essentially all of the core conserved features of vertebrate cellular
cartilage forms transiently during metamorphosis of this species. We also present evidence that a key
regulator of collagenous cellular cartilage development, SoxE, gained new cis-regulatory sequences
during vertebrate evolution to direct its novel expression in NCC. Together these results suggest that
the origin of the vertebrate head skeleton was not dependent on the evolution of a qualitatively new
skeletal tissue, but on the spread of this tissue throughout the head. We further propose that the
evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major
factor in the evolution of the vertebrate head skeleton.
26 Ophiuroid skeletogenesis in development and regeneration
Oliveri, Paola (University College London)
Calcitic endoskeleton is a fundamental character of echinoderms. In all adults it is represented as
mesh-like ossicle units, while in larvae it is prominently formed as radiate spicules only in two classes:
Echinoidea and Ophiuroidea. The regulatory program for the development of the sea urchin larval
skeleton has been extensively studied and provides an excellent framework to study evolution of
regulatory networks governing cell type specification. Very little is known about skeletogenesis in
ophiuroids. I will describe the effort we are undertaking to develop molecular resources and
experimental approaches to study skeletogenesis during the development and adult arm regeneration of
the ophiuroid Amphiura filiformis. From a developmental transcriptome, we isolated and studied the
spatio-temporal expression of more than 25 genes orthologous of sea urchin developmenatal
skletogenic (SM) and non skeletogenic mesodermal (NSM) genes. By early blastula stage, a group of
cells, within the vegetal plate, show a SM molecular signature by expressing genes such as alx1 and
p19. Later they will be the first ingressing cells and will secrete two bilaterally arrayed spicules. Despite
a rather similar embryonic development compared to sea urchin, A. filiformis shows several differences
in regulatory states of both SM and NSM cells, suggesting different network linkages. During arm
regeneration different skeletal elements are formed at different moment of the regeneration process.
Cells in sub-epithelial position in the blastema, where skeleton primordial are formed, show similar
regulatory states to the embryonic SM cells, indicating the usage of a similar embryonic SM network
module. On the contrary, at later stages of regeneration different skeletal elements have different
regulatory states.
27 The role of Nodal signaling in axis formation of the hemichordate Saccoglossus kowalevskii
Lowe, Chris (Stanford University); Wlizla, Marcin (University of Chicago); Gonzalez, Paul (Stanford
University); Darras, Sebastien (Observatoire Oceanologique de Banyuls)
The role of Nodal signaling during the early embryonic development of chordates and echinoderms is
well characterized. This ligand plays key roles in the early establishment of the embryonic axes in both
groups. While there are similarities in the role of Nodal signaling in dorsoventral and left/right patterning
between groups, further comparative data from hemichordates are required for a more comprehensive
understanding of the evolution of the this signaling pathway during early deuterostome evolution. We
present functional data on the role of Nodal signaling in the direct-developing hemichordate
Saccoglossus kowalevskii during early axis formation. There are three Nodal ligands in S. kowalevskii
representing a recent diversification of this ligand family. Two of the ligands are expressed in the
endomesodermal precursors at early blastula, and a third in the prospective posterior ectoderm in later
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blastula and through gastrulation. We present both specific knockdown by RNAi and inhibitor
experiments, and over-expression by mRNA injection and protein incubations, to disrupt Nodal signaling
and assay effects on early axis formation.
28 Cis-reg logic of Ese and Prox genes underlying output in blastocoelar mesoderm founders.
Ransick, Andrew (Caltech); Davidson, Eric (Caltech)
In euechinoids, the induction mechanism which initially specifies the chromogenic mesoderm founders
occurs relatively early compared to establishment of the other indispensible non-skeletogenic
mesodermal cell types of the pluteus larva, which include the pleisiomorphic mesodermal lineages for
muscle cells, blastocoelar mesenchyme and coeloms. Two significant consequences of this specification
sequence for the architecture of the mesodermal GRN are, firstly, that primary non-skeletogenic
mesodermal territory founders all activate the regulators GCM and GataE, which initially biases them
toward the pigment cell fate. Second, mechanism(s) must exist to redirect specification of a portion of
these non-skeletogenic mesodermal founders toward the other non-pigment cell fates mentioned above.
Cis-regulatory analyses of the regulators Ese and Prox, which are expressed exclusively in this
secondarily established cohort of non-pigment cell mesodermal founders, are providing insights into the
early mesoderm GRN that directs establishment of mesodermal sub-lineages.
29 Asymmetrical stabilization of hypoxia inducible factor a during sea urchin embryogenesis
Su, Yi-Hsien (Institute of Cellular and Organismic Biology, Academia Sinica)
Establishment of the dorsal-ventral axis during early development is a crucial step for bilaterians to build
their bilateral symmetric bodies. In sea urchin embryos, unequal distribution of mitochondria is important
for patterning their oral/ventral-aboral/dorsal axis. The prospective oral side contains more mitochondria
and is more oxidizing than the aboral side. Hypoxia has been shown to impair the specification of the
axis and result in a radially symmetric embryo. Hypoxia inducible factor α (Hifα) is a transcription factor
that is stabilized when cells are exposed to hypoxia whereas it is degraded under normaxia when its
proline residues are hydroxylated by hydroxylase enzymes. Previously we demonstrated that sea urchin
Hifα (SpHifα) initiates the gene regulatory network controlling the specification of aboral ectoderm.
However, SpHifα transcript is maternally deposited and distributed ubiquitously in the early embryo and
how its gene product selectively activates the aboral genes is unknown. Here we show that SpHifα is
asymmetrically stabilized and activated on the aboral side of the blastula embryo. Hypoxia and
hydroxylase inhibitor DMOG both abolish the asymmetrical distribution of SpHifα. SpHifα proteins also
become symmetrically distributed when their proline residues are mutated. Within embryo clusters that
the redox gradient is re-established from the inside to the outside, SpHifα proteins are predominantly
localized in the inside that later tends to become the aboral side of the embryo. These results suggest
that the hydroxylation and degradation machinery acting on SpHifα is activated on the oral side of the
embryo and the asymmetrical stabilization of SpHifα on the aboral side is regulated by the redox
gradient. The differential activation of Hifα in the sea urchin embryo thus provides an example of its
fundamental role during normal embryogenesis.
30 Gene Regulatory Network Governing 2-Dimensional Expression Patterns in the Sea Urchin
Ectoderm
Li, Enhu (Caltech); Cui, Miao (California Institute of Technology); Peter, Isabelle (California Institute of
Technology); Davidson, Eric (California Institute of Technology)
Regulatory state boundary formation is a general process in early development, in which embryonic
territory is divided up into spatial domains which express distinct sets of regulatory genes. We establish
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the mechanistic principles by which multiple orthogonal boundaries of this kind are progressively formed
on the oral side of the sea urchin embryo, according to an encoded genomic program. These boundaries
separate prospective endoderm from ectoderm domains, neurogenic from non-neurogenic domains,
ciliated band from oral ectoderm domains, and produce an orthogonal grid of regulatory states.
Boundary formation invariably depends on spatial transcriptional repression superimposed on more
widespread domains of transcriptional activation.
31 A global assessment of Wnt family functions in the sea urchin embryonic gene regulatory
networks
Cui, Miao i. (Caltech)
Wnt signaling affects cell fate specification processes throughout development. Here we take advantage
of the well-studied gene regulatory networks (GRNs) for sea urchin embryogenesis to investigate the
regulatory functions of the Wnt signaling system during pre-gastrular development. We show that all five
expressed wnt genes display similar features of expression: spatially dynamic early expression, later
restriction to posterior endoderm and/or vegetal ectoderm and complete absence from the animal half of
the embryo. Expression of frizzled genes is largely non-overlapping, in sum covering most domains of
the embryo. Wnt signaling is not required during early development, due to maternal nuclear b-catenin,
but later specifically regulates endodermal and apical neurogenic GRNs, as shown by monitoring
genome-wide regulatory gene expression in embryos treated with the C59 Wnt signaling inhibitor.
Morpholino perturbations of individual Wnts demonstrates their functional divergence, such that Wnt8
restricts apical neurogenic fates and Wnt1 and Wnt16 activates endodermal regulatory genes. A
positive feedback circuit between wnt1, wnt16 and hox11/13b highlights the importance of Wnt signaling
in the posterior endoderm. In addition, we show the specific regulatory functions of Wnt ligands in
embryonic patterning along the primary vegetal-animal axis. This work thus reveals both comprehensive
and particular functions of Wnt signaling in the global regulatory context of this embryo.
32 Carmen Andrikou
33 New Genes in Dorsal-Ventral Skeletal Patterning
Bradham, Cynthia (Biology Department, Boston University); Piacentino, Michael (Biology
Department, Boston University); Zuch, Daniel (Biology Department, Boston University); Hewitt, Finnegan
(Biology Department, Boston University); Ramachandran, Janani (Biology Department, Boston
University); Chung, Oliver (Biology Department, Boston University); Reyna, Arlene (Biology Department,
Boston University); Hameeduddin, Hajerah (Biology Department, Boston University); Li, Christy (Biology
Department, Boston University); Yu, Jia (Biology Department, Boston University); Patel, Vijeta (Biology
Department, Boston University); Chaves, James (Biology Department, Boston University); Ferrell, Patrick
(Biology Department, Boston University); Bardot, Evan (Biology Department, Boston University); Lee,
David (Biology Department, Boston University); Shaw, Scott (Biology Department, Boston University);
Cho, Ah Ra (Biology Department, Boston University); Core, Amanda (Biology Department, Boston
University); Tse, Matt (Biology Department, Boston University); Olenik, Ekaterina (Scientific Computing
and Visualization, Boston University); Keenan, Jessica (Program in Bioinformatics, Boston University);
Hogan, J.D. (Program in Bioinformatics, Boston University); Luo, Lingqi (Program in Bioinformatics,
Boston University); Coulomb-Huntington, Jasmin (Program in Bioinformatics, Boston University);
Poutska, Albert J. (Max-Planck Institut fuer Molekulare Genetik, Berlin)
Skeletal patterning in sea urchin embryos is regulated by cues within the ectoderm that direct the
position of the skeleton-secreting PMCs. We performed an RNA seq-based screen to identify those cues
based on the hypothesis that patterning genes are absent from the ectoderm in embryos treated with
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either nickel chloride or SB203580, and identified candidates as genes down-regulated by both
treatments compared to controls. Using loss of function (LOF) analysis, we tested 10 candidates and
found that each is required for normal PMC positioning and skeletal patterning but not skeletogenesis,
while none impacted ectodermal DV specification, as assessed by ciliary band labeling. We identified
the ventrally-expressed sulfate transporter, LvSLC, as required for ventral and anterior PMC positioning
and ventral/anterior skeleton formation, and show that a ventral-to-dorsal gradient of sulfated
proteoglycans (sPGs) is present in control embryos and correlates spatially with SLC expression. This
gradient is flattened by SLC LOF, suggesting that sPGs are an attractive cue for ventral and anterior
PMCs. We also identified ventrally expressed Notch2 as required for the dorsal localization of PMCs
and dorsal skeleton formation, suggesting that ventral Notch2 functions to repel PMCs dorsally. These
results suggest that together, SLC and Notch2 define the initial PMC distribution. We tested this
hypothesis with combined SLC and Notch2 LOF, and the results show that double morphants exhibit
disrupted PMC positioning at late gastrula, which does not resolve at later timepoints, and a failure to
secrete a skeleton. Double morphants possess a restricted ciliary band, indicating the ectodermal
dorsal-ventral specification is intact. Together, these data identify SLC and Notch2 as specifically
mediating initial dorsal-ventral PMC positioning.
34 G protein regulation of cell shape change in the early sea urchin embryo
Shuster, Charles b. (New Mexico State University); Ellis, Andrea (New Mexico State University);
Sepulveda, Silvia (New Mexico State University); Alvarez, Anthony (New Mexico State University); De La
Rosa, Richard (New Mexico State University)
The actin cytoskeleton is the primary determinant of cell shape and the driving agent of shape change in
animal cells. As blastomeres of the early embryo undergo cycles of cell division, they also undergo a
gradual transition from a spherical to an epithelial morphology, and we are focused on understanding
the cytoskeletal dynamics underlying these processes. Using live cell probes, we now know that in
addition to cortical and microvillar actin, there are cytoplasmic populations of actin in sea urchin eggs,
and all of these filament populations undergo dynamic changes during the cell cycle. These actin
populations are presumably regulated by Rho family GTPases, and while Rho is known to be the master
regulator of contractile ring formation during cytokinesis, the roles of Rac and Cdc42 in the early embryo
are less well defined. Expression of activated mutants of both Rac and Cdc42 lead to cytokinesis failure.
Interestingly, activated Rac also resulted in exaggerated blastomere cohesion, suggesting that Rac
activity may be involved in the spherical to epithelial shape change that occurs in later divisions.
Dominant-negative mutants of Rac and Cdc42 had no effects on early cleavages, consistent with the
notion that these factors are not required for cell division. However, co-expression of T17N mutants of
Rac and Cdc42 led to dramatic alterations in blastomere organization, suggesting that these factors play
partially redundant roles in regulating spindle orientation. Current efforts are focused on defining the
molecular basis by which Rac and Cdc42 antagonize the cytokinetic apparatus, as well as explore the
roles of these G proteins in the regulation of cell adhesion and cell polarity in the early embryo.
35 Three cell behaviors involved in shaping the ascidian gastrula : cell cycle duration, unequal
cleavage, and oriented cell division.
McDougall, Alex (Sorbonne Universities/CNRS); Dumollard, Remi (Sorbonne Universities/CNRS);
Chenevert, Janet (Sorbonne Universities/CNRS); Costache, Vlad (Sorbonne Universities/CNRS);
Hebras, Celine (Sorbonne Universities/CNRS)
Cell cycle control mechanisms affect cell number, size and position in the ascidian embryo. Cell number
is controlled by a gene regulatory network (GRN) initiated by b-catenin at two key points during
embryogenesis. First, at the 16 cell stage nuclear b-catenin in endomesodermal cells causes them to
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cycle faster than the ectoderm cells lacking b-catenin giving rise to the 24 cell stage. Two cell cycles
later b-catenin slows down endodermal cells giving rise to a 112 cell embryo at the time of gastrulation.
During this time from the 8 to the 64 cell stage two posterior vegetal cells undergo three successive
rounds of unequal cleavage generating two small posterior cells at the 64 cell stage that will form the
germ lineage. Here a maternal mechanism leads to the formation of a cortical structure that causes
unequal cleavage by displacing the spindle from the cell center and reorienting it. In all other cells up to
the gastrula stage (112 cells) the mitotic spindle is oriented parallel to the outside of the embryo.
Comparing the mathematical prediction of cell division plane with actual cell division planes reveals that
cells divide according to the long-axis rule (with an apical constraint), unless they display unequal
cleavage. The precise topographical positioning of most blastomeres up to the gastrula stage is
therefore defined by an apical long-axis rule. These three mechanisms partly explain how ascidian
embryos modulate the cell cycle to generate their invariant cleavage pattern up to the gastrula stage.
We have gone some way to explain the invariant cleavage pattern that was formalized by a blastomere
nomenclature system developed by Conklin in 1905.
36 David Burgess
37 Functional analysis of microRNAs in development
Song, Jia L. (University of Delaware); Stepicheva, Nadezda (University of Delaware); Nigam, Priya
(University of Delaware)
microRNAs (miRNAs) are 22-nucleotide RNAs that are expressed in both animals and plants. In animal
cells, they fine tune gene expression by pairing to the 3’ untranslated region of protein coding mRNAs to
repress their translation and/or induce mRNA degradation. Relatively little is known about the function of
specific miRNAs in animal cells because of the difficulty in miRNA target prediction as well as the
presence of miRNA families with redundant target genes/function in mammals. The majority of the
miRNA gene families in Strongylocentrotus purpuratus contain a single member which makes it a
tractable model to examine the function of individual miRNAs. While we have a rich knowledge on
transcription factor based regulation in cell specification, virtually nothing is known about the regulatory
role of miRNAs in early development. Therefore, we use the sea urchin gene regulatory network as a
strong basis to fill the knowledge gap of miRNA function in developmental pathways. We integrate the
regulatory roles of miRNAs into the canonical Wnt signaling pathway, which is highly conserved and
essential for the specification of endoderm and mesoderm in metazoans. Using luciferase reporter
constructs and site-directed mutagenesis, we found Dishevelled and β-catenin to be directly regulated
by at least one shared miRNA. Blocking miRNA regulation of the β-catenin gene resulted in significant
increase in β-catenin protein accumulation, increased transcript levels of Wnt responsive endodermal
regulatory genes, and aberrant gut development. Results from this study elucidate the critical regulatory
roles of miRNAs in early development, identify conserved miRNAs that modulate the Wnt pathway, and
provide unprecedented insight into the functional role of miRNAs in the developing embryo.
38 The ectoderm-mesoderm connection and the upstream regulation of VEGF and VEGFR
Ben-Tabou de-Leon, Smadar (The University of Haifa, Israel); Gildor, Tsvia (The University of Haifa,
Israel)
The sea urchin larval skeleton is formed by the skeletogenic mesoderm (SM) cells through epithelial to
mesenchymal transition, cell migration, cell fusion and biomineralization. The communication between
the ectoderm and the SM cells through VEGF signaling is necessary for the skeleton formation.
According to the current model, the SM specification and the ectoderm patterning are controlled by
independent regulatory mechanisms and the coordinated activation of VEGF and VEGFR are simply due
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to the sequential activation of regulatory genes in each tissue. Yet, various signaling pathways and
regulatory genes are active at both the ectoderm and the SM and some of them could be part of the
SM-ectoderm coordination device. Here we seek to determine whether the upstream regulation of VEGF
is coordinated with or independent of the upstream regulation of VEGFR. To that end, we perturb the
expression of key ectodermal regulatory genes and study the effect on VEGF and VEGFR
spatio-temporal expression pattern. To verify our perturbation analysis results we use VEGF
recombinant GFP BAC to identify a minimal cis -regulatory region that recapitulates VEGF expression
pattern. This minimal region contains binding sites of predicted ectodermal regulators. We believe that
our studies will determine the mechanisms that mediate the coordinated expression of VEGF and
VEGFR and illuminate the basic principles of inter-tissue communication during organ formation.
39 Late Alk4/5/7 activity is required for anterior skeletal patterning in sea urchin embryos
Piacentino, Michael (Boston University); Ramachandran, Janani (Boston University); Bradham,
Cynthia (Boston University)
Skeletal patterning in the sea urchin embryo requires a conversation between the skeletogenic primary
mesenchyme cells (PMCs) and the overlying pattern-dictating ectoderm; however, our understanding of
the molecular basis for this process remains incomplete. Here, we show that TGF-ß-receptor signaling is
required during gastrulation to pattern the anterior skeleton. Treatment with SB431542 (SB43), a
specific inhibitor of the TGF-ß type I receptor Alk4/5/7, during gastrulation blocks anterior PMC
positioning in the oral hood and the formation of the animal skeleton, but does not perturb ciliary band
restriction or neural development. SB43 treatment during gastrulation does not perturb dorsal-ventral
specification, but does perturb left-right axis specification, as expected. Feeding experiments show that,
while Alk4/5/7 inhibition does not prevent the formation of a mouth, SB43-treated plutei display reduced
feeding ability, presumably due to the loss of the anterior skeleton. Both Univin and Nodal are potential
ligands for Alk4/5/7; however, Nodal is unilaterally expressed on only the right side, while Univin is
bilaterally expressed in the ectoderm adjacent to the animal skeleton during the relevant time period.
Our results demonstrate that Univin is necessary and sufficient for secondary skeletal patterning,
consistent with the hypothesis that Univin is the relevant Alk4/5/7 ligand for anterior skeletal patterning.
Taken together, our data demonstrate that Alk4/5/7 signaling during gastrulation is required to direct
PMCs to the oral hood, and suggest that the relevant ligand for this signaling event is Univin.
40 Segregation of pigment and blastocoelar cells depends on the interplay between the lineage
specific transcription factors ESE and GCM
Molina Jiménez, Mª Dolores (Institut de Biologie Valrose (iBV), UMR7277, CNRS/UNSA); Lepage,
Thierry (Institut de Biologie Valrose (iBV), UMR7277, CNRS/UNSA)
The non-skeletogenic mesoderm (NSM) is initially specified by Delta/Notch signalling as a unique
population of gcm-positive mesodermal precursors that will eventually differentiate into two main cell
types: blastocoelar cells, which form on the ventral side and lose expression of gcm, and pigment cells,
which form on the dorsal side and retain expression of gcm. This dorsal–ventral patterning depends on
the antagonistic interplay between Nodal and BMP2/4 signals: Nodal signals emanating from the ventral
ectoderm promote specification of blastocoelar cells and repress formation of pigment cells while
dorsally, BMP2/4 counteracts the activity of Nodal, allowing pigment cell differentiation. The transcription
factor Not represses gcm expression in the ventral NSM downstream of Nodal signalling, allowing
expression of blastocoelar lineage specific transcription factors such as gataC, ese, and scl, and
specification of blastocoelar cell fates. Here, we show that the blastocoelar cell specific transcription
factor ese and the pigment cell specific transcription factor gcm appear initially coexpressed in the
precursors of the immunocytes at the hatching blastula stage. Importantly, Nodal and BMP2/4 are not
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required for this early ese and gcm expression but to resolve and pattern a population of cells with an
initial mixed identity. By the time of delamination of the primary mesenchymal cells, segregation of ese
and gcm expression and DV patterning of the NSM occurs. Ventral NSM cells retain expression of ese
and lose expression of gcm, while dorsal NSM lose expression of ese and retain expression of gcm.
Perturbation and overexpression experiments indicate that Ese is necessary and sufficient to promote
expression of blastocoelar cells lineage specific transcription factors such as gataC, prox1 and scl on
the ventral population of NSM as well as to restrict gcm expression to the dorsal population of NSM
precursors. Thus, we identify Ese as an key component of the gene regulatory network responsible of
blastocoelar cell specification.
41 SoxC functions in neural precursor cells in sea urchin embryo neurogenesis
Wei, Zheng (NIH/NIDCR); Angerer, Lynne (NIH/NIDCR); Angerer, Robert (NIH/NIDCR)
Previously we showed that Six3 was necessary for development of all nerves in the sea urchin embryo
(Wei et al., 2009). The expression of Six3 begins during cleavage stages broadly throughout the embryo
and then is confined to patches of contiguous cells; but it is not expressed in individual neural
precursors. This suggests that Six3 is involved in establishing neuroectoderm territories but is not
directly involved in initiating neural differentiation. To look for Six3-dependent genes that might execute
Six3's function in neurogenesis, we carried out a microarray-based screen at the mesenchyme blastula
stage when expression of proneural factor orthologs begins to appear in individual cells. Among the set
of Six3-dependent transcription factors expressed in individual cells, SoxC has a unique function in
neural precursor cells. By in situ hybridization, we found that SoxC is expressed in individual cells in
neuroectoderm. More importantly, when SoxC was knocked down with morpholino oligos, development
of all neurons was greatly reduced as is also the case in Six3 morphants. However, in a double in situ
with Synaptagmin B, which is a marker for almost all neurons, no cells expressed both. This observation
suggested that SoxC-expressing cells could be in an intermediate state and other factors mediate their
further differenciation. We carried out a RNA-Seq screen for potential candidates. Among the affected
genes, we tested Z167 and Brn1/2/4 and found that they both are partially co-expressed with SoxC and
partially co-expressed with Synaptagmin B or Tph, a marker for Serotonin cells. Therefore, in sea urchin
embryo, neurogenesis is a multistep process and SoxC functions in neural precursors, which is a
conserved function observed in other systems.
42 Suppression of nodal expression in prospective dorsal cells of the early sea urchin embryo by
the Hbox12 homeodomain regulator
Cavalieri, Vincenzo (University of Palermo)
Dorsal/Ventral (DV) axis formation in the sea urchin embryo depends upon the expression of nodal on
the ventral side, which behaves as a DV organizing centre. However, only fuzzy clues are known as to
the early symmetry-breaking steps that lead to the positioning of such an organizer. An extremely
interesting candidate for this role is the hbox12 homeobox-containing gene. In Paracentrotus lividus ,
hbox12 expression is antecedent and complementary with respect to that of nodal , being confined in
prospective dorsal cells. We show that ectopic expression of Hbox12 provokes DV abnormalities and
attenuates nodal as well as nodal -dependent gene transcription. By blastomere transplantation, we also
establish that DV defects arise from hbox12 misexpression in the animal hemisphere. To impair Hbox12
function we expressed ubiquitously a truncated form of the protein, encoding for the homeodomain. Such
a perturbation disrupts DV axis formation by allowing ectopic expression of nodal across the embryo.
Moreover, clonal loss-of-function imposed by either blastomere transplantation or gene transfer assays
highlights that Hbox12 action in prospective dorsal cells is necessary for DV polarization. Remarkably,
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the localized knock-down of nodal restores DV polarity of embryos lacking hbox12 function. Finally, we
show that hbox12 is involved in the dorsal-specific inactivation of the p38 MAPK, which is known to be
required for nodal expression. Altogether, our results indicate that Hbox12 prevents the ectopic
activation of nodal transcription within the future dorsal side of the early sea urchin embryo.
43 Evolution of ectoderm-mesoderm communication during skeletal patterning in echinoid larvae
Lyons, Deirdre (Duke University); Martik, Megan (Duke University); Kimura, Julian (Duke University);
McClay, David (Duke University)
The echinoid larval endoskeleton is a classic model for studies of cell differentiation and
morphogenesis. It is also a model for the evolution of embryonic patterning, since specific parts of the
skeleton can vary between species. Two of the most variable elements are the recurrent and posterior
connecting rods. The developmental basis of this variation is not well understood. By comparing the
sea urchin Lytechinus variegatus , which forms a small recurrent rod and no posterior connecting rod,
with the sand dollar Mellita quinquiesperforata , which forms both, we found that the difference in their
skeletal patterning is already obvious in the arrangement of primary mesenchyme cells (PMCs) at
gastrula stages. Mellita embryos possess an antero-dorsal chain of PMCs between the tips of the
longitudinal strands, which emanate from the ventro-lateral clusters. These PMCs contribute to the
recurrent rod and the anastomosed posterior connecting rod. No antero-dorsal chain exists in
Lytechinus , but at prism stages it is from an analogous location that the recurrent rods form from a
branching event at the tip of each longitudinal strand. Because the position of the PMCs is dictated by
ectodermal patterning in sea urchins, we investigated the role of ectodermal patterning in Mellita PMC
arrangement. We found that Nodal signaling controls the position of the antero-dorsal chain in the
oral/aboral axis. Ectodermal signals that control the spatiotemporal pattern of underlying PMCs have
diverged between these two echinoids. These data provide the framework for studies in both species
that address the details of these two patterning systems at the molecular level.
44 Experimental approach to divergence in test organization between euechinoid and cidaroid
sea urchins
Gao, Feng (California Institute of Technology); Erkenbrack, Eric (California Institute of Technology);
Petsios, Elizabeth (University of Southern California); Thompson, Jeffrey (University of Southern
California); Bottjer, David (University of Southern California); Davidson, Eric (California Institute of
Technology)
The two extant crown groups of echinoids are the subclasses Cidaroidea and Euechinoidea, which
diverged around P/T boundary and differs strikingly in several aspects of skeletal morphogenesis. Such
divergence thus represents a compelling opportunity for understanding the evolutionary mechanisms of
specific body plan innovations in echinoderms. Our initial focus is on the difference of their test
pattering, a major phylogenetic character as revealing from their development and fossil records.
Embryos from Strongylocentrotus purpuratus (Euechinoid) and Eucidaris tribuloides (Cidaroid), were
cultured in the lab to the stage six weeks after metamorphosis, and samples were collected at different
time points. The whole animal and extracted skeletal elements were scanned under SEM and micro-CT
to know how their tests are built differently in these two species by addition of successive rows of body
plates at the start of adult skeletogenesis. The difference on their test patterning was first visible from
one week after metamorphosis with compound plates in Sp vs simple plates in Et from the ambulacral
region while no discernible difference from the interambulacral region. WMISH was done on genes from
the gene regulatory network underlying the development of the skeletogenic lineage of Echinoderm to
find what accounts for the clade–specific differences in the spatial deployment of this GRN on the
ambulacral region in Sp and Et. Gene regulatory analysis and synthetic experimental evolution will be
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done in the future, and our final goal is to understand the mechanistic origin of the Euechonoid
compound platting at GRN level.
45 Evolution of pancreatic cell types: insights from the sea urchin Strongylocentrotus purpuratus
Perillo, Margherita (Stazione Zoologica Anton Dohrn di Napoli, Naples, Italy); Arnone, Maria Ina
(Stazione Zoologica Anton Dohrn di Napoli, Naples, Italy)
The proper pancreas of vertebrates is a gland composed essentially of digestive enzymes-producing
acinar cells and hormone-producing endocrine cells. Although the pancreatic hormone insulin is well
characterized in vertebrates, little information is reported about insulin-like peptides (ILPs) in
non-chordate deuterostomes. In order to fill this critical gap in pancreas evolution, our aim is to
characterize the homologues of pancreatic cell types and ILPs in the embryos and larvae of
Strongylocentrotus purpuratus, so far one of the best non-chordate deuterostome for evolutionary
comparison studies. To this end, the spatial expression of ILPs found in the sea urchin genome (two
paralogues named SpILP1 and SpILP2) and their predicted receptor (SpInsr) has been extensively
studied. Remarkably, SpILP1 has been found to be localized in a group of cells of the larval gut in a
feeding-dependent fashion. Moreover, to characterize homologues of pancreatic cell types, we
characterized the spatial expression of the orthologues of pancreatic transcriptional factors (SpNgn,
SpNeuroD, SpIsl, SpHnf1, SpPtf1a, SpMist1) and exocrine pancreas terminal differentiation genes
(SpCpa2L, SpPnlp, SpAmy3), which expression increases after feeding. Notably, perturbation analysis
experiments also demonstrated that in the sea urchin the link between SpHnf1, SpPtf1a and the
pancreatic digestive enzymes is conserved, thus allowing us identifying an acinar-like cell type in the
upper stomach of the sea urchin larva. In addition, the already identified SpLox&SpBrn1/2/4+ cells have
been further characterized and we found that it is a neurosecretory cell type that produces a novel
neuropeptide. Furthermore, comparing the above outcome together with available data in other animal
models, we propose a model of pancreatic cell types evolution across metazoan. Concluding,
comparison of the ILPs characterized in the sea urchin with proteins from other bilaterians were helpful
to carry out a phylogenetic study aimed to explain how molecules of the insulin family evolved.
46 Regulatory gene use within the gastrulating sea urchin
Valencia, Jon E. (Caltech)
Our goal is to produce a regulatory gene expression dataset that will aid in the systems-level
understanding of cell-type specification and organ formation in the post-gastrula embryo. A complete
understanding of the regulatory genes that comprise the regulatory states formed during development is
the first step in building GRNs. Previous regulatory gene surveys analyzed temporal and spatial
expression up to the onset early gastrulation. We present a complete spatio-temporal characterization
(from 36 to 72 hours post fertilization) of gene expression patterns for all known and predicted
transcription factors genes in the sea urchin genome. The genome contains 368 transcription factors
genes (non zinc fingers and identified zinc fingers) and only 284 of those are significantly expressed
during the hours of gastrulation as determined to be relevant by RNA-seq data. We have analyzed gene
expression patterns by whole-mount in-situ hybridization for 80% of the regulatory genes expressed at
four time-points during and post gastrulation and have annotated these patterns into the six major
domains of the developing sea urchin. Interestingly, our analysis of this dataset has revealed that the
non-skeletal mesoderm, endoderm, oral ectoderm and apical ectoderm each expresses around 50% of
the regulatory genes considered, whereas skeletal mesoderm and aboral ectoderm each expresses less
than 20%. These results not only show an enormous regulatory complexity but also that most regulatory
genes are expressed in multiple domains. Additionally, our results offer many insights into the global use
of regulatory genes expressed during sea urchin development.
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47 Delta-Notch signaling and HesC mediate the spatial confinement of the skeletogenic-specific
regulatory gene alx1 to micromere-descendants in Eucidaris tribuloides
Erkenbrack, Eric (Caltech); Davidson, Eric (Caltech)
The early restriction of skeletogenic regulatory genes—namely alx1 , delta , ets1 , tbrain —to
micromere-descendants via the Pmar1-HesC double-negative gate is a widespread piece of GRN
circuitry in euechinoids, having been described in at least three euechinoid Orders. Evidence for this
specific circuit is absent outside of euechinoids, raising the evolutionary question of how and when it
was installed. We undertook GRN analysis of skeletogenic regulatory genes in a cidaroid sea urchin,
Eucidaris tribuloides ( Et ), as it is a representative of a distantly-related clade from euechinoids (~275
mya) and manifests several significant developmental differences with euechinoids. Repeated BLAST
searches of genomic and transcriptomic data sets of Et yielded no significant hits for pmar1 , supporting
the supposition that pmar1 is a euechinoid novelty. In Et , delta is first zygotically expressed in
micromere-descendants at early blastula stage, rather than during cleavage as in euechinoids. HesC is
maternal and is zygotically transcribed shortly after delta transcription begins. However, rather than
being expressed everywhere but the skeletogenic micromeres as in euechinoids, this repressor is
expressed exclusively in mesodermal cells immediately surrounding micromere-descendants. Alx1 is
zygotically expressed exclusively in micromere-descendants; while ets1 and tbrain are general
mesodermal drivers--the latter being maternal. We found that the zygotic expression of hesc is
Delta-dependent. The absence of HesC (and also of Delta presentation) causes the alx1 domain to
expand to surrounding mesoderm cells, but not to the whole embryo as in euechinoids. Thus the spatial
confinement of alx1 to micromere-descendants in Et is Delta-Notch dependent. Evolutionarily,
HesC-mediated repression of alx1 is likely an ancestral linkage in the echinoid skeletogenic GRN. The
global function of preventing skeletogenesis by HesC in euechinoids is not required in Et ; rather its only
skeletogenic role is to separate skeletogenic from non-skeletogenic mesodermal fates during
embryogenesis.
48 Modularity in DNA Binding Preference of a Tbrain Transcription Factor May Allow for More
Versatile Transcriptional Responses and Increased Evolvability
Cheatle Jarvela, Alys (Carnegie Mellon University)
In the sea star (Pm), Tbrain (Tbr), a t-box transcription factor, carries out a variety of ancestral roles in
the endomesoderm and ectoderm. However, in the sea urchin (Sp), Tbr has a singular and well-defined
role in the skeletogentic gene regulatory network. The DNA binding regions of these proteins contain
differences in critical DNA-contacting amino acids, suggesting a protein level change could be
responsible, but it is thought that this type of change must be incredibly rare owing to the potentially
lethal pleiotropic effects of altering a multifunctional protein. However, novel techniques have allowed for
far more sensitive assays of transcription factor function. One such technique, Protein-Binding
Microarrays, has indicated that DNA binding is more complex than originally indicated, in that the same
transcription factor can recognize multiple binding sites, and only a subset of these might be conserved
among closely related paralogs (Badis and Bulyk 2009). Here, we first demonstrate that this type of
complexity also applies to orthologous transcription factors. While both Tbr orthologs recognize the
same primary motif, only PmTbr also has a secondary binding motif. While affinity for the primary
binding site is conserved, affinity for the secondary binding motif is more evolutionarily labile. We have
verified the effects of binding specificity and affinity changes in vivo using a dual Otxb1/2 CRM reporter
system. Our in vivo assays demonstrate that differences in transcriptional responses governed by
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primary vs. secondary sites may allow for greater evolution in timing of regulatory control. This uncovers
a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We
hypothesize that division of transcriptional functions between multiple binding sites may allow orthologs
to evolve new secondary binding preferences rapidly, as the conserved primary site reduces pleiotropic
effects.
49 Germ cells Rock!
Wessel, Gary M. (Brown University)
They really do....
50 Michael Whitaker
51 Translatome analysis following fertilization
Morales, Julia (equipe TCCD, UMR8227 CNRS-UPMC, Station Biologique de Roscoff); Chassé,
Héloïse (equipe TCCD, UMR8227 CNRS-UPMC, Station Biologique de Roscoff); Boulben, Sandrine
(equipe TCCD, UMR8227 CNRS-UPMC, Station Biologique de Roscoff); Corre, Erwann (ABiMS,
FR2424 CNRS-UPMC, Station Biologique de Roscoff); Le Corguillé, Gildas (ABiMS, FR2424
CNRS-UPMC, Station Biologique de Roscoff); Glippa, Virginie (equipe TCCD, UMR8227 CNRS-UPMC,
Station Biologique de Roscoff); Coson, Bertrand (equipe TCCD, UMR8227 CNRS-UPMC, Station
Biologique de Roscoff); Belle, Robert (equipe TCCD, UMR8227 CNRS-UPMC, Station Biologique de
Roscoff); Mulner-Lorillon, Odile (equipe TCCD, UMR8227 CNRS-UPMC, Station Biologique de Roscoff);
Cormier, Patrick (equipe TCCD, UMR8227 CNRS-UPMC, Station Biologique de Roscoff)
Half of the variation in proteins concentration in a cell can be attributed to differences in translation rates
as determined by global quantification of gene expression. Therefore, protein synthesis represents an
import step in gene expression regulation. Early embryogenesis is highly dependent on translational
regulatory cascades, and relies on the use of maternally stored mRNA in the cytoplasm. Sea urchin
embryos offer an elegant model to elucidate the network of actors involved in translational control during
early development. Protein synthesis, low in unfertilized eggs and stimulated rapidly following
fertilization, is necessary for the onset of the first cell divisions. We have shown previously that
translational machinery is highly regulated and activated at fertilization, acting upon translation initiation
and elongation steps. Translatome, a subset of the transcriptome comprising polysomes-associated
mRNAs, corresponds to the totality of mRNAs translated into proteins thus reflecting the functional
readout of the genome. A complete examination of translatome is made possible by carrying out
polysome profiling coupled with high-throughput sequencing technologies. We have undertaken the
analysis of the translatome following fertilization and showed selective recruitment of mRNAs encoding
translation factors and RNA binding proteins. From these data on translatome analysis and our previous
results on translation machinery activation at fertilization, we suggest that a translational regulatory
cascade is important for the first few hours of development of the embryo.
52 Gene regulatory networks in the real world: stress, robustness, and evolution
Wray, Gregory (Duke University)
Development produces a reproductively competent adult despite a wide range of genetic and
environmental perturbations. At the same time, development retains the flexibility to to evolve in
response to environmental change. We are actively investigating how sea urchins balance these
conflicting demands on development gene regulatory networks. We compared the effects of three kinds
of real world perturbations: natural genetic variation and two environmental stressors linked to global
warming, pH and temperature. Both projected environmental stressors have measurable impacts on
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developmental phenotypes and on proxies for fitness. Nonetheless, development is able to buffer both
environmental stressors at least as well as they are able to buffer the consequences of natural genetic
variation. Overall, our results indicate that early development in sea urchins is highly robust to genetic
and environmental perturbation, but that specific gene interactions are more sensitive and may allow
adaptive changes in organismal traits.
53 A prelude to the present: Paleontological perspectives on 450 million years of echinoid
evolution
Thompson, Jeffrey R. (University of Southern California); Petsios, Elizabeth (University of Southern
California); Bottjer, David (University of Southern California)
Echinoids are a morphologically diverse group of echinoderms with a robust fossil record dating back to
the Upper Ordovician period, about 450 million years ago. The fossil record of echinoids provides
valuable data to both paleontologists and neontologists interested in evolutionary processes. But what
data are available to neontologists through study of the fossil record? The fossil record of the
Echinoidea allows for the testing of evolutionary hypotheses and provides insight into how large-scale
events such as global climate and sea level change govern evolution and diversification processes.
Following the Permian-Triassic mass extinction, 252 million years ago, echinoid diversity underwent a
bottleneck and concomitant reduction in morphological disparity. The surviving bauplans, with only two
columns of plates in each interambulacral and ambulacral column became the template for all
post-Paleozoic, and thus recent, echinoids. Using a paleogenomic approach, it is possible to place
changes in the GRN and genome within this context of evolutionary time. The fossil record provides the
only evidence available to directly observe trait reversals, especially in relation to extinct stem-groups
and also provides observable evidence for the morphologic manifestation of changes in the skeletogenic
gene regulatory network circuitry. Fossil and molecular developmental approaches are now being used
to understand the divergence between cidaroids and the euechinoids, the two clades that comprise all
post-Paleozoic echinoids. Thus far, fossil data has elucidated better estimates for the timing of this
divergence and for the timing of the first appearance of ordinal and family level traits, and the associated
regulatory circuitry.
54 Pattern and process during gut morphogenesis: an evolutionary perspective
Arnone, M. Ina (Stazione Zoologica Anton Dohrn)
It can be argued that one of the first developmental patterning systems to evolve was the molecular
network that orchestrates the formation of the digestive system thus releasing multi-cellular organisms
from body size constraints and allowing the further evolution of highly specialized internal structures.
The antero-posterior patterning of the embryonic gut represents one of the most intriguing biological
processes in development. A dynamic control of gene transcription regulation and cell movement is
perfectly orchestrated shaping a functional gut in distinct specialized parts. Two ParaHox genes, Xlox
and Cdx, play key roles in vertebrate and sea urchin gut patterning through molecular mechanisms that
are still mostly unclear. We combined functional analysis methodologies with high resolution imaging
and RNA-seq to investigate Xlox and Cdx regulation and function in the sea urchin embryo. We revealed
part of the regulatory machinery responsible for the onset of Cdx transcription, uncovered a Wnt10
signal mediating Xlox repression in the intestinal cells and provided evidence of Xlox control of the
stomach differentiation. A role for the retinoic acid mediated signalling to initiate this process was also
uncovered. Our findings offer a novel mechanistic explanation of how the control of transcription is
linked to cell differentiation and morphogenesis for the development of the sea urchin larval gut. A
comparison with vertebrates showed a striking conservation of topology of gene expression and
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signalling events between sea urchin and mouse, thus revealing some key aspects of deep homology
that are most probably shared by all bilaterian guts. Preliminary analyses, comparing sea urchin and sea
star gut patterning, suggest that this conservation does not extended to the level of gene interactions.
55 Differentiation of immunocytes and the emergence of the larval immune system
Rast, Jonathan (University of Toronto); Buckley, Katherine (University of Toronto); Ho, Eric (University
of Toronto); Schrankel, Catherine (University of Toronto); Wang, Guizhi (University of Toronto)
Animal immunity is an organism-wide phenomenon involving specialized immune cells, barrier epithelia
and other tissues. In the gut associated immune system of Bilateria, interactions among the lumenal
microbiota, the gut epithelium, dedicated immunocytes and peripheral tissues have broad effects on
throughout the host. The feeding larva of the purple sea urchin provides a deuterostome model that is
well-suited to investigate these processes. Larvae are morphologically simple and transparent, which
allows organism-wide imaging at single-cell resolution. Upon exposure to Vibrio diazotrophicus, several
classes of specialized immune cells exhibit stereotypic changes in morphology, undergo migration to the
gut epithelium and specific interactions among themselves. These behavioral changes coincide with
changes in gene expression in the gut. Aspects of this response are synchronous among individuals and
reproducible. Transcriptome data from larvae exposed to bacteria at several time points and treatments
provide a system-wide measure of transcriptional changes. Notably, expression of several IL-17
homologs is quickly induced in the gut epithelium. These signals are tightly regulated, expressed
exclusively in presence of bacteria and are the most strongly upregulated in the genome. Deletion
analysis of IL-17 transgenes localizes response and attenuation elements. Antisense perturbation of
IL-17 receptors identifies candidate downstream genes. Later in the response, transcription levels
change throughout the organism for genes encoding transcription factors (e.g., NfkB, PU.1, and Irf),
signaling molecules (e.g., TNF and Mif), pattern recognition receptors (e.g., PGRPs) and immune
effectors (e.g., 185/333 genes). The molecular complexity of this inflammatory response within the
context of the simple morphology of the sea urchin larva provides an ideal system in which to
characterize the fundamental programs of immune response mediated by the gut epithelium.
56 A GRN for neurogenesis in the sea star P. miniata
Hinman, Veronica (Carnegie Mellon U)
Nervous systems are centralized in various taxa, distributed throughout the animal tree of life. It has
been of great interest to understand how such complex structures can evolve from diffuse neural nets
and whether the transition had a single origin. The evolution of the extraordinary complex vertebrate
nervous system has been especially puzzling to understand, in part because their sister taxa, the
hemichordates and echinoderms have very simple nervous systems. Although sea stars had featured
prominently in early hypotheses of vertebrate neural origins, an increasing understanding of molecular
mechanism of development have suggested alternative hypotheses that imply that the sea star, and
echinoderms generally have derived nervous systems. We have developed extensive gene regulatory
networks that explain how the sea star Patiria miniata larval nervous system is specified and patterned.
This work reveals an extraordinary similarity in regulatory mechanism of development between these
echinoderms and vertebrates. More significantly, these analyses demonstrate how the modular nature of
these regulatory networks, and in particular the independence between patterning and specification, can
explain how nervous systems patterning can readily evolve.
57 Spiralian embryogenesis at your fingertips: the quest for an early annelid GRN
Schneider, Stephan (Iowa State University); Pruitt, Margaret (Iowa State University); Bastin, Benjamin
(Iowa State University); Chou, Hsien-chao (Iowa State University)
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Although conserved features among developmental gene regulatory networks (GRNs) between various
deuterostome embryos are emerging, a comparison to protostomes remains difficult due to the highly
derived nature of early embryogenesis and the genomes in the protostome models C. elegans and
Drosophila (Ecdysozoa). To fill this gap we focus on early developmental mechanisms in the protostome
Platynereis dumerilii (Annelida, Lophotrochozoa). Platynereis has retained a more ancestral gene set
without the extensive gene loss and gain observed in the other protostome models. In addition, early
development of Platynereis is attractive as it exhibits a series of invariant, stereotypic asymmetric cell
divisions, allowing individual embryonic cells to be recognized by size and position. Furthermore,
Platynereis exhibits a global reiterative beta-catenin mediated cell fate specification mechanism in early
development that appears to convey lineage-specific binary cell fate decisions. To gain insights into the
early embryonic GRNs, and the contribution of reiterative beta-catenin asymmetries to specify cell fates,
we deployed a variety of RNA-seq based approaches. Comparing normal and manipulated embryos we
have identified embryonic transcripts from the zygote through the mid gastrula (1 cell stage through
~330 cell stage) that include developmental regulators of the beta-catenin pathway. We have
subsequently mapped the expression of developmental regulators including beta-catenin pathway
components (ligands, receptors, intracellular components) into distinct cell lineages. Our approaches
capture stage-specific transcriptional snapshots of Platynereis embryos, and thereby provide the first
comprehensive view of temporal inputs and outputs into annelid GRNs that utilize a spiral-mode of cell
divisions to segregate cell fates.
58 Developmental plasticity: A broad utilization of germ line molecules in multipotent cells of the
sea urchin
Yajima, Mamiko (Brown University); Wessel, Gary (Brown University)
Echinoderms are classically known for having remarkable regenerative capabilities. Adults can
regenerate entire segments such as arms, internal organs and even gonads, and embryos maintain
multipotency until late in their development: Cells from other lineages can transfate and compensate for
a missing part of the embryo. The mechanisms that regulate this amazing plasticity of echinoderm cells
are not yet clear, but we recently found that a transient use of germ line molecule may be taking a critical
role in facilitating cellular multi-potentiality in the multipotent cells of the sea urchin embryo. In this talk,
we focus on a conserved germ line molecule, Vasa, and demonstrate how it is transiently expressed,
control cell cycle progression, and further regulate general protein synthesis necessary for the
embryogenesis, developmental reprogramming and/or regeneration. From the results showed in this
talk, we propose Vasa functions broadly in mRNA regulation to enable proper distribution and efficient
translation of mRNAs that contribute to developmental plasticity of the sea urchin embryo.
59 Maintenance of the anterior neurogenic ectoderm in the sea urchin embryo
Yaguchi, Junko (University of Tsukuba); Yaguchi, Shunsuke (University of Tsukuba); Yamazaki,
Atsuko (University of Tsukuba); Yamamoto, Akane (University of Tsukuba); Inaba, Kazuo (University of
Tsukuba)
In normal sea urchin embryos, the anterior neurogenic ectoderm is restricted to the anterior end
although almost all of cell fate will be specified as this region in pre-signaling condition. Previous works
suggested that a sequence of canonical and non-canonical Wnt signal-dependent mechanisms regulate
the restriction of the neurogenic ectoderm, but it is still unclear how the restricted neurogenic ectoderm
is stably maintained during the early embryogenesis. Here we show that a maternally expressed
transcription factor, Meis, is required for the maintenance of the anterior neurogenic ectoderm.
Cis-regulatory analysis of FoxQ2, a transcription factor essential for the formation of anterior neurogenic
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ectoderm, revealed an important module for the maintenance of its expression. Among transcription
factors that might bind to the region, we focused on Meis homeodomain protein in this study. When we
block the translation of Meis by morpholino anti-sense oligonucleotides, the expression of foxQ2 was not
maintained after hatching nevertheless the initial expression of foxQ2 was not affected. As a
consequence, the morphant lost the anterior neurogenic ectoderm and serotonergic neurons. These
results indicated that Meis is required for the maintenance of anterior neurogenic ectoderm. In addition,
the expression pattern of univin, a lateral ectoderm marker, and PMC pattern shift towards more anterior
side in Meis morphants than those in control embryos, indicating that Meis is also required for the
precise anteroposterior patterning of the sea urchin embryo.
60 Coelomocytes and the Sea Urchin Immune System
Smith, L Courtney (George Washington University)
Annotation of the sea urchin genome identified and highlighted the complexity of the innate immune
system in this invertebrate, which includes several large families of immune response genes. The
coelomocytes are the primary mediators of this system in the adult sea urchin and are characterized as
phagocytes, spherule cells, and vibratile cells. There are four morphotypes of phagocytes including
polygonal, discoidal and small phagocytes, plus a newly identified type. Only the phagocytes express
immune genes such as SpC3, SpBf, SpNFkB, SpTLR, and Sp185/333. The Sp185/333 gene family
encodes a wide array of highly diverse but structurally similar Sp185/333 proteins that have core
functions in the immune system. One recombinant (rSp0032) shows specific binding to Vibrio and yeast,
but not to Bacillus, whereas separated sub-regions of rSp0032 show deregulated binding. rSp0032
binding to bacteria and yeast is mediated by LPS, flagellin and glucan, but not by peptidoglycan. Binding
to LPS is specific with high affinity. rSp0032 reduces the growth rate of E. coli but not Vibrio, and does
not induce phagocytosis. On the other hand, native Sp185/333 proteins are secreted from phagocytes,
bind to and opsonize bacteria, retard the growth of most bacteria, and induce phagocytosis. These
results suggest that the various Sp185/333 isoforms have a range of functions and that they act
synergistically in the coelomic fluid. We analyzed the Sp185/333 gene expression in single phagocytes
using single cell RT-PCR and amplicon sequencing. Surprisingly, individual phagocytes express a single
gene of the ~50 member gene family. This intriguing result infers a much more complex and
sophisticated regulatory system for controlling Sp185/333 gene expression than has been imagined
previously.
61 Shifts in the Expression of Developmental Regulatory Genes Involved in the Evolution of a
Novel Life History Difference
Wygoda, Jennifer (Duke University); Byrne, Maria (University of Sydney); McClay, David (Duke
University); Wray, Gregory (Duke University)
Developmental mode can influence dispersal, gene flow, speciation and extinction rates in marine taxa
and thus can have important consequences for micro- and macroevolutionary processes. While the
ancestral developmental mode of sea urchins is indirect through a feeding larval stage (planktotrophic),
non-feeding development (lecithotrophic) has evolved independently multiple times. In order to identify
evolutionary changes in gene expression underlying this ecologically significant shift in life history, we
used Illumina RNA-seq to measure expression dynamics across development in three sea urchin
species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris
tuberculata (3 myr), and an out-group planktotroph Lytechinus variegatus (50 myr). Our analyses draw
on a well-characterized developmental gene regulatory network (GRN) in sea urchins to understand how
the ancestral developmental program was altered during the evolution of lecithotrophic development.
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Our results suggest that changes in gene expression profiles were more numerous during the evolution
of lecithotrophy than during the persistence of planktotrophy, and this contrast is even stronger when
only GRN genes are considered. We found evidence for both conservation and divergence of GRN
linkages in H. erythrogramma, as well as significant changes in the expression of genes with known
roles in patterning the larval skeleton and gut, which are greatly modified in lecithotrophs. Collectively,
these results indicate that the transition from planktotrophic to lecithotrophic development involved a
surprising number of changes to key developmental processes over a short evolutionary timescale.
62 The Developmental Transcriptome of the Ophiuroid Amphiura filiformis
Dylus, David V. (University College London); Oliveri, Paola (Unviersity College London)
Ophiuroids form a larval skeleton similar to echinoids and represent an evolutionarily interesting class.
Unfortunately, so far sequence resources were limited for this group of animals. With recent advances in
high throughput sequencing it is now possible to create cheaper and faster sequence resources for
non-model organisms. Moreover, the development of new graph-theoretical software approaches
enables to easily assemble sequences without prior genome availability. These facts allowed us to
perform mRNA sequencing on key developmental stages (Cleavage, Blastula, Mesenchyme Blastula
and Gastrula) for the brittle star Amphiura filiformis without the need of a reference genome. The
sequencing was carried out on the Illumina HiSEQ 2500 platform using 2 lanes with 100bp paired end
reads resulting in ~700 million reads. In order to perform a de novo transcriptome assembly, we used
digital normalization followed by assembly with the trinity software package. Using all available reads
our reference transcriptome resulted in ~600.000 sequences (N50: 1094). In these sequences, we found
~15.000 transcripts with high similarity to other echinoderm genes. These include 80% of transcription
factors present in S. purpuratus. When re-aligning the reads for time-course expression levels we found
a high correlation between our QPCR data and the transcriptome. Focusing on 231 larval skeletogenic
genes identified in sea urchin, only 171 could be recognized in A. filiformis, including a cohort of genes
that could not be found when conducting a similar analysis with Patiria miniata genomic sequences.
Since asteroids do not form larval skeleton, this suggests the existence of a unique set of genes utilized
during larval skeletogenesis in echinoids and ophiuroids.
63 Sequencing Echinoderm Genomes
Cameron, R. Andrew (California Institute of Technology)
The sequencing enterprise for echinoderm genomes is now changing directions as the efforts of large
sequencing centers wanes while that of individual laboratories increases. In light of these changes, I will
present a synopsis of the present work to decode echinoderm genomes. I will describe the breadth and
nature of the various genome and transcriptome projects available in public sites and some in
preparation. I will compare the quality of the various datasets and how they can be used. And lastly, I will
discuss future directions for our community efforts in genome and transcriptome sequencing and
annotation.
64 GRIP-Seq: Genome-wide Regulatory element Immunoprecipitation coupled with Next
Generation Sequencing
Tulin, Sarah L. (MBL); Bocconcelli, Carlo (Falmouth Academy); Smith, Joel (MBL)
We describe a new method for identifying cis -regulatory elements. Our protocol combines elements of
chromatin conformation capture (e.g., 3C, 4C), chromatin immunoprecipitation, and paired-end
high-throughput sequencing. By modifying these existing methods, we enrich for active cis -regulatory
elements across the genome. Our pioneer dataset, available to the community, derives from S.
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purpuatus embryos collected at 24 hours post fertilization. We benchmark our results against
independent findings, i.e., rigorously tested cis- elements previously identified. We find good
congruence, and in particular we find a strong signal-to-noise ratio for known distal elements, marking
an improvement over existing methods. A second goal of this new method is to identify which
transcription start site corresponds with which element: on this score, our findings show only a modest
signal, though it is too early to say how our protocol might be adjusted to yield more reliable results. In
conclusion, we report a promising protocol for the genome-wide identification of active regulatory
elements, a rate-limiting step in determining comprehensive, predictive network models.
65 Experimental measurement of embryonic regulatory states
Nam, Jongmin (Rutgers University-Camden); Erkenbrack, Eric M. (Caltech)
Regulatory state is defined as the total set of active transcription factors presented in a given cell. As an
expansion of the original concept, embryonic regulatory state is defined as the sum of regulatory
activities in an entire set of cells in a given embryo. Because regulatory activities of a given set of
transcription factors is determined by the information present in cis-regulatory modules (CRMs), one can
use a large number of CRM::reporter constructs as a reading device for embryonic regulatory states. In
this study, we used a set of 120 CRMs isolated from 42 sea urchin regulatory genes to quantitatively
measure regulatory states in developing embryos of the purple sea urchin, the pencil sea urchin, and the
zebrafish. We will present our findings on the developmental and evolutionary relationships of the
regulatory states measured in the three species.
66 PARtisan Development
Moorhouse, Kathleen (Boston College); Gudejko, Heather (Boston College); Burgess, David (Boston
College)
Establishment and maintenance of cell polarity has become an increasingly interesting biological
question in a diversity of cell types and has been found to play a role in variety of biological functions.
Previously, it was thought that the sea urchin embryo remained relatively unpolarized until the first
asymmetric division at the 16cell stage of development. However, there is mounting evidence to suggest
that polarity is established much earlier. We analyzed roles of the cell polarity regulators, the PAR
complex proteins, and how their disruption in early development affects later developmental milestones
such as blastula and gastrula formation. We found that PAR6 along with aPKC and CDC42 localize to
the apical cortex (free surface) as early as the 2-cell stage of development and this localization is
retained through the gastrula stage. Uniquely, PAR1 also colocalizes with these apical markers through
the gastrula stage, despite the formation of a polarized epithelium. Additionally, PAR1 was found to be
in complex with aPKC, but not PAR6, during these developmental stages. PAR6, aPKC, and CDC42 are
anchored in the apical cortex by myosin assembly. Myosin assembly was also found to be necessary to
maintain proper PAR6 localization through subsequent cleavage divisions. Interference with myosin
assembly prevented the embryos from reaching the blastula stage, while transient disruptions of either
actin or microtubules did not have this effect. Additionally, inhibition of myosin assembly effected actin,
but not myosin, localization. These observations suggest that disruptions of the polarity complex in the
early embryo can have a significant impact on the ability of the embryo to reach later critical stages in
development.
67 Germ Cell Associated Gene Expression is Regulated by Nodal Signaling in the Sea Star Patiria
Miniata
Fresques, Tara (Brown University, Wessel Lab); Zazueta, Vanesa (Brown University, Wessel Lab);
Reich, Adrian (Brown University, Wessel Lab); Wessel, Gary (Brown University, Wessel Lab)
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In many animals, germ cells are specified by cell-to-cell signaling events. In some of these animals,
conserved germ cell markers accumulate in broad embryonic domains in early development. Successive
rounds of restriction of the germ cell markers to smaller and smaller embryonic domains result through
development. In these animals it is unknown which signaling pathways are involved in restricting germ
cell markers to increasingly smaller embryonic domains. We noticed that in the development of the sea
star P. miniata , the germ cell factor Vasa similarly becomes sequentially restricted to smaller domains,
and becomes asymmetrically restricted in regards to the Dorsal/Ventral and Left/Right axis. We
hypothesize that the conserved Dorsal/Ventral and Left/Right signaling pathway ligand, Nodal, is
involved in restricting germ cell factors in the sea star, and thereby instructs the other cells to become
the germ line. We performed RNA in situ localizations with the known germ cell marker Vasa and known
Nodal signaling pathways members to test if they become asymmetrically expressed in opposite
embryonic domains at the same time. We also cultured embryos in a pharmacological inhibitor designed
to inhibit Nodal signaling to test if disrupting Nodal signaling causes aberrant accumulation of the germ
cell marker Vasa. We found that Vasa and Nodal become asymmetry expressed at the same time in
development and that disruption of the Nodal signaling pathway causes ectopic accumulation of Vasa.
These preliminary data suggest Nodal is required for repression of the germ cell fate, and the germ cell
mRNAs, in the ventral and right side of the embryo and identifies the first inhibitory signaling ligand that
may be involved in the inductive specification of germ cells via a sequential restriction mechanism.
68 Hedgehog Signaling Requires Motile Cilia in the Sea Urchin
Warner, Jacob (Duke University); Morris, Robert L. (Wheaton College); McCarthy, Ali (Wheaton
College); McClay, David (Duke University)
A relatively small number of signaling pathways govern the early patterning processes of metazoan
development. The architectural changes over time to these signaling pathways offer unique insights into
their evolution. In the case of Hedgehog (Hh) signaling, two very divergent mechanisms of pathway
transduction have evolved. In vertebrates, signaling relies on trafficking of Hh pathway components to
nonmotile specialized primary cilia. In contrast, protostomes do not use cilia of any kind for Hh signal
transduction. How these divergent lineages adapted such dramatically different ways of activating the
signaling pathway is an unanswered question. Here, we present evidence that in the sea urchin, a basal
deuterostome, motile cilia are required for embryonic Hh signal transduction, and the Hh receptor
Smoothened (Smo) localizes to cilia during active Hh signaling. This is the first evidence that Hh
signaling requires motile cilia and the first case of an organism requiring cilia outside of the vertebrate
lineage.
69 Tissue homeostasis, regeneration and negligible senescence: insight from the sea urchin
Bodnar, Andrea (Bermuda Institute of Ocean Sciences); Lortie, Mae (Bermuda Institute of Ocean
Sciences); Du, Colin (Bermuda Institute of Ocean Sciences); Parsons, Rachel (Bermuda Institute of
Ocean Sciences); Coffman, James (Mount Desert Island Biological Laboratory)
Sea urchins exhibit a very different life history from humans and short-lived model animals and therefore
provide the opportunity to gain new insight into the complex process of aging. Sea urchins grow
indeterminately, regenerate damaged appendages, reproduce throughout their lifespan and some
species show no increase in mortality rate at advanced ages. Further, different species of sea urchins
have very different reported lifespans, thus providing a unique model to investigate mechanisms
underlying both lifespan determination and negligible senescence. Studies to date have demonstrated
maintenance of telomeres, maintenance of antioxidant and proteasome enzyme activities and little
accumulation of oxidative cellular damage with age in tissues of sea urchin species with different
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lifespans. Gene expression studies using Strongylocentrotus purpuratus indicate that key cellular
pathways involved in energy metabolism, protein homeostasis and tissue regeneration are maintained
with age. Quantitative analyses of cell proliferation (BrdU incorporation) and apoptosis (assessed by
TUNEL and the Apo ssDNA™ assays) in sea urchin tissues (muscle, nerve, esophagus and
coelomocytes) indicates a low level of tissue renewal that is maintained with age in species with different
lifespans ( S. franciscanus , S. purpuratus and Lytechinus variegatus ). Expression of genes involved in
cell proliferation and/or pluripotency (i.e. pcna , tert , soxB1 , oct1/2 , myc , vasa , piwil2 ) is maintained
with age in these tissues. Regenerative capacity, assessed by measuring the regrowth of amputated
tube feet and spines in L. variegatus , is maintained with age. We postulate that long-term maintenance
of mechanisms that sustain tissue homeostasis and regenerative capacity are essential for
indeterminate growth and negligible senescence and an understanding of these mechanisms may reveal
effective strategies to prevent the degenerative decline with age.
70 Deuterostome origins of gastric pH regulation
Stumpp, Meike (Academia Sinica (ICOB)); Hu, Marian Y. (Academia Sinica (ICOB)); Tseng, Yung-Che
(National Taiwan Normal University); Su, Yi-Hsien (Academia Sinica (ICOB)); Yu, Jr-Kai (Academia
Sinica (ICOB)); Hwang, Pung-Pung (Academia Sinica (ICOB))
The highly acidic gastric environment (pH 2 to 3.5) in vertebrates created by the secretion of
hydrochloric acid (HCl) mainly functions to kill bacteria and to aid digestion by solubilizing food. The pH
regulatory mechanisms in vertebrate stomachs are well understood. Until present, though, the gastric pH
regulation in early deuterostome invertebrate larvae (such as echinopluteus and hemichordate tornaria)
has been of little interest. A contrasting feature of early deuterostome larval stomachs is their highly
alkaline gastric pH between 9.5 (echinopluteus) and 10 (tornaria) and well adapted digestive enzymes
(proteases and phosphatases). As determined using pharmacological, electrophysiological and
molecular tools, the cellular mechanisms responsible for gastric alkalization are mainly based on proton
excretion from the stomach lumen via the gastric cells to the extracellular matrix. In Strongylocentrotus
purpuratus , Na + /K + -ATPase (NKA) and H + -pump (VHA) are driving secondary active transporters
such as the Na + /H + -exchanger (NHE) and the K + /H + -exchanger (KHE). Hemichordate Ptychodera
flava larvae additonally employ bicarbonate secreting transporters such as Na + /HCO 3 - -cotransporter
(NBC3) and anion exchanger (AE) to archive an even higher pH within the stomach lumen than
echinopluteus larvae. Another interesting difference between both larvae is the use of H + /K + -ATPase
(HKA, detected by the alkalization inhibition by the specific inhibitor omeprazol) in P. flava instead of
VHA – and the presence of a protein in the stomach epithelium that is detectable by a human HKA
antibody. In vertebrates, HKA has evolved to the driving force and the major contributor to achieving
such a strong acidic environment within the stomach. Here we provide evidence that basic
characteristics of vertebrate gastric pH regulation are already established in echinoid and hemichordate
larval stages.
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Poster Abstracts
71 The small GTPase Arf6 is essential for the development of the sea urchin larval gut
Ahiakonu, Priscilla (University of Delaware); Stepicheva, Nadezda (University of Delaware); Dumas,
Megan (University of Delaware); Song, Jia L. (University of Delaware)
Arf6 is a small GTPase that acts as a molecular switch, cycling between the plasma membrane in its
active GTP bound form and endosomal compartments in its inactive GDP bound form. While a rich
knowledge exists in the cellular functions of Arf6, relatively little is known about its physiological role in
development. This study examines the function of Arf6 in mediating actin remodeling and membrane
trafficking in early development using the purple sea urchin as a model. We found that perturbation of
Arf6 in the form of its knockdown, the constitutively activated GTPase defective Arf6 mutant Arf6 (Q67L),
and the dominant active Arf6 mutant Arf6 (T27N), resulted in severe gastrulation defects. Arf6
knockdown led to a range of dose-dependent severity of developmental defects including a delay in
development, vegetal cell attachment, and exogastrulation. We tested perturbation of Arf6 during
gastrulation by examining its effect in the migration of endodermal cells. Results indicate that Arf6
perturbed embryos have wider gut size, blastopore and disorganized endodermal cells, leading to
severe defects in larval gut structures and function. Overall our results demonstrate that Arf6 is essential
for proper morphogenic movements of endodermal cells during gastrulation and the development and
function of the larval gut.
72 1-MA signaling and Hox cluster in the crown-of-thorns Acanthaster planci starfish.
Baughman, Ken (OIST); Satoh, Nori (OIST); Shoguchi, Eiichi (OIST)
AUTHORS: Kenneth Baughman, Nori Satoh, and Eiichi Shoguchi Crown-of-Thorns starfish Acanthaster
planci are corallivorous starfish, known for their consumption and devastation of hard corals in the
Pacific and Indian Oceans 1 . Investigation of A. planci developmental biology may be useful for
understanding how to mitigate the damage A. planci causes to coral reefs. First, we explored A. planci
1-methyl-adenine (1-MA) signaling in oocyte meiotic resumption 2,3 , and mapped 1-MA signaling using
our own A. planci sequencing data. We confirmed that 1-MA signaling functions in A. planci by using a
1-MA protocol to synchronize and fertilize eggs. We observed that 1-MA resulted in egg ejection from
female gonad tissue. Subsequent fertilization in the presence of 1-MA, but not in its absence, confirmed
1-MA function in A. planci. Based on the existing starfish literature and our own genome and
transcriptome data, we mapped 1-MA oocyte resumption in a Systems Biology Graphical Notation
(SBGN) diagram 4 . We find acceptable candidate genes for all components in our SBGN diagram.
Second, using our sequencing data, we identified the Hox cluster in A. planci , and explored its
phylogenetic consequences. Based on our Hox cluster data, we conclude that A. planci retains the
overall organization of the Hox cluster found in hemichordates 5 , but retains the uniquely echinoderm
organization of Hox13a-c. 1. Chesher, R. H. Destruction of Pacific corals by the sea star Acanthaster
planci. Science 165, 280–283 (1969). 2. Kishimoto, T. A primer on meiotic resumption in starfish
oocytes: The proposed signaling pathway triggered by maturation-inducing hormone. Mol. Reprod. Dev.
78, 704–707 (2011). 3. Kishimoto, T. & Kanatani, H. Cytoplasmic factor responsible for germinal vesicle
breakdown and meiotic maturation in starfish oocyte. Nature 260, 321–322 (1976). 4. Le Novère, N. et
al. The Systems Biology Graphical Notation. Nat. Biotechnol. 27, 735–741 (2009). 5. Freeman, R. et al.
Identical Genomic Organization of Two Hemichordate Hox Clusters. Current Biology 22, 2053–2058
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(2012).
73 A transcriptomic strategy for identifying novel mediators of the sea urchin larval immune
response
Buckley, Katherine M. (University of Toronto, Sunnybrook Research Institute); Ho, Eric (University
of Toronto, Sunnybrook Resear); Rast, Jonathan (University of Toronto, Sunnybrook Resear)
The purple sea urchin genome encodes a complex repertoire of genes involved in the immune response,
including large gene families of pathogen recognition receptors, as well as signaling and effector
molecules. Recently available transcriptome data add a further dimension by identifying expressed
sequences more accurately than gene model predictions. Genes involved in the immune response are
difficult to identify in a non-targeted screen for two reasons: (1) Many immune genes are transcriptionally
activated only in response to specific pathogens. (2) Genes involved in immunity tend to evolve quickly
and are therefore difficult to identify computationally based on sequence similarity. To identify genes that
mediate the sea urchin immune response, we have generated RNA-Seq data from larvae responding to
a gut-associated bacterial challenge. Larvae were exposed to the marine bacteria Vibrio diazotrophicus
and collected at several time points. These data were used to assess expression levels of known
transcripts throughout the course of infection. Several transcriptionally regulated genes were identified
that are known to have important homologs in vertebrate immunity (e.g., IL-1 receptor, members of the
Socs family, Nf-?B and PU.1). Additionally, we assembled the reads to generate the immune activated
transcriptome. From this assembly, we have identified 184 previously unknown transcripts that are
strongly activated in response to bacteria. Among these are a divergent homolog of IL-17, which is a
key regulator of vertebrate epithelial immunity, and a transcript that encodes a peptidoglycan binding
domain. Novel immune effectors and signaling molecules have been difficult to identify outside of
vertebrates and this approach offers a new strategy for their discovery.
74 Expression of the karyopherin-alpha family of nuclear transport proteins in Lytechinus
variegatus.
Byrum, Christine (College of Charleston); Smith, Jason (College of Charleston); Easterling, Marietta
(College of Charleston); Bridges, M. Catherine (College of Charleston)
Among eukaryotes, karyopherin-alpha (KAP-α) importins act in concert with the KAP-β protein KPNB1 to
transfer transcription factors and other molecules across the nuclear pore complex. Although many
importins are ubiquitously distributed, differential expression of the KAP-α family has been reported in
several instances and two of these, KPNA1 and KPNA2, play important roles in neural differentiation of
mouse embryonic stem cells (Yasuhara et al., 2007 and 2012). To investigate potential roles of these
nuclear transport proteins in developmental processes, we identified three members of the KAP-α family
and examined mRNA distribution of these importins in embryos of the sea urchin Lytechinus variegatus.
We found that LvKPNA1/5/6 and LvKPNA3/4 were clearly expressed in cleaving embryos. By the
blastula and gastrula stages, expression was most evident in the vegetal plate and archenteron, and by
the prism/pluteus stages, faint staining was observed only in the oral surface and/or gut. A third
karyopherin, LvKPNA2/7, exhibited a different expression pattern and was first observed in the vegetal
plate and in patches of ectodermal cells within the mesenchyme blastula. By the gastrula stage, it was
found in the archenteron and several additional patches of ectodermal cells. In prism/pluteus stage
embryos, LvKPNA2/7 was restricted to the gut and was localized to subsets of cells along the ciliary
band. Similar patterns have been reported for the neural marker Synaptotagmin and for another gene
that influences neural differentiation in vertebrates, LvBrn1/2. Our study is an important first step
towards better understanding roles of these nuclear transport proteins in embryogenesis.
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75 Transcriptome Analysis of Late Gastrula Pigment Cells
Calestani, Cristina (Valdosta State University); Barsi, Julius C (California Institute of Technology); Tu,
Qian (California Institute of Technology); Ortiz, Antonio (Valdosta State University); Buckley, Kate M
(University of Toronto); Stearnes, Ariel (Valdosta State University); Rast, Jonathan P (University of
Toronto); Davidson, Eric H (California Institute of Technology)
Pigment cells derive from the non-skeletogenic mesenchyme and migrate during early gastrula to the
aboral ectoderm. Several evidences support a role of pigment cells in the immune-defense of the larva.
In order to identify a comprehensive set of genes required for pigment cells development, with a
particular focus on differentiation genes and their direct upstream regulators, we sequenced the
transcriptome of a late gastrula pigment cell enriched sample (39-46 hours p.f.). The transcriptome
sequence has also the potential to shed more light on pigment cells function. The RNA-seq approach
involved the comparison of a pigment cell enriched with a pigment cell depleted sample. Pigment cells
were labeled by injecting fertilized eggs with a BAC containing gfp driven by the pigment cell specific
promoter of glial cell missing (gcm). Embryos were disaggregated and GFP positive cells were isolated
by FACS. The GFP negative fraction was also collected. RNA and cDNA libraries were prepared from
both the GFP positive and the GFP negative fractions. The cDNA libraries were then sequenced. An
estimate of the pigment cell genes enrichment was provided by the ratio between the transcript number,
expressed as fragments per kilobase of transcript per million mapped reads (FPKM), of the GFP positive
over the GFP negative fraction. Genes known to be exclusively expressed in pigment cells, such as gcm,
six1/2, sulfotransferase1, flavin monooxygenase1, 2 and 3 were very effectively enriched, within a range
of 50 to 250 times. Genes enriched at least 10 times belong to a wide range of functional categories
including: cell adhesion, defensome, GPCR Rhodopsins, immunity, metalloproteases, nervous system,
cell signaling, and transcription factors.
76 mTOR regulation of polysomal recruitment at fertilization
Chassé, Héloïse (équipe TCCD, UMR 8227, Station Biologique de Roscoff, CNRS/UPMC); Boulben,
Sandrine (équipe TCCD, UMR 8227, Station Biologique de Roscoff, CNRS/UPMC); Cormier, Patrick
(équipe TCCD, UMR 8227, Station Biologique de Roscoff, CNRS/UPMC); Morales, Julia (équipe TCCD,
UMR 8227, Station Biologique de Roscoff, CNRS/UPMC)
In sea urchin eggs, fertilization triggers a 10-fold increase in protein synthesis rates while initiating the
synchronous divisions of early development. Sea urchin eggs contain the initiation factor eIF4E
associated to its inhibitor 4EBP. Fertilization induces a rapid dissociation of eIF4E from 4EBP, eIF4E
then associates to the scaffold protein eIF4G, initiating the formation of a functional initiation complex.
These events are dependent upon the activation of the mTOR pathway. Incubation of sea urchin eggs
with mTOR inhibitors such as rapamycin or PP242, inhibits protein synthesis and the onset of cell cycle.
The translatome corresponds to the subset of maternal mRNAs actively engaged in translation, which
are recruited into polysomal fractions. We have analysed the role of the mTOR pathway on the
translatome at fertilization using PP242 inhibitor: we show that the polysomal recruitment of some
mRNAs are sensitive to mTOR inhibition, while others mRNAs are still translated despite the inhibition of
the mTOR pathway. Among the translated mRNAs strongly dependent on mTOR activity are the mRNAs
encoding proteins involved in the cell cycle.
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77 SLC and Notch2 regulate dorsal-ventral PMC positioning and skeletal patterning
Chung, Oliver (Boston University); Piacentino, Michael (Boston University); Hewitt, Finnegan (Boston
University); Patel, Vijeta (Boston University); Ferrell, Patrick (Boston University); Chaves, James (Boston
University); Li, Christy (Boston University); Hameeduddin, Hajerah (Boston University); Poutska, Albert
(Max Planck Institute); Bradham, Cynthia (Boston University)
In the early stages of sea urchin embryonic development, primary mesenchyme cells (PMCs) secrete a
calcium carbonate skeleton in response to patterning cues from the adjacent ectoderm. To determine the
molecular mechanism underlying skeletal patterning, we performed an RNA-seq-based screen, which
identified several ectodermal patterning gene candidates, including the sulfate transporter, SLC26a2
(SLC), and the receptor, Notch2. SLC loss-of-function (LOF) results in PMC localization to the dorsal
hemisphere, and results in ventral and anterior skeletal patterning defects. SLC is expressed in the
ventral and anterior ectoderm, suggesting that SLC functions to provide an attractive cue to PMCs.
Interestingly, Notch2 LOF promotes the reciprocal phenotype, with ventral PMC localization and dorsal
skeletal patterning defects. Like SLC, Notch2 is also expressed in the ventral ectoderm. These data
suggest that SLC establishes a gradient in sPGs that provides an attractive cue, while Notch2 functions
as a molecular switch to direct a subset of PMCs to the dorsal territory. To test this model, we performed
combined SLC LOF and Notch2 LOF experiments. Loss of both SLC and Notch2 produces embryos that
lack an organized PMC ring; instead, the PMCs adopt an arbitrary and disorganized pattern within the
embryonic blastocoel. These embryos typically fail to form a skeleton, or produce only small,
rudimentary skeletal elements. Co-injection of control MO with either SLC or Notch2 MO does not
provoke a similar outcome, but instead such embryos resemble the corresponding single MO
phenotypes. These results support the model in which SLC and Notch2 function together to direct
primary PMC migration and skeletal patterning.
78 Mesoderm and Micromere Development in Eucidaris tribuloides
Coots, Ashley D. (Rochester Institute of Technology); Covington, Rae Ann (Rochester Institute of
Technology); Lung, Kara (Rochester Institute of Technology); Wood, Maureen (Rochester Institute of
Technology); Sweet, Hyla (Rochester Institute of Technology)
Sea urchins are grouped into cidaroids (pencil urchins) and euechinoids (including pin-cushion type
urchins, sand dollars, and heart urchins), which diverged about 250 million years ago. These groups
have differences in both adult and embryo morphology. In the field of sea urchin development,
euechinoids have been commonly studied, however, cidaroid development has not. The purpose of this
study is to further document the normal development of the cidaroid sea urchin, Eucidaris tribuloides.
The development of skeletogenic cells and pigment cells was examined. Both cell types accumulate
slowly during development to about sixteen cells per embryo; however, the number of cells is quite
variable. Micromere development during cleavage was also examined. In euechinoid embryos, four
micromeres form at the 16-cell stage and form large micromeres and small micromeres at the next
cleavage. The large micromeres develop into the skeleton, while the small micromeres contribute to the
coelomic pouches. The micromeres also send signals to surrounding cells to make pigment cells and
other cell types. In cidaroid embryos, the number of micromeres is variable in that they may have 0, 1, 2,
3, or 4 micromeres in one embryo. The micromeres of the 16-cell stage embryo do divide to form both
large micromeres and small micromeres. The number of micromeres contained in cidaroid embryos was
found to be directly correlated with the number of skeletogenic cells in later stage embryos. In future
studies, further research will be done on how the variability of micromeres affects cell signaling in the
cidaroid embryo.
79 Expression of embryonic skeletal development genes during adult arm regeneration of the
brittle star Amphiura filiformis
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Czarkwiani, Anna (University College London); Dylus, David (University College London); Oliveri,
Paola (University College London)
The brittle star Amphiura filiformis is becoming an important model for regeneration research due to the
emerging molecular toolbox which will complement already existing anatomical and ecological data. Its
arms regenerate within a few weeks and re-pattern all the essential components such as skeleton,
podia, radial water canal and the nerve. Initially after amputation and wound epithelialisation, a blastema
(mass of undifferentiated cells) will be formed. Then during regeneration the small arm will elongate and
begin to differentiate metameric units following the invasion of the radial water canal. Our lab has
already shown that gene expression analysis like qPCR and whole-mount in situ hybridization (WMISH)
can be successfully employed to study genes involved in brittle star arm regeneration. We use this novel
model system to understand the relationship between regeneration and embryonic development using
skeletogenesis as a proxy for studying cell specification, morphogenesis and molecular mechanisms.
Using the WMISH technique we have characterized the expression pattern of approximately 20
important gene candidates from the embryonic skeletogenesis GRN during arm regeneration in A.
filiformis. We found that most of the genes, which are expressed in the skeletal lineage of the brittle star
embryo, are also localized to the skeletal domains in adult arm regeneration. During the early stage of
regeneration those genes are expressed in a subepithelial layer of mesenchymal cells, which is where
spicules first appear, and then become restricted to different skeletal elements during differentiation.
Altogether this data shows that the regeneration molecular network underlying skeletogenesis might be
similar to the embryo skeletogenic network, consistent with the idea of the co-option of the larval
skeleton form the adult.
80 Polarity of small micromeres and its impact on localization of plasma membrane proteins
Espinoza, Jose A. (University of California, San Diego); Campanale, Joseph (Scripps Institution of
Oceanography); Gokirmak, Tufan (Scripps Institution of Oceanography); Hamdoun, Amro (Scripps
Institution of Oceanography)
Micromeres (Mics) undergo a reduction in ATP binding cassette (ABC) transporter activity, presumably
related to their later epithelial-mesenchymal transition (EMT). Overexpression of constitutively active
Cdc42 causes accumulation of the fluorescent ABC-transporter substrate calcein in the Mics to decrease
from 1.6 to 1.07 fold (Mics/rest of the embryo). Calcein accumulation in the whole embryo remained
unchanged, suggesting that ABC-transporter down-regulation is linked to cellular polarity. Next, we
investigated cellular polarity in small micromeres (Smics) by expressing fluorescent pleckstrin homology
domain (PH) of phospholipase C-delta, which binds to phosphatidylinositol 4,5-bisphosphate (PIP2).
Smics accumulate nearly 80% more PH fluorescence on their basal surface than macromeres and
mesomeres, indicating that cellular polarity in the Smics is altered. To examine relations between
reduction in ABC-transporter activity and cellular polarity we co-expressed Sp -Vasa with a suite of
ABC-transporters with robust apical and basolateral membrane polarity. We compared fluorescence of
each transporter in Smics to controls in cleavage stage embryos. The apical transporters Sp -ABCB1a
and Sp -ABCB4a, basolateral transporter Sp -ABCC1, and a neutral membrane marker were 15 to 50%
less fluorescent on apical membranes. The apical transporter Sp -ABCG2a and the basolateral
transporter Sp -ABCC1ß were 50 and 5% brighter, respectively. A 50-80% increase in fluorescence on
the basal surface of Smics was measured for all transporters. Finally, we immunolocalized ABCB1a and
Vasa protein at 48 HPF and found reduced ABCB1a localization on the apical surface of the Smics,
suggesting that protein accumulation remains low and cellular polarity remains altered through
migration. In the future, we plan to study interactions between polarity generating molecules in Smics,
including cdc42 and phosphatidylinositol lipids, and ABC-transporter activity.
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81 Ophioplocus esmarki Embryo and Larvae Swimming Behavior
Freyn, Alec W. (Rochester Institute of Technology); Coots, Ashley (Rochester Institute of
Technology); Sweet, Hyla (Rochester Institute of Technology)
Brittle stars can be distinguished based on mode of reproduction as brooding or non-brooding species.
Brooding brittle stars keep developing young inside bursal pouches until the juvenile stage, while
non-brooders allow embryos and larvae to project into the water column, where ocean currents can
transport them away from their parents. Brittle star larvae are further characterized as feeding or
non-feeding. In feeding larvae, ciliary bands are used to obtain nutrients from the environment.
Non-feeding larvae also have ciliary bands, but primarily use them for swimming. The main purpose of
this study was to observe swimming patterns and calculate swimming speeds of the embryos and larvae
of the brooding, non-feeding brittle star, Ophioplocus esmarki . We hypothesize that swimming behavior
will differ in comparison to those displayed in non-brooding brittle star species. Embryos and larvae were
collected from bursal pouches of adult brittle stars and their swimming behavior was observed up to
eight days after fertilization. Unlike other non-feeding brittle stars, the embryos and larvae of this
brooding species do not swim vertically into the water column. Swimming patterns consisted of four
categories: circular, linear, hovering, and still, which are similar to what has been found in other
non-feeding brittle stars. Swimming speeds were calculated by distance traveled (in mm) per second. It
was found that there was a difference in swimming patterns over time; the embryos were more active
early in development (swimming in circular and linear patterns) rather than later. There was also a
statistically significant decrease in swimming speed over the six-day period. The changes observed in
swimming behavior may correspond to changes in ciliary band, skeleton, or nervous system
development.
82 Identifying and measuring voltage gradients in normal and perturbed embryos
Hadyniak, Sarah E. (Boston University); Schatzberg, Daphne (Boston University); Lawton, Matthew L.
(Boston University); Bishop, Jacob (Boston University); Beane, Wendy (Tufts University); Levin, Michael
(Tufts University); Bradham, Cynthia (Boston University)
Embryonic patterning requires the formation of endogenous bioelectrical gradients in several
developmental model organisms. Voltage gradients also play a significant role in wound healing and
regeneration. To identify changes in the relative voltage of the embryo, we used DiBAC 4 , a
bis-barbituric acid oxonol dye that exhibits enhanced fluorescence in depolarized cells. In control
embryos, DiBAC 4 fluorescence decreases steadily over time, indicating a relative hyperpolarization as
development proceeds. This change is consistent with prior observations that differentiated cells are
hyperpolarized compared to undifferentiated cells. In addition, the PMCs are relatively hyperpolarized
compared to ectoderm in control embryos. The embryo also exhibits a gradient of electrical activity
across the ectoderm. To determine the orientation of this gradient, we injected Nodal mRNA plus a
fluorescent dye into one blastomere at the four cell stage, which biases the labeled blastomere to a
ventral fate, then imaged the ventral label in conjunction with DiBAC 4 . The results show that control
embryos possess a dorsal-ventral gradient of polarization within the ectoderm at hatched blastula stage.
We performed a pharmacological screen for ion channel inhibitors, and identified SCH28080 as an
inhibitor of skeletogenesis, and Concanamycin A (ConA) as an inhibitor of dorsal-ventral specification.
DiBAC 4 analysis shows that SCH28080, an H + /K + antiport inhibitor, generally depolarizes the
embryo, including the PMCs, although only the PMCs are developmentally affected by SCH28080. We
will also report the effect of ConA, a V-ATPase inhibitor, on the DiBAC 4 distribution overall and within
the ectoderm. Together these data establish the existence of voltage differences within the embryo, and
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an important role for voltage differences within the PMCs and the ectoderm during normal development.
83 Semi-dry sea urchin experiment using preserved egg and sperm supply
Kiyomoto, Masato (Ochanomizu University)
The sea urchin is a wonderful material for observing animal development. Here, I propose a supply of
preserved egg and sperm to realize a semi-dry sea urchin experiment, neither temperature-controlled
aquarium nor a large volume of seawater needed. Sea urchin gametes were preserved for one month in
some cases. Spawned eggs could be kept for the time with fertilizing ability in antibiotics seawater as
Epel reported. It is also possible to lengthen the preserved time of dry sperm by the addition of
antibiotics. Then, the provider, such as the marine laboratories, collects and prepares them, each
laboratory and school just receive and do experiment. We had supported high schools from the two
projects supported by CoREF (Consortium for Renovating Education of the Future), one was an easy
and simple fertilization experiment without taking care of urchins for the beginners and the other was a
larval culture in a small bottle by each student to observe the metamorphosis to juveniles. We started
new projects, one is for the experimental courses in the universities (Supply of Marine Bioresource
supported by the Ministry of Education, Culture, Sports, Science & Technology in Japan) and the other
is for all elementary and secondary educations (as A Gift from the Sea supported by The Nippon
Foundation). Both include gametes, embryos, larvae and juveniles to observe important stages.
84 Development of the nervous system in a sea cucumber, Apostichopus japonicus: shift from
bilateral to pentaradial symmetry
Kondo, Mariko (The Univ. of Tokyo); Nagai, Akiko (The Univ. of Tokyo); Kikuchi, Mani (The Univ. of
Tokyo); Omori, Akihito (The Univ. of Tokyo); Akasaka, Koji (The Univ. of Tokyo)
The five-fold radial, or pentaradial symmetry in the adult body plan is a well-known feature of
echinoderms. In contrast, the larval body displays bilateral symmetry, and the body plan changes at the
metamorphosis stage. In an attempt to investigate how pentaradial symmetry is formed, we are using a
sea cucumber Apostichopus japonicus as our model. The sea cucumber does not produce an adult
rudiment before metamorphosis, so the whole larval body is modified into the adult body. We are
especially interested in the formation of neural tissues, since it is relatively easy to observe its
development, and could possibly provide clues to how pentaradial symmetry is formed; during early
development, a larval nervous system is generated, but a separate adult nervous system with five radial
nerves appears at metamorphosis, which is probably one of the earliest examples of pentaradial
symmetry. However, the mechanism how the nervous system or an overall pentaradial body is
established is unknown. In addition, the developmental process of the sea cucumber is the same as sea
lilies that are considered the most basal group in echinoderms, so this type of development may be the
ancestral form of echinoderms. Therefore, the study on sea cucumber development may reveal the
evolution of echinoderm body patterning. We have cloned several neural tissue marker genes including
musashi (a marker for neurogenesis) and soxB1 (marker for stem cells or precursor cells in the central
nervous system). Using these markers for expression analysis and anti-neural protein antibodies to
perform immunohistochemistry, we are aiming to identify in which developmental stage and from where
the adult nervous system arises. We expect that this work will provide insights to the molecular basis of
the shift from bilateral to pentaradial symmetry.
85 Expression of germ cell markers in hemichordate Ptychodera flava: implication to the
embryonic origin of PGCs in Ambulacraria
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Lin, Ching-Yi (Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan); Yu, Jr-Kai
(Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan); Su, Yi-Hsien (Institute of Cellular
and Organismic Biology, Academia Sinica, Taiwan)
Specification of primordial germ cells (PGCs) during development is an essential step for animals to
produce next generation. Hemichordates are the sister group of echinoderms, which together are
referred to as the Ambulacraria, that is closely related to chordates. According to previous studies, the
PGCs of echinoderms develop from the coelom of larva except some sea urchin species, which develop
PGCs from small micromere lineage. To understand how the common ancestor of Ambulacraria
developed their PGCs, we study the expression of germline specific genes vasa and nanos in a
hemichordate acorn worm Ptychodera flava . We found maternal vasa and nanos transcripts are
expressed ubiquitously during the cleavage stages; subsequently vasa and nanos transcripts are
co-localized to the dorsal-vegetal region of the archenteron after gastrulation. However, we found
cytoplasmic vasa protein signals distributed near the vegetal pole of the unfertilized eggs and
subsequently localized to the 8 vegetal blastomeres of the 32-cell stage embryo. After 64-cell stage, the
distribution of vasa protein becomes peri-nuclear in the most vegetal-tier blastomeres. During
gastrulation, the number of vasa-positive cells decreases, and these cells are localized to the
dorsal-vegetal region of the archenteron, where the vasa and nanos are expressed zygotically. In
tornaria larvae, these cells start to proliferate and later contribute to the developing adult coelom. Our
results show the evolutionary consistency of PGCs marker genes expression patterns between
echinoderms and hemichordate P. flava . However, the enrichment of vasa protein in the vegetal
blastomeres appears to be much earlier in P. flava , suggesting there are some variations in vasa
protein regulation between echinoderms and hemichordates during early embryogenesis.
86 BioTapestry: Interacting With the Network Directly in the Web Browser
Longabaugh, William (Institute for Systems Biology); Paquette, Suzanne (Institute for Systems
Biology); Leinonen, Kalle (Institute for Systems Biology)
BioTapestry is a well-established tool for building, visualizing, and sharing models of gene regulatory
networks (GRNs), with particular emphasis on the GRNs that drive development. The Java-based
BioTapestry Viewer has been used to provide an interactive online version of the sea urchin
endomesoderm network since 2003. Newer web-browser technologies such as HTML5 Canvas have
made it possible to provide an interactive graphical network model directly in a web browser, and we
have now created a version of the BioTapestry Viewer using these technologies. At the same time, this
new software architecture continues to support the traditional BioTapestry Editor desktop application.
87 How pluteus arms were evolved?
Morino, Yoshiaki (University of Tsukuba); Koga, Hiroyuki (University of Tsukuba); Wada, Hiroshi
(University of Tsukuba)
Some lineage of the echinoderms experienced an amazing evolution in their larval forms, namely
acquisition of the pluteus arms. In sea urchin, interaction between epidermis and mesenchyme was
shown to be essential for development of the pluteus arms. We also reported that a similar interaction is
probably essential for the development of larval arms of ophiopluteus. Thus, the interaction between
epidermis and mesenchyme is key issue for understanding the origin of pluteus larvae. Based on the
common gene expression pattern of transcription factors between larval and adult skeletogenesis, it was
suggested that evolution of larval skeleton was achieved in part by co-option of the specification
program of adult skeletogenic cells were co-opted to embryonic stage. Here, we asked whether the
interaction between epidermis and mesenchyme was also co-opted for the pluteus evolution. We
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focused the development of direct-type developer sea urchin Peronella japonica . We examined
expression pattern of alx1 , fgfA / fgfr2 , vegf / vegfr , pax2/5/8 , otp , pea3 , tetraspanin , wnt5 and P4
antigens in several developmental stages from gastrula to juvenile. Based on these dates, we discuss
multiple steps required for the evolution of pluteus arm.
88 5-LOX is Required for Skeletal Patterning in Sea Urchin Embryos
Murray, Ian S. (Boston University); Patel, Vijeta (Boston University); Li, Christy (Boston University); Yu,
Annie (Boston University); Hameeduddin, Hajerah (Boston University); Hewitt, Finnegan (Boston
University); Poustka, Albert (Max-Planck Institut für Molekulare Genetik); Bradham, Cynthia (Boston
University)
Skeletal patterning in sea urchins requires ectodermal instruction of the skeleton-secreting primary
mesenchymal cells (PMCs). At late gastrula stage, the PMCs migrate to form a ring-and-cords pattern
and begin to secrete the skeleton in the form of two triradiates. A comprehensive screen for ectodermal
genes involved in the direction of PMC migration and positioning identified multiple skeletal patterning
genes, including lipoxygenase (LvLOX). LOX is a leukotriene generator which indirectly impacts multiple
signaling cascades. LvLOX was knocked down using an antisense morpholino-substituted
oligonucleotide (MO), and skeletal patterning defects were systematically evaluated. The resulting
embryos exhibit defects in triradiate orientation about the DV axis, defects in 2° skeletogenesis on the
left side, and loss of midline elements. LOX exists in three primary isoforms in mammalian cells (5-LOX,
12-LOX, and 15-LOX), but the sea urchin ortholog cannot be definitively classified by Bayesian
phylogeny. To better classify LvLOX, embryos were treated with three isoform-specific LOX inhibitors
and a pan-LOX inhibitor, and the skeletal defects of the resulting perturbants were compared to that of
LvLOX morphants. The embryos treated by the 5-LOX inhibitor MK886 exhibited similar rotational
defects as well as loss of midline and left side elements, while the other isoform-specific drugs did not
mimic the LvLOX MO. The pan LOX inhibitor produced more severe defects than either MK886 or
LvLOX MO. Together these data demonstrate that leukotrienes are required for normal skeletal
patterning, particularly in the midline and the left side, and for regulating triradiate orientation about the
DV axis.
89 Histological and molecular biological analysis on the adult nervous system of the feather star
Oxycomanthus japonicus
Omori, Akihito (Misaki Marine Biological Station); Kurokawa, Daisuke (Misaki Marine Biological
Station); Akasaka, Koji (Misaki Marine Biological Station)
Adult nervous system of feather stars is subdivided into three systems; oral, deeper-oral, and aboral
systems. Although previous studies show the shapes and possible functions of neural cells in each
nervous system, studies about detailed whole-body cell distributions of these nervous systems is still
limited. To confirm whole-body structures of these nervous systems, we examined immunolocalizations
and gene expression patterns of some neural markers as well as observing detailed histological
structures in a feather star Oxycomanthus japonicus . Histological stainings revealed a large ganglion of
aboral nervous system with highly condensed cell bodies. Synaptotagmin immunoreactivity was detected
in the ganglion, the nerve rings, and the radial nerves of all three nervous systems. We found that the
expression of pan-neural marker gene Elav is not expressed in cells around articulations in the aboral
brachial nerves, which indicates the existence of non-neural cells in the aboral nervous system. We also
found that the expression of neural stem cell marker gene musashi is limited in the oral nervous system,
suggesting the existence of immature neural cells in the oral nerves.
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90 Circadian clock in the S. purpuratus larva: a diverged time-keeping mechanism that drives 24h
rhythmicity?
Petrone, Libero (UCL); Lerner, Avigdor (UCL); Oliveri, Paola (UCL)
A circadian clock is a 24h time-keeping mechanism that synchronizes several biological processes with
local environment. In metazoans the circadian system is driven by a regulatory network of so called
"clock genes" interconnected in a transcriptional-translational feedback loops (TTO) that generates
rhythmicity at mRNA and protein level. The TTO is entrained to environmental cues such as light.
Evolution of circadian clocks provides an ideal system to understand evolution of regulatory networks.
Sea urchin and its molecular tools can facilitate the comprehension of the evolution of the time-keeping
mechanism in bilaterians. For this purpose we identified and analyzed the expression of orthologous
clock genes in the sea urchin larvae. Genome survey identifies almost all clock genes known in
protostomes and deuterostomes, with exception of period and p-like cry, indicating that the last common
ancestor of all bilaterians had a complex clock toolkit. Furthermore, protein domain analysis shows
evolution of proteins consistent with the presence/absence of members of the clock toolkit. Quantitative
gene expression data reveal that the circadian clock begins to oscillate consistently in the free-living
larva. Spu-vCry and Spu-tim show an opposite 24h oscillation in both light/dark (L/D) and free running
(D/D) conditions; several other genes consistently show oscillation in L/D condition only; while, neither
Spu-clock, nor Spu-bmal have rhythmic expression. Interestingly, in-situ hybridization of all sea urchin
clock genes together with cell markers (e.g. serotonin) suggest the presence of light perceiving cells in
the apical organ with a molecular mechanism similar to the protostome one. Importantly, our study
highlights differences in the architecture and gene regulation of the sea urchin larval circadian clock
compared to other metazoan clocks.
91 Appearance of Order Level Traits in the Triassic Echinoid Fossil Record
Petsios, Elizabeth (University of Southern California); Thompson, Jeffery (University of Southern
California); Bottjer, David (University of Southern California)
Echinoid diversity in the Triassic (252-201 million years ago) is poorly understood, impeding detailed
study of the evolutionary history of this group during this crucial time period following the appearance of
the two modern subclasses of regular echinoids, cidaroids and euechinoids. Fossil representatives of
early cidaroids and euechinoids are scarce, making interpretation of the evolutionary timing of
appearance of crown group skeletal characters difficult. Diversification of these two groups appears to
have occurred sometime during the Permian, arising from the family Archaeocidaridae, though fossil
representatives from this time are limited. Stem group cidaroids of the family Miocidaridae are known
from the Permian and Early Triassic, but it is not until the Late Triassic that the first definitive euechinoid
is found. Known Triassic echinoid diversity tracks the occurrences of rare sites of exceptional echinoid
fossil preservation (Lagerstatten), where whole or partially complete tests can be found. However, a
large distribution of disaggregated echinoid material can be found throughout Triassic deposits,
representing an untapped pool of potential diversity information. Though species level differentiation of
specimens requires considerable preservation of articulated test material, ordinal level characters can
be identified allowing for distinction between ancestral cidaroids and euechinoids and the identification
of character states. Presented here is a survey of known Triassic echinoid diversity from the literature
and the earliest known occurrences of ordinal level cidaroid and euechinoid traits. Future work will aim
to uncover additional disarticulated fossil material from deposits that may record the occurrences of
ordinal level characters from previously unknown time periods.
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92 Concanamycin A perturbs dorsal-ventral specification in sea urchin embryos
Reidy, Patrick (Boston University); Bishop, Jacob (Boston University); Schatzberg, Daphne (Boston
University); Zushin, Peter (Boston University); Ross, Erik (Boston University); Carney, Tamara (Boston
University); Bradham, Cynthia (Boston University)
Bioelectrical gradients are important for normal development, wound healing, and regeneration.
Bioelectrical changes are mediated by differential ion channel activity. In a pharmacological screen for
ion channel inhibitors, we found Concanamycin A (ConA), a V-ATPase inhibitor, blocks dorsal-ventral
specification in sea urchin embryos. ConA treatment results in morphological and skeletal radialization,
and ConA-treated embryos exhibit an unrestricted ciliary band that occupies most of the ectoderm.
Timecourse experiments demonstrate that embryos remain sensitive to ConA until after mesenchyme
blastula stage, indicating that the effect of ConA is subsequent to the onset of Nodal and BMP2/4
expression. In situ hybridization experiments show that ConA-treated embryos express BMP2/4 mRNA,
but do not express BMP2/4 targets, while Nodal and Nodal targets exhibit expanded expression profiles.
Taken together, these data indicate that ConA treatment ventralizes embryos and prevents BMP2/4
target gene expression.
93 LvBMP5-8 is required for normal skeletal patterning but not dorsal-ventral specification in the
sea urchin embryo
Ramachandran, Janani (Boston University); Chung, Oliver (Boston University); Piacentino, Michael
(Boston University); Reyna, Arlene (Boston University); Yu, Jia (Boston Uniersity); Hameeduddin,
Hajerah (Boston University); Poustka, Albert (Max Planck Institute); Bradham, Cynthia (Boston
University)
Skeletal patterning in the sea urchin embryo is established by the communication between the
pattern-dictating ectoderm and the skeletogenic primary mesenchyme cells (PMCs); however, the
molecular basis for this process remains unknown. To identify skeletal patterning genes, we performed a
three-way differential RNA-seq screen. One candidate gene identified by this screen is bone
morphogenetic protein 5-8 (BMP5-8), a member of the TGF-β superfamily of signaling ligands. At late
gastrula stage, LvBMP5/8 is expressed in the ventral ectoderm. We performed BMP5-8 loss-of-function
(LOF) analysis by morpholino antisense oligonucleotide (MO) microinjection, and found that BMP5-8
morphant plutei display rotational and left-sided skeletal patterning defects. Dorsal-ventral (DV) axis
specification and neural development are normal in BMP5-8 LOF embryos, indicating that LvBMP5-8 is
not required for dorsal specification. Consistent with that conclusion, combined injections of LvBMP2/4
and LvBMP5-8 MO appear equivalent to LvBMP2/4 MO alone. Interestingly, BMP5-8 gain-of-function
(GOF) results in radialization and loss of skeletal elements, similar to the effects of BMP2/4 GOF, except
that BMP5-8 GOF embryos exhibit an apical bellcap. BMP5-8 GOF results in a complete loss of ventral
Chordin expression and a radialization of dorsal Tbx2/3 expression, indicating that BMP5/8 is sufficient
for dorsalization. Immunostaining in BMP5-8 GOF embryos show that the ciliary band is restricted to the
apical plate, and serotonergic neurons are restricted to the apical bellcap. Synaptotagmin B-positive
neurons extend processes radially around the ectoderm in a circumferential belt, and occasionally
occupy the endoderm in BMP5-8 GOF embryos. Thus, LvBMP5-8 is required for left-right but not
dorsal-ventral specification.
94 H+/K+ antiport activity is required for PMC differentiation and skeletogenesis
Schatzberg, Daphne (Boston University); Lawton, Matthew (Boston University); Hadyniak, Sarah
(Boston University); Bishop, Jacob (Boston University); Ross, Erik (Boston University); Carney, Tamara
(Boston University); Beane, Wendy (Tufts University); Levin, Michael (Tufts University); Bradham,
Cynthia (Boston University)
Page 47 of 51
The bioelectrical signatures associated with regeneration, wound healing, development, and cancer are
low amplitude changes in the polarization state of the cell, which persist over long durations. To identify
ion channels required for bioelectrical changes during normal development of the sea urchin Lytechinus
variegatus , a pharmacological screen was performed . We identified SCH28080, a competitive inhibitor
of the H + /K + antiport ATPase, as a potent inhibitor of skeletogenesis in the developing larva. The
ectoderm is correctly specified and differentiated in SCH28080-treated embryos, indicating that
SCH28080 exerts a PMC-specific effect on skeletogenesis. A panel of fluorescent dyes was used to
asses the ion distributions in PMCs within SCH28080-treated embryos. Compared to controls, the PMCs
in SCH28080-treated embryos are relatively depolarized and exhibit a relatively high proton
concentration at time points corresponding to PMC ingression and early migration, but return to levels
comparable to controls during gastrulation . Sodium and chloride ion concentrations exhibit
comparatively late changes in SCH28080-treated embryos that are likely compensatory. In control
embryos, overexpression of the transcription factor Pmar is sufficient to convert all the cells in the
embryo to PMCs. However, this effect is incomplete in SCH28080-treated embryos, indicating that
SCH28080 impairs PMC differentiation. While PMC ingression is delayed but otherwise normal in
SCH28080-treated embryos, the PMCs fail to undergo their final cell cleavage, to migrate to their typical
positions, and to secrete a skeleton. SCH28080-treated embryos also exhibit defective PMC syncytium
formation. These data indicate that activity of the H + /K + antiport ATPase is required for the set of
changes that hallmark PMC differentiation following ingression.
95 Genome-wide analysis of the skeletogenic gene regulatory network of sea urchins
Shashikant, Tanvi (Carnegie Mellon University); Rafiq, Kiran (Carnegie Mellon University); Ettensohn,
Charles (Carnegie Mellon University)
A central challenge of developmental and evolutionary biology is to understand the transformation of
genetic information into morphology. Elucidating the connections between genes and anatomy will
require model morphogenetic processes that are amenable to detailed analysis of cell/tissue behaviors
and to systems-level approaches to gene regulation. The formation of the calcified endoskeleton of the
sea urchin embryo is a valuable experimental system for developing such an integrated view of the
genomic regulatory control of morphogenesis. A transcriptional gene regulatory network (GRN) that
underlies the specification of skeletogenic cells (primary mesenchyme cells, or PMCs) has recently been
elucidated. In this study, we carried out a genome-wide analysis of mRNAs encoded by effector genes in
the network and uncovered transcriptional inputs into many of these genes. We used RNA-seq to
identify >400 transcripts differentially expressed by PMCs during gastrulation, when these cells undergo
a striking sequence of behaviors that drives skeletal morphogenesis. Our analysis expanded by almost
an order of magnitude the number of known (and candidate) downstream effectors that directly mediate
skeletal morphogenesis. We carried out genome-wide analysis of (1) functional targets of Ets1 and Alx1,
two pivotal, early transcription factors in the PMC GRN, and (2) functional targets of MAPK signaling, a
pathway that plays an essential role in PMC specification. These studies identified transcriptional inputs
into >200 PMC effector genes. Our work establishes a framework for understanding the genomic
regulatory control of a major morphogenetic process and has important implications for reconstructing
the evolution of biomineralization in metazoans.
96 Expression of the ATP-Binding Cassette transporter Sp-ABCC5a in pigment cells is required
for sea urchin gastrulation
Shipp, Lauren E. (Scripps Institution of Oceanography); Hill, Rose (Scripps Institution of
Oceanography); Moy, Gary (Scripps Institution of Oceanography); Gokirmak, Tufan (Scripps Institution of
Oceanography); Hamdoun, Amro (Scripps Institution of Oceanography)
Page 48 of 51
ATP-binding cassette (ABC) transporters are synthesized during development with roles in
morphogenesis and protection from xenobiotics. In embryos of the sea urchin ( Strongylocentrotus
purpuratus ), one such transporter is Sp -ABCC5a, whose expression in a subset of mesodermal cells is
required for orientation and development of the endodermal hindgut. Transcripts of the ABCC5a gene
are first detected at hatching (21 hpf) and are most abundant during the gastrula stage (42 hpf).
Expression is limited to a subset of aboral non-skeletogenic mesenchyme (NSM) cells that become
pigment cells. ABCC5a protein expression is first detected during early gastrulation (34 hpf) and peaks
at the prism stage (50 hpf). ABCC5a expression is controlled by Delta-Notch signaling emanating from
the skeletogenic mesenchyme, as treatment with DAPT at 3 hpf, but not 17 hpf, blocks its induction.
Morpholino knockdown of ABCC5a (~210 kDa) does not appear to affect differentiation of pigment and
blastocoelar cells, or production of echinochrome pigment, but instead results in abnormal archenteron
formation. ABCC5a-morphants develop elongated archenterons and fused mouths, but following
gastrulation, their hindguts protrude out from the blastopore to form a prolapse of the vegetal/posterior
pole. This is observed in 90% of knockdown embryos by the late prism stage (~60 hpf). We hypothesize
that efflux of signaling molecules, possibly cyclic nucleotides, from ABCC5a is required for complete
invagination of the endoderm. In the absence of ABCC5a, hindgut-precursor cells remain on the vegetal
pole of the embryo throughout gastrulation. This would suggest that pigment cell precursors help orient
the endoderm cells during gut formation. We are currently addressing this hypothesis by further
characterizing the ABCC5a-knockdown phenotype with live-imaging and cell-type labeling, and by
probing the potential substrates of ABCC5a with efflux and migration assays.
97 microRNA-31 Regulates Skeletogenesis of the Sea Urchin Embryo
Stepicheva, Nadezda (University of Delaware); Song, Jia (University of Delaware)
The microRNAs (miRNAs) are small non-coding RNAs that regulate the translation and stability of target
mRNAs. miRNAs play an important role in early developmental processes in many organisms. However,
the molecular mechanism of miRNA-mediated regulation in the embryo is difficult to study. Here we
examine miR-31, which is a highly evolutionary conserved miRNA that was shown to play a role in
human cancer and bone formation, in early development using the sea urchin as a model. Our data
indicate that loss-of-function of miR-31 in sea urchin embryos disrupts the function and localization of
primary mesenchyme cells (PMCs), which are responsible for the formation of skeletal spicules. Using
luciferase reporter constructs and site-directed mutagenesis, we identified miR-31 to directly regulate
Pmar1 , Alx1 and Cyclophilin 1 ( Cyp1 ) within the skeletogenesis gene regulatory network. Blocking the
specific regulation of miR-31 on Alx1 in the developing embryo with miRNA target protector morpholino
antisense oligonucleotides (miRNA TP MASO) resulted in mislocalization of PMCs. These results
indicate that miR-31 mediated regulation Alx1 gene is sufficient to disrupt proper skeletogenesis. This
study contributes to an understanding of the novel regulatory role of miR-31 in the developing embryo
and incorporates post-transcriptional regulation by miR-31 into the skeletogenesis GRN.
98 miR-124 Regulation of the Delta/ Notch signaling pathway
Suarez, Santiago N. (University of Delaware); Song, Jia (University of Delaware)
microRNAs (miRNAs) play a crucial role in cell differentiation. The brainspecific miR-124 is highly
conserved in both invertebrates and vertebrates. We propose to use the purple sea urchin,
Strongylocentrotus purpuratus, to examine the function of miR-124. The Delta/Notch is a conserved
signaling pathway that regulates neural differentiation and it also activates GCM that is critical for the
specification of mesodermally-derived pigment cells. We bioinformatically identified Delta, Notch and
GCM to contain potential miR-124 binding sites. We hypothesize that miR-124 regulates the Delta/Notch
Page 49 of 51
signaling pathway important for neurogenesis and mesodermal specification. We will examine
loss-of-function and gain-of-function of miR-124 induced phenotypes and identify cell types regulated by
miR-124 using various molecular markers. We observed a significantly larger midgut width in larval
stage embryos in miR-124 knockdown embryos as compared to the control embryos. We will investigate
the direct regulation of miR-124 on Delta, Notch, and GCM by cloning their 3’untranslated regions
downstream of luciferase reporter constructs. This study elucidates the function of miR-124 by
identifying its direct gene targets and contributes to our understanding of neural development and
mesodermal specification.
99 Spatial Regulation of Gene Expression in the Skeletogenic Mesenchyme by Extrinsic Cues
Sun, Zhongling (Carnegie Mellon University); Ettensohn, Charles (Carnegie Mellon University)
Short-range signals from ectodermal territories regulate gene expression in the primary mesenchyme
cells (PMCs) of the sea urchin embryo. Previous studies have shown asymmetric gene expression in
PMCs after gastrulation despite the fact that PMCs are homogeneous when they are first specified.
Recent studies have demonstrated the critical role of the VEGF and MAPK pathways in skeletogenesis.
In this study, we use whole mount in situ hybridization to examine the expression patterns of 34 highly
expressed PMC-specific/enriched mRNAs in Strongylocentrotus purpuratus embryos at the late gastrula,
early prism and pluteus stages. The expression patterns of these 34 genes at each stage can be
classified into 3-4 categories. Most of these mRNAs are expressed at higher levels at sites of rapid
biomineral deposition. Furthermore, we use a VEGFR inhibitor, axitinib and a MAPK inhibitor, U0126 to
show that the VEGF and MAPK pathways regulate gene expression in the PMCs at the tips of extending
arms at the pluteus stage in a similar way, suggesting that the MAPK pathway acts downstream of
VEGF. Both pathways are essential for maintaining high expression level in the PMCs at the tips of the
anterolateral rods and the postoral rods. Gene expression in the PMCs at the tips of the body rods on
the dorsal side of the embryo is not affected by axitinib or U0126 treatment. We propose that the
coordinate, localized expression pattern of suites of effector genes reflects regulation via a common
signaling pathway. Our results indicate that multiple signaling pathways are involved in regulating gene
expression in the PMCs: VEGF/MAPK signaling on the ventral side and a separate, unidentified pathway
on the dorsal side. In addition, we find that the expression of individual effector genes can be
sequentially regulated by multiple signaling pathways during development.
100 A probabilistic modeling of the cell lineage highlights interindividual variability in
Paracentrotus lividus early development
villoutreix, paul (cnrs); Rizzi, Barbara (CNRS); Delile, Julien (CNRS); Duloquin, Louise (CNRS); Faure,
Emmanuel (CNRS); Savy, Thierry (CNRS); Bourgine, Paul (CNRS); Peyriéras, Nadine (CNRS)
Assessing the question of inter-individual specific differences and intra-individual cell differentiation at
the single cell resolution is renewed by novel live imaging and reconstruction techniques. The sea urchin
Paracentrotus lividus is used as a model to describe, measure and quantify it. We studied a cohort of
five digitally reconstructed developing embryos from the 32 cells stage (4hpf) to hatching (around 500
cells, 10 hpf) which consist in the automatic reconstruction of the spatio-temporal cell lineage (3D+time)
and the membrane segmentation of each cells from 2-photon microscopy processed by the
BioEmergences platform. A first level of interindividual macroscopic variability is captured by a linear
spatio-temporal rescaling. A finer level of description was defined by clustering cells by common cell
type and cell cycle, allowing interindividual comparison. These groups were the basis for a probabilistic
modeling reproducing the cellular proliferation, volume and surface macroscopic dynamics. This model
show the independency of the life length characteristics between a cell and its mother, and the
Page 50 of 51
decomposition of the cellular volume and surface into a stochastic and deterministic part. We produced
a prototypical model able to predict the behavior of a normal blastula by merging the parameters value
from each specimen of the cohort. The corresponding artificial cell lineage could be embedded in space
with a biomechanical model. In addition to testing mechanical assumptions, this spatio-temporal
prototype may be used as a spatial template for a gene expression atlas integrating individual cell
variability. This work is one the first attempt to study the variability from the individual cell to the whole
organism, thus contributing to the foundation of the emerging field of quantitative developmental biology.
101 The embryonic transcriptome for Lytechinus variegatus
Zuch, Daniel (Boston University); Hogan, J.D. (Boston University); Keenan, Jessica (Boston
University); Luo, Lingqi (Boston Univesity); Saji, Akhil (Boston University); Sundermeyer, Mary Ann
(Boston Univesity); Piacentino, Michael (Boston Univesity); Schatzberg, Daphne (Boston Univesity);
Azzizi, Elham (Boston Univesity); Zhang, Shile (Boston Univesity); Heilbut, Adrien (Boston Univesity);
Poustka, Albert (Max Planck Institute, Berlin Germany); Bradham, Cynthia (Boston Univesity)
The transcriptome for L. variegatus (Lv) was sequenced at 11 developmental timepoints using the
Illumina platform, then assembled using SOAPdenovo trans and annotated by BLAST analysis against
S. purpuratus (Sp) gene models and nr. The N50 for the assembled scaffolds was 638. The timepoints
were normalized using quantile normalization, and the expression data were validated using qPCR
analysis. We identified sequences that correspond with 18825 S. purpuratus gene models. We
evaluated the stage of onset for known endomesodermal and ectodermal gene regulatory network
genes, and find that the onsets for the majority of these genes in Lv agrees with predictions from both Sp
and P. lividus, suggesting that the overall network logic is preserved among these species. K-means
clustering identified numerous clusters within the overall transcriptome as well as among the
developmentally expressed transcription factors, including clusters expressed primarily at single stages,
and clusters with broader expression profiles. Principal component (PC) analysis identified the transition
from early blastula to hatched blastula as the transition accounting for the most variation (PC1), followed
by the transition from late gastrula to early pluteus (PC2), then the transitions from mesenchyme blastula
through early, mid and late gastrula (PC3). Together, the first three PCs account for 71% of the variation
in the expression data. Finally, we analyzed the vertebrate phylotypic period genes identified by Irie and
Kuratani (Nature Communications 2011). Of the 109 genes, we identified 93, and found that the largest
number are co-expressed at "late" pluteus stage (48 hours post-fertilization), suggesting that sea urchin
plutei correspond most closely to the vertebrate pharyngula stage in terms of gene expression profiles.
Page 51 of 51
Participants
Priscilla Ahiakonu
University of Delaware
pkahiako@udel.edu
KOJI AKASAKA
University of Tokyo
kojiaka@mmbs.s.u-tokyo.ac.jp
Carmen Andrikou
Stazione Zoologica 'Anton Dohrn' di
Napoli
carmen.andrikou@szn.it
M. Ina Arnone
Stazione Zoologica Anton Dohrn
miarnone@szn.it
Julius Barsi
Caltech
barsi@caltech.edu
Ken Baughman
OIST
kbaughman@oist.jp
Smadar Ben-Tabou de-Leon
The University of Haifa, Israel
sben-tab@univ.haifa.ac.il
Andrea Bodnar
Bermuda Institute of Ocean Sciences
andrea.bodnar@bios.edu
Cynthia Bradham
Boston University
cbradham@bu.edu
Bruce Brandhorst
Simon Fraser University
brandhor@sfu.ca
Katherine Buckley
University of Toronto, Sunnybrook
Research Institute
kbuckley@sri.utoronto.ca
David Burgess
Boston College
david.burgess@bc.edu
Robert Burke
University of Victoria
rburke@uvic.ca
Christine Byrum
College of Charleston
byrumc@cofc.edu
Cristina Calestani
Valdosta State University
ccalestani@valdosta.edu
R. Andrew Cameron
California Institute of Technology
acameron@caltech.edu
Joseph Campanale
Scripps Institution of Oceanography
jpcampan@ucsd.edu
Gregory Cary
CMU
gregorycary@gmail.com
Vincenzo Cavalieri
University of Palermo
vincenzo.cavalieri@unipa.it
Héloïse Chassé
équipe TCCD, UMR 8227, Station
Biologique de Roscoff, CNRS/UPMC
hchasse@sb-roscoff.fr
Alys Cheatle Jarvela
Carnegie Mellon University
acheatle@andrew.cmu.edu
Oliver Chung
Boston University
chungman27@gmail.com
Ashley Coots
Rochester Institute of Technology
adc5793@rit.edu
Jenifer Croce
CNRS
jeni.croce@obs-vlfr.fr
Miao Cui
Caltech
miaocui@caltech.edu
Anna Czarkwiani
University College London
a.czarkwiani@ucl.ac.uk
Eric Davidson
California Institute of Technology
davidson@caltech.edu
David Dylus
University College London
david.dylus.10@ucl.ac.uk
Allison Edgar
Duke University
ae75@duke.edu
Susan Ernst
Tufts University
susan.ernst@tufts.edu
Eric Erkenbrack
Caltech
erkenbra@caltech.edu
Jose Espinoza
University of California, San Diego
j1espino@ucsd.edu
Charles Ettensohn
Dept. of Biol. Sci., Carnegie Mellon
University
ettensohn@andrew.cmu.edu
Roberto Feuda
Caltech
rfeuda@caltech.edu
Constantin Flytzanis
University of Patras
kostas@bcm.tmc.edu
Adam Foote
Carnegie Mellon University
afoote@andrew.cmu.edu
Tara Fresques
Brown University, Wessel Lab
tara_fresques@brown.edu
Alec Freyn
Rochester Institute of Technology
awf1659@g.rit.edu
Feng Gao
California Institute of Technology
gaofeng@caltech.edu
Sarah Hadyniak
Boston University
hadyniak@bu.edu
John Henson
Dickinson College
henson@dickinson.edu
Veronica Hinman
Carnegie Mellon U
veronica@cmu.edu
Eric Ingersoll
Penn State Abington
epi1@psu.edu
Masato Kiyomoto
Ochanomizu University
kiyomoto.masato@ocha.ac.jp
Mariko Kondo
The Univ. of Tokyo
konmari@mmbs.s.u-tokyo.ac.jp
Oliver Krupke
University of Victoria
okrupke@uvic.ca
Patrick Lemaire
CRBM, UMR5237, CNRS/University
Montpellier; Institut de Biologie
Computationnelle, Montpellier, France
patrick.lemaire@crbm.cnrs.fr
Thierry Lepage
CNRS
tlepage@unice.fr
Enhu Li
Caltech
enhuli@caltech.edu
Ching-Yi Lin
Academic Sinica
sunbeank@gmail.com
William Longabaugh
Institute for Systems Biology
wlongabaugh@systemsbiology.org
Chris Lowe
Stanford University
clowe@stanford.edu
Deirdre Lyons
Duke University
dcl.duke@gmail.com
Megan Martik
Duke University
megan.martik@duke.edu
David McClay
Duke University
dmcclay@duke.edu
Alex McDougall
CNRS
dougall@obs-vlfr.fr
Daniel Medeiros
University of Colorado, Boulder
daniel.medeiros@colorado.edu
Artemis Michail
University of Patras
amichail@upatras.gr
Dolores Molina
Institut de Biologie Valrose (iBV),
UMR7277, CNRS/UNSA
loli.molina.jimenez@gmail.com
Kathleen Moorhouse
Boston College
moorhous@bc.edu
Julia Morales
equipe TCCD, UMR8227 CNRS-UPMC,
Station Biologique de Roscoff
morales@sb-roscoff.fr
Yoshiaki Morino
University of Tsukuba
yoshiaki.morino@gmail.com
Robert Morris
Wheaton College
rmorris@wheatonma.edu
Ian Murray
Boston University
ismurray@bu.edu
Jongmin Nam
Rutgers University-Camden
jn322@camden.rutgers.edu
Paola Oliveri
University College London
p.oliveri@ucl.ac.uk
Akihito Omori
Misaki Marine Biological Station
omori@mmbs.s.u-tokyo.ac.jp
Nathalie Oulhen
Brown University
nathalie_oulhen@brown.edu
Margaret Peeler
Susquehanna University
mpeeler@susqu.edu
Margherita Perillo
Stazione Zoologica Anton Dohrn di
Napoli, Naples, Italy
margherita.perillo@szn.it
Isabelle Peter
Caltech
ipeter@caltech.edu
Libero Petrone
UCL
libero.petrone.11@ucl.ac.uk
Elizabeth Petsios
University of Southern California
petsios@usc.edu
Nadine Peyrieras
CNRS
nadine.peyrieras@inaf.cnrs-gif.fr
Michael Piacentino
Boston University
mpiacent@bu.edu
Dominic Poccia
Amherst College
dlpoccia@amherst.edu
Janani Ramachandran
Boston University
janani93@bu.edu
Andrew Ransick
Caltech
andyr@caltech.edu
Ryan Range
Mississippi State University
range@biology.msstate.edu
Jonathan Rast
University of Toronto
jrast@sri.utoronto.ca
Patrick Reidy
Boston University
preidy@bu.edu
Laura Romano
Denison University
romanol@denison.edu
Daphne Schatzberg
Boston University
daph@bu.edu
Stephan Schneider
Iowa State University
sqs@iastate.edu
Catherine Schrankel
University of Toronto
c.schrankel@gmail.com
Nick Schuh
Sunnybrook Health Science Center
nick.w.schuh@gmail.com
Tanvi Shashikant
Carnegie Mellon University
tshashik@andrew.cmu.edu
Lauren Shipp
Scripps Institution of Oceanography
lshipp@ucsd.edu
Charles Shuster
New Mexico State University
cshuster@nmsu.edu
Stephen Small
New York University
sjs1@nyu.edu
L Courtney Smith
George Washington University
csmith@gwu.edu
Peter Smith
University of Southampton
p.j.smith@soton.ac.uk
Jia Song
University of Delaware
jsong@udel.edu
Andriana Stamopoulou
University of Patras
andriana.stamopoulou@gmail.com
Nadezda Stepicheva
University of Delaware
nstepich@udel.edu
Jonathon Stone
Rochester Institute of Technology
jjs8236@rit.edu
Meike Stumpp
Academia Sinica (ICOB)
meike.stumpp@gmail.com
Yi-Hsien Su
Institute of Cellular and Organismic
Biology, Academia Sinica
yhsu@gate.sinica.edu.tw
Santiago Suarez
University of Delaware
santisua@udel.edu
Zhongling Sun
Carnegie Mellon University
zhonglin@andrew.cmu.edu
S. Zachary Swartz
Brown University Molec Biology, Cell
Biology & Biochemistry
steven_swartz@brown.edu
Hyla Sweet
Rochester Institute of Technology
hxssbi@rit.edu
Jeffrey Thompson
University of Southern California
thompsjr@usc.edu
Sarah Tulin
MBL
stulin@mbl.edu
Jon Valencia
Caltech
jev@caltech.edu
Zheng Wei
NIH/NIDCR
zhwei@mail.nih.gov
Michael Whitaker
Newcastle University
michael.whitaker@ncl.ac.uk
Jennifer Wygoda
Duke University
jaw61@duke.edu
paul villoutreix
cnrs
paul.villoutreix@inaf.cnrs-gif.fr
Gary Wessel
Brown University
rhet@brown.edu
Athula Wikramanayake
University of Miami
athula@miami.edu
Dongdong Xu
Brown university
xudong0580@163.com
Shunsuke Yaguchi
University of Tsukuba
yag@kurofune.shimoda.tsukuba.ac.jp
Mamiko Yajima
Brown University
mamiko_yajima@brown.edu
Vanesa Zazueta-Novoa
Brown University
vanesa_zazueta-novoa@brown.edu
Minyan Zheng
Carnegie Mellon University
minyanz@andrew.cmu.edu
Leonard Zon
HHMI/Boston Children's Hospital
zon@enders.tch.harvard.edu
Daniel Zuch
Boston University
dtzuch@bu.edu