Jason Manley
Drs. Andrea Bodnar &
Helena Reinardy
3 October 2014
Insight from the sea urchin
Bermuda Institute of Ocean Sciences
Why sea urchins?
 More closely related to humans than other invertebrate model
organisms
 Molecular tools are available for their study
 Regeneration of spines & tube feet provide a useful model for
muscle, nerve and skin regeneration
 Searching for pathways required for tissue regeneration
 Understanding regeneration could explain how they maintain
tissues over their incredible life span
SEM of sea urchin tube foot
Lytechinus variegatus
askabiologist.asu.edu/sites/default/files/tubefeetsem.jpg
Goal #1:
Investigate the mechanisms of
regeneration in L. variegatus
using chemical and genetic inhibitors.
Regeneration Assay
Goal #2:
Measure expression of stem cell marker
genes in L. variegatus tube feet.
RNA Extraction & qRT-PCR
http://bitesizebio.s3.amazonaws.com/wp-content/uploads/2011/11/pipetting-RSI.jpg
Tube Feet Regeneration Assay
DAPT:
chemical inhibitor
of γ-secretase;
indirect Notch
inhibitor
Notch signaling has been shown to be important for maintaining
cellular proliferation in mice, humans, and during fin regeneration in
zebrafish (Moellering, Conboy & Grotek)
http://cdn.zmescience.com/wp-content/uploads/2013/02/jagged1.jpg
% Regrowth with DAPT
Tube Feet
Student’s t-test
compared to control:
* P<0.05
** P<0.01
Spines
Vivo-morpholinos: Genetic inhibition
 Morpholinos knock down genes: bind to mRNA to prevent
translation into proteins
 Designed to readily enter cell membranes
https://biochemist01.files.wordpress.com/2013/04/central-dogma.jpg
Regrowth with vMO
100%
Tube Feet
90%
Spines
Regrowth (Mean & ± SD)
80%
Control
70%
Delta
60%
50%
SuH
40%
Delta + SuH (n=1)
30%
20%
10%
0%
8
15
Days post amputation (dpa)
20
8
15
Days post amputation (dpa)
20
Tissue
Collection
RNA
Extraction
RNA
Reverse
Transcription
cDNA
qRT-PCR
Amplified DNA
Data: relative levels of
gene expression
http://www.thermoscientific.de/com/CMA/Images/Image_44186.jpg
Stem Cell & Cell Proliferation Markers
in Homeostatic Tube Feet
Relative Gene Expression (ΔCt)
10
Low expression
9
8
7
6
High expression
5
4
3
2
1
0
Vasa
Oct
SoxB1
Myc
PCNA
Piwi
TERT
Regeneration, Notch & Stem Cells
 Validation of this novel sea urchin regeneration assay
 Use of chemical and genetic inhibitors of Notch signaling
showed that Notch is critical for regeneration in L. variegatus
 Early indication of the presence of stem cells in L. variegatus,
when they have never been characterized in echinoderm
 Looking forward: advances in human regenerative medicine;
understanding how we age (and how we can prevent it!)
THANKS!
Drs. Andrea Bodnar & Helena Reinardy