Axis Formation and Gastrulation II
The Xenopus Oocyte is Asymmetric
“Animal”
VegT
Vg1
Maternal Determinants
“Vegetal”
VegT: Transcription factor
-Promotes “vegetal” identity (endoderm)
-Activates mesoderm inducers (nodals/TGF-ß’s)
Vg1: Another nodal/ TGF-ß’s
Sperm Entry Point Determines D/V Axis in Xenopus
“Animal”
An
Cortical Rotation
D
Wnt11
VegT
Wnt11
“Vegetal”
Maternal Determinants
VegT: Transcription Factor
Promotes “vegetal” identity
(endoderm)
Wnt11: Signaling Ligand
Promotes Dorsal identity
V
VegT
Veg
Specifying the Germ Layers
Mesoderm Inducers are TGF-ß Family Members
(Vg1, Nodals (Xnr’s), Activin)
-higher in dorsal mesoderm
Wnt11
Specifying The Spemann-Mangold Organizer
V
Wnt11
Nodals (TGF-ß’s)
D
V
V
D
D
Transplant Dorsal Blastopore Lip
Donor: Pigmented (newt)
Host: Unpigmented (newt)
Spemann and Mangold, 1924
V
V
D
D
Form Second Body Axis
Spemann and Mangold, 1924
Normal Body Axis
V
V
D
D
Second Body Axis
Second Body Axis:
Dorsal Mesoderm (Notocord)--Donor Cells
Neural Plate--Host Cells
Other mesoderm--mix of Donor and Host
Donor tissue “organized” host tissue to
take on new cell identities
Blastopore lip = an Organizer
Spemann and Mangold, 1924
Also two sites of gastrulation
Specifying The Speman-Mangold Organizer
V
D
V
D
wnt11
nodals
The Organizer:
-Patterns the mesoderm along the D/V axis
-Determines the site of gastrulation
-Allows for induction of the nervous system (neuroectoderm)
Fate Map of the Xenopus Blastula
Xenopus
An
V
D
Veg
Hypothesis: Neural Induction
The dorsal lip secretes a signaling molecule that patterns the mesoderm and
induces neural plate specification
Search for molecules that can mimic organizer activity
Find secreted INHIBITORS of TGF-ß ligands (Chordin, Noggin, Follistatin)
Neural is DEFAULT state and TGF-ß signaling is required to INDUCE ectoderm
TGF-ß (BMP, Dpp)
Frog
Xolloid/Tolloid
Fly
D
Chordin, Sog
xolloid
sog
Note: BMP4 is different type of TGF-ß than nodals
-nodals form early gradient that is high in dorsal regions
-BMPs form later gradient that is high in ventral regions
V
Fish and Frog Embryos Appear Very Different
Yolk Cell
Fish and Frogs Do Things Similarly
-The oocyte has an Animal-Vegetal Axis
-The Wnt pathway initiates the D/V Axis
(unclear how--doesn’t seem to be sperm)
-BMPs and Wnts pattern the mesoderm
V
V
D
D
BMPs
V
V
nodals
Organizer
D(Shield)
-Nodal (and FGF) signaling specifies the mesoderm
(and endoderm) with a dorsal bias
-Dorsal mesoderm makes the organizer (shield)
D
V
Wnts
D
Fate Mapping (Lineage Tracing) to Investigate
Cell Identity and Developmental Potential
Xenopus
An
V
D
Veg
Zebrafish
It is not birth, marriage or death,
but gastrulation, which is truly the
most important time in your life.
- Lewis Wolpert (1986)
Gastrulation
Cell Movements Relevant for Gastrulation
Getting cells inside
Spreading tissues out
Moving cells around
Making tissues longer
Convergence/extension
Xenopus Gastrulation Initiates at the Organizer
(aka Dorsal Blastopore Lip)
Xenopus Gastrulation
Zebrafish Fate Map
Schier and Talbot, 2005
Zebrafish Gastrulation
Early Zebrafish Cleavages
Zebrafish Epiboly Transforms the Embryo Into a Hemisphere
Solnica-Krezel, 2006
Cells Involute and Ingress to Form the Dosal Shield
Cells Migrate Anteriorly After Involution
Solnica-Krezel, 2002
Convergence/Extension Also Contributes to A/P Axis Formation
Extension of cells along the A/P axis
Convergence of lateral cells toward the midline of the embryo
Convergence/Extension Requires
the PCP Pathway
Solnica-Krezel, 2006
The Anterior-Posterior Axis is Also Coupled to Gastrulation
The developmental potential and inducing properties of cells in the dorsal
blastopore lip change with time:
-Cells in the lip early become anterior mesoderm and induce anterior neural tissue
-Latter cells become posterior and induce more posterior neural structures
-A gradient of Wnt activity is high in posterior and low in anterior
Chick Embryos Look Different but Act Similarly
The Chick Embryo Forms as a Flattened
Disc of Cells On the Yolk
The primitive streak/node is the organizer and
expresses goosecoid and chordin
The posterior marginal zone initiates primitive
streak/organizer formation and expresses Vg1 and
Wnt8
The primitive streak elongates with the
node/organizer at the leading edge
Hensen’s Node is the Avian Organizer
Similar to Xenopus blastopore and Fish dorsal shield in terms of both patterning and
gastrulation movements
The node can induce nervous system development and a secondary axis when transplanted
The node expresses BMP antagonists, like the Xenopus and Zebrafish organizers
All Vertebrate Embryos Have a Spemann-Mangold Organizer
Organizer
Bird
Fish
Dorsal Shield
Frog
Blastopore Lip
Bird
Henson’s Node
Mouse
Node
Chick Gastrulation Movements
-In the Node and Primitive Streak, cells delaminate from the epiblast and
ingress to form the endoderm and mesoderm
-Epiblast cells continue to enter the streak from lateral regions
-Once they have ingressed in the streak,
newly formed endoderm and mesoderm move laterally again
Gastrulation and Patterning Follow the Same Logic as Fish and Frogs
As with fish and frogs, the first cells gastrulating through the chick organizer (node) become anterior
endoderm and prechordal plate mesoderm
The next cells through will form notochord
These first cells also induce the nervous system from the overlying ectoderm
Cells gastrulating through more posterior primitive streak become other mesoderm and endoderm derivatives
BUT, note that the organizer MOVES as the primitive streak first advances and then regresses
More posterior regions of notocord are formed from cells migrating through node as it regresses
The A/P Axis is “laid down” During
Primitive Steak Regression
This is in contrast to the active extension of
the A/P axis seen in frog and fish embryos
(although some active mechanisms do
further elongate the chick A/P axis)
Consequently, posterior development is
delayed relative to anterior development
“Laying down” the notocord during
regression of Henson’s node
Human Embryos Gastrulate Like Chick Embryos
Different Embryos, Common Themes
Activin (TGF-ß): A morphogen for the mesoderm
Animal cap assay
(After Green et al., 1992)
(After Asashima, 1994)
Lineage Analysis and Fate Mapping
Goal: Identify which cells in the early embryo give
rise to particular cells in the later embryo or adult
Lineage Analysis and Fate Mapping
Approaches:
1) Just watch closely
Lineage Analysis and Fate Mapping
Approaches:
1) Just watch closely
2) Cell transplantation
3) Use a Lineage Tracer
-Inject into single cells or few cells
-Activate in single cells or few cells
Fate Mapping by Single Cell Injection of Lineage Tracer
e.g. Woo and Fraser, 1995
Laser Activation of Lineage Tracer
“Caged” fluorescein:
e.g. Jopling and den Hertog, 2005
Photo-activatable GFP
Absorbance
spectrum
before PA
Absorbance
spectrum
after PA
Patterson and Lippincott-Schwartz, 2002
Lineage Analysis and Fate Mapping
Approaches:
1) Just watch closely
2) Cell transplantation
3) Use a Lineage Tracer
-Inject into single cells or few cells
-Activate in single cells or few cells
4) Genetic labeling
-Random labeling
-Labeling a specific lineage
Genetic Mosaics
Genetic mosaics can be created by mitotic recombination
induced by X-rays or a site-specific recombinase
Genetic Tracking of Specific Lineages
Question: What do cells that express YFG at time X develop into?
Actin Promoter
YFG
STOP
GFP
loxP
CRE-ER
Add tamoxofen at time X
Cells expressing YFG at that time permanently
labeled with GFP
Some things to worry about:
-Can you identify and label your cells of interest?
-Does your labeling technique interfere with normal
development?
-Is your lineage tracer stable over time?
-Is your lineage tracer diluted by cell division?