1. Axis Formation and Gastrulation II

2. The Xenopus Oocyte is Asymmetric
“Animal”
VegT
Vg1
“Vegetal”
Maternal Determinants
VegT: Transcription factor
-Promotes “vegetal” identity (endoderm)
-Activates mesoderm inducers (nodals/TGF-ß’s)
Vg1: Another nodal/ TGF-ß’s
Just like for Drosophila, Xenopus egg is being patterned during oogenesis
Animal--Vegetal axis established (not really AP or DV)
VegT is transcription factor
Maternal VegT RNA localized to vegetal side and promotes vegetal or endoderm identity

3. Sperm Entry Point Determines D/V Axis in Xenopus “Animal” An
Cortical Rotation D
VegT
Wnt11
Maternal Determinants
VegT: Transcription Factor
Promotes “vegetal” identity
(endoderm)
Wnt11: Signaling Ligand
Promotes Dorsal identity V
“Vegetal”
Veg
Sperm entry point detemines future ventral side
Induces MT driven “cortical rotation” where cortex of zygote rotates relative to rest of cytoplasm
Opposite point is dorsal--grey cresent
Wnt11 thought to rotate and induce dorsal
Wnt11 acts through canonical wnt pathway (Tao Heasman, 2005, Heasman lab 2007)
Basically--whole idea is to make a vegetal (VegT) to dorsal (Wnt11) axis orthogonal to An-Veg axis

4. Specifying the Germ Layers
Mesoderm Inducers are TGF-ß Family Members
(Vg1, Nodals (Xnr’s), Activin)
-higher in dorsal mesoderm
Wnt11

5. Specifying The Spemann-Mangold OrganizerV D
Wnt11
Nodals (TGF-ß’s)

6. Transplant Dorsal Blastopore Lip
Donor: Pigmented (newt) V D V D
Host: Unpigmented (newt)
Spemann and Mangold, 1924

7. V D V D
Form Second Body Axis
Spemann and Mangold, 1924

8. Normal Body Axis Second Body Axis Spemann and Mangold, 1924 V D V D
Also two sites of gastrulation
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

9. Specifying The Speman-Mangold OrganizerV 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)

10. Fate Map of the Xenopus BlastulaAn V D
Veg

11. D V Fly Frog sog 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
sog D V
Fly
TGF-ß (BMP, Dpp)
Chordin, Sog
Xolloid/Tolloid
xolloid
Frog
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

12. Fish and Frog Embryos Appear Very Different
Yolk Cell

13. 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 D V D
BMPs V D
Also get nuclear accumulation of b-catenin in future dorsal region of fish embryo
Not known how this is regulated
-does not seem to be due to sperm entry point (which is fixed at micropyle)
-initial cleavages do not appear to predict D/V axis
-but Gore 2005 show localization of squint (nodal) at early cleaveages and eventually do dorsal side
-Scheir and Talbot show nodal ligands not important for D/V
-BUT this still indicates early cleavages define D/V, in conflict with lineage analysis
-also, disruption of microtubules soon after fertilization does disrupt D/V--also in contrast to lineage work
-also, localization of germ plasm clearly indicates that machinery for post-fertilization localization of determinants exists
MVD: could cytoplasmic bridges affect early fate map so identity is really more restricted early than these studies would suggest??
Organizer
(Shield) V
nodals D V D
Wnts
-Nodal (and FGF) signaling specifies the mesoderm
(and endoderm) with a dorsal bias
-Dorsal mesoderm makes the organizer (shield)

14. Fate Mapping (Lineage Tracing) to Investigate Cell Identity and Developmental Potential
Xenopus An V D
Veg
Zebrafish

15. It is not birth, marriage or death, but gastrulation, which is truly the most important time in your life.
              - Lewis Wolpert (1986)
Gastrulation

16. Cell Movements Relevant for Gastrulation
Moving cells around
Getting cells inside
Spreading tissues out
Making tissues longer
Convergence/extension

17. Xenopus Gastrulation Initiates at the Organizer
(aka Dorsal Blastopore Lip)

18. Xenopus Gastrulation

19. Zebrafish Fate Map
Schier and Talbot, 2005

20. Zebrafish Gastrulation

21. Early Zebrafish Cleavages

22. Zebrafish Epiboly Transforms the Embryo Into a Hemisphere
Solnica-Krezel, 2006

23. Cells Involute and Ingress to Form the Dosal Shield
Cells Migrate Anteriorly After Involution
Solnica-Krezel, 2002

24. Convergence/Extension Requires the PCP Pathway
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

25. 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

26. 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

27. 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

28. All Vertebrate Embryos Have a Spemann-Mangold Organizer
Bird
Fish
Frog
Bird
Mouse
Henson’s Node
Dorsal Shield
Blastopore Lip
Node

29. 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

30. 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

31. 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

32. Human Embryos Gastrulate Like Chick Embryos

33. Different Embryos, Common Themes

34. Activin (TGF-ß): A morphogen for the mesoderm
Animal cap assay
(After Green et al., 1992)
(After Asashima, 1994)

35. Lineage Analysis and Fate Mapping
Goal: Identify which cells in the early embryo give rise to particular cells in the later embryo or adult
In order to understand how a particular cell type develops, you have to know where it comes from

36. Lineage Analysis and Fate Mapping
Approaches:
1) Just watch closely

38. Lineage Analysis and Fate Mapping
Approaches:
Just watch closely
Cell transplantation
Use a Lineage Tracer
-Inject into single cells or few cells
-Activate in single cells or few cells

39. Fate Mapping by Single Cell Injection of Lineage Tracer
Single cell ionophoretic injection of fluoroscein dextran
e.g. Woo and Fraser, 1995

40. 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
Can be hard to inject into single cells
Get more control using laser activation
Also, with PA-GFP don’t even have to inject
PA GFP
-wt EGFP actually does some photo conversion but still absorbs at 488 prior to PA (so high background)
-found mutant with no absorbance at 488 prior to PA
-after PA see 100 fold increase in fluorescence after excitation with 488

41. Lineage Analysis and Fate Mapping
Approaches:
Just watch closely
Cell transplantation
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

42. Genetic Mosaics
Genetic mosaics can be created by mitotic recombination
induced by X-rays or a site-specific recombinase

43. Genetic Tracking of Specific Lineages
Question: What do cells that express YFG at time X develop into?
Actin Promoter
STOP
GFP
loxP
YFG
CRE-ER
Add tamoxofen at time X
Cells expressing YFG at that time permanently labeled with GFP

44. 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?