1. Overview of gastrulation
Lecture 3
Axis determination.
Overview of gastrulation
Establishment of the anterior-posterior (AP) axis
The primitive streak and the node
Dorso-Ventral and left-right axes
Specification of the germ line
You should understand
The role of extraembryonic tissues in specification of the A-P axis
The contribution of key signalling pathways influencing gastrulation (BMP, nodal and Wnt)
Relationship of germ layers to tissues of the developing embryo
Specification of the germ cells.
Where am I?
Anterior (Head)
Dorsal (Back)
Ventral (Front)
Left
Right
Posterior (Tail)

2. of the developing embryo
Germ layers, Ectoderm, Mesoderm, and Endoderm, give rise to all tissues
of the developing embryo
Blastocyst

3. Development of the egg cylinder

4. Onset of gastrulation
Brachyury expression marks
the primitive streak
What determines the site of initiation of the primitive streak?

5. Major signalling pathways; BMP and Nodal/Activin
BMPs and Nodal signal by binding type I/II receptor and activating ser/thr kinase to phosphorylate Smads
BMP signal transduced by phosphorylating Smad 1,5, or 8 and Nodal through Smad 2 or 3
Phospho Smads bind Smad4, translocate to the nucleus and activate target genes

6. Major signalling pathways; Nodal fine tuning
Nodal can be regulated at the level of conversion of pro-nodal to nodal by Furin/PACE4
Cer1 and Lefty 1 are diffusible antagonists of nodal.

7. Major signalling pathways; canonical Wnt
Binding of Wnt ligand to frizzled/LRP stabilises b-catenin by blocking activity of the destruction
complex comprising Axin, Dvl, and the kinases CK1 gamma and GSK beta.
Stabilised b-catenin translocates to the nucleus, binds to TCF family proteins and activates expression
In the absence of b-catenin TCF proteins repress target genes.
In the absence of wnt ligand, b-catenin is phosphorylated by CK1 and GSK3 and degraded
DKK1 anagonises Wnt signalling by sequestering and internalising LRP
WIF1 and sFRP are frizzled related proteins that bind and sequester Wnt ligands

8. Onset of gastrulation
Brachyury expression marks
the primitive streak
What determines the site of initiation of the primitive streak?

9. Establishment of proximal-distal assymetry
The distal visceral endoderm (dve) ?
At E5.5 Nodal is expressed in epiblast cells of early egg cylinder embryos.
At the proximal rim the ExE provides two positive feedback loops (Bmp4 and pro-nodal cleavage)
that enhance nodal signalling in the proximal epiblast.
Visceral endoderm cells at the distal tip of the egg cylinder (the dve) respond to Nodal
signalling from the epiblast by producing a cocktail of Nodal and Wnt inhibitors, further enhancing the
nodal gradient
Unknown inhibitory signals from ExE block nodal responsiveness in other regions of the VE.

10. Rotation of VE moves DVE cells to anterior position
(AVE) thus converting P-D axis into A-P axis.

11. Courtesy of Shankar Srinivas, DPAG

12. Signalling from the AVE determines A-P axis orientation.
DVE and then AVE cells secrete diffusible antagonists of Nodal (Lefty1 and Cer-l)
and Wnt (DKK1) signalling
Confers anterior characteristics to underlying epiblast (expresses neuroectoderm markers),
confirmed by analysis of AVE transplants.
Conversely, epiblast cells retaining high Nodal and Wnt signalling are specified to become
mesoderm, marking the initiation of gastrulation.
Nodal and Wnt3 target genes include Fgf8 that triggers epitheilial to mesenchymal
transition (EMT) in nascent mesoderm. EMT leads to delamination of nascent mesoderm
required for cell movements during primitive streak migration.
Thomas and Beddington (1996) Curr Biol. 6, ; Brennan et al (2001) Nature 411, p

13. Evidence from mutants Nodal mutants fail to specify mesoderm and
neuroectoderm
Smad2 in VE required for anterior specification.
Mutants specify mesoderm only, eg express brachyury
throughout epiblast

14. Development of the germline

15. Specification of Primordial Germ Cells (PGCs)
High levels of BMP signalling instruct epiblast cells to adopt
PGC fate – Blimp1 expression (single cell transcriptomics).
Blimp1 (with Prmt5) and Prdm14 repress somatic fate (eg hoxb1) and
promotes pluripotent fate (Oct4, Sox2 and Nanog).
In Smad2 mutants lacking A-P polarity, PGCs are specified in random
Patches along the rim of the epiblast.
Ohinata et al (2005), Nature 436, p

16. Embryonic Germ (EG) cell lines can be derived from PGCs
+ LIF + bFGF + Steel factor
Nearly indistinguishable from ES cells
Contribute to all tissues and germline in chimeras
Differences at imprinted loci in some lines
Note PGCs like ES cells are alkaline phosphatase positive
Matsui et al (1992) Cell 70, p841-7.

17. Gastrulation

18. The primitive streak extends bidirectionally into
extraembryonic ectoderm and epiblast regions

19. Mesoderm contributes to extraembryonic tissues

20. The primitive streak extends laterally as well as proximo-distally

21. Mesoderm subpopulations that are temporally specified
give rise to distinct tissues
Muscle and blood of extraembryonic tissues
gut
definitive endoderm
Mesenchyme of viscera, connective tissue of limbs,blood.
Mesendoderm
Somites – vertebrae, muscle blocks ….
Notochord, head process
dorso-ventral patterning is established by location of anterior streak lineages

22. The node is a specialised signalling centre

23. Left-right assymetry

24. Left right assymetry and the node
Nodal mRNA is initially expressed equally in cells on either side of the node
Rotation of cilia in node sets up leftward flow of extracellular fluid
Either immotile cilia (red) act as mechanosensors and increase Nodal transcription via
Ca2+ flux, or flow sets up left/right morphogen gradient that induces Nodal on the left.
Nonaka et al (2002) Nature 418, p96-9.

25. End Lecture 3