Transcript lecture 3 modified

Lecture 3
Where am I?
Anterior (Head)
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
Right
Dorsal (Back)
Ventral (Front)
Left
Posterior (Tail)
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.
Germ layers, Ectoderm, Mesoderm, and Endoderm, give rise to all tissues
of the developing embryo
Blastocyst
Development of the egg cylinder
Onset of gastrulation
Brachyury expression marks
the primitive streak
What determines the site of initiation of the primitive streak?
Major signalling pathways; BMP and Nodal/Activin
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BMPs and Nodal signal by binding type I/II receptor and activating ser/thr kinase to phosphorylate Smads
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BMP signal transduced by phosphorylating Smad 1,5, or 8 and Nodal through Smad 2 or 3
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Phospho Smads bind Smad4, translocate to the nucleus and activate target genes
Major signalling pathways; Nodal fine tuning
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Nodal can be regulated at the level of conversion of pro-nodal to nodal by Furin/PACE4
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Cer1 and Lefty 1 are diffusible antagonists of nodal.
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.
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Stabilised b-catenin translocates to the nucleus, binds to TCF family proteins and activates expression
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In the absence of b-catenin TCF proteins repress target genes.
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In the absence of wnt ligand, b-catenin is phosphorylated by CK1 and GSK3 and degraded
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DKK1 anagonises Wnt signalling by sequestering and internalising LRP
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WIF1 and sFRP are frizzled related proteins that bind and sequester Wnt ligands
Onset of gastrulation
Brachyury expression marks
the primitive streak
What determines the site of initiation of the primitive streak?
Establishment of proximal-distal assymetry
The distal visceral endoderm (dve)
?
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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.
Rotation of VE moves DVE cells to anterior position
(AVE) thus converting P-D axis into A-P axis.
Courtesy of Shankar Srinivas, DPAG
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, 1487-1496; Brennan et al (2001) Nature 411, p965-969
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
Development of the germline
Specification of Primordial Germ Cells (PGCs)
• High levels of BMP signalling instruct 30-40 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, p207-213
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.
Gastrulation
The primitive streak extends bidirectionally into
extraembryonic ectoderm and epiblast regions
Mesoderm contributes to extraembryonic tissues
The primitive streak extends laterally as well as proximo-distally
Mesoderm subpopulations that are temporally specified
give rise to distinct tissues
Muscle and blood of extraembryonic tissues
gut
definitive endoderm
Mesendoderm
Notochord, head process
Mesenchyme of viscera, connective tissue of limbs,blood.
Somites – vertebrae, muscle blocks ….
• dorso-ventral patterning is established by location of anterior streak lineages
The node is a specialised signalling centre
Left-right assymetry
Left right assymetry and the node
E8.5
• 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.
End Lecture 3