Presentation on theme: "Overview of gastrulation"— Presentation transcript:
1 Overview of gastrulation Lecture 3Axis determination.Overview of gastrulationEstablishment of the anterior-posterior (AP) axisThe primitive streak and the nodeDorso-Ventral and left-right axesSpecification of the germ lineYou should understandThe role of extraembryonic tissues in specification of the A-P axisThe contribution of key signalling pathways influencing gastrulation (BMP, nodal and Wnt)Relationship of germ layers to tissues of the developing embryoSpecification of the germ cells.Where am I?Anterior (Head)Dorsal (Back)Ventral (Front)LeftRightPosterior (Tail)
2 of the developing embryo Germ layers, Ectoderm, Mesoderm, and Endoderm, give rise to all tissuesof the developing embryoBlastocyst
4 Onset of gastrulationBrachyury expression marksthe primitive streakWhat 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 SmadsBMP signal transduced by phosphorylating Smad 1,5, or 8 and Nodal through Smad 2 or 3Phospho 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/PACE4Cer1 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 destructioncomplex 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 expressionIn 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 degradedDKK1 anagonises Wnt signalling by sequestering and internalising LRPWIF1 and sFRP are frizzled related proteins that bind and sequester Wnt ligands
8 Onset of gastrulationBrachyury expression marksthe primitive streakWhat 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 Nodalsignalling from the epiblast by producing a cocktail of Nodal and Wnt inhibitors, further enhancing thenodal gradientUnknown 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.
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) signallingConfers 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 becomemesoderm, marking the initiation of gastrulation.Nodal and Wnt3 target genes include Fgf8 that triggers epitheilial to mesenchymaltransition (EMT) in nascent mesoderm. EMT leads to delamination of nascent mesodermrequired 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 neuroectodermSmad2 in VE required for anterior specification.Mutants specify mesoderm only, eg express brachyurythroughout epiblast
15 Specification of Primordial Germ Cells (PGCs) High levels of BMP signalling instruct epiblast cells to adoptPGC fate – Blimp1 expression (single cell transcriptomics).Blimp1 (with Prmt5) and Prdm14 repress somatic fate (eg hoxb1) andpromotes pluripotent fate (Oct4, Sox2 and Nanog).In Smad2 mutants lacking A-P polarity, PGCs are specified in randomPatches 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 factorNearly indistinguishable from ES cellsContribute to all tissues and germline in chimerasDifferences at imprinted loci in some linesNote PGCs like ES cells are alkaline phosphatase positiveMatsui et al (1992) Cell 70, p841-7.
20 The primitive streak extends laterally as well as proximo-distally
21 Mesoderm subpopulations that are temporally specified give rise to distinct tissuesMuscle and blood of extraembryonic tissuesgutdefinitive endodermMesenchyme of viscera, connective tissue of limbs,blood.MesendodermSomites – vertebrae, muscle blocks ….Notochord, head processdorso-ventral patterning is established by location of anterior streak lineages
24 Left right assymetry and the node Nodal mRNA is initially expressed equally in cells on either side of the nodeRotation of cilia in node sets up leftward flow of extracellular fluidEither immotile cilia (red) act as mechanosensors and increase Nodal transcription viaCa2+ flux, or flow sets up left/right morphogen gradient that induces Nodal on the left.Nonaka et al (2002) Nature 418, p96-9.