Lecture 3 - Morphogenesis Flashcards
-present two cell behaviours that drive animal morphogenesis 1) convergent extension 2) epitheilial to mesenchymal transition Some of the molecular mechanisms underlying these morphogenic events will also be presented including; the non-canoonical Wnt/Planar cell polarity pathway that regulated convergent extension and the transcriptional repressor Snail that is essential for EMT
What is covergent extension?
When cells in a developing embryo come together and elongate
-polarized cell behavior drives mediolateral intercalcation in Xenopus mesoderm
What is the process of covergent extension?
- Transient lamellipodia are in a random orientation
- these proterusions become stable and polarised along the mediolateral axis
- stable, polarised lamllipodia attach to neighborouring cells and generate traction
- this is followed by the process of boundary capture
What is covergent extension important in?
Gastrulation/neurolation
What is the process of boundary capture in covergent extension ?
- One a polarised cell meets a boundary (such as the matrix between the forming notochord and forming stomites)
- cell attaches stably to this boundary and that side of the cell ceases its protrusive action
- The other side of the captured cell continues to exert traction on neighboring cells pulling them towards the boundary
- all boundary captured cells exert traction medially bringing boundaries closer together
- results in the elongation of the axis
- allows convergent extension to occur
Give an example of where covergent elongation and boundary capture occurs
Drosophila bristles
- position and oritenation determined by cell polarity
- planar cell polarity mutants have randomly oritentated bristles
What is the pathway of cell polarity?
-ultimately done by passing signals from the outside of the cell through the cell surface
WNT signalling pathways:
- canonical
- non-canonical
What is the cannonical Wnt signalling pathway?
- Fz is the receptor (acts alongside LRP co-receptors)
- Wnt is the ligand that binds to the Fz receptor
1. Activation of the dishevelled gene results in the inhibition of GSK3-APC-axin complex (in the absense of Wnt this complex would phosphorylate beta-catenin resulting in its degradation)
2. Inhibition of Gsk3 results in a stabilisation of beta-catenin
3. beta-catenin moves to the nucleus and forms a complex with the transcription factor TCF/LEF to affect gene expression - causes cancer and DOES not result in polarity
- non-canonical Wnt signalling i.e the planar cell polarity pathway does
What is the process of non-canonical Wnt signalling-planar cell polarity pathway?
- Wnt ligand activated Fz receptors
- This interacts with dsh and dsh is activated
- this results in the activation of small GTPases - Rho and Rac
- These mediate cytoskeleton reorgansisation and the formation of lamellopodia
What gene is common to both cannonical and non-cannonical pathways?
Dishevelled gene
-the branchpoint between canonical and PCP pathways
What are the domains of the dsh gene?
DIX
-dimerises with Axin
DDZ
-for canonical pathway: binds casein kinase, GSK3, FRODO
-For PCP pathway: binds pickle, strabisums, DAAM1
DEP
-associated with small GTPases Rac and JNK
-moves Dsh to the cell membrane
What is the purpose of polarity in cells in the Drosophila cuticle?
outgrowth of bristles is coordinated
What are the drosophila genes involved in the drosophila cuticle cell polarity pathway
Dsh, Frz, Stabismum, Prickle and JNK
-homolgous of these genes are expressed in Xenopus cells undergoing covergent extension
What does over-expression or loss-of-function of PCP genes result in?
loss of cell polarity
What is the sub-cellular localisation of Dsh?
-Dsh accumulates in lamellipodia (once has been localised to the membrane) which are normally polarised during covergent extension
What do mutations in DEP domain of Dsh result in?
impaired membrane localisation of Dsh and impaired PCP signalling
What happens if Dsh is disrupted?
this randomises the normally polarisaed protrusions
-in PCP mutants dsh is on all cells so there is no direction and no gastrulation
What is the epithelial to mesenchymal transition? Give examples of EMT in embryos
- cells lose contact with each other and become disorganised mesenchymal cells though the Epithelial to Mesenchymal transition
e. g. - gastrulation in chicks (amniotes)
- neural crest delamination in the dorsal neural tube
- ingression of the primary mesenchymal cells at the onset of gastrulation in sea urchins
What are the features of gastrulation in amniotes?
- depends on the epithelial to mesenchymal transition where migratory mesenchyme form progressively through the primitive streak
- regulated by FGF (promotes expression of snail)
- EMT in these cells is also driven by canonical wnt signalling through Beta-catenin stabilisation
What are the features of neural crest cell generation?
-generated in dorsal region of the neural tube
-where cells undergo an epithelial-mesenchymal transition then delaminate from the neural tube and migrate to the periphery
(pigment cells doing this in tigers forms stripes)
What are the features of primary mesenchymal cell ingression in sea urchins?
- occurs in the early blastula in sea urchin
- EMT in the vegetal plate of sea urchin blastula
- includes cells undertaking EMT to drive ingression
- primary mesynchymal cells are derived from micromeres and go on to form larval skeleton exclusively
- results in cellular changes:
- down regulated cell-cell adhesion
- increases motility
- changes cell shape
- epithelial become mesenchymal cells that migrate
What is the epithelium?
- sheet of cells closely attached with adherens jucntions
- junctions contain the proten E-cadherin
- EMT is thought to occur by down regulation of E.cadherin
- E.cad repressors include slug, snail and twist (transcription factors) which bind to the E.cadherin promoter inactivating transcription
- introducing snail to cells can cause EMT
- introducing E.cadherin can cause MET
How were the transcription factors controlled by E.caderin discovered?
-observed expression in insitu hybridisation
-used morpholino antisense oligonucleotide targeted against endogenous Snail mRNA to inhibit the expression and function of the gene product post transcription
-PMCs in snail knock outs fail to undergo EMT and fail to down regulate E. cadherin expression
Therefore: snail represses E.cadherin and this activates EMT
