Vertebrate development Flashcards
Transcription in xenopus oogenesis
Lampbrush chromosomes
High rate of transcription
18S 28S rRNAs amplified in free DNA circles within the nucleus
oocyte-specific 5S rRNA genes
Rna pol transcribes many genes in a single transcript
Early transport of mRNA in xenopus oocyte
Early pathway: Xcat2, MT-independent
Entrapped in mitochondrial cloud
Vegetal transport together with cloud fragments
Later pathway of mRNA transport
Vg1, VegT mRNA - MT-dependent transport
RNA accumulate around nucleus, concentrate on basal side by associating with ER
Transport to vegetal pole
Anchored in vegetal pole in MT-independent manner
Selectively repressing translation
Only translated after fertilisation (eg. VegT)
Only translated when properly localised (Vg1, VegT)
Short poly(A) tail represses translation.
Maskin binds to RNA, prevents elongation of poly(A) and inhibits translation
MBT
After 12 cleavage divisions
Cell cycle slows down - becomes asynchronous and with more conventional Cyclin-CDK
Zygotic transcription can occur
Cortical Rotation is necessary for D-V formation
UV irradiation - inhibits MT alignment, inhibits dorsal development
Can rescue by tipping embryo
Cortical rotation necessary for Wnt localisation
Injection of Wnt can form secondary D-V axis, or rescue UV-irradiated embryo
how is B-catenin stabilised in dorsal pole?
Dsh - fast via microtubules
Wnt11 mRNA - slower via cortical rotation
Inhibits GSK3B, stabilises B-catenin
B-catenin with Tcf3 forms NC
NC - expresses?
Expresses:
Twin, Siamois,
Strongly induced to express Xnr’s via TGFB-signals and B-catenin stabilisation combined
Endo- meso- ectoderm patterning
TGFB morphogen gradient
Intermediate activin –> Xbra in animal caps
High activin - expresses goosecoid in the dorsal
How SO is formed, and what it expresses
Relies on:
Wnt signalling stabilisation, which causes expression of Siamois and Twin
TGFB/Xnr signalling from the vegetal side and the NC, which activates Smad2
Expresses:
Goosecoid
Inhibitor molecules (noggin, chordin, follistatin, Cerberus,
Specification map vs fate map
Lateral mesoderm is specified to form blood and mesenchyme, but goes on to form somites
Specification map vs fate map
Lateral mesoderm is specified to form blood and mesenchyme, but goes on to form somites
TGFB signalling is necessary and sufficient for animal cap cells to form mesoderm,
Animal Cap assay
Identify components of XTC tissue culture supernatant that can induce mesoderm formation of animal cap cells
Veg1, derriere, Xnr’s, activin
Dominant-negative TGFB receptor - loss of mesoderm
Depletion of VegT causes loss of mesoderm
TGFB also plays a role in anterior structure formation and dorsalisation (via SO formation)
Vg1 or activin depletion by morpholinos –> reduction of anterior and dorsal structures
VegT activity
T-box transcription factor in the vegetal half
Induces Xnr’s and derriere and other TGFB family members
Together with B-catenin, strongly potentiates Xnr expression
Show that both NCand tgfb is necessary for SO formation
Grafting of NC or injection of siamois –> secondary D-V axis (or rescue of UV embryo)
Morpholinos against activin –> Loss of Gsc and organiser formation
How is Ectoderm and Mesoderm ventralised/dorsalised
BMP4 expressed in ventrolateral region of mesoderm and ectoderm - induces ventral fate.
SO produces BMP inhibitors - noggin chordin follistatin, allowing dorsalisation.
Dorsal ectoderm - neural tissue
Lateral Mesoderm - forms somite
Evidence that BMP-4 induces ventral tissue, and antagonists are secreted by S.O
Overexpression of BMP-4 causes ventralisation
Truncated dominant-negative BMP-4 abolishes BMP-4 signalling
Injection of Noggin, Chordin, Follistatin morpholinos results in loss of neural tissue
And the individual screens done by De Robertis and Harland
De RObertis and Harland
De RObertis - cDNA specifically expressed in isolated SOs.
Harland - identified genes that could rescue embryos after UV-irradiation, or induce secondary D-V axis in WT-embryos
Identified inhibitors of BMP-4 (noggin, chordin, follistatin)
Anterior posterior patterning
Temporal regulation of Wnt-signalling
First cells to involute receive Frzb, Dkk, Cerberus (are Xwnt8 inhibitors) hence do not receive Wnt signals, hence forms anterior tissue
BMP also weakly induces its own inhibition to confer stability and resistance to stochastic variation
Inhibitors secreted by S.O.
Cerberus inhibits Xwnt8, BMP,s and Xnrs
Frzb - soluble form of Frizzled, binds and inhibits Xwnt8.
Purpose and steps of gastrulation
Precisely position 3 germ layers in the embryo
Involution
Convergent extension
Epiboly
Cell migration via fibronectin
Fibronectin localised on blastocoel roof, provides cue for cell migration.
Before involution, mesodermal cells can attach but not migrate on fibronectin.
After involution, mesodermal cells can migrate on fibronectin
Cell rearrangement
Xbra induces Xwnt11
Xwnt11 planar polarity pathway.
Dominant-negative Xbra or DN-Xwnt11 blocks gastrulation mutant
DN-Xbra formed by fusing engrailed repressor to Xbra DNA-binding domain
Planar polarity pathway
Dsh similarly activated, but result in Rho and Rac GTPase activation, rather than B-catenin
Planar polarity: Uses PDZ and DEP domains Canonical: DIX and PDZ
Addition of N-terminal truncated Dsh (without DIX domain) can still rescue DN-Xwnt11 mutants
Addition of B-catenin activates canonical pathway, but does NOT rescue DN-Xwnt11
Addition of DN-Tcf3 would block canonical Wnt pathway, but does not affect gastrulation.
MyoD
Basic HLH TF, dimerises with partner E proteins
Master regulator of myogenesis (with Myf5)
Other bHLH TFs act sequentially to induce terminal differentiation (myogenin, MRF4)
How MyoD regulated?
Can only drive muscle differentiation upon cell cycle exit
Cell cycle proteins upregulate Id
CycC-CDK4 phosphorylates Rb, which inhibits Rb (a transcriptional coactivator to MyoD)
Low expression of CDK inhibitors that stabilise MyoD
Phosphorylation of MyoD by CDKs promotes ub-mediated degradation
Cellular Reprogramming
Fusion of rat muscle cell with human liver cell - muscle differentiation programme dominates
MyoD: Fibroblasts to muscle
Expression of AscI1, Brn2, MyT1 - turns on neural programme
