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CDB > Vertebrate development > Flashcards

Vertebrate development Flashcards

1
Q

Transcription in xenopus oogenesis

A

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

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2
Q

Early transport of mRNA in xenopus oocyte

A

Early pathway: Xcat2, MT-independent

Entrapped in mitochondrial cloud

Vegetal transport together with cloud fragments

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3
Q

Later pathway of mRNA transport

A

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

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4
Q

Selectively repressing translation

A

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

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5
Q

MBT

A

After 12 cleavage divisions

Cell cycle slows down - becomes asynchronous and with more conventional Cyclin-CDK

Zygotic transcription can occur

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6
Q

Cortical Rotation is necessary for D-V formation

A

UV irradiation - inhibits MT alignment, inhibits dorsal development

Can rescue by tipping embryo

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7
Q

Cortical rotation necessary for Wnt localisation

A

Injection of Wnt can form secondary D-V axis, or rescue UV-irradiated embryo

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8
Q

how is B-catenin stabilised in dorsal pole?

A

Dsh - fast via microtubules
Wnt11 mRNA - slower via cortical rotation
Inhibits GSK3B, stabilises B-catenin

B-catenin with Tcf3 forms NC

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9
Q

NC - expresses?

A

Expresses:
Twin, Siamois,

Strongly induced to express Xnr’s via TGFB-signals and B-catenin stabilisation combined

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10
Q

Endo- meso- ectoderm patterning

A

TGFB morphogen gradient

Intermediate activin –> Xbra in animal caps

High activin - expresses goosecoid in the dorsal

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11
Q

How SO is formed, and what it expresses

A

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,

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12
Q

Specification map vs fate map

A

Lateral mesoderm is specified to form blood and mesenchyme, but goes on to form somites

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13
Q

Specification map vs fate map

A

Lateral mesoderm is specified to form blood and mesenchyme, but goes on to form somites

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14
Q

TGFB signalling is necessary and sufficient for animal cap cells to form mesoderm,

A

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

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15
Q

TGFB also plays a role in anterior structure formation and dorsalisation (via SO formation)

A

Vg1 or activin depletion by morpholinos –> reduction of anterior and dorsal structures

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16
Q

VegT activity

A

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

17
Q

Show that both NCand tgfb is necessary for SO formation

A

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

18
Q

How is Ectoderm and Mesoderm ventralised/dorsalised

A

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

19
Q

Evidence that BMP-4 induces ventral tissue, and antagonists are secreted by S.O

A

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

20
Q

De RObertis and Harland

A

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)

21
Q

Anterior posterior patterning

A

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

22
Q

Inhibitors secreted by S.O.

A

Cerberus inhibits Xwnt8, BMP,s and Xnrs

Frzb - soluble form of Frizzled, binds and inhibits Xwnt8.

23
Q

Purpose and steps of gastrulation

A

Precisely position 3 germ layers in the embryo

Involution
Convergent extension
Epiboly

24
Q

Cell migration via fibronectin

A

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

25
Q

Cell rearrangement

A

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

26
Q

Planar polarity pathway

A

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.

27
Q

MyoD

A

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)

28
Q

How MyoD regulated?

A

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

29
Q

Cellular Reprogramming

A

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

CDB (6 decks)

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