Mod 8

Question 1

Introductory Developmental Biology and Embryology

Zebrafish is a good “genetic” organism
Its characteristics?

Answer 1

Zebrafish is a good “genetic” organism

large broods
breed all year
easy to maintain
transparent embryos develop outside mother
embryogenesis completed in 24h
microinjection of blastomeres permits cell labelling experiments or transgene expression

The first vertebrate system extensively studied by mutagenesis screens.

Mutations have been isolated in different genes (here pigmentation defects).

Question 2

See slide 4 nd 5

Question 3

Zebrafish primary oocytes are highly transcriptionally active during the?

In early stages of growth what is visible ?– this structure is thought to be associated with?

The developing oocyte is surrounded by?

One follicle cell maintains contact with the?
and this creates a small hole in the?
and it is through this hole that sperm enter during fertilization – the structure is known as the?

Answer 3

Zebrafish primary oocytes are highly transcriptionally active during the growth phase (transcriptional activity during the diplotene stage of meiosis results in cytologically distinctive lampbrush chromosomes).

In early stages of growth the Balbiani body (aka the mitochondiral cloud) is visible – this structure is thought to be associated with maternal RNA localization

The developing oocyte is surrounded by follicle cells – these secrete the vitelline envelope.

One follicle cell maintains contact with the oocyte – and this creates a small hole in the vitelline envelope – and it is through this hole that sperm enter during fertilization – the structure is known as the micropyle (recall that Drosophila have a micropyle too).

Question 4

Zebrafish - Following fertilization:
What happens?

Answer 4

there is a cortical granule reaction-(CGR) (as there is in sea urchin and other systems)

following CGR chorion (formerly known as the vitelline envelope) lifts from the surface of the egg forming the fertilization envelope

Pronounced cytoplasmic streaming (associated with actin/myosin based contraction) occurs – resulting in a non-yolky cytoplasm located at the animal pole – a clear “island” of yolk-free cytoplasm – this is called the cytoplasmic cap or the blastodisc.

This is where cleavage divisions (meroblastic) will occur. Following cleavage divisions, the blastodisc becomes the blastoderm.

Question 5

See slide 11 for Early Zebrafish Development
See slide 17 diagram

Question 6

Maternal Effect Mutations – have shown that maternally contributed mRNA and proteins are required for?

maternal mutants that disrupt microtubules also disrupt?

Answer 6

Maternal Effect Mutations – have shown that maternally contributed mRNA and proteins are required for:

establishment of embryonic polarity
cell divisions and cleavage

maternal mutants that disrupt microtubules also disrupt pattern of cleavage divisions

like Xenopus - morphogenetic determinants probably travel along microtubules

Question 7

~ 10th division – the midblastula transition occurs - three distinct cell populations?

Answer 7

~ 10th division – the midblastula transition occurs - three distinct cell populations:
yolk syncytial layer (YSL) (extra-embryonic)
enveloping layer (EVL) (extra-embryonic)
deep cells (embryo proper)

Question 8

early blastoderm cells are not determined – there is much cell mixing during?
– any early blastomere can become ?
– but a fate map can be generated on?

Answer 8

early blastoderm cells are not determined – there is much cell mixing during cleavage – any early blastomere can become any number of different tissues – but a fate map can be generated on the blastoderm just before gastrulation

Question 9

See slides 21-23 for diagram

Question 10

Fish gastrulation starts with?
involves radial intercalation of?

enveloping layer (EVL) is joined to the?
and it gets dragged along ?

expansion of the yolk syncytial layer is dependent on?

during epiboly which side becomes thicker?

Answer 10

Fish gastrulation starts with epiboly of blastoderm cells over the yolk - involves radial intercalation of deep cells with more superficial cells

enveloping layer (EVL) is joined to the yolk syncytial layer (YSL) – and it gets dragged along (if EVL/YSL attachments are experimentally severed enveloping layer (EVL) and deep cells spring back to top of yolk)

expansion of the yolk syncytial layer is dependent on microtubules

during epiboly one side becomes thicker – future dorsal side

Question 11

at halfway point of epiboly – thickening throughout the margin of the epibolizing blastoderm called? Forms?

germ ring consists of ?

Answer 11

at halfway point of epiboly – thickening throughout the margin of the epibolizing blastoderm called the germ ring – forms as a synchronous wave

germ ring consists of :

epiblast (superficial layer)

hypoblast (inner layer) – in fish contains precursors for mesoderm and endoderm

Question 12

The yolk (Yolk syncytial layer) can induce?.

Region of yolk beneath the presumptive dorsal region is acting like the?

Answer 12

The yolk (Yolk syncytial layer) can induce mesoderm.

Region of yolk beneath the presumptive dorsal region is acting like the Nieuwkoop centre in Xenopus.

Question 13

Convergence movements produce a local accumulation of cells at?

Epiboly undergoes a temporary pause while the shield forms, but continues afterwards?

Answer 13

Convergence movements produce a local accumulation of cells at one position along the germ ring – the embryonic shield.

Epiboly undergoes a temporary pause while the shield forms, but continues afterwards and the blastoderm margin completely covers the yolk.

Question 14

In the region of the embryonic shield, cells converge and extend?
– narrowing along the?

Answer 14

In the region of the embryonic shield, cells converge and extend anteriorly – narrowing along the dorsal midline – this is the precursor of the notochord.

Question 15

Convergence and Extension in the zebrafish gastrula
See diagram slide 34

Question 16

shows arrangement of mesoderm during gastrulation

mesoderm is surrounding the?

“sometimes called ?” because?

Answer 16

shows arrangement of mesoderm during gastrulation

mesoderm is surrounding the yolk

“sometimes called “endomesoderm” because endoderm and mesoderm are here mixed or “intermingled”– they separate out later

Question 17

Based on transplantation experiments, the embryonic shield:

is critical in establishing?

can convert lateral and ventral mesodem (precursors of blood and connective tissues) into?

can cause the ectoderm to become?

Answer 17

Based on transplantation experiments, the embryonic shield:

is critical in establishing the dorsal-ventral axis

can convert lateral and ventral mesodem (precursors of blood and connective tissues) into dorsal mesoderm (notochord and somites)

can cause the ectoderm to become neural rather than epidermal

Question 18

Exactly like the amphibian blastopore lip, the embryonic shield:

forms the?

precursors of the prechordal plate and notochord are responsible for?

mechanism is very similar to?

Answer 18

Exactly like the amphibian blastopore lip, the embryonic shield:

forms the prechordal plate & the notochord

precursors of the prechordal plate and notochord are responsible for the induction of ectoderm to become neural ectoderm

mechanism is very similar to amphibians (involves BMPs and Wnt signaling) – anterior vs. posterior depends on the levels and combination of signals

Question 19

BMPs and Wnts are made in the?

the developing notochord (just like in frogs) secretes factors ?that block?

chordamesoderm secretes a protein called ? It binds?

mutants lacking Chordino do not develop a?

Answer 19

BMPs and Wnts are made in the ventral and lateral regions of the embryo (BMP2B induces ventral and lateral fate & Wnt8 ventralizes, lateralizes, and posteriorizes tissues).

the developing notochord (just like in frogs) secretes factors (BMP antagonists) that block induction, allowing ectoderm to become neural

chordamesoderm secretes a protein called Chordino - Chordino binds and inactivates BMP2B – it is an extracellular BMP antagonist

mutants lacking Chordino do not develop a neural tube properly

Question 20

zebrafish have a shield = “organizer” that functions in a very similar way to what we saw in?

in frogs the organizer was specified due to?

In Zebrafish – the nuclei of the yolk syncytial layer beneath the the cells that will become the ?

Answer 20

zebrafish have a shield = “organizer” that functions in a very similar way to what we saw in Xenopus

in frogs the organizer was specified due to the spatially restricted accumulation of β-catenin synthesized from maternal messages (and activation of Wnt signalling) in the endodermal cells beneath the dorsal blastopore lip (the Nieuwkoop centre)

In Zebrafish – the nuclei of the yolk syncytial layer beneath the the cells that will become the embryonic shield also accumulate β-catenin (dorsal YSL has nuclear β-catenin)

Question 21

inducing β-catenin accumulation on the ventral side (where it is not normally expressed) leads to?

β-catenin in the shield combines with Tcf3 to form a?

mesoderm patterning genes include?

Bozozok can? Functions…

Answer 21

inducing β-catenin accumulation on the ventral side (where it is not normally expressed) leads to dorsalization and a second embryonic axis ( = siamese twin fish)

β-catenin in the shield combines with Tcf3 to form a transcription factor – activates genes required for mesoderm patterning

mesoderm patterning genes include Squint (a Nodal-like paracrine factor) and Bozozok (homeodomain similar to Siamois in Xenopus)

Bozozok can:
repress BMP and Wnt genes that promote ventral functions
suppress a transcriptional inhibitor and allow organizer genes to function
along with Squint can activate the chordino gene (plus other “organizer” genes)

Question 22

Chordin (Xenopus)/Chordino (Zebrafish) is a?.

Chordin: A paracrine factor with organizer activity. Chordin binds directly to?

See table also on 43

Answer 22

Chordin (Xenopus)/Chordino (Zebrafish) is a BMP antagonist.

Chordin: A paracrine factor with organizer activity. Chordin binds directly to BMP4 and BMP2 and prevents their complexing with their receptors, thus inducing dorsal ectoderm to become neural tissue.

Question 23

See slide 44

Question 24

Frogs and Fish – early development is very similar..but different

In Xenopus the sperm entry point was the main factor that “broke” the?
… but in zebrafish the sperm must enter through the?

Yolk ablation experiments suggest that there is a migration of a?

Experimental evidence: treatment with microtubule depolymerizing drug nocodazole suppresses ?
Dorsal axis formation is also suppressed in ?

And what is ichabod?

Answer 24

Frogs and Fish – early development is very similar..but different

In Xenopus the sperm entry point was the main factor that “broke” the asymmetry of the egg.…… but in zebrafish the sperm must enter through the micropyle located at the animal pole.… and there is no obvious cortical rotation in zebrafish.

So, the immediate cause of dorsoventral symmetry breaking in fish is uncertain.
(creating asymmetry…this is a key point for multicellular life – and possibly greater implications for life, the universe and everything….)

Yolk ablation experiments suggest that there is a migration of a dorsal determinant (probably wnt8 mRNA) from the vegetal pole to the dorsal side.

Experimental evidence: treatment with microtubule depolymerizing drug nocodazole suppresses axis formation.

Dorsal axis formation is also suppressed in embryos laid by ichabod- females.

And what is ichabod?
…the ichabod gene encodes a β-catenin and there is a detectable increase of nuclear β-catenin on the dorsal side of the early blastoderm stage embryo.

Question 25

The Balbiani body is a spherical membraneless structure that forms in?
Its constant components are?

The Balbiani body (AKA mitochondrial cloud) in Xenopus transports?

The localization of Wnt8a mRNA in Zebrafish oocytes is associated with the?

Subsequent movement of Wnt8a mRNA from the vegetal region to the?

Answer 25

The Balbiani body is a spherical membraneless structure that forms in contact with the oocyte nucleus in early oogenesis, and fragments and disperses in late oogenesis. Its constant components are mitochondria, endoplasmic reticulum (ER), membranous vesicles and lipid droplets.

The Balbiani body (AKA mitochondrial cloud) in Xenopus transports localized RNA to the vegetal pole of the oocyte (remember VegT and Vg1?).

The localization of Wnt8a mRNA in Zebrafish oocytes is associated with the Balbiani body.

Subsequent movement of Wnt8a mRNA from the vegetal region to the DORSAL REGION is not understood but is thought to be microtubule dependent.

Question 26

During epiboly, cells at the border undergo ?

See diagram also slide 47

Answer 26

During epiboly, cells at the border undergo structural changes and involute. As they do, mesodermal genes (red above) are activated.

Question 27

EARLY FISH DEVELOPMENT: SUMMARY
cleavage is?

future dorsal side hypoblast and epiblast intercalate – forming a?

this is the embryonic shield =?

neural ectoderm will form where?
the embryonic shield secretes?

the shield is established by?

Answer 27

cleavage is meroblastic

enveloping layer/ yolk syncytical layer / blastoderm deep cells
migration over yolk - epiboly
germ ring hypoblast and epiblast
future dorsal side hypoblast and epiblast intercalate – forming a thickening

this is the embryonic shield = blastopore lip of Xenopus and has ORGANIZER qualities -

neural ectoderm will form where BMP-mediated induction of epidermis is PREVENTED

the embryonic shield secretes BMP antagonists

the shield is established by β-catenin and underlying endodermal cells that secrete Nodal-related paracrine factors