zebrafish development

Question 1

describe what the initial cleavage of the embryo is like

Answer 1

it is discoidal and meroblastic. the yolk- free cytoplasm forms a blastodisc and the yolk- rich forms a vegetal yolk

Question 2

describe the stages of blastomere joining and the impact of this

Answer 2

initially the blastodiscs are joined to each other and to the yolk by cytoplasmic bridges. after the 16 cell stage only the inner cells are now joined to the yolk. these joins allow the diffusion of molecules larger than would be allowed by normal gap junctions

Question 3

how does cleavage change at different cell stages

Answer 3

up to 32 cells the cleavage is radial and forms a sheet of cells. from the 64 to 512 cell stage the cleavage becomes irregular but is still synchronous

Question 4

describe the mid blastula transition MBT

Answer 4

it happens at the 1000 cell stage (division 10) and marks the end of synchronous division. cells begin to differentiate and the zygotic genome is activated

Question 5

what layers form at the MBT and what will these layers become

Answer 5

superficial animal cells form the enveloping layer EVL which will form the periplasm. deep cells lose their connection, gaps form and they will develop into the embryo. nuclei of around 20 marginal cells migrate into the yolk, divide and form the yolk syncytial layer YSL which is split into internal and external

Question 6

what is epiboly and what drives it

Answer 6

the first morphogenetic movement at which the yolk domes and the blastoderm engulfs it. it is driven by the eYSL which moves downwards, pulling the EVL as it is joined by adherins and tight junctions

Question 7

when does gastrulation happen and what takes place

Answer 7

it happens at 50% epiboly, halting it. some cells begin to internalise which forms the hypoblast (involuting cells which will become the endoderm and mesoderm) and the epiblast (external cells which will form the ectoderm)

Question 8

what is the shield and what does it result in

Answer 8

it is the dorsal thickening of the germ ring due to the dorsal migration of surrounding cells. after its formation epiboly resumes

Question 9

where does the AP axis form and why

Answer 9

the AP axis forms dorsally as dorsal hypoblast cells form the axial mesendoderm (PCP and notochord). lateral hypoblast cells move dorsally due to convergent extension and they form the paraxial and lateral mesoderm

Question 10

what is special about somitogenesis and neurogenesis

Answer 10

they happen simultaneously at around 10.5 hours

Question 11

describe the hatching process

Answer 11

from day 2 the embryos can hatch from the chorion. by day 5 they have used up most of the yolk so they develop swim bladders in order to feed

Question 12

how does EVL division change and why does this alter cellular composition of the layers

Answer 12

initially the EVL undergoes both parallel and delamination division, hence some EVL cells contribute to the deep cell layer. after the late blastula stage they only divide parallel and form the periderm. deep cells cant enter the superficial EVL

Question 13

why can’t a fate map be made at the gastrula stage

Answer 13

there is deep cell mixing so different and cells can form any germ layer

Question 14

when does deep cell tissue restriction begin and what is the broad rule for determining fates

Answer 14

restriction begins at the shield stage where cell latitude shows some correlation with the germ layer of the progeny

Question 15

what tissues arise from the ectoderm

Answer 15

neural tissue is the default but epidermal tissue can arise if BMP signals are received

Question 16

what forms the mesendoderm and what tissues does it give rise to

Answer 16

it forms from the dorsal and lateral hypoderm and forms the PCP (gsc marker) and the notochord (tbxta/ntl/bra markers)

Question 17

how does the endoderm for the gut tube

Answer 17

it arises form marginal cells which undergo EMT to become mesenchymal stem cells. these converge at the midline at somitogenesis to form the gut tube

Question 18

how is germ cell fate decided

Answer 18

by vasa mRNA which encodes a specific germ cell protein. it localises at part of the 1st/2nd cleavage planes and then in four cells. these divide to form tissues which migrate to the gonads

Question 19

how has it been shown that maternal molecules can act as morphogens

Answer 19

by the removal of the yolk which contains the maternal molecules. this caused mis development of dorsal/ anterior structures eg no head, no notochord etc

Question 20

describe the movement of Wnt8a mRNA

Answer 20

it is a maternal molecule which is localised at the vegetal pole but is transported dorsally by microtubules

Question 21

describe the effects of Wnt8a protein

Answer 21

it is secreted out of the yolk into dorsal cells in which is activates the Wnt pathway. some nuclei migrate to form the dorsal YSL (Nieuwkoop equivalent) and others form the shield (Spemann’s organiser equivalent). the Wnt pathway also gives rise to BMP which promotes default neural ectoderm

Question 22

what is beta catenin and describe its mutants

Answer 22

it is a maternal mRNA, therefore mutants are maternal as opposed to zygotic. eg mich mutants which lose dorsoanterial structures and lack PCP/notochord markers

Question 23

how has it been shown that the shield is the equivalent to the amphibian Spemann’s organiser

Answer 23

ventral transplantation saw the generation of a second axis on the ventral side

Question 24

how is the zygotic genome activated

Answer 24

before the MBT the genome is blocked by histone pools which prevent transcription factors binding to DNA. after the MBT there is more DNA being synthesised but the number of histones is constant so they can no longer compete

Question 25

what are the two methods of degrading maternal molecules

Answer 25

either with miR430 which works in association with RISC factors or by tagging them with m6A which is targeted and degraded by Ythdf2 proteins

Question 26

how does are YSL genes activated and what do they do

Answer 26

they are activated by TF mxtx2 which is induced by TF nanog. YSL genes induce the mesoderm and endoderm

Question 27

how are mesoderm and endoderm genes activated

Answer 27

they are activated by nodal related mRNA ndr1 and ndr2. these activate mesoderm at low concentration and endoderm at high concentration. when the concentration is the same, brief exposure induces bra/ntl and long exposure induces bra/ntl and gsc

Question 28

what happens when ndr proteins are removed and how do phenotypes vary

Answer 28

their removal causes squint and cyclops mutants. single mutants are mild but double have no ntl/bra/tbxta/gsc in the shield and therefore no endoderm or axial mesoderm. there is some tail cells that still form due to the presence of ventroleteral ntl

Question 29

what is one eyed pinhead oep and how do mutants show the role of genomes in nodal signalling

Answer 29

oep is a nodal co-receptor, where the Zoep mutation is embryonic lethal but can be rescued by injecting the embryo with oep mRNA. they discovered by the maternal and zygotic genomes are involved in nodal sequencing due to the role of compensation. in Moep the zygotic compensated, in Zoep the maternal mostly compensates but in MZoep there is almost no nodal signalling so there is no mesoderm or endoderm

Question 30

what are the regulators of ntl and how are they regulated

Answer 30

ntl has two regulators E1 and E2. without E2 there is a loss of dorsal ntl expression, the same as when there is no nodal signalling, indicating that E2 is nodal-regulated. without E1 there is a loss of ventral ntl expression

Question 31

what is a morpholino and how are they different to nucleotides

Answer 31

they are modified oligonucleotides. they have morpholine rings instead of pentose sugars and they have phosphorodiamidate bonds instead of phosphodiester

Question 32

what are the two types of morpholino and how do they differ

Answer 32

there is 5’UTR/ATG which bind at the 5’UTR and prevent the initiation of translation by ribosomes. there is also splice which bind to the splice acceptor/donor, alter the transcript and produce abnormal or truncated proteins

Question 33

what phenotypes do the morpholino types give

Answer 33

5’UTR/ATG gives MZoep and splice gives Zoep because the maternal mRNA is already spliced

Question 34

what does TF MyoD do

Answer 34

it is expressed by skeletal cells in the paraxial mesoderm

Question 35

what does TF Gata1 do

Answer 35

it is expressed by erythrocytes in the lateral mesoderm

Question 36

what does vasa mRNA do

Answer 36

it encodes germ cell specific proteins which localise in parts of the cleavage planes

Question 37

what does Wnt8a mRNA do

Answer 37

it is localised in the vegetal pole but is transported dorsally by microtubules. then the Wnt8a protein is secreted from the yolk cell and activates the Wnt pathway in certain cells

Question 38

what does chordin do

Answer 38

its a BMP antagonist which promotes the development of default neural ectoderm

Question 39

what does BMP do

Answer 39

its a bone morphogenic protein which causes the ectoderm to give rise to epidermis as opposed to default neural tissue

Question 40

what does beta catenin do

Answer 40

it is an mRNA needed to induce dorsoanterial structures

Question 41

what does nodal do

Answer 41

it is a protein which regulates mesendoderm formation

Question 42

what does gsc do

Answer 42

it marks the prechordal plate in the axial mesendoderm

Question 43

what does tbxta/ntl/bra do

Answer 43

it marks the notochord in the axial mesendoderm