Zebrafish Development Genetics

Question 1

Methods of getting picture of genes involved in development of zfish

Answer 1

Systematic mutagenesis

Systematic phenotype screening

Cloning mutated genes

Question 2

Benefits of zebra fish model?

Answer 2

Easy husbandry - keep in tanks
Embryos develop externally (in egg)
Embryos are translucent
Rapid development (first 18hrs can be filmed under microscope - can clearly see ball turn into patterned embryo, eg gastrulation)
Accurate staging - particular structure assign embryo to diff stages -important for finding if mutant phenotype is slowing development
Well defined fate map FROM GASTRULATION ONWARDS
Embryo manipulation is easy

Question 3

Basic stages of zfish embryo

Answer 3

4 cell on yolk - connected to yolk syncytial by cytoplasmic bridges
Sphere formed from cell divisions

Then development of axes and organs
Shield
80% Epiboly
1 somite
Somite development
19 somites

Question 4

Zfish embryo manipulation

Answer 4

Morpholino oligonucleotides
Rescue via ectopic expression
Cell transplantation
Targeted mutagenesis using crispr/cas9
Random mutagenesis

Question 5

Morpholono oligonucleotides

Answer 5

Antisense RNA
Leads to degradation and/or inhibition of target mRNA

Antisense RNA binds to target mRNA
Morpholino groups cause the mRNA to be unrecognisable by embryo’s RNases

Question 6

Random mutagenesis

Answer 6

Hits random genes
Perform Systematically - so hopefully will hit all genes involved in development

Uses:
N-ethyl N-nitrosourea - a point mutagen
Transfers ethyl group to O or N
Results in mispairing and basepair substitution
Up to 1 in 1000 mutation rate
82% mutations involve AT base pairs to either TA or GC

64% missense mutations - can substitute AA for another
10% nonsense - new STOP
26% splicing errors

Question 7

Mutagenesis screens

Answer 7

Tank of males
Add ENU
All cells mutated incl spermatogonia
Introduce these males to WT females and mate them

Some fish in F1 are heterozygous for certain mutant
Mate heterozygous F1 with WT fish

In F2 all fish now have heterozygote fish male and female
Take F2 in random pairs

Some of progeny F3 will be homozygous for the mutation

Question 8

Random mutagenesis screen saturation

Answer 8

Means that every possible gene able to be hit by this method was

Presence of single alleles in screen means that saturation was not hit

every new gene hit after saturation would be similar to ones already hit

Also nonspecific phenotypes - may have contained some additional genes

Question 9

Epiboly

Answer 9

Cells that sit on top of top of zfish egg
Divide to about 1000 cells - ball stage

Then suddenly these cells crawl down surface of yolk
Once 50% of yolk - cells begin to go in and gastrulation occurs

Question 10

Number of mutants in category in screen significance

Answer 10

No of mutants in each category is partlyaffected by how easily the phenotype is recognised
So there is some subjectivity
Eg would see more pigmentation mutant phenotypes vs internal organ ones as they are easier to recognise

Question 11

Candidate approach to isolating genes

Answer 11

When screens were done in zfish
Genome sequencing was not so good compared to other species human and mouse eg

No inbred strains and few polymorphisms for mapping
Cloning genes was difficult

Genes were identified through candidate approach
See phenotype in zfish that is similar to phenotype in another organism
Know the gene that causes that in other organism
Gives candidates to look at in zfish

Question 12

Mutants affecting zfish axial elongation

Answer 12

No tail: a T-box containing TF

Pipetail: a signalling molecule Wnt5

Knypeck: cell surface molecule that acts as a cofactor for signaling, glypican 4 or 6

Question 13

No Tail expression

Answer 13

Expressed in posterior end of tail bud
And in the notochord

And in the chords neural hinge structure

Question 14

Wnt5 (pipetail) and Knypeck expression

Answer 14

Expressed in tail end as axis elongates from ant-post

Actors in tail development found in the post end

Question 15

No tail, Pipetail and Knypeck mutant phenotypes

Answer 15

Similar
Squished at tail end
Axis fails to elongate

Question 16

No tail interactions with Knypeck and Pipetail

Answer 16

No tail Cross mutant is more messed up than single mutants
Indicates linked genetic pathways

Question 17

Knypeck and no tail mutant effect on cell movements

Answer 17

Don’t affect differentiation
But affect cell movements
Vessel there that should’ve moved out in elongation are expressing genes characteristic of where they currently are in the mutant
Suggests cell movement is important for changing gene expression

Question 18

Mouse T(brachyury) mutants and no tail mutants similarities

Answer 18

Resemble no tail mutants
Disrupt the orthodoxies of no tail

Disrupts mesoderm movement in mouse gastrulation and axis elongation
Cells with mutant protein from this gene have issues in cells dispersing/migrating and elongation

Good equivalence if functions between mouse and zfish mutations
Mouse Wnt5a (similar to zfish wnt5) has similar phenotype too -elongation disrupted short embryo

Question 19

Mouse elongation mutants and no tail mutants differences

Answer 19

Loss of brachuary in mouse is more detrimental to embryo than no tail loss in zfish

Mouse Wnt5a affects outgrowth of all appendages, probably by reducing proliferation. Only the axis is affected in Pipetail (wnt5) mutants

No mutations in mlise glypicans have yet been shown to affect axial elongation

Question 20

Grumpy and Sleepy mutants

Answer 20

Notochord differentiation mutants
Mutant embryos shorter than WT
Notochord forms but fails to differentiate into rod like supporting structure (important structure in fish embryos as they need the support for early swimming)

Question 21

Grumpy and Sleepy mutants affected genes

Answer 21

Truncated Lamin B1 (grumpy) and C1 (sleepy) chains (components of laminin 1 an ECM protein around the notochord)

These genes mRNA is expressed around the notochord

Phenotype can be replicated with Morpholino oligonucleotides

The missing chain can be supplied by wt notochord or somites in organiser grafts
Rescues notochord differentiation

Question 22

Relevance if grumpy and sleepy to other vertebrates

Answer 22

No lamin mutations affect notochord morphogenesis in mouse
Notochord provides structural support for fish embryos but not mammal
Mammals don’t need to move in uterus so developed notochord not needed so early

So may not be relevant for mammals

Question 23

Zebra fish genomics

Answer 23

Mammals mostly have 4 hox clusters
Zfish had 7
And has 5 shh family members jnstead of 3

Sometimes is unclear which gene is the orthodoxies of a mouse gene
-glypican 4 or 6 (knypeck) - similar to both
-Tbx16 (spadetail) - no true orthologue

Question 24

Why the difference in genomics between zfish and mammals

Answer 24

After fish and rest of vertebrates split
Evolutionary event in fish - so rest of vertebrates unaffected

Variable genome loss and genome duplication
So reason for why genome is different