Zebrafish Development Genetics Flashcards
Methods of getting picture of genes involved in development of zfish
Systematic mutagenesis
Systematic phenotype screening
Cloning mutated genes
Benefits of zebra fish model?
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
Basic stages of zfish embryo
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
Zfish embryo manipulation
Morpholino oligonucleotides
Rescue via ectopic expression
Cell transplantation
Targeted mutagenesis using crispr/cas9
Random mutagenesis
Morpholono oligonucleotides
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
Random mutagenesis
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
Mutagenesis screens
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
Random mutagenesis screen saturation
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
Epiboly
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
Number of mutants in category in screen significance
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
Candidate approach to isolating genes
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
Mutants affecting zfish axial elongation
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
No Tail expression
Expressed in posterior end of tail bud
And in the notochord
And in the chords neural hinge structure
Wnt5 (pipetail) and Knypeck expression
Expressed in tail end as axis elongates from ant-post
Actors in tail development found in the post end
No tail, Pipetail and Knypeck mutant phenotypes
Similar
Squished at tail end
Axis fails to elongate
No tail interactions with Knypeck and Pipetail
No tail Cross mutant is more messed up than single mutants
Indicates linked genetic pathways
Knypeck and no tail mutant effect on cell movements
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
Mouse T(brachyury) mutants and no tail mutants similarities
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
Mouse elongation mutants and no tail mutants differences
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
Grumpy and Sleepy mutants
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)
Grumpy and Sleepy mutants affected genes
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
Relevance if grumpy and sleepy to other vertebrates
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
Zebra fish genomics
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
Why the difference in genomics between zfish and mammals
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