Zebrafish

Question 1

no tail (ntl)

Answer 1

Zebrafish, 2 alleles; ntl160 and ntl195. defect in mesoderm development, particularly affecting the posterior somites and notochord. Phenotype homolog in mice is T-brachyury.

Question 2

nacre (nac) phenotype

Answer 2

mutants grew to adulthood but lacked melanocytes but had normal amount of melanin in the eyes. Phenotype is similar to Steel, c-kit and Mitf in mice (white but black eyed)

Question 3

c-kit and Mitf

Answer 3

act cell autonomously; wild type cells injected into mutant embryos could give rise to pigmented melanocytes

Question 4

Steel

Answer 4

Wild type cells injected into steel embryos die because Steel encodes a secreted survival factor.

Question 5

Nac/nac embryos

Answer 5

Wild type cells injected into nac/nac embryos produced melanocytes; nac is a cell autonomous factor like c-kit and Mitf, not secreted factor like Steel

Question 6

What is the homology between no tail and T-brachyury?

Answer 6

69.7% identical amino acid sequence and similar expression pattern. Zf-T and ntl closely linked on chromosome 19

Question 7

What phenotype do T-brachyury mice exhibit?

Answer 7

failure to make posterior somites and no differentiation of the notochord.

Question 8

ntl160 and ntl195

Answer 8

Antibody staining = no detectable Zf-T protein sequence
ntl160 sequence analysis = frame shift mutation in exon six
ntl195 sequence analysis = 1544 insertion in exon 2
Both mutations = truncated protein, therefore ntl encodes zebrafish homolog of T-brachyury

Question 9

Zebrafish homolog of Mitf

Answer 9

maps close to nac on chr 6. mRNA of nac expressed in melanocytes in wild type and mutant embryos.
nac mRNA in mutants = nonsense mutation not affecting mRNA expression but would lead to a truncated protein.
Functional complementation of wild type nacre DNA into nac/nac embryos rescues phenotype.

Question 10

mini fin

Answer 10

partial/full loss of the tail fin; loss of ventral pattern elements from tail bud in embryo. dpp and tolloid mutatns in drosophila have equivalent D-V patterning phenotype.

Question 11

dpp and tolloid related genes in the zebrafish have been mapped to which chromosomes

Answer 11

2 dpp related genes; chromosome 11 and 20.
Single tolloid related gene: chromosome 1
However, mfn shows linkage with DNA markers on chromosome 1, eliminating the dpp related genes as candidates

Question 12

Sapje mutant phenotype

Answer 12

develop skeletal muscle lesions, resemble human muscular dystrophies (caused by genetic defects in components of Dystrophin Associated Protein Complex)

Question 13

How many zebrafish orthologues of the mammalian dystrophin and utrophin have been uncovered?

Answer 13

A single ortholog; zebrafish dystrophin (dmd) localised to embryonic muscle attachments like that of its mammalian counterpart and a nonsense mutation in the 4th exon segregated with the sap phenotype. (Duchenne muscular dystrophy is caused by equivalent mutation in exon of human gene).

Question 14

What does injection of zebrafish dmd morpholino do?

Answer 14

dmd morpholino (antisense RNA) causes specific reduction in dmd mRNA levels, producing similar defects to sap mutants; confirms sapje corresponds to zebrafish orthologue of dystrophin.