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Fig. 3. Positional cloning of lsn. (A)
lsnw24 maps on to linkage group 12 between markers z9891
and z4373. No recombinants were found between lsnw24 and a
SSCP marker in the 3' UTR of an EST (Fa05b04) that encodes for a
zebrafish trap100. We isolated a full-length cDNA corresponding to
trap100 from homozygous-lsn mutant and wild-type embryos and
12 independent clones were completely sequenced in both directions. A T to A
(encoding Y to stop) mutation was identified in trap100 cDNAs derived
from homozygous-lsn mutants. (B) A schematic illustrating the
overall structure and percentage similarity between human, mouse and zebrafish
Trap100, and the predicted truncated Trap100 in lsn mutants.
(C) Sequence tracing of one of the 12 independent clones derived from
genomic PCR of genomic DNA from mutant and wild-type embryos showing TAT (Y)
to TAA (stop) mutation. (D) A bar graph showing rescue of the
lsn homozygous mutant enteric phenotype by injection of wild-type
trap100 mRNA. Embryos derived from an incross of heterozygote
lsn fish were injected with 50 pg of trap100 mRNA, fixed and
stained with anti-Hu antibody at 96 hpf and genotyped. Control represents
genotyped uninjected embryos from the same cross. Total number of enteric
neurons in a 10-somite segment of the intestine were counted. Numbers
represent the mean number of enteric neurons in this 10-somite
segment±s.e.m. for 15 embryos of each genotype for each condition.
*Significantly different from control (Student's t-test
P<0.001).
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