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Fig. 1. A mutation in vam6 causes hypopigmentation and hepatomegaly in
lbk. (A-D) Bright-field images show the hypopigmentation
of skin melanocytes and the RPE, and a brownish discolouration of the
intestinal tract (B,D, arrows) in lbk. Asterisks in A,C indicate the
swim bladders in wild-type larva. (E-H) Dark-field images show an
absence of iridophore reflections in the lbk skin and eye (G,H;
arrows). Note the translucency of the lbk RPE in H. Arrow in F
indicates the intestinal tract. Asterisk in E indicates the lac of swim
bladder. (I,J) lbk larvae display an enlarged liver
(insets in I,J; outlined areas) and lack swim bladders (asterisk). (K-O) The
gene for Vam6p is mutated in lbk. (K) Genetic mapping of
lbk resulted in linkage to SSLP markers z9692 and z4053 on chromosome
17. RH mapping of zebrafish vam6 resulted in linkage to EST
fc27c07.x1, which was found to be located close to z9692 on the T51 RH map and
thus served as an anchor to link the two maps. (L) Sequencing of cDNA
from lbk-/- and homozygous wild-type larvae revealed a
nucleotide exchange at position 1066 from C
T (arrowheads), resulting in
a premature STOP codon. (M) Domain structures of the wild-type Vam6p
(bottom) and the protein truncated in lbk lacking the C-terminal 520
amino acids (top), including the clathrin homology (CLH) repeat domain and
Ypt7p-interacting sequences. (N) Sequence alignment of human, mouse,
chicken and zebrafish Vam6p proteins shows the high level of conservation in
the region affected by the mutation (red box). (O) Sequencing of the
complete vam6 cds from the lbk* mutant revealed
that this larva was heterozygous at two positions: the CT nucleotide
exchange identified in lbk (right panel, lower trace; arrowhead) and
a novel TA exchange at position 374 of the vam6 cds (left
panel, lower trace). The latter results in the exchange of a conserved
methionine (M) at amino acid position 125 for a lysine (K) in the N-terminal
citron homology (CNH) domain (see Figs S2, S3 in the supplementary material).
Sequencing of the complete vam6 cds from lbk*
siblings showed the heterozygous presence of the lbk mutation (right
panel, upper trace; arrowhead), but the absence of the TA exchange at
position 374 (left panel, upper trace).
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