DEV006098 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. External phenotype of lbk mutant and sibling embryos at 48 hours of development. Clutch of embryos from a pair of lbk^+/− heterozygous zebrafish at 2 dpf. Although some embryos display lower levels of melanin pigmentation in skin melanocytes and in the RPE, the differences are gradual and allow no reliable distinction between wild-type and lbk^−/− mutant individuals at this stage.

• Supplemental Figure S2 -

  Fig. S2. Vam6p is highly conserved in vertebrate species. Alignments of the human, mouse, chicken and zebrafish Vam6p amino acid sequences. Residues shaded in blue are conserved between all species. The overall homology of the zebrafish Vam6p to the human, mouse and chicken proteins is over 80%. The consensus sequence of all four sequences is given above the alignment. Homologous domains predicted by SMART (http://smart.embl-heidelberg.de) are indicated with coloured bars: magenta, N-terminal citron homology (CNH) domain; orange, clathrin homology (CLH) repeat domain. The mutations identified for the lbk and lbk* mutants are indicated by red and green boxes, respectively.

• Supplemental Figure S3 -

  Fig. S3. Identification of an additional allele of lbk in an allele screen. (A,B) External phenotype of the compound heterozygous lbk* mutant larva identified in the allele screen. Like lbk^−/− larvae, the 5 dpf lbk* larva shows a hypopigmentation of the RPE and skin melanocytes (A), dull iridiophores (B) and lacks a swim bladder (A, asterisk). The lbk* phenotype is, however, less severe than that of lbk^−/− larvae, indicating that the second allele is a hypomorph. (C,D) The TrA exchange at position 374 of the vam6 cds identified in the lbk* mutant larva (Fig. 1O) results in the exchange of a conserved methionine (M) at amino acid position 125 for a lysine in the N-terminal citron homology (CNH) domain. (E,F) Sequencing of vam6 alleles from wild-type zebrafish. To provide further evidence that the mutation identified in lbk* disrupts the function of Vam6p, we prepared genomic DNA from 71 adult wild-type zebrafish of the same genetic background as that of the fish used in the allele screen and sequenced the corresponding 142 vam6 alleles. Although the sequencing did detect polymorphisms in the vam6 alleles between these fish (letters marked in red in panel A), none of the sequences contained the TrA point mutation identified for lbk*, which leads to the exchange of a highly conserved methionine residue for a lysine. This indicates that the TrA point mutation in lbk* is indeed the cause of the observed trans-heterozygote phenotype (see Fig. 6E,F). (E) Alignment of all sequences derived from the 71 wild-type zebrafish. The thymine (T) that is mutated to adenine (A) in lbk* is indicated by a red box. (F) Four representative sequencing traces showing the region harbouring the TrA point mutation identified for lbk*. All of these sequences show the wild-type T.

• Supplemental Figure S4 -

  Fig. S4. Transplantation experiments show that the defect in lbk is cell-autonomous. Bright-field images of the dorsal head region (A) and tail (B) of four dark-adapted 5 dpf larvae. The skin melanocytes that have developed into normally pigmented melanocytes in an lbk^−/− mutant background (arrows). Inset in B is a higher magnification dorsal view of four melanocytes with wild-type appearance indicated by arrows in B. Melanocytes displaying the severe hypopigmentation characteristic of lbk (white arrowheads) can be seen next to cells showing wild-type levels of melanin (black arrows).

• Supplemental Figure S5 -

  Fig. S5. Skin melanocytes are hypopigmented in lbk. Skin melanocytes in wild-type (A,B) and lbk mutant larvae (C,D) at 5 dpf. (B,D) Higher magnifications of regions in A and C, respectively. Melanosome number is substantially reduced in the lbk melanocytes (C), which leads to the hypopigmentation characteristic of these mutants, and the abnormally shaped melanosomes (D, arrowheads). Despite a majority of abnormally small and misshapen melanosomes, lbk skin melanocytes contain some melanosomes displaying wild-type size, shape and levels of melanin pigmentation (arrow). Scale bars: 5 µm in A,C; 1 µm in B,D.

• Supplemental Figure S6 -

  Fig. S6. Retinal Müller glia cells show a normal morphology in lbk mutants. Cryosections of 5 dpf wild-type and lbk eyes were stained with DAPI (blue; panels B and E) to show the position of the nuclei in the retina and an antibody against the glia-specific enzyme glutamine synthetase to label the retinal Müller glia cells (red; arrowheads in C and F). Phase-contrast images of the sections of wild-type and lbk eyes are shown in A and D, respectively. No obvious differences in the number, arrangement and morphology of the Müller glia cells were observed between wild-type and lbk larvae. ON, optic nerve. Scale bars: 50 µm.

• Supplemental Figure S7 -

  Fig. S7. The intestine of lbk mutant larvae shows an increase in small vesicles. Transversal TEM sections of the anterior intestine of 7 dpf wild-type (A) and lbk mutant (B) larvae show an increase in the number and decrease in the size of vesicles in the cells lining the intestinal lumen in lbk. The images are overviews comprising the higher magnifications shown in Fig. 4K,L, respectively. L, intestinal lumen; N, nucleus. Scale bars: 10 µm in A,B.

• Supplemental Figure S8 -

  Fig. S8. vam6 mRNA is widely expressed during zebrafish development. Whole-mount in situ hybridisation detecting vam6 mRNA expression at different stages of development (A-K) and in adult tissues (L,M) of wild-type zebrafish. (A) Shield stage embryo (6 hpf); vam6 mRNA expression is comparatively weak in all cells of this stage. (B) 24 hpf embryo; at this stage vam6 is widely expressed with the highest levels in the developing brain. (C-F) 48 hpf embryo; vam6 expression has increased compared with earlier stages. Expression is highest in the head region, the region of the developing liver and intestinal tract (D; arrowheads) and in the skin melanocytes (E,F; arrowheads). (G-K) 5 dpf larva; ventral views (G,J) and lateral views (H,I,K). As in earlier stages, vam6 mRNA is widely expressed, but at higher levels than before. Strong expression can be detected in the peripheral retina (region of the RPE; arrowheads in G, H and J), the liver (H; arrow), the melanocytes of the skin (I; arrowheads) and the intestinal tract (K; arrowheads). (L,L′) Expression of vam6 mRNA in the adult zebrafish liver. Panel L′ shows the corresponding sense probe. (M,M′) vam6 mRNA expression in the cells lining the intestinal tract in the adult zebrafish. M′ shows the corresponding sense probe.