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Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates

Kirsten A. Dutton^1,*, Angela Pauliny^1,*, Susana S. Lopes¹, Stone Elworthy¹, Tom J. Carney¹, Jörg Rauch², Robert Geisler², Pascal Haffter² and Robert N. Kelsh^1,

¹ Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
² Max-Planck-Institut für Entwicklungsbiologie, Spemannstraße 35/III, D-72076 Tübingen, Germany
^* These authors contributed equally to this work and are to be considered joint first authors

View larger version (54K): [in a new window]   Fig. 1. A zebrafish sox10 homologue maps to the region of the cls locus. (A) Sequence comparison of predicted zebrafish Sox10 homologue (44, 45 and 60% identity to mouse Sox8, Sox9 and Sox10, respectively). Blocks of identity corresponding to all proposed functional domains can be seen, including the HMG domain (red underline; 95% amino acid identity), N-terminal synergy domain (1-105; 48% identity), dimerisation domain (66-105; 78% identity), C-terminal transcriptional activation domain (395-485; 76% identity) and a domain C-terminal to the HMG domain corresponding to a putative protein-protein interaction domain (234-325; 64% identity) (Bondurand et al., 2000; Kuhlbrodt et al., 1998a; Kuhlbrodt et al., 1998b; Liu et al., 1999; Peirano and Wegner, 2000). (B) Maximum likelihood phylogenetic tree of subgroup E Sox genes. Zebrafish sox10 clusters within the Sox10 clade of vertebrate Sox genes. The Accession Numbers for the sequences are as follows: chicken Sox8 (AF228664); trout SoxP1 (D83256); mouse Sox8 (AF191325); human SOX8 (AF226675); frog Sox9a (AB035887); alligator Sox9 (AF106572); trout Sox9 (AB006448); zebrafish sox9a (AF277096); zebrafish sox9b (AF277097); chicken Sox9 (AB012236); pig Sox9 (AF029696); human SOX9 (Z46629); zebrafish sox10 (AF402677); chicken Sox10 (AF152356); mouse Sox10 (AF047389); rat Sox10 (AJ001029); human SOX10 (NM_006941). (C) Mapping using the LN54 panel placed sox10 on LG 3 in the region of the cls locus identified using microsatellite markers (we found four recombinants between cls and z13387 in 274 meioses). Note that z8492 was not polymorphic and could not be analysed in the mapping cross. (D) Schematic to illustrate changes in Sox10 mutant proteins. In clsm618 a T425A substitution results in a non-conservative change (Leu142Gln) within the HMG domain (red). In clstw2 and clstw11, a A1126T substitution introduced a Stop codon truncating the protein just N-terminal to the transactivation domain (blue). Insertion of a 1.4 kb transposon at the site indicated by the arrow in A disrupts sox10 in clst3 and introduces a C-terminal extension of eight novel amino acids before premature truncation N-terminal to the HMG domain (yellow). (E) Chromatogram traces to show nucleotide changes affecting sox10-coding regions in clsm618 and clstw2.

View larger version (48K): [in a new window]   Fig. 2. cls phenotype is rescued by ectopic sox10 expression. Wild-type embryos show many large, strongly pigmented melanophores at 48 hpf (A; close-up of anal region in D), while hs>sox10(L142Q)-injected clsm618 mutants (B) show only tiny melanised spots in position of premigratory NCCs (arrowheads). (C,E,F) By contrast, clsm618 embryos injected with hs>sox10 and heat-shocked, show mosaic rescue of melanophores. This embryo showed one rescued melanophore in the dorsal stripe (* in C), two in the ventral stripe (arrows in C; close-up in E) and one on the yolk sac (F). Scale bar: 125 µm in A-C; 70 µm in D-F.

View larger version (89K): [in a new window]   Fig. 3. Embryonic sox10 expression in wild-type and cls embryos. (A,B) sox10 is expressed in most cranial (A; five-somite stage) and trunk (B; 14-somite stage) premigratory NCCs; double mRNA in situ hybridisation with fkd6 (O, purple) and sox10 (P, green) in a six-somite stage embryo reveals extensive overlap, although some individual NCCs lack sox10 (arrowheads). (C) 18-somite stage embryo shows strong expression in premigratory (black arrow) and migrating (asterisk) NCCs and otic vesicle (o). (D,E) Double mRNA in situ hybridisation with dlx2 (D, purple) and sox10 (E, green) in 29 hpf stage embryos shows absence of sox10 expression in developing branchial arches (1-5). (F) By 24 hpf in wild types, strong expression is associated with cranial ganglia (white arrowheads) and posterior lateral line nerve (black arrowhead; enlarged in inset), otic vesicle (o), migrating NCCs throughout trunk (asterisks) and in premigratory crest (arrow). (G) At 24 hpf in cls mutants, expression in the head is clustered (white asterisks) and cranial ganglia have reduced labelling (white arrowheads). Cells expressing sox10 extend along the posterior lateral line nerve (arrowhead and inset). Rostral trunk shows some migrating cells (black asterisk), but sox10-positive cells are clustered dorsal to the neural tube (arrows) in trunk and tail. (H,I) Combined sox10 in situ hybridisation (purple) and anti-Hu antibody labelling (orange) shows strong sox10 expression associated with wild-type (H) posterior lateral line ganglion (g), much reduced in cls mutant (I). (J) In transverse section of wild-type ganglion (approximate position indicated by white line in H), sox10 expression is strong peripherally (non-neuronal cells), but absent centrally (neurones). (K,L) 36 hpf wild-type embryos show weak expression in some melanophores, but not all. Thus, weak expression (arrowhead) is seen in some cells of the dorsal stripe (K), but not in cells on the yolk sac (L). (M,N) Number of sox10-expressing NCCs in different locations (premigratory, pre; migrating on medial pathway, MP; migrating on lateral pathway, LP) of trunk and anterior tail (somites 1-20) at 24 (M) and 30 hpf (N) in WT and cls mutants. (Q,R) Segmentally arranged lines of sox10-positive cells lying adjacent to the notochord (no), presumably glia, are abundant in wild type (Q) and only weakly affected in cls mutants (R) at 40 hpf. (S,T) Transverse section of trunk of 60 hpf wild-type embryo (S) shows enteric nervous system expression (arrowheads) lateral to the gut (g), absent in cls mutant (T). e, eye; m, muscle; s, somite; vs, ventral stripe melanophores. All images are lateral views, rostral towards the left, dorsal upwards, except dorsal views of A,K,O,P. Scale bar: 200 µm in A; 50 µm in B,H-I,K-L; 120 µm in C; 75 µm in D,E; 150 µm in F,G; 35 µm in J; 65 µm in O,P; 55 µm in Q,R; 45 µm in S,T.

View larger version (102K): [in a new window]   Fig. 4. NCC death in cls embryos. (A-C) NCC clones die by an apoptotic mechanism in cls embryos. Lateral views of 40 hpf cls embryo in which two daughters of a single labelled NCC contributed to the posterior lateral line ganglion, lying just posterior to the otic vesicle (o). In the live embryo, both cells show blebbed morphology typical of apoptotic cells when viewed with Nomarski optics (A). After fixation and processing for TUNEL (B) and detection of the biotinylated-dextran lineage-tracer (C), visible TUNEL signal of these clonal cells indicates DNA fragmentation characteristic of apoptotic cells. (D-K) Whole-mount TUNEL shows NCC apoptosis in cls embryos. Lateral views of dorsal spinal cord (sc) in tail of 30 (D,H), 35 (E,I), 40 (F,J) and 45 (G,K) hpf embryos show apoptotic NCCs immediately dorsal to the spinal cord from 35 hpf in cls (arrowheads, D-G), but not wild-type (H-K), embryos. Scattered TUNEL-positive cells are prominent in dorsal spinal cord (*) of cls embryos (E-G); these are occasionally seen in wild-type siblings at these stages (data not shown). df, dorsal fin. (L) Time-course of labelled single cranial NCC clone survival in cls mutants and their wild-type siblings. Percentage of surviving clones is given at each of the five standard time points when embryos were examined. The time points correspond to approximately 16, 32, 40, 56 and 64 hpf, respectively. The first time point includes only single labelled NCCs based on examination within a few hours after labelling. See text for further details. (M,N) Wild-type xanthophores (arrows) at 48 hpf have a very flattened, thin morphology and are only weakly coloured (M), while a dying cls xanthophore (arrow) shows characteristic apoptotic morphology and concentrated yellow coloration, which was usually visible by 35 hpf (N). Scale bar: 100 µm in A-C; 50 µm in D-K; 75 µm in M,N.

View larger version (130K): [in a new window]   Fig. 5. cls mutants lack nac and spa expression. Lateral views of caudal trunk of 25 hpf wild-type and cls embryos after in situ hybridisation with nac (A,B), spa (E,F) and dct (G,H) probes. (C,D) nac/mitf expression is decreased weakly (C) or strongly (D) after injection with either a low or high dose, respectively, of sox10 morpholino. n, neural tube; y, yolk sac. Scale bar: 75 µm.

View larger version (16K): [in a new window]   Fig. 6. Model of role of cls/sox10 in melanophore specification. Formal genetic interactions between selected genes known to function in zebrafish melanophore development are schematised. In cls mutants, failure to activate nac expression (and thus to specify melanophore fate) results in absence of gene products critical for melanophore survival, migration and differentiation. For details, see main text.