Somatolactin selectively regulates proliferation and morphogenesis of neural-crest derived pigment cells in medaka

Abstract

Species-specific colors and patterns on animal body surfaces are determined primarily by neural-crest-derived pigment cells in the skin (chromatophores). However, even closely related species display widely differing patterns. These contrasting aspects of chromatophores (i.e., the fixed developmental control within species and extreme diversity among species) seem to be a curious and suitable subject for understanding evolution and diversity of organisms. Here we identify a gene responsible for medaka "color interfere" mutants by positional cloning. These mutants do not show any obvious morphological and physiological defects other than defects in chromatophore proliferation and morphogenesis. The mutation has been identified as an 11-base deletion in somatolactin, which causes truncation 91 aa upstream of the C terminus of the protein's 230 aa. Somatolactin transcription changed dramatically during morphological body color adaptation to different backgrounds. This genetic evidence explains somatolactin function. Studying this mutant will provide further insights into the development and regulation of chromatophores and clues for reassessing other functions of somatolactin suggested in other fish.

Fig. 2.

Responses of ci chromatophores to chemicals and backgrounds. (a–d) Chromatosome movements within leucophores and melanophores on ci mutant scales. (a) Movements in Ringer's solution. (b) Movements in 60 mM KCl Ringer's solution. (c) Movements in 10^–5 M noradrenaline. (d) Movements in 10^–7 M forskolin. Aggregation and dispersion of melanosomes and leucosomes were normal in ci mutants. We also screened α-melanophore-stimulating hormone, melanophore-concentrating hormone, and melatonin, none of which caused abnormal aggregation or dispersion of leucosomes or melanosomes. (Scale bar, 0.1 mm.) (e) Cell size of scale chromatophores of background-adapted fish. Melanophore size increased or decreased in response to black or white backgrounds, respectively, in both the wild type (black) and ci (white). In contrast, leucophores decreased or increased in response to black or white backgrounds, respectively, in the wild type. Leucophores in the ci mutant did not decrease on a black background (asterisk). A similar result was obtained for cell numbers under these conditions (data not shown).

Fig. 1.

Appearance of wild-type and ci fish. (a) Lateral views of male adult wild-type (upper) and ci (lower) fish. (b and c) Larger magnification of body surfaces of wild-type (b) and ci (c) fish. Increased number and more dendritic shape of leucophores are observed. Fewer xanthophores are visible. No apparent differences are observed in melanophores. (d–g) When the scales are peeled off with forceps, epidermis and chromatophores are also removed. Dramatically increased number of scale leucophores could be observed in ci mutants (e) compared with wild type (d). Under the scales, scattered xanthophores in wild type (f) are mostly invisible in ci (g). (h) F₂ intercross siblings with phenotype of ci (upper) and wild type (lower). When the larvae reached this size (8 mm), they could be distinguished without the microscope because of their bright color. Note slightly more dendritic leucophores in ci. The phenotype appears even before this stage, although it is more difficult to recognize (data not shown). (Scale bar = 1cmin a; 0.5 mm in b–e; and 1 mm in f and g.)

Fig. 3.

Positional cloning of the ci gene. (a) A high-resolution recombination map around the ci locus on linkage group 13. The location of Fli was unclear in this map, although another map (constructed between Northern HNI and Southern AA2) elucidated its location between OLb01.12f and stEM31–6 (data not shown). Numbers under the map are the number of recombinations detected between neighboring markers (among 886 meioses). (b) BAC contig between the ci locus and OLa27.03d. The BAC 031N10 contains the ci mutation candidate region. (c) Diagram of a part of 031N10 insert sequences determined by shotgun sequencing. Assembly was disturbed by multiple copies of repetitive sequences (light blue), including reverse-transcriptase-, transposase-, and small interspersed nuclear element-like sequences (dark blue). Only one ORF (pink) was identified by blastx analyses. (d) Higher magnification of the contig, including the ci candidate gene, somatolactin. Exons are indicated by pink. (e) Partial sequences of the fourth exon of somatolactin (Upper, wild type; Lower, ci). The ci mutation (deleted 11 bases) is boxed and its position in the ci allele is indicated by an arrowhead. A newly created in-frame stop codon caused by the frameshift is underlined.

Fig. 4.

The medaka somatolactin and phylogenetic analyses with related proteins. (a) Genomic sequence of the medaka ci locus. Red indicates five exons of somatolactin identified by 3′ and 5′ RACEs. Splice donor and acceptor consensus sequences are underlined. Light blue shows the two putative poly(A) signal sequences (see text). Dark blue shows the deleted 11 bases in the ci allele. Translation initiation and stop codons are boxed. Putative TATA box is indicated by green. (b) The amino acid sequence of medaka somatolactin and its alignment to those of other fish. Residues conserved among the majority of them are shown in red. N-glycosylation sequences are shown by light blue, and seven cysteine residues relatively conserved among the somatolactins are indicated by dark blue. Boxed residues are deleted and substituted with seven different amino acids (EQDQVGV) in medaka ci. (c) The phylogenetic relationship among growth hormone (GH), prolactin (PL), and somatolactin (SL) is redrawn according to the result from the clustalw software. Only fish from which these three hormone sequences are available on the GenBank database are shown, except for medaka and lungfish. Fish somatolactins create a distinct clade to other GH-PL members in higher vertebrates (including placental lactogens and somatostatins, data not shown). Note the similar phylogenetic relationship in fish between growth hormone and prolactin, but not with somatolactin.

Fig. 5.

Expressional analyses of somatolactin mRNA. (a) Expression of the medaka somatolactin mRNA in adult organs. (Upper) Diagram of mRNA. The broad line shows translated regions and the vertical lines show boundaries between exons. Arrows show positions of primers. (Lower) Strong expression was detected only in brain, including pituitary; much weaker expression was detected in gonads and embryos (data not shown). (b) Somatolactin RT-PCR using brain cDNAs of Northern inbred (HNI), Southern inbred (Hd-rR), and ci. No product was detectable in ci. Identical primers to those in a were used. (c) Genomic and RT-PCR using reverse primers designed at two putative poly(A) signal sequences (Fig. 4a). Their forward primers (black) are identical. The reverse primer at the first poly(A) signal (black) amplified products of the expected length from both genomic DNA (g) and brain cDNA (c), but the reverse primer at the second poly(A) signal (gray) amplified only from genomic DNA, indicating few, if any, longer transcripts in medaka. (d) Somatolactin transcription during background adaptation. Identical primers to those shown in a were used. Wild-type fish were adapted to black or white backgrounds for 0, 1, 2, 3, 6, and 10 days, as indicated on top. The total RNAs in brain were quantified by spectrophotometry and gel electrophoreses, and RT-PCR was performed for somatolactin and β-actin using the same amount of template. Although β-actin band intensity did not differ among these fish, somatolactin transcripts gradually and markedly decreased during white-background adaptation. No obvious change was observed during black-background adaptation.