Interspecies Difference in the Regulation of Melanocyte Development by SOX10 and MITF

Interspecies difference in the regulation of melanocyte development by SOX10 and MITF Ling Hou*†, Heinz Arnheiter‡, and William J. Pavan*† *Genetic Disease Research Branch, National Human Genome Research Institute, and ‡Mammalian Development Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-4472 Communicated by Francis S. Collins, National Institutes of Health, Bethesda, MD, April 17, 2006 (received for review March 10, 2006) There is increasing indication that interspecific phenotypic differ- gous for a targeted mutation in Sox10 (12). In humans, SOX10 ences result from variations in gene-regulatory interactions. Here mutations are associated with Waardenburg–Hirschsprung dis- we provide evidence that mice differ from zebrafish in the way ease [Online Mendelian Inheritance in Man (OMIM) accession they use homologous key components to regulate pigment cell no. 277580] (13) and a complex neurocristopathy that combines differentiation. In both zebrafish and mice, one transcription peripheral demyelinating neuropathy, central leukodystrophy, factor, SOX10, controls the expression of another, MITF (microph- Waardenburg syndrome, and Hirschprung disease (PCWH, thalmia-associated transcription factor), which in turn regulates a OMIM accession no. 609136) (14). MITF, on the other hand, is set of genes critical for pigment cell development and pigmenta- a basic helix–loop–helix leucine zipper transcription factor tion. Mutations in either Sox10 or Mitf impair pigment cell devel- whose mutations lead to pigmentary abnormalities and, depend- opment. In Sox10-mutant zebrafish, experimentally induced ex- ing on the species, deafness and eye abnormalities but none of pression of Mitf fully rescues pigmentation. Using lineage-directed the intestinal or demyelinating and dysmyelinating symptoms gene transfer, we show that, in the mouse, Mitf can rescue seen with Sox10 mutations (6, 15). Mitf is mutated in nacre Sox10-mutant precursor cells only partially. In fact, retrovirally zebrafish (9), in microphthalmia mice (6, 15), and in humans with mediated, Sox10-independent Mitf expression in mouse melano- Waardenburg syndrome type 2A (OMIM accession no. 193510) blasts leads to cell survival and expression of a number of pigment (16) and Tietz syndrome (OMIM accession no. 103500) (17). biosynthetic genes but does not lead to expression of tyrosinase, BIOLOGY During pigment cell development, both SOX10 and MITF act the rate-limiting pigment gene which critically depends on both DEVELOPMENTAL cell-autonomously because they are normally expressed in pig- Sox10 and Mitf. Hence, compared with fish, mice have evolved a ment cell precursors (6, 7, 10, 18–21), can rescue the respective regulation of tyrosinase expression that includes feed-forward loops between Sox10 and tyrosinase regulatory regions. The mutant pigment cells after gene transfer (this report), and are results may help to explain how some embryos, such as zebrafish, functionally linked in a common pathway. In fact, in vitro, SOX10 can achieve rapid pigmentation after fertilization, whereas others, regulates, in cooperation with other factors, the expression of such as mice, become pigmented only several days after birth. Mitf through binding sites in the melanocyte-specific M pro- moter of Mitf (22–25). Interestingly, in zebrafish, Sox10 is rapidly neural crest ͉ pigmentation ͉ transcription ͉ cell fate ͉ neurocristopathy down-regulated in pigment cell precursors when they start migrating from the neural crest (10, 26). In contrast, in mice, Sox10 expression lasts much longer in developing melanocytes igmentation patterns of the skin and its appendages such as (called melanoblasts) and persists during the cells’ migration and Pfeathers and hairs have always served as convenient markers for the distinction of one species from another. Still, relatively homing into skin (27, 28). little is known about the mechanisms that control this species- The finding of spatial-temporal differences in the expression specific pigmentation from its earliest manifestation during patterns of Sox10 between zebrafish and mice may suggest that development to the mature pattern of the adult. Given that there are species-specific differences in the regulatory link transcription factors function as critical regulators of cell lineage between Sox10 and Mitf. Recently, it has been shown that determination and development, it is not surprising that a experimentally induced expression of Mitf can fully rescue number of distinct transcription factors have also been found to melanocyte development and pigmentation in Sox10 mutant regulate the development of pigment cells (1–4). SOX10 and zebrafish (26). This finding indicates that the major if not only MITF (microphthalmia-associated transcription factor), for in- function of Sox10 in zebrafish melanocyte development is the stance, are both DNA-binding proteins involved in the devel- induction of Mitf. Here, we find that melanocyte development in opment of vertebrate melanocytes, which are pigment-bearing mice does not follow a similarly simple pathway. Using a recently cells that are generated in the neural crest and migrate to various designed method of lineage-directed gene transfer in cultured destinations including skin, eye, and inner organs (5). In fact, if neural crest cells (29), we show that in contrast to zebrafish, either SOX10 or MITF is missing, the pigment lineage is aborted MITF can rescue survival and partial differentiation but not full (6–10). Conceivably, subtle interspecific differences in the func- pigmentation of Sox10-deficient mouse melanoblasts. Our find- tions of these two factors might influence the spatial-temporal ings indicate that the regulatory circuits that link Sox10, Mitf, and development of melanocytes and hence determine adult specific downstream pigmentation genes differ between ze- pigmentation. brafish and mice and may hence explain why the developmental SOX10 is a member of the SRY-box containing high mobility onset of pigmentation is distinct in these two species. group DNA-binding proteins (11). Its role in melanocyte development is evident from the effects of spontaneous and induced mutations. In zebrafish, for instance, several alleles of colorless Conflict of interest statement: No conflicts declared. are due to mutations in Sox10 (10). In mice, a spontaneous, Abbreviations: En, embryonic day n; RCAS, replication-competent avian sarcoma-leukosis truncating mutation in Sox10 is found in Dominant megacolon (7, virus long terminal repeat with splice acceptor; TVA, gene product of tv-a, encoding the receptor for subgroup A avian leucosis virus-derived retrovirus; HA, hemagglutinin; DCT, 8). Heterozygotes with this mutation can have white belly spots dopachrome tautomerase; TYR, tyrosinase. and always show a loss of neural crest-derived enteric ganglia, †To whom correspondence may be addressed at: National Human Genome Research which severely reduces the motility of the large intestine (7). A Institute, National Institutes of Health, 49 Convent Drive, Building 49͞Room 4A82, similar pigmentation͞colon phenotype is seen in mice heterozy- Bethesda, MD 20892. E-mail: [email protected] or [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603114103 PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 ͉ 9081–9085 Downloaded by guest on October 1, 2021 nance of Sox10 expression, we also tested cultures from embryos heterozygous or homozygous for a functional null allele of Mitf, Mitf mi-ew, whose protein product lacks part of the basic domain (15) and accumulates poorly in mutant cells (18, 21). In fact, SOX10 protein was readily seen in cultures from Mitf mi-ew͞ϩ control embryos (Fig. 1C) as well as in cultures from Mitf mi-ew/mi-ew embryos (Fig. 1D). Thus, the genetic hierarchy between Sox10 and Mitf, suggested from in vivo observations and molecular analyses (12, 20, 23, 24, 26), was maintained in primary cultures, and cells positive for ␤-gal (representing Sox10 expression) or endogenous SOX10 survived at least initially despite the absence of either functional SOX10 or MITF. Sox10 Acts Through Mitf to Induce Downstream Target Genes. On the basis of the above experiments and recent results in zebrafish Fig. 1. Expression of MITF in primary neural crest cell cultures depends on (26), we reasoned that deliberate expression of SOX10 in Sox10, but SOX10 expression does not depend on Mitf. Cultures were estab- melanocyte precursors should rescue Mitf expression and pig- lished from E9.5 embryos of the indicated genotypes and stained for SOX10 mentation in Sox10lacZ/lacZ cells and that expression of MITF and MITF expression 2 days later. ␤-gal͞MITF double-positive cells in alone should likewise fully rescue pigment cell development. To lacZ͞ϩ ͞ Sox10 cultures are shown (white arrows in A), and SOX10 MITF double- achieve lineage-directed gene transfer, we used a method de- mi-ew͞ϩ positive cells in Mitf cultures are shown (white arrows in C). In contrast, scribed in ref. 29 that is based on the RCAS (replication- Sox10lacZ/lacZ cultures show only ␤-gal- but not MITF-labeled cells (white arrows in B), whereas Mitfmi-ew/mi-ew cultures show SOX10- but not MITF-labeled competent avian sarcoma-leukosis virus long terminal repeat cells (D). with splice acceptor) system (31) and is composed of two principal components. First, the receptor for the virus RCAS, TVA (receptor for subgroup A Rous sarcoma virus), is ex- Results pressed from a transgene under the control of regulatory regions

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