Analysis of SOX10 function in neural crest-derived melanocyte development: SOX10-dependent transcriptional control of dopachrome tautomerase.

SOX10 is a high-mobility-group transcription factor that plays a critical role in the development of neural crest-derived melanocytes. At E11.5, mouse embryos homozygous for the Sox10(Dom) mutation entirely lack neural crest-derived cells expressing the lineage marker KIT, MITF, or DCT. Moreover, neural crest cell cultures derived from homozygous embryos do not give rise to pigmented cells. In contrast, in Sox10(Dom) heterozygous embryos, melanoblasts expressing KIT and MITF do occur, albeit in reduced numbers, and pigmented cells eventually develop in nearly normal numbers both in culture and in vivo. Intriguingly, however, Sox10(Dom)/+ melanoblasts transiently lack Dct expression both in culture and in vivo, suggesting that during a critical developmental period SOX10 may serve as a transcriptional activator of Dct. Indeed, we found that SOX10 and DCT colocalized in early melanoblasts and that SOX10 is capable of transactivating the Dct promoter in vitro. Our data suggest that during early melanoblast development SOX10 acts as a critical transactivator of Dct, that MITF, on its own, is insufficient to stimulate Dct expression, and that delayed onset of Dct expression is not deleterious to the melanocyte lineage.

The Sox10(Dom) mouse: modeling the genetic variation of Waardenburg-Shah (WS4) syndrome.

Hirschsprung disease (HSCR) is a multigenic neurocristopathy clinically recognized by aganglionosis of the distal gastrointestinal tract. Patients presenting with aganglionosis in association with hypopigmentation are classified as Waardenburg syndrome type 4 (Waardenburg-Shah, WS4). Variability in the disease phenotype of WS4 patients with equivalent mutations suggests the influence of genetic modifier loci in this disorder. Sox10(Dom)/+ mice exhibit variability of aganglionosis and hypopigmentation influenced by genetic background similar to that observed in WS4 patients. We have constructed Sox10(Dom)/+ congenic lines to segregate loci that modify the neural crest defects in these mice. Consistent with previous studies, increased lethality of Sox10(Dom)/+ animals resulted from a C57BL/6J locus(i). However, we also observed an increase in hypopigmentation in conjunction with a C3HeB/FeJLe-a/a locus(i). Linkage analysis localized a hypopigmentation modifier of the Dom phenotype to mouse chromosome 10 in close proximity to a previously reported modifier of hypopigmentation for the endothelin receptor B mouse model of WS4. To evaluate further the role of SOX10 in development and disease, we have performed comparative genomic analyses. An essential role for this gene in neural crest development is supported by zoo blot hybridizations that reveal extensive conservation throughout vertebrate evolution and by similar Northern blot expression profiles between mouse and man. Comparative sequence analysis of the mouse and human SOX10 gene have defined the exon-intron boundaries of SOX10 and facilitated mutation analysis leading to the identification of two new SOX10 mutations in individuals with WS4. Structural analysis of the HMG DNA-binding domain was performed to evaluate the effect of human mutations in this region.

Sox10 mutation disrupts neural crest development in Dom Hirschsprung mouse model.

Hirschsprung disease (HSCR, MIM #142623) is a multigenic neurocristopathy (neural crest disorder) characterized by absence of enteric ganglia in a variable portion of the distal colon. Subsets of HSCR individuals also present with neural crest-derived melanocyte deficiencies (Hirschsprung-Waardenburg, HSCR-WS, MIM #277580). Murine models have been instrumental in the identification and analysis of HSCR disease genes. These include mice with deficiencies of endothelin B receptor (Ednrb(s-l); refs 1,2) endothelin 3 (Edn3(ls): refs 1,3) the tyrosine kinase receptor cRet and glial-derived neurotrophic factor. Another mouse model of HSCR disease, Dom, arose spontaneously at the Jackson Laboratory. While Dom/+ heterozygous mice display regional deficiencies of neural crest-derived enteric ganglia in the distal colon, Dom/Dom homozygous animals are embryonic lethal. We have determined that premature termination of Sox10, a member of the SRY-like HMG box family of transcription factors, is responsible for absence of the neural crest derivatives in Dom mice. We demonstrate expression of Sox10 in normal neural crest cells, disrupted expression of both Sox10 and the HSCR disease gene Ednrb in Dom mutant embryos, and loss of neural crest derivatives due to apoptosis. Our studies suggest that Sox10 is essential for proper peripheral nervous system development. We propose SOX10 as a candidate disease gene for individuals with HSCR whose disease does not have an identified genetic origin.

Coordinate control and variation in X-linked gene expression among female mice.

In normal female mammals, one of the two X Chromosome (Chr) homologs per cell is silenced coordinately during early embryogenesis. The genes located on the inactivated X homolog are predicted to be influenced by the same underlying repression mechanism. To test the uniformity of cis-acting gene repression, 32 genetically identical F1 female mice were analyzed for differential expression of homologous alleles at three X-linked genes-Otc, Atp7a (= Mottled), and Hprt. Gene expression was assayed by the single-nucleotide primer extension (SNuPE) method, thereby allowing the three genes to be quantitated from the same RNA sample. Although variable between individual animals, the relative expression of the two alleles (allelic expression ratio) of the genes is significantly correlated within each steady-state RNA pool. When examined by animal age (3 months to 12 months), no statistically significant differences were observed in the mean or variance of allelic expression ratio. Together, the results confirm that X inactivation is coordinately controlled and is stable across the early- to mid-adult life span.

Disparate tissue-specific expression of members of the tissue kallikrein multigene family of the rat.

To understand the regulatory diversity of the rat family of linked kallikrein genes, we have assayed the expression of family members in 20 major organs. Reverse transcription-polymerase chain reaction analysis using primers and hybridization probes specific for each of the 10 expressed kallikrein genes showed that no two family members share the same organ-specific pattern of expression. The only common site of expression for all 10 known active genes is the submandibular gland. The presence of the mRNA for at least one family member is detected in 19 of these 20 organs (liver excepted), from as few as three organs to as many as 18 for individual family members. For individual genes there can be more than a 10(5)-fold variation in mRNA levels among organs, from a limit of detection of slightly less than 1 mRNA molecule/10 cells to more than 10,000 mRNA molecules/cell. Despite high sequence conservation and close linkage, the members of this family are expressed in very different and complex patterns. A gradient of diversity of expression corresponds to the order of the genes within the kallikrein family locus.