SOX10 mutations in patients with Waardenburg-Hirschsprung disease.

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) and Hirschsprung's disease (HSCR; aganglionic megacolon) are congenital disorders caused by defective function of the embryonic neural crest. WS and HSCR are associated in patients with Waardenburg-Shah syndrome (WS4), whose symptoms are reminiscent of the white coat-spotting and aganglionic megacolon displayed by the mouse mutants Dom (Dominant megacolon), piebald-lethal (sl) and lethal spotting (ls). The sl and ls phenotypes are caused by mutations in the genes encoding the Endothelin-B receptor (Ednrb) and Endothelin 3 (Edn3), respectively. The identification of Sox10 as the gene mutated in Dom mice (B.H. et al., manuscript submitted) prompted us to analyse the role of its human homologue SOX10 in neural crest defects. Here we show that patients from four families with WS4 have mutations in SOX10, whereas no mutation could be detected in patients with HSCR alone. These mutations are likely to result in haploinsufficiency of the SOX10 product. Our findings further define the locus heterogeneity of Waardenburg-Hirschsprung syndromes, and point to an essential role of SOX10 in the development of two neural crest-derived human cell lineages.

Protein zero gene expression is regulated by the glial transcription factor Sox10.

Myelinating glia express high levels of a unique set of genes which code for structural proteins of the myelin sheath. Few transcription factors have so far been implicated in the regulation of any myelin gene. Here we show that the protein zero (P(0)) gene, a myelin gene exclusively expressed in the Schwann cell lineage of the peripheral nervous system, is controlled in its expression by the high-mobility-group domain protein Sox10 both in tissue culture and in vivo. Induction of wild-type Sox10, but not of other transcription factors or Sox10 mutants, strongly increased endogenous P(0) expression in tissue culture. This activation was mediated by the P(0) promoter, which was stimulated by Sox10 in transient transfections. Detailed analyses revealed the involvement of a proximal and a distal promoter region. The distal region functioned only in conjunction with the proximal one and contained a single Sox consensus binding site, which accounted for most of its activity. In contrast, the proximal region mediated Sox10 responsiveness on its own. It contained multiple binding sites for Sox proteins, with two high-affinity sites being the most significant. P(0) expression also depended on Sox10 in vivo, as evident from the analysis of Schwann cell precursors in mouse embryos with Sox10 mutation at day 12.5 of embryogenesis. To our knowledge this is the most conclusive link to date between a glial transcription factor and cell-specific activation of myelin gene expression.

Development and degeneration of dorsal root ganglia in the absence of the HMG-domain transcription factor Sox10.

The HMG-domain transcription factor Sox10 is essential for the development of various neural crest derived lineages including glia and neurons of the peripheral nervous system (PNS). Within the PNS the most striking defect is the complete absence of glial differentiation whereas neurogenesis seemed initially normal. A degeneration of motoneurons and sensory neurons occurred later in development. The mechanism that leads to the dramatic effects on the neural crest derived cell lineages in the dorsal root ganglia (DRG), however, has not been examined up to now. Here, we provide a detailed analysis of proliferation and apoptosis in the DRG during the time of their generation and lineage segregation (between E 9.5 and E 11.5). We show that both increased apoptosis as well as decreased proliferation of neural crest cells contribute to the observed hypomorphism.

Expression of the SOX10 gene during human development.

SOX10, a new member of the SOX gene family, is a transcription factor defective in the Dom (Dominant megacolon) mouse and in the human Shah-Waardenburg syndrome. To help unravel its physiological role during human development, we studied SOX10 gene expression in embryonic, fetal, and adult human tissues by Northern blot and in situ hybridization. As in mice, the human SOX10 gene was essentially expressed in the neural crest derivatives that contribute to the formation of the peripheral nervous system, and in the adult central nervous system. Nevertheless, it was more widely expressed in humans than in rodents. The spatial and temporal pattern of SOX10 expression supports an important function in neural crest development.

Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling.

The transcription factor Sox10 is required for proper development of various neural crest-derived cell types. Several lineages including melanocytes, autonomic and enteric neurons, and all subtypes of peripheral glia are missing in mice homozygous for Sox10 mutations. Moreover, haploinsufficiency of Sox10 results in neural crest defects that cause Waardenburg/Hirschsprung disease in humans. We provide evidence that the cellular basis to these phenotypes is likely to be a requirement for Sox10 by neural crest stem cells before lineage segregation. Cell death is increased in undifferentiated, postmigratory neural crest cells that lack Sox10, suggesting a role of Sox10 in the survival of neural crest cells. This function is mediated by neuregulin, which acts as a survival signal for postmigratory neural crest cells in a Sox10-dependent manner. Furthermore, Sox10 is required for glial fate acquisition, as the surviving mutant neural crest cells are unable to adopt a glial fate when challenged with different gliogenic conditions. In Sox10 heterozygous mutant neural crest cells, survival appears to be normal, while fate specifications are drastically affected. Thereby, the fate chosen by a mutant neural crest cell is context dependent. Our data indicate that combinatorial signaling by Sox10, extracellular factors such as neuregulin 1, and local cell-cell interactions is involved in fine-tuning lineage decisions by neural crest stem cells. Failures in fate decision processes might thus contribute to the etiology of Waardenburg/Hirschsprung disease.

Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome.

Waardenburg syndrome (WS) is an autosomal dominant disorder with an incidence of 1 in 40 000 that manifests with sensorineural deafness and pigmentation defects. It is classified into four types depending on the presence or absence of additional symptoms. WS1 and WS3 are due to mutations in the PAX3 gene whereas some WS2 cases are associated with mutations in the microphthalmia-associated transcription factor (MITF) gene. The WS4 phenotype can result from mutations in the endothelin-B receptor gene (EDNRB), in the gene for its ligand, endothelin-3 (EDN3), or in the SOX10 gene. PAX3 has been shown to regulate MITF gene expression. The recent implication of SOX10 in WS4 prompted us to test whether this transcription factor, known to cooperate in vitro with PAX3, is also able to regulate expression from the MITF promoter. Here we show that SOX10, in synergy with PAX3, strongly activates MITF expression in transfection assays. Analyses revealed that PAX3 and SOX10 interact directly by binding to a proximal region of the MITF promoter containing binding sites for both factors. Moreover, SOX10 or PAX3 mutant proteins fail to transactivate this promoter, providing further evidence that the two genes act in concert to directly regulate expression of MITF. In situ hybridization experiments carried out in the dominant megacolon (DOM:) mouse, confirmed that SOX10 dysfunction impairs MITF: expression as well as melanocytic development and survival. These experiments, which demonstrate an interaction between three of the genes that are altered in WS, could explain the auditory-pigmentary symptoms of this disease.

The transcription factor Sox10 is a key regulator of peripheral glial development.

The molecular mechanisms that determine glial cell fate in the vertebrate nervous system have not been elucidated. Peripheral glial cells differentiate from pluripotent neural crest cells. We show here that the transcription factor Sox10 is a key regulator in differentiation of peripheral glial cells. In mice that carry a spontaneous or a targeted mutation of Sox10, neuronal cells form in dorsal root ganglia, but Schwann cells or satellite cells are not generated. At later developmental stages, this lack of peripheral glial cells results in a severe degeneration of sensory and motor neurons. Moreover, we show that Sox10 controls expression of ErbB3 in neural crest cells. ErbB3 encodes a Neuregulin receptor, and down-regulation of ErbB3 accounts for many changes in development of neural crest cells observed in Sox10 mutant mice. Sox10 also has functions not mediated by ErbB3, for instance in the melanocyte lineage. Phenotypes observed in heterozygous mice that carry a targeted Sox10 null allele reproduce those observed in heterozygous Sox10(Dom) mice. Haploinsufficiency of Sox10 can thus cause pigmentation and megacolon defects, which are also observed in Sox10(Dom)/+ mice and in patients with Waardenburg-Hirschsprung disease caused by heterozygous SOX10 mutations.