HOXB5 cooperates with NKX2-1 in the transcription of human RET.
ABSTRACT: The enteric nervous system (ENS) regulates peristaltic movement of the gut, and abnormal ENS causes Hirschsprung's disease (HSCR) in newborns. HSCR is a congenital complex genetic disorder characterised by a lack of enteric ganglia along a variable length of the intestine. The receptor tyrosine kinase gene (RET) is the major HSCR gene and its expression is crucial for ENS development. We have previously reported that (i) HOXB5 transcription factor mediates RET expression, and (ii) mouse with defective HOXB5 activity develop HSCR phenotype. In this study, we (i) elucidate the underlying mechanisms that HOXB5 mediate RET expression, and (ii) examine the interactions between HOXB5 and other transcription factors implicated in RET expression. We show that human HOXB5 binds to the promoter region 5' upstream of the binding site of NKX2-1 and regulates RET expression. HOXB5 and NKX2-1 form a protein complex and mediate RET expression in a synergistic manner. HSCR associated SNPs at the NKX2-1 binding site (-5G>A rs10900296; -1A>C rs10900297), which reduce NKX2-1 binding, abolish the synergistic trans-activation of RET by HOXB5 and NKX2-1. In contrast to the synergistic activation of RET with NKX2-1, HOXB5 cooperates in an additive manner with SOX10, PAX3 and PHOX2B in trans-activation of RET promoter. Taken together, our data suggests that HOXB5 in coordination with other transcription factors mediates RET expression. Therefore, defects in cis- or trans-regulation of RET by HOXB5 could lead to reduction of RET expression and contribute to the manifestation of the HSCR phenotype.
Project description:Neural crest cells (NCCs) migrate from different regions along the anterior-posterior axis of the neural tube (NT) to form different structures. Defective NCC development causes congenital neurocristopathies affecting multiple NCC-derived tissues in human. Perturbed Hoxb5 signaling in vagal NCC causes enteric nervous system (ENS) defects. This study aims to further investigate if perturbed Hoxb5 signaling in trunk NCC contributes to defects of other NCC-derived tissues besides the ENS. We perturbed Hoxb5 signaling in NCC from the entire NT, and investigated its impact in the development of tissues derived from these cells in mice. Perturbation of Hoxb5 signaling in these NCC resulted in Sox9 downregulation, NCC apoptosis, hypoplastic sympathetic and dorsal root ganglia, hypopigmentation and ENS defects. Mutant mice with NCC-specific Sox9 deletion also displayed some of these phenotypes. In vitro and in vivo assays indicated that the Sox9 promoter was bound and trans-activated by Hoxb5. In ovo studies further revealed that Sox9 alleviated apoptosis induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Sox9 expression in chick NT. This study demonstrates that Hoxb5 regulates Sox9 expression in NCC and disruption of this signaling causes Sox9 downregulation, NCC apoptosis and multiple NCC-developmental defects. Phenotypes such as ENS deficiency, hypopigmentation and some of the neurological defects are reported in patients with Hirschsprung disease (HSCR). Whether dysregulation of Hoxb5 signaling and early depletion of NCC contribute to ENS defect and other neurocristopathies in HSCR patients deserves further investigation.
Project description:The Hirschsprung disease (HSCR) is a complex congenital disorder, arising from abnormalities in enteric nervous system (ENS) development. There is a gender disparity among the patients, with the male to female ratio as high as 5 : 1. Loss-of-function mutations of HSCR genes and haploinsufficiency of their gene products are the primary pathogenic mechanisms for disease development. Recent studies identified over half of the HSCR disease susceptibility genes as targets for the sex-determining factor SRY, suggesting that this Y-encoded transcription factor could be involved in sexual dimorphism in HSCR. Among the SRY targets, the tyrosine kinase receptor RET represents the most important disease gene, whose mutations account for half of the familial and up to one-third of the sporadic forms of HSCR. RET is regulated by a distal and a proximal enhancer at its promoter, in which PAX3 and NKX2-1 are the resident transcription factors respectively. We show that the SRY-box 10 (SOX10) co-activator interacts and forms transcriptional complexes with PAX3 and NKX2-1 in a sequence-independent manner and exacerbates their respective transactivation activities on the RET promoter. SRY competitively displaces SOX10 in such transcription complexes and represses their regulatory functions on RET. Hence SRY could be a Y-located negative modifier of RET expression; and if it is ectopically expressed during ENS development, such SRY repression could result in RET protein haploinsufficiency and promotion of HSCR development, thereby contributing to sexual dimorphism in HSCR.
Project description:Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.
Project description:Hirschsprung's disease (HSCR) results from failed colonization of the embryonic gut by enteric neural crest cells (ENCCs); colonization requires RET proto-oncogene (RET) signaling. We sequenced RET to identify coding and splice-site variants in a population-based case group and we tested for associations between HSCR and common variants in RET and candidate genes (ASCL1, homeobox B5 (HOXB5), L1 cell adhesion molecule (L1CAM), paired-like homeobox 2b (PHOX2B), PROK1 and PROKR1) chosen because they are involved in ENCC proliferation, migration and differentiation in animal models. We conducted a nested case-control study of 304 HSCR cases and 1215 controls. Among 38 (12.5%) cases with 34 RET coding and splice-site variants, 18 variants were previously unreported. We confirmed associations with common variants in HOXB5 and PHOX2B but the associations with variants in ASCL1, L1CAM and PROK1 were not significant after multiple comparisons adjustment. RET variants were strongly associated with HSCR (P-values between 10(-3) and 10(-31)) but this differed by race/ethnicity: associations were absent in African-Americans. Our population-based study not only identified novel RET variants in HSCR cases, it showed that common RET variants may not contribute to HSCR in all race/ethnic groups. The findings for HOXB5 and PHOX2B provide supportive evidence that genes regulating ENCC proliferation, migration and differentiation could be risk factors for HSCR.
Project description:The enteric nervous system (ENS) is composed of neurons and glial cells, organized as interconnected ganglia within the gut wall, which controls peristalsis of the gut wall and secretions from its glands. The Ret receptor tyrosine kinase is expressed throughout enteric neurogenesis and is required for normal ENS development; humans with mutations in the RET locus have Hirschsprung disease (HSCR, an absence of ganglia in the colon), and mice lacking Ret have total intestinal aganglionosis. The Ret mutant mouse provides a tool for identifying genes implicated in development of the ENS. By using RNA from WT and Ret mutant (aganglionic) gut tissue and DNA microarrays, we have conducted a differential screen for ENS-expressed genes and have identified hundreds of candidate ENS-expressed genes. Forty-seven genes were selected for further analysis, representing diverse functional classes. We show that all of the analyzed genes are expressed in the ENS and that the screen was sensitive enough to identify genes marking only subpopulations of ENS cells. Our screen, therefore, was reliable and sensitive and has identified many previously undescribed genes for studying ENS development. Moreover, two of the genes identified in our screen Arhgef3 and Ctnnal1, have human homologues that map to previously identified HSCR susceptibility loci, thus representing excellent candidates for HSCR genes. This comprehensive profile of ENS gene expression refines our understanding of ENS development and serves as a resource for future developmental, biochemical, and human genetic studies.
Project description:Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in ret heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in ret mutants, and partially impaired in ret heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant ret heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated mapk10 loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of mapk10 mutations into ret heterozygotes enhanced the ENS deficit, supporting MAPK10 as a HSCR susceptibility locus. Our studies demonstrate that ret heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.
Project description:BACKGROUND: The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. PRINCIPAL FINDINGS: In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. CONCLUSIONS: Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFR?1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.
Project description:Disruptions in gene regulatory networks (GRNs), driven by multiple deleterious variants, potentially underlie complex traits and diseases. Hirschsprung disease (HSCR), a multifactorial disorder of enteric nervous system (ENS) development, is associated with at least 24 genes and seven chromosomal loci, with RET and EDNRB as its major genes. We previously demonstrated that RET transcription in the ENS is controlled by an extensive GRN involving the transcription factors (TFs) RARB, GATA2 and SOX10 and other HSCR genes. We now demonstrate, using human and mouse cellular and animal models, that EDNRB is transcriptionally regulated in the ENS by GATA2, SOX10 and NKX2.5 TFs. Significantly, RET and EDNRB expression is regulated by their shared use of GATA2 and SOX10, and in turn, these TFs are controlled by EDNRB and RET in a dose-dependent manner. This study expands the ENS development GRN to include both RET and EDNRB, uncovers the mechanistic basis for RET-EDNRB epistasis and emphasizes how functionally different genes associated with a complex disorder can be united through a common GRN.
Project description:Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses-often de novo-contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease.
Project description:In rare families RET tyrosine kinase receptor substitutions located in exon 10 (especially at positions 609, 618, and 620) can concomitantly cause the MEN 2A (multiple endocrine neoplasia type 2A) or FMTC (familial medullary thyroid carcinoma) cancer syndromes, and Hirschsprung's disease (HSCR). No animal model mimicking the co-existence of the MEN 2 pathology and HSCR is available, and the association of these activating mutations with a developmental defect still represents an unresolved problem. The aim of this work was to investigate the significance of the RET(C620R) substitution in the pathogenesis of both gain- and loss-of-function RET-associated diseases. We report the generation of a line of mice carrying the C620R mutation in the Ret gene. Although Ret(C620R) homozygotes display severe defects in kidney organogenesis and enteric nervous system development leading to perinatal lethality. Ret(C620R) heterozygotes recapitulate features characteristic of HSCR including hypoganglionosis of the gastrointestinal tract. Surprisingly, heterozygotes do not show any defects in the thyroid that might be attributable to a gain-of-function mutation. The Ret(C620R) allele is responsible for HSCR and affects the development of kidneys and the enteric nervous system (ENS). These mice represent an interesting model for studying new therapeutic approaches for the treatment of HSCR disease.