Search

Basic helix-loop-helix (bHLH) transcription factors control developmental decisions in a wide range of embryonic cell types. The HLH motif mediates homo- and heterodimerization, which juxtaposes the basic regions within the dimeric complex to form a bipartite DNA binding domain that recognizes a DNA consensus sequence known as an E-box. eHAND and dHAND (also known as HAND1 and HAND2) are closely related bHLH proteins that control cardiac, craniofacial and limb development. Within the developing limb, dHAND expression encompasses the zone of polarizing activity in the posterior region, where it has been shown to be necessary and sufficient to induce the expression of the morphogen sonic hedgehog. Misexpression of dHAND in the anterior compartment of the limb bud induces ectopic expression of sonic hedgehog, with resulting preaxial polydactyly and mirror image duplications of posterior digits. To investigate the potential transcriptional mechanisms involved in limb patterning by dHAND, we have performed a structure-function analysis of the protein in cultured cells and ectopically expressed dHAND mutant proteins in the developing limbs of transgenic mice. We show that an N-terminal transcriptional activation domain, and the bHLH region, are required for E-box-dependent transcription in vitro. Remarkably, however, digit duplication by dHAND requires neither the transcriptional activation domain nor the basic region, but only the HLH motif. eHAND has a similar limb patterning activity to dHAND in these misexpression experiments, indicating a conserved function of the HLH regions of these proteins. These findings suggest that dHAND may act via novel transcriptional mechanisms mediated by protein-protein interactions independent of direct DNA binding.

Neural crest cells play a key role in craniofacial development. The endothelin family of secreted polypeptides regulates development of several neural crest sublineages, including the branchial arch neural crest. The basic helix-loop-helix transcription factor dHAND is also required for craniofacial development, and in endothelin-1 (ET-1) mutant embryos, dHAND expression in the branchial arches is down-regulated, implicating it as a transcriptional effector of ET-1 action. To determine the mechanism that links ET-1 signaling to dHAND transcription, we analyzed the dHAND gene for cis-regulatory elements that control transcription in the branchial arches. We describe an evolutionarily conserved dHAND enhancer that requires ET-1 signaling for activity. This enhancer contains four homeodomain binding sites that are required for branchial arch expression. By comparing protein binding to these sites in branchial arch extracts from endothelin receptor A (EdnrA) mutant and wild-type mouse embryos, we identified Dlx6, a member of the Distal-less family of homeodomain proteins, as an ET-1-dependent binding factor. Consistent with this conclusion, Dlx6 was down-regulated in branchial arches from EdnrA mutant mice. These results suggest that Dlx6 acts as an intermediary between ET-1 signaling and dHAND transcription during craniofacial morphogenesis.

The Hairy-related transcription-factor (HRT) genes encode three related basic helix-loop-helix transcription factors that show sequence similarity to the Hairy and Enhancer of split family of transcriptional repressors. HRT proteins are expressed in specific regions of the developing heart, vasculature, pharyngeal arches and somites, and the periodicity of their expression in somitic precursors mirrors that of Notch signaling-related molecules. In the present study, we show that the intracellular domain of the Notch1 receptor (Notch1 IC), which is constitutively active, up-regulates HRT2 expression in 10T(1/2) fibroblasts. Luciferase reporter assays using the regulatory regions of the mouse HRT genes revealed that transcription of all three genes is stimulated by Notch1 IC. The promoters of the HRT genes share homology in a binding site for Suppressor of Hairless [Su(H)], a transcriptional mediator of Notch signaling. A dominant-negative Su(H) mutant abolished Notch-activated HRT2 expression, and mutation of the conserved Su(H) consensus site in the HRT2 promoter attenuated transcriptional activation by Notch. Ectopic expression of HRT proteins also blocked activation of HRT2 expression by Notch1 IC through a mechanism requiring the basic region, but not the conserved carboxyl-terminal YQPW-TEVGAF motif of HRT2. These findings identify HRT genes as downstream targets for Notch signaling and reveal a negative autoregulatory loop whereby HRT proteins repress their own expression through interference with Notch signaling.

Heart formation in vertebrates is believed to occur in a segmental fashion, with discreet populations of cardiac progenitors giving rise to different chambers of the heart. However, the mechanisms involved in specification of different chamber lineages are unclear. The basic helix-loop-helix transcription factor dHAND is expressed in cardiac precursors throughout the cardiac crescent and the linear heart tube, before becoming restricted to the right ventricular chamber at the onset of looping morphogenesis. dHAND is also expressed in the branchial arch neural crest, which contributes to craniofacial structures and the aortic arch arteries. Using a series of dHAND-lacZ reporter genes in transgenic mice, we show that cardiac and neural crest expression of dHAND are controlled by separate upstream enhancers and we describe a composite cardiac-specific enhancer that directs lacZ expression in a pattern that mimics that of the endogenous dHAND gene throughout heart development. Deletion analysis reduced this enhancer to a 1.5 kb region and identified subregions responsible for expression in the right ventricle and cardiac outflow tract. Comparison of mouse regulatory elements required for right ventricular expression to the human dHAND upstream sequence revealed two conserved consensus sites for binding of GATA transcription factors. Mutation of these sites abolished transgene expression in the right ventricle, identifying dHAND as a direct transcriptional target of GATA factors during right ventricle development. Since GATA factors are not chamber-restricted, these findings suggest the existence of positive and/or negative coregulators that cooperate with GATA factors to control right ventricular-specific gene expression in the developing heart.

Limb outgrowth and patterning of skeletal elements are dependent on complex tissue interactions involving the zone of polarizing activity (ZPA) in the posterior region of the limb bud and the apical ectodermal ridge. The peptide morphogen Sonic hedgehog (SHH) is expressed specifically in the ZPA and, when expressed ectopically, is sufficient to mimic its functions, inducing tissue growth and formation of posterior skeletal elements. We show that the basic helix-loop-helix transcription factor dHAND is expressed posteriorly in the developing limb prior to Shh and subsequently occupies a broad domain that encompasses the Shh expression domain. In mouse embryos homozygous for a dHAND null allele, limb buds are severely underdeveloped and Shh is not expressed. Conversely, misexpression of dHAND in the anterior region of the limb bud of transgenic mice results in formation of an additional ZPA, revealed by ectopic expression of Shh and its target genes, and resulting limb abnormalities that include preaxial polydactyly with duplication of posterior skeletal elements. Analysis of mouse mutants in which Hedgehog expression is altered also revealed a feedback mechanism in which Hedgehog signaling is required to maintain the full dHAND expression domain in the developing limb. Together, these findings identify dHAND as an upstream activator of Shh expression and important transcriptional regulator of limb development.

The basic helix-loop-helix (bHLH) transcription factors, Hand1 and Hand2 (refs 1,2), also called eHand/Hxt/Thing1 and dHand/Hed/Thing2 (refs 3,4), respectively, are expressed in the heart and certain neural-crest derivatives during embryogenesis. In addition, Hand1 is expressed in extraembryonic membranes, whereas Hand2 is expressed in the deciduum. Previous studies have demonstrated that Hand2 is required for formation of the right ventricle of the heart and the aortic arch arteries. We have generated a germline mutation in the mouse Hand1 gene by replacing the first coding exon with a beta-galactosidase reporter gene. Embryos homozygous for the Hand1 null allele died between embryonic days 8.5 and 9.5 and exhibited yolk sac abnormalities due to a deficiency in extraembryonic mesoderm. Heart development was also perturbed and did not progress beyond the cardiac-looping stage. Our results demonstrate important roles for Hand1 in extraembryonic mesodermal and heart development.