Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in C. elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. Two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naive blastomeres to muscle when overproduced ectopically in the embryo. In addition, genetic null mutants were used to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type (Fukushige, 2006).
The extensive cell lineage information and streamlined genome of the ascidian, Ciona intestinalis has been exploited to investigate heart development in a basal chordate. Several cardiac genes were analyzed, including the sole Ciona ortholog of the Drosophila tinman gene, and tissue-specific enhancers were isolated for some of the genes. Conserved sequence motifs within these enhancers facilitated the isolation of a heart enhancer for the Ciona Hand-like gene. Altogether, these studies provide a regulatory framework for the differentiation of the cardiac mesoderm, beginning at the 110-cell stage, and extending through the fusion of cardiac progenitors during tail elongation. The cardiac lineage shares a common origin with the germ line, and zygotic transcription is first detected in the heart progenitors only after its separation from the germ line at the 64-cell stage. It is proposed that germ-line determinants influence the specification of the cardiac mesoderm, both by inhibiting inductive signals required for the development of noncardiac mesoderm lineages, and by providing a localized source of Wnt-5 and other signals required for heart development. The possiblility is discussed that the germ line also influences the specification of the vertebrate heart (Davidson, 2003).
dHAND and eHAND are related basic helix-loop-helix transcription factors that are expressed in the cardiac mesoderm and in numerous neural crest-derived cell types in chick and mouse. To better understand the evolutionary development of overlapping expression and function of the HAND genes during embryogenesis, the zebrafish and Xenopus orthologues were cloned. Comparison of dHAND sequences in zebrafish, Xenopus, chick, mouse and human demonstrated conservation throughout the protein. Expression of dHAND in zebrafish is seen in the earliest precursors of all lateral mesoderm at early gastrulation stages. At neurula and later stages, dHAND expression is observed in lateral precardiac mesoderm, branchial arch neural crest derivatives and posterior lateral mesoderm. At looping heart stages, cardiac dHAND expression remains generalized with no apparent regionalization. Interestingly, no eHAND orthologue was found in zebrafish. In Xenopus, dHAND and eHAND are co-expressed in the cardiac mesoderm without the segmental restriction seen in mice. Xenopus dHAND and eHAND are also expressed bilaterally in the lateral mesoderm without any left-right asymmetry. Within the branchial arches, XdHAND is expressed in a broader domain than XeHAND, similar to their mouse counterparts. Together, these data demonstrate conservation of HAND structure and expression across species (Angelo, 2000).
The precursors of several organs reside within the lateral plate mesoderm of vertebrate embryos. The zebrafish hands off locus is essential for the development of two structures derived from the lateral plate mesoderm -- the heart and the pectoral fin. hands off mutant embryos have defects in myocardial development from an early stage: they produce a reduced number of myocardial precursors, and the myocardial tissue that does form is improperly patterned and fails to maintain tbx5 expression. A similar array of defects is observed in the differentiation of the pectoral fin mesenchyme: small fin buds form in a delayed fashion, anteroposterior patterning of the fin mesenchyme is absent and tbx5 expression is poorly maintained. Defects in these mesodermal structures are preceded by the aberrant morphogenesis of both the cardiogenic and forelimb-forming regions of the lateral plate mesoderm. Molecular analysis of two hands off alleles indicates that the hands off locus encodes the bHLH transcription factor Hand2, which is expressed in the lateral plate mesoderm starting at the completion of gastrulation. Thus, these studies reveal early functions for Hand2 in several cellular processes and highlight a genetic parallel between heart and forelimb development (Yelon, 2000).
The ventrally expressed secreted polypeptide endothelin1 (Edn1) patterns the skeleton derived from the first two pharyngeal arches into dorsal, intermediate and ventral domains. Edn1 activates expression of many genes, including hand2 and Dlx genes. It was of interest to know how hand2/Dlx genes might generate distinct domain identities. Differential expression of hand2 and Dlx genes was shown to delineate domain boundaries before and during cartilage morphogenesis. Knockdown of the broadly expressed genes dlx1a and dlx2a results in both dorsal and intermediate defects, whereas knockdown of three intermediate-domain restricted genes dlx3b, dlx4b and dlx5a results in intermediate-domain-specific defects. The ventrally expressed gene hand2 patterns ventral identity, in part by repressing dlx3b/4b/5a. The jaw joint is an intermediate-domain structure that expresses nkx3.2 and a more general joint marker, trps1. The jaw joint expression of trps1 and nkx3.2 requires dlx3b/4b/5a function, and expands in hand2 mutants. Both hand2 and dlx3b/4b/5a repress dorsal patterning markers. Collectively, this work indicates that the expression and function of hand2 and Dlx genes specify major patterning domains along the dorsoventral axis of zebrafish pharyngeal arches (Talbot, 2010).
Extracellular matrix (ECM) remodeling is critical for organogenesis, yet its molecular regulation is poorly understood. In zebrafish, asymmetric migration of the epithelial lateral plate mesoderm (LPM) displaces the gut leftward, allowing correct placement of the liver and pancreas. To observe LPM migration at cellular resolution, EGFP was transgenically expressed under the control of the regulatory sequences of the bHLH transcription factor gene hand2. Laminin was found to be distributed along the LPM/gut boundary during gut looping, and it appears to become diminished by the migrating hand2-expressing cells. Laminin diminishment is necessary for LPM migration and is dependent on matrix metalloproteinase (MMP) activity. Loss of Hand2 function causes reduced MMP activity and prolonged laminin deposition at the LPM/gut boundary, leading to failed asymmetric LPM migration and gut looping. This study reveals an unexpected role for Hand2, a key regulator of cell specification and differentiation, in modulating ECM remodeling during organogenesis (Yin, 2010).
Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors. Two bHLH genes, dHAND and eHAND, have now been isolated that are expressed in the bilateral heart primordia and subsequently throughout the primitive tubular heart and its derivatives during chick and mouse embryogenesis. Incubation of stage 8 chick embryos with dHAND and eHAND antisense oligonucleotides revealed that either oligonucleotide alone has no effect on embryonic development, whereas together they arrested development at the looping heart tube stage. Thus, dHAND and eHAND may play redundant roles in the regulation of the morphogenetic events of vertebrate heart development (Srivastava, 1995).
During embryonic development in amniotes, the extraembryonic mesoderm, where the earliest hematopoiesis and vasculogenesis take place, also generates smooth muscle cells (SMCs). It is not well understood how the differentiation of SMCs is linked to that of blood (BCs) and endothelial (ECs) cells. This study shows that, in the chick embryo, the SMC lineage is marked by the expression of a bHLH transcription factor, dHand. Notch activity in nascent ventral mesoderm cells promotes SMC progenitor formation and mediates the separation of SMC and BC/EC common progenitors marked by another bHLH factor, Scl. This is achieved by crosstalk with the BMP and Wnt pathways, which are involved in mesoderm ventralization and SMC lineage induction, respectively. These findings reveal a novel role of the Notch pathway in early ventral mesoderm differentiation, and suggest a stepwise separation among its three main lineages, first between SMC progenitors and BC/EC common progenitors, and then between BCs and ECs (Shin, 2009).
The precise function of the Notch pathway in the process of muscle and BC/EC lineage separation remains to be elucidated. The data suggest that, during chick ventral mesoderm differentiation, the Notch pathway acts together with the BMP and Wnt pathways, and that it plays a 'permissive', rather than an 'instructive', role in mediating the separation of SMCs and BC/ECs. The Notch pathway does not control the induction of but rather the balance between these two populations. Evidence is provided that the induction of these lineages is controlled by the activities of both the BMP pathway, as a general ventral mesoderm inducer, and the canonical Wnt pathway, as a strong SMC lineage inducer. Ectopic activation of the BMP pathway can induce both SMC and BC/EC lineages, with the balance of SMCs and BC/ECs being regulated by Notch activity. It is not clear whether the induction of SMCs by the BMP pathway is a direct or indirect process, or whether it requires an active Wnt pathway. In this analysis, a stronger and wider ectopic dHand induction was observed by CA-β-Catenin than by CA-ALK6 around the anterior primitive streak where BMP antagonists are highly expressed, suggesting that the induction of SMCs by the Wnt pathway does not require active BMP signaling. A recent in vitro study suggested that Notch activity promotes the degradation of Scl by facilitating its ubiquitination, and that this process requires the transcriptional regulation of Notch pathway activity through Suppressor of Hairless. Although there is no direct evidence in support of a similar phenomenon in the current system, it could in principle act as a possible mechanism for the Notch activity-mediated segregation of SMCs and BC/ECs. Furthermore, Nrarp (an ankyrin-repeat protein that is transcriptionally regulated by the Notch signaling pathway), in addition to serving as a Notch-activity readout and a feedback regulator of the Notch pathway, has also been shown to positively regulate the canonical Wnt pathway by blocking the ubiquitination and increasing the stability of Lef1 in zebrafish. This might also serve as a possible mechanism for the Notch and Wnt pathway-mediated SMC specification observed in this system (Shin, 2009).
The yeast two-hybrid technique was employed to screen a mouse embryo cDNA library for novel tissue-specific Class B basic helix-loop-helix (bHLH) transcription factors that heterodimerize with the ubiquitously expressed Class A bHLH protein E12. From this screen, a novel bHLH protein was cloned, which was named eHAND. Its low sequence identity with other bHLH family members and unique expression pattern during development suggest that eHAND defines a new subclass of Class B bHLH proteins. eHAND was expressed at high levels in trophoblast cells and extraembryonic membranes throughout development. The first site of eHAND expression in embryos is the heart, where it is expressed at high levels between 8.5 and 10.5 days post coitum, after which transcript levels declined abruptly. By 13.5 d.p.c., eHAND expression in the heart is localized to regions of valve formation. Expression in other regions of the embryo is confined to tissues with a substantial neural crest component. eHAND is expressed in the first branchial arch and its derivatives, in the sympathoadrenal lineage, and in the enteric systems. The expression pattern of eHAND during development is distinct from that of other bHLH genes and suggests that it has a role in formation of extraembryonic tissues, heart, and neural crest derivatives (Cserjesi, 1995).
dHAND and eHAND are basic helix-loop-helix (bHLH) transcription factors expressed during embryogenesis and are required for the proper development of cardiac and extraembryonic tissues. HAND genes, like the myogenic bHLH genes, are classified as class B bHLH genes, which are expressed in a tissue-restricted pattern and function by forming heterodimers with class A bHLH proteins. Myogenic bHLH genes are shown not to form homodimers efficiently, suggesting that their activity is dependent on their E-protein partners. To identify HIPs (HAND-interacting proteins) that regulate the activity of the HAND genes, a 9.5-10.5-day-old mouse embryonic yeast two-hybrid library was screened with eHAND. Several HIPs held high sequence identity to eHAND, indicating that eHAND could form and function as a homodimer. Based on the high degree of amino acid identity between eHAND and dHAND, it is possible that dHAND can also form homodimers and heterodimers with eHAND. Yeast and mammalian two-hybrid assays as well as biochemical pull-down assays showed that eHAND and dHAND are capable of forming both HAND homo- and hetero-dimers in vivo. To investigate whether HAND genes form heterodimers with other biologically relevant bHLH proteins, HAND heterodimerization with the recently identified Hairy-related transcription factors, HRT1-3, was demonstrated. This finding is exciting, because both HRT and HAND genes are coexpressed in the developing heart and limb and both have been implicated in establishing tissue boundaries and pattern formation. Moreover, competition gel shift analysis demonstrates that dHAND and eHAND can negatively regulate the DNA binding of MyoD/E12 heterodimers in a manner similar to MISTI and Id proteins, suggesting a possible transcriptional inhibitory role for HAND genes. Taken together, these results show that dHAND and eHAND can form homo- and hetero-dimer combinations with multiple bHLH partners and that this broad dimerization profile reflects the mechanisms by which HAND genes regulate transcription (Firulli, 2000).
The basic helix-loop-helix (bHLH) factor Hand1 plays an essential role in cardiac morphogenesis, and yet its precise function remains unknown. Protein-protein interactions, involving Hand1, provide a means of determining how Hand1-induced gene expression in the developing heart might be regulated. Hand1 is known to form either heterodimers with near-ubiquitous E-factors and other lineage-restricted class B bHLH proteins or homodimers with itself in vitro. To date, there have been no reported Hand1 protein interactions involving non-bHLH proteins. Heterodimer-versus-homodimer choice is mediated by the phosphorylation status of Hand1; however, little is known about the in vivo function of these dimers or, importantly, how they are regulated. In an effort to understand how Hand1 activity in the heart might be regulated postdimerization, tertiary Hand1-protein interactions with non-bHLH factors was studied. A novel interaction of Hand1 with the LIM domain protein FHL2, a known transcriptional coactivator and corepressor expressed in the developing cardiovascular system, is described. FHL2 interacts with Hand1 via the bHLH domain and is able to repress Hand1/E12 heterodimer-induced transcription but has no effect on Hand1/Hand1 homodimer activity. This effect of FHL2 is not mediated either at the level of dimerization or via an effect of Hand1/E12 DNA binding. In summary, these data describe a novel differential regulation of Hand1 heterodimers versus homodimers by association of the cofactor FHL2 and provide insight into the potential for a tertiary level of control of Hand1 activity in the developing heart (Hill, 2004).
dHAND is a transcription factor belonging to the class B basic helix-loop-helix protein family and is expressed during embryogenesis in the heart, branchial arches, limb buds, and neural crest derivatives. Despite much study, the molecular mechanisms involved in the regulation of dHAND activity are not well understood. Yeast two-hybrid screening was performed using full-length dHAND as bait; this led to identification of several dHAND-binding proteins, including three E-proteins: E2A, ME2, and ALF1. Subsequent analysis revealed that although their heterodimerization and transcriptional activities were similar, dHAND/E-protein heterodimers bind to an E-box element with differing affinities, suggesting they have distinct DNA binding specificities. Moreover, in situ hybridization showed that E-protein genes are expressed fairly ubiquitously among embryonic tissues, including the branchial arches and limb buds. By contrast, little signal was detected in the heart, suggesting that dHAND complexes with partners other than E-proteins in cardiac tissue (Murakami, 2004b).
An intricate array of cell-specific multiprotein complexes participate in programs of cell-specific gene expression through combinatorial interaction with different transcription factors and cofactors. The dHAND basic helix-loop-helix (bHLH) transcription factor, which is essential for heart development and extra embryonic structures, is thought to regulate cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted transcription factors such as GATA4 and NKX2.5. dHAND also interacts with the myocyte enhancer binding factor-2c (MEF2C) protein, a MADS-box transcription factor that is essential for heart development. dHAND and MEF2C synergistically activate expression of the atrial naturetic peptide gene (ANP) in transfected HeLa cells. GST-pulldown and immunoprecipitation assay demonstrate that full-length MEF2C protein is able to interact with dHAND in vitro and in vivo, just like MEF2A and bHLH transcription factors MyoD in skeletal muscle cells. In addition, electrophoretic mobility shift assays (EMSAs) demonstrate that MEF2C and dHAND do not influence each other's DNA binding activity. Using chromatin immunoprecipitation (ChIP) analysis in H9c2 cells it has been shown that dHAND interacts with MEF2C to form protein complex and binds A/T sequence in the promoter of ANP. These results suggest the existence of large multiprotein transcriptional complex with core DNA binding proteins that physically interact with other transcriptional factors to form favorable conformation to potentiate transcription (Zang, 2004).
HAND2/dHAND is a basic helix-loop-helix transcription factor expressed in the heart and neural crest derivatives during embryogenesis. Although dHAND is essential for branchial arch, cardiovascular and limb development, its target genes have not been identified. The regulatory mechanisms of dHAND function also remain relatively unknown. Akt/PKB, a serine/threonine protein kinase involved in cell survival, growth and differentiation, phosphorylates dHAND and inhibits dHAND-mediated transcription. AU5-dHAND expressed in 293T cells becomes phosphorylated, possibly at its Akt phosphorylation motif, in the absence of kinase inhibitors, whereas the phosphatidylinositol 3-kinase inhibitor wortmannin and the Akt inhibitor NL-71-101, but not the p70 S6 kinase inhibitor rapamycin, significantly reduce dHAND phosphorylation. Coexpression of HA-Akt augments dHAND phosphorylation at multiple serine and threonine residues mainly located in the bHLH domain and, as a result, decreases the transcriptional activity of dHAND. Consistently, alanine mutation mimicking the nonphosphorylation state abolishes the inhibitory effect of Akt on dHAND, whereas aspartate mutation mimicking the phosphorylation state results in a loss of dHAND transcriptional activity. These changes in dHAND transcriptional activity are in parallel with changes in the DNA binding activity rather than in dimerization activity. These results suggest that Akt-mediated signaling may regulate dHAND transcriptional activity through the modulation of its DNA binding activity during embryogenesis (Murakami, 2004a).
dHAND and eHAND are related basic helix-loop-helix (bHLH) transcription factors that are expressed in mesodermal and neural crest-derived structures of the developing heart. In contrast to their homogeneous expression during avian cardiogenesis, during mouse heart development dHAND and eHAND are expressed in a complementary fashion and are restricted to segments of the heart tube fated to form the right and left ventricles, respectively. dHAND and eHAND represent the earliest cardiac chamber-specific transcription factors yet identified. Targeted gene deletion of dHAND in mouse embryos resulted in embryonic lethality at embryonic day 10.5 from heart failure. This description of the cardiac phenotype of dHAND mutant embryos is the first demonstration of a single gene controlling the formation of the mesodermally derived right ventricle and the neural crest-derived aortic arches and reveals a novel cardiogenic subprogramme for right ventricular development (Srivastava, 1997).
The basic helix-loop-helix (bHLH) transcription factors, Hand1 and Hand2, also called eHand/Hxt/Thing1 and dHand/Hed/Thing2, 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. A germline mutation was generated 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. These results demonstrate important roles for Hand1 in extraembryonic mesodermal and heart development (Firulli, 1998).
dHAND and eHAND are basic helix-loop-helix transcription factors that play critical roles in cardiac development. The HAND genes have a complementary left-right cardiac asymmetry of expression with dHAND predominantly on the right side and eHAND on the left side of the looped heart tube. Although eHAND is asymmetrically expressed along the anterior-posterior and dorsal-ventral embryonic axes, it is symmetrically expressed along the left-right axis at early stages of embryonic and cardiac development. After cardiac looping, dHAND and eHAND are expressed in the right (pulmonary) and left (systemic) ventricles, respectively. The left-right (LR) sidedness of dHAND and eHAND expression is demonstrated to be anatomically reversed in situs inversus (inv/inv) mouse embryos; however, dHAND expression persists in the pulmonary ventricle and eHAND in the systemic ventricle regardless of anatomic position, indicating chamber specificity of expression. dHAND-null mice fail to form a right-sided pulmonary ventricle. Mice homozygous for the dHAND and inv mutations are demonstrated to have only a right-sided ventricle [which is morphologically a left (systemic) ventricle]. These data suggest that the HAND genes are involved in development of segments of the heart tube that give rise to specific chambers of the heart during cardiogenesis, rather than controlling the direction of cardiac looping by interpreting the cascade of LR embryonic signals (Thomas, 1998).
Nkx2.5/Csx and dHAND/Hand2 are conserved transcription factors that are coexpressed in the precardiac mesoderm and early heart tube and control distinct developmental events during cardiogenesis. To understand whether Nkx2.5 and dHAND may function in overlapping genetic pathways, mouse embryos lacking both Nkx2.5 and dHAND were generated. Mice heterozygous for mutant alleles of Nkx2.5 and dHAND are viable. Although single Nkx2.5 or dHAND mutants have a morphological atrial and single ventricular chamber, Nkx2.5;dHAND double mutants have only a single cardiac chamber which was molecularly defined as the atrium. Complete ventricular dysgenesis was observed in Nkx2.5;dHAND double mutants; however, a precursor pool of ventricular cardiomyocytes was identified on the ventral surface of the heart tube. Because Nkx2.5 mutants failed to activate eHAND expression even in the early precardiac mesoderm, the double mutant phenotype appears to reflect an effectively null state of dHAND and eHAND. Cell fate analysis in dHAND mutants suggests a role of HAND genes in survival and expansion of the ventricular segment, but not in specification of ventricular cardiomyocytes. These molecular analyses also reveal the cooperative regulation of the homeodomain protein, Irx4, by Nkx2.5 and dHAND. These studies provide the first demonstration of gene mutations that result in ablation of the entire ventricular segment of the mammalian heart, and reveal essential transcriptional pathways for ventricular formation (Yamagishi, 2001).
HAND2 is an essential transcription factor for cardiac, pharyngeal arch, and limb development. Apoptosis in the HAND2-null embryo causes hypoplasia of the right ventricle and pharyngeal arches leading to lethality by embryonic day (E)10.0 from heart failure. In order to investigate the role of apoptosis in inducing the HAND2-null phenotype, mouse embryos were generated lacking both HAND2 and Apaf-1, a central downstream mediator of mitochondrial damage-induced apoptosis. In contrast to HAND2-/- embryos, HAND2-/-Apaf-1-/- embryos at E10.5-11.0 had well-developed pharyngeal arches, aortic arch arteries, and no signs of cardiac failure. TUNEL analysis through pharyngeal arches of HAND2-/-Apaf-1-/- embryos revealed decreased apoptosis and the embryos had clearly patent aortic arch arteries. However, ventricular hypoplasia and cell death were unchanged in these animals compared to HAND2-/- embryos, resulting in growth arrest at E11.0. This study suggests that loss of HAND2 in the pharyngeal arch mesenchyme leads to apoptosis in an Apaf-1-dependent fashion and that, while loss of aortic arch integrity contributes to the early lethality, the ventricular defects are independent of arch development (Aiyer, 2005).
The basic helix-loop-helix transcription factors Hand1 and Hand2 display dynamic and spatially restricted expression patterns in the developing heart. Mice that lack Hand2 die at embryonic day 10.5 from right ventricular hypoplasia and vascular defects, whereas mice that lack Hand1 die at embryonic day 8.5 from placental and extra-embryonic abnormalities that preclude analysis of its potential role in later stages of heart development. To determine the cardiac functions of Hand1, mice were generated harboring a conditional Hand1-null allele and the gene was excised by cardiac-specific expression of Cre recombinase. Embryos homozygous for the cardiac Hand1 gene deletion displayed defects in the left ventricle and endocardial cushions, and exhibited dysregulated ventricular gene expression. However, these embryos survived until the perinatal period when they died from a spectrum of cardiac abnormalities. Creation of Hand1/2 double mutant mice revealed gene dose-sensitive functions of Hand transcription factors in the control of cardiac morphogenesis and ventricular gene expression. These findings demonstrate that Hand factors play pivotal and partially redundant roles in cardiac morphogenesis, cardiomyocyte differentiation and cardiac-specific transcription (McFadden, 2005).
The basic helix-loop-helix (bHLH) transcription factor Hand2 is required for growth and development of the heart, branchial arches and limb buds. To determine whether DNA binding is required for Hand2 to regulate the growth and development of these different embryonic tissues, mutant mice were generated in which the Hand2 locus was modified by a mutation (referred to as Hand2EDE) that abolished the DNA-binding activity of Hand2, leaving the remainder of the protein intact. In contrast to Hand2 null embryos, which display right ventricular hypoplasia and vascular abnormalities, causing severe growth retardation by E9.5 and death by E10.5, early development of the heart appeared remarkably normal in homozygous Hand2EDE mutant embryos. These mutant embryos also lacked the early defects in growth of the branchial arches seen in Hand2 null embryos and survived up to 2 to 3 days longer than did Hand2 null embryos. However, Hand2EDE mutant embryos exhibited growth defects in the limb buds similar to those of Hand2 null embryos. These findings suggest that Hand2 regulates tissue growth and development in vivo through DNA binding-dependent and -independent mechanisms (Liu, 2009).
Targeted deletion of the bHLH DNA-binding protein Hand2 in the neural crest, impacts development of the enteric nervous system (ENS), possibly by regulating the transition from neural precursor cell to neuron. This hypothesis was tested by targeting Hand2 deletion in nestin-expressing neural precursor (NEP) cells. The mutant mice showed abnormal ENS development, resulting in lethal neurogenic pseudo-obstruction. Neurogenesis of neurons derived from NEP cells identified a second nestin non-expressing neural precursor (NNEP) cell in the ENS. There was substantial compensation for the loss of neurons derived from the NEP pool by the NNEP pool but this was insufficient to abrogate the negative impact of Hand2 deletion. Hand2-mediated regulation of proliferation affected both neural precursor and neuron numbers. Differentiation of glial cells derived from the NEP cells was significantly decreased with no compensation from the NNEP pool of cells. These data indicate differential developmental potential of NEPs and NNEPs; NNEPs preferentially differentiate as neurons, whereas NEPs give rise to both neurons and glial cells. Deletion of Hand2 also resulted in complete loss of NOS and VIP and a significant decrease in expression of choline acetyltransferase and calretinin, demonstrating a role for Hand2 in neurotransmitter specification and/or expression. Loss of Hand2 resulted in a marked disruption of the developing neural network, exemplified by lack of a myenteric plexus and extensive overgrowth of fibers. Thus, Hand2 is essential for neurogenesis, neurotransmitter specification and neural network patterning in the developing ENS (Lei, 2011).
One of the first morphological manifestations of left/right (L/R) asymmetry in mammalian embryos is a pronounced rightward looping of the linear heart tube. The direction of looping is thought to be controlled by signals from an embryonic L/R axial system. Morphological L/R asymmetry in the murine heart first becomes apparent at the linear tube stage as a leftward displacement of its caudal aspect. Beginning at the same stage, the basic helix-loop-helix (bHLH) factor gene eHand is expressed in a strikingly left-dominant pattern in myocardium, reflecting an intrinsic molecular asymmetry. In embryo hearts lacking the homeobox gene Nkx2-5, which does not loop, left-sided eHand expression is abolished. The data predict that eHand expression is enhanced in descendants of the left heart progenitor pool as one response to inductive signaling from the L/R axial system, and that eHand controls intrinsic morphogenetic pathways essential for looping (Biben, 1997).
Basic helix-loop-helix (bHLH) transcription factors control developmental decisions in a wide range of embryonic cell types. The HLH motif mediates homo- and hetero-dimerization; this 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, a structure-function analysis was performed of the protein in cultured cells and dHAND mutant proteins were ectopically expressed in the developing limbs of transgenic mice. 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 (McFadden, 2000).
Sonic hedgehog (Shh) signals via Gli transcription factors to direct digit number and identity in the vertebrate limb. This study characterized the Gli-dependent cis-regulatory network through a combination of whole-genome chromatin immunoprecipitation (ChIP)-on-chip and transcriptional profiling of the developing mouse limb. These analyses identified approximately 5000 high-quality Gli3-binding sites, including all known Gli-dependent enhancers. Discrete binding regions exhibit a higher-order clustering, highlighting the complexity of cis-regulatory interactions. Further, Gli3 binds inertly to previously identified neural-specific Gli enhancers, demonstrating the accessibility of their cis-regulatory elements. Intersection of DNA binding data with gene expression profiles predicted 205 putative limb target genes. A subset of putative cis-regulatory regions were analyzed in transgenic embryos, establishing Blimp1 as a direct Gli target and identifying Gli activator signaling in a direct, long-range regulation of the BMP antagonist Gremlin. In contrast, a long-range silencer cassette downstream from Hand2 likely mediates Gli3 repression in the anterior limb. These studies provide the first comprehensive characterization of the transcriptional output of a Shh-patterning process in the mammalian embryo and a framework for elaborating regulatory networks in the developing limb (Vokes, 2008).
HAND2 (dHAND) is a basic helix-loop-helix (bHLH) transcription factor expressed in numerous tissues during development including the heart, limbs, and a subset of neural crest derivatives. Functional analysis has shown that HAND2 is involved in development of the branchial arches, heart, limb, vasculature, and nervous system. Although it is essential for development of numerous tissues, little is known about its mode of action. To this end, HAND2 transcriptional regulatory mechanisms have been characterized. Using mammalian one-hybrid analysis it has been shown that HAND2 contains a strong transcriptional activation domain in the amino-terminal third of the protein. Like most tissue-restricted bHLH factors, HAND2 heterodimerizes with the broadly expressed bHLH factors, the E-proteins. The consensus DNA binding site of HAND2 was determined; HAND2 binds a subset of E-boxes as a heterodimer with E12. Yeast two-hybrid screening of a neuroblastoma cDNA library for HAND2-interacting proteins selected HAND2 and numerous additional members of the E-protein family. Although HAND2 homodimer formation was confirmed by in vitro analysis, HAND2 fails to homodimerize in a mammalian two-hybrid assay but demonstrates robust HAND2/E12 interaction. It is concluded that HAND2 functions as a transcription activator by binding a subset of E-boxes as a heterodimer with E-proteins (Dai, 2002).
The bHLH protein eHAND plays an important role in the development of extraembryonic, mesodermal, and cardiac cell lineages, presumably through heterodimerization with other HLH proteins and DNA binding. A novel transcriptional activity of eHAND has been identified. In transient transfection assays, eHAND is a potent inhibitor of activation by some but not all bHLH proteins. eHAND can prevent E-box DNA binding by these bHLH proteins. Interestingly, eHAND can also strongly inhibit transactivation activity by a MyoD approximately E47 tethered dimer, which suggests a distinct mechanism of action. eHAND also inhibits MyoD-dependent skeletal muscle cell differentiation and expression of the muscle-specific myosin heavy chain protein. In addition, eHAND can repress activity of the natural p75LNGFR promoter, whose expression overlaps that of eHAND and dHAND. The inhibitory activity of eHAND may be attributed to multiple mechanisms, such as the ability to act as a corepressor, the presence of a repression domain, and its ability to sequester E proteins in an inactive complex. Based upon its inhibitory effect on bHLH proteins and cellular differentiation, it is proposed that eHAND may function by several mechanisms to promote placental giant cell proliferation by negatively regulating the activities of the bHLH protein MASH-2 (Bounpheng, 2000).
Reported here is the isolation and characterization of murine and human cDNAs encoded for by Irx4 (Iroquois homeobox gene 4). Mouse and human Irx4 proteins are highly conserved (83%) and their 63-aa homeodomains are more than 93% identical to those of the Drosophila Iroquois patterning genes. Human IRX4 maps to chromosome 5p15.3, which is syntenic to murine chromosome 13. Irx4 transcripts are present in the developing central nervous system, skin, and vibrissae, but are predominantly expressed in the cardiac ventricles. In mice at embryonic day (E) 7.5, Irx4 transcripts are found in the chorion and at low levels in a discrete anterior domain of the cardiac primordia. During the formation of the linear heart tube and its subsequent looping (E8.0 -8.5), Irx4 expression is restricted to the ventricular segment and is absent from both the posterior (eventual atrial) and the anterior (eventual outflow tract) segments of the heart. Throughout all subsequent stages in which the chambers of the heart become morphologically distinct (E8.5-11) and into adulthood, cardiac Irx4 expression is found exclusively in the ventricular myocardium. Irx4 gene expression has also been assessed in embryos with aberrant cardiac development: mice lacking RXRalpha or MEF2c have normal Irx4 expression, but mice lacking the homeobox transcription factor Nkx2-5 (Csx) have markedly reduced levels of Irx4 transcripts. dHand-null embryos initiate Irx4 expression, but cannot maintain normal levels. These data indicate that the homeobox gene Irx4 is likely to be an important mediator of ventricular differentiation during cardiac development downstream of Nkx2-5 and dHand (Bruneau, 2000).
Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate the specification and differentiation of numerous cell types during embryonic development. Hand1 and Hand2 are expressed by a subset of neural crest cells in the anterior branchial arches and are involved in craniofacial development. However, the precise mechanisms by which Hand proteins mediate biological actions and regulate downstream target genes in branchial arches is largely unknown. This study reports that Hand2 negatively regulates intramembranous ossification of the mandible by directly inhibiting the transcription factor Runx2, a master regulator of osteoblast differentiation. Hand proteins physically interact with Runx2, suppressing its DNA binding and transcriptional activity. This interaction is mediated by the N-terminal domain of the Hand protein and requires neither dimerization with other bHLH proteins nor DNA binding. Partial colocalization of Hand2 and Runx2 was observed in the mandibular primordium of the branchial arch, and downregulation of Hand2 precedes Runx2-driven osteoblast differentiation. Hand2 hypomorphic mutant mice display insufficient mineralization and ectopic bone formation in the mandible due to accelerated osteoblast differentiation, which is associated with the upregulation and ectopic expression of Runx2 in the mandibular arch. This study shows that Hand2 acts as a novel inhibitor of the Runx2-DNA interaction and thereby regulates osteoblast differentiation in branchial arch development (Funato, 2009).
Hand genes encode basic helix-loop-helix transcription factors that are expressed in the developing gut, where their function is unknown. Enteric Hand2 expression is limited to crest-derived cells, whereas Hand1 expression is restricted to muscle and interstitial cells of Cajal. Hand2 is developmentally regulated and is intranuclear in precursors but cytoplasmic in neurons. Neurons develop in explants from wild-type but not Hand2-/- bowel, although, in both, crest-derived cells are present and glia arise. Similarly, small interfering RNA (siRNA) silencing of Hand2 in enteric crest-derived cells prevents neuronal development. Terminally differentiated enteric neurons do not develop after conditional inactivation of Hand2 in migrating crest-derived cells; nevertheless, conditional Hand2 inactivation does not prevent precursors from expressing early neural markers. It is suggested that enteric neuronal development occurs in stages and that Hand2 expression is required for terminal differentiation but not for precursors to enter the neuronal lineage (D'Autreaux, 2007).
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