bHLH Transcription Factor Hand2 Research Articles

Identification and characterization of Hand2 upstream genomic enhancers active in developing stomach and limbs.

The bHLH transcription factor HAND2 plays important roles in the development of the embryonic heart, face, limbs, and sympathetic and enteric nervous systems. To define how and when HAND2 regulates these developmental systems, requires understanding the transcriptional regulation of Hand2. Remarkably, Hand2 is flanked by an extensive upstream gene desert containing a potentially diverse enhancer landscape. Here, we screened the regulatory interval 200 kb proximal to Hand2 for putative enhancers using evolutionary conservation and histone marks in Hand2-expressing tissues. H3K27ac signatures across embryonic tissues pointed to only two putative enhancer regions showing deep sequence conservation. Assessment of the transcriptional enhancer potentialof these elementsusing transgenic reporter lines uncovered distinct in vivo enhancer activities in embryonic stomach and limb mesenchyme, respectively. Activity of the identified stomach enhancer was restricted to the developing antrum and showed expression within the smooth muscle and enteric neurons. Surprisingly, the activity pattern of the limb enhancer did not overlap Hand2 mRNA but consistently yielded a defined subectodermal anterior expression pattern within multiple transgenic lines. Together, these results start to uncover the diverse regulatory potential inherent to the Hand2 upstream regulatory interval.

Osr1 couples intermediate mesoderm cell fate with temporal dynamics of vessel progenitor cell differentiation.

Transcriptional regulatory networks refine gene expression boundaries to define the dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that establish the boundary between the IM and neighboring vessel progenitors are poorly understood. Here, we delineate roles for the zinc-finger transcription factor Osr1 in kidney and vessel progenitor development. Zebrafish osr1 mutants display decreased IM formation and premature emergence of lateral vessel progenitors (LVPs). These phenotypes contrast with the increased IM and absent LVPs observed with loss of the bHLH transcription factor Hand2, and loss of hand2 partially suppresses osr1 mutant phenotypes. hand2 and osr1 are expressed together in the posterior mesoderm, but osr1 expression decreases dramatically prior to LVP emergence. Overexpressing osr1 during this timeframe inhibits LVP development while enhancing IM formation, and can rescue the osr1 mutant phenotype. Together, our data demonstrate that osr1 modulates the extent of IM formation and the temporal dynamics of LVP development, suggesting that a balance between levels of osr1 and hand2 expression is essential to demarcate the kidney and vessel progenitor territories.

Hand2 inhibits kidney specification while promoting vein formation within the posterior mesoderm.

Proper organogenesis depends upon defining the precise dimensions of organ progenitor territories. Kidney progenitors originate within the intermediate mesoderm (IM), but the pathways that set the boundaries of the IM are poorly understood. Here, we show that the bHLH transcription factor Hand2 limits the size of the embryonic kidney by restricting IM dimensions. The IM is expanded in zebrafish hand2 mutants and is diminished when hand2 is overexpressed. Within the posterior mesoderm, hand2 is expressed laterally adjacent to the IM. Venous progenitors arise between these two territories, and hand2 promotes venous development while inhibiting IM formation at this interface. Furthermore, hand2 and the co-expressed zinc-finger transcription factor osr1 have functionally antagonistic influences on kidney development. Together, our data suggest that hand2 functions in opposition to osr1 to balance the formation of kidney and vein progenitors by regulating cell fate decisions at the lateral boundary of the IM.

Distinct roles of hand2 in developing and adult autonomic neurons.

The bHLH transcription factor Hand2 is essential for the acquisition and maintenance of noradrenergic properties of embryonic sympathetic neurons and controls neuroblast proliferation. Hand2 is also expressed in embryonic and postnatal parasympathetic ganglia and remains expressed in sympathetic neurons up to the adult stage. Here, we address its function in developing parasympathetic and adult sympathetic neurons. We conditionally deleted Hand2 in the parasympathetic sphenopalatine ganglion by crossing a line of floxed Hand2 mice with DbhiCre transgenic mice, taking advantage of the transient Dbh expression in parasympathetic ganglia. Hand2 elimination does not affect Dbh expression and sphenopalatine ganglion size at E12.5 and E16.5, in contrast to sympathetic ganglia. These findings demonstrate different functions for Hand2 in the parasympathetic and sympathetic lineage. Our previous Hand2 knockdown in postmitotic, differentiated chick sympathetic neurons resulted in decreased expression of noradrenergic marker genes but it was unclear whether Hand2 is required for maintaining noradrenergic neuron identity in adult animals. We now show that Hand2 elimination in adult Dbh-expressing sympathetic neurons does not decrease the expression of Th and Dbh, in contrast to the situation during development. However, gene expression profiling of adult sympathetic neurons identified 75 Hand2-dependent target genes. Interestingly, a notable proportion of down-regulated genes (15%) encode for proteins with synaptic and neurotransmission functions. These results demonstrate a change in Hand2 target genes during maturation of sympathetic neurons. Whereas Hand2 controls genes regulating noradrenergic differentiation during development, Hand2 seems to be involved in the regulation of genes controlling neurotransmission in adult sympathetic neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1111-1124, 2016.

Nfat and miR-25 cooperate to reactivate the transcription factor Hand2 in heart failure

Although aberrant reactivation of embryonic gene programs is intricately linked to pathological heart disease, the transcription factors driving these gene programs remain ill-defined. Here we report that increased calcineurin/Nfat signalling and decreased miR-25 expression integrate to re-express the basic helix-loop-helix (bHLH) transcription factor dHAND (also known as Hand2) in the diseased human and mouse myocardium. In line, mutant mice overexpressing Hand2 in otherwise healthy heart muscle cells developed a phenotype of pathological hypertrophy. Conversely, conditional gene-targeted Hand2 mice demonstrated a marked resistance to pressure-overload-induced hypertrophy, fibrosis, ventricular dysfunction and induction of a fetal gene program. Furthermore, in vivo inhibition of miR-25 by a specific antagomir evoked spontaneous cardiac dysfunction and sensitized the murine myocardium to heart failure in a Hand2-dependent manner. Our results reveal that signalling cascades integrate with microRNAs to induce the expression of the bHLH transcription factor Hand2 in the postnatal mammalian myocardium with impact on embryonic gene programs in heart failure.

Interaction of Hand2 and E2a is important for transcription of Phox2b in sympathetic nervous system neuron differentiation

Transcription factors play a crucial role in the development of various tissues. In particular, the transcription factors of the basic helix-loop-helix (bHLH) family are crucial regulators of neurodifferentiation. Previous studies suggested that the bHLH transcription factor Hand2 is essential for sympathetic nervous system neuron differentiation in vivo, but the molecular mechanisms involved have not been well elucidated. It is important for understanding their mode of action in cellular differentiation to clarify how these bHLH factors regulate distinct transcriptional targets in a temporally and spatially controlled manner. Recent reports on ES cell differentiation suggested that its molecular mechanism mimics that of in vivo neurogenesis. However, the diverse nature of ES cell populations has prevented efficient analysis. To address this issue, we previously established a cell line in P19 embryonal carcinoma (EC) cells. Efficient sympathetic nervous system (SNS) neuron differentiation is induced in the cell line. Using this cell line, we succeeded in showing that the interaction of bHLH transcription factor Hand2 with E2a is required for transcription of Phox2b, which is essential for autonomic nervous system neuron development, and this binding activates this expression in SNS differentiation. Moreover, we also demonstrated that Hes5 regulated the transcription of Phox2b as a negative regulator and it inhibited the SNS differentiation. These findings have enabled us to determine the novel regulatory mechanism of Phox2b in SNS differentiation.

O30. Hepatopancreatic development and function in zebrafish

Remodeling of the extracellular matrix (ECM) plays crucial roles in both tumor progression and organ development. However, the molecular mechanisms that regulate ECM remodeling are not fully understood. In zebrafish, asymmetric migration of the epithelial lateral plate mesoderm (LPM) displaces the gut to the left, leading to the correct placement of the liver and pancreas. The bHLH transcription factor Hand2 is expressed in the anterior LPM as well as its mesenchymal derivatives. In order to observe these tissues at higher resolution, we generated a transgenic line that expresses GFP under the control of the hand2 regulatory regions. By confocal imaging, we show that during the process of gut looping, Laminin is distributed along the LPM/gut boundary, and appears to be degraded by the Hand2-expressing cells as they undergo asymmetric migration. Laminin degradation is required for LPM migration and is dependent on matrix metalloprotease (MMP) activity. Loss of Hand2 function causes reduced MMP activity and prolonged Laminin deposition at the LPM/gut boundary. Consequently, asymmetric LPM migration and leftward gut looping fail to occur in these embryos. Our study reveals an unexpected role for Hand2, a transcription factor that controls cell specification and differentiation, in modulating ECM remodeling during organ morphogenesis.

Hand2 ensures an appropriate environment for cardiac fusion by limiting Fibronectin function

Heart formation requires the fusion of bilateral cardiomyocyte populations as they move towards the embryonic midline. The bHLH transcription factor Hand2 is essential for cardiac fusion; however, the effector genes that execute this function of Hand2 are unknown. Here, we provide in zebrafish the first evidence for a downstream component of the Hand2 pathway that mediates cardiac morphogenesis. Although hand2 is expressed in cardiomyocytes, mosaic analysis demonstrates that it plays a non-autonomous role in regulating cardiomyocyte movement. Gene expression profiles reveal heightened expression of fibronectin 1 (fn1) in hand2 mutant embryos. Reciprocally, overexpression of hand2 leads to decreased Fibronectin levels. Furthermore, reduction of fn1 function enables rescue of cardiac fusion in hand2 mutants: bilateral cardiomyocyte populations merge and exhibit improved tissue architecture, albeit without major changes in apicobasal polarity. Together, our data provide a novel example of a tissue creating a favorable environment for its morphogenesis: the Hand2 pathway establishes an appropriate environment for cardiac fusion through negative modulation of Fn1 levels.

Hand2 Regulates Extracellular Matrix Remodeling Essential for Gut-Looping Morphogenesis in Zebrafish

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, we transgenically expressed EGFP under the control of the regulatory sequences of the bHLH transcription factor gene hand2. We found that laminin is distributed along the LPM/gut boundary during gut looping, and that 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. Our study reveals an unexpected role for Hand2, a key regulator of cell specification and differentiation, in modulating ECM remodeling during organogenesis.

The bHLH transcription factor Hand2 regulates the expression of nanog in ANS differentiation

The basic helix-loop-helix transcription factor Hand2 is induced by bone morphogenetic proteins (BMPs) in neural crest-derived precursor cells during the early stage of development of the autonomic nervous system (ANS). Previous studies showed that Hand2 was essential for the ANS differentiation. However, regulatory mechanism of pluripotent genes has not been elucidated in ANS differentiation. Here, we show that Hand2 regulated nanog expression in ANS differentiation. Our studies demonstrated that the forced expression of Hand2 promoted the ANS differentiation program in P19 embryonal carcinoma (EC) cells without aggregation. Furthermore, our results suggested that Hand2 bound to the promoter of nanog, a gene required for embryonic stem cells self-renewal, and suppressed nanog expression after Hand2 induction. The rapid downregulation of nanog mRNA during ANS differentiation correlated with the Hand2 transcriptional activity and nanog promoter methylation. These findings are evidence for a presence of the novel regulatory mechanism of nanog in ANS differentiation.

The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons

The basic helix–loop–helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-β-hydroxylase ( DBH) and tyrosine hydroxylase ( TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter ( VAChT) and choline acetyltransferase ( ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.

HAND transcription factors are required for neonatal sympathetic neuron survival

Expression of the basic helix-loop-helix transcription factor HAND2 begins early in sympathetic neuron development and is essential for the differentiation of noradrenergic neurons. Here, we show that the expression of HAND2 and related HAND1 are maintained in sympathetic neurons throughout fetal and postnatal development when these neurons depend on target-derived nerve growth factor (NGF) for survival. Short interfering RNA knockdown of endogenous HAND2 and, to a lesser extent, HAND1 in neonatal sympathetic neurons cultured with NGF, reduced the expression of the NGF receptor tyrosine kinase TrkA (tropomyosin-related kinase A), as well as neuronal survival. Chromatin immunoprecipitation analysis showed that NGF promotes HAND2 binding to the TrkA minimal enhancer and that transfection of sympathetic neurons with a TrkA expression plasmid rescued the neurons from HAND knockdown. These findings show that HAND transcription factors have a crucial function in sustaining the survival of neonatal sympathetic neurons with NGF by a feed-forward loop that maintains the expression of TrkA.

Dysregulation of Cardiogenesis, Cardiac Conduction, and Cell Cycle in Mice Lacking miRNA-1-2

MicroRNAs (miRNAs) are genomically encoded small RNAs used by organisms to regulate the expression of proteins generated from messenger RNA transcripts. The in vivo requirement of specific miRNAs in mammals through targeted deletion remains unknown, and reliable prediction of mRNA targets is still problematic. Here, we show that miRNA biogenesis in the mouse heart is essential for cardiogenesis. Furthermore, targeted deletion of the muscle-specific miRNA, miR-1-2, revealed numerous functions in the heart, including regulation of cardiac morphogenesis, electrical conduction, and cell-cycle control. Analyses of miR-1 complementary sequences in mRNAs upregulated upon miR-1-2 deletion revealed an enrichment of miR-1 "seed matches" and a strong tendency for potential miR-1 binding sites to be located in physically accessible regions. These findings indicate that subtle alteration of miRNA dosage can have profound consequences in mammals and demonstrate the utility of mammalian loss-of-function models in revealing physiologic miRNA targets.

Twoendothelin 1effectors,hand2andbapx1,pattern ventral pharyngeal cartilage and the jaw joint

A conserved endothelin 1 signaling pathway patterns the jaw and other pharyngeal skeletal elements in mice, chicks and zebrafish. In zebrafish, endothelin 1 (edn1 or sucker) is required for formation of ventral cartilages and joints in the anterior pharyngeal arches of young larvae. Here we present genetic analyses in the zebrafish of two edn1 downstream targets, the bHLH transcription factor Hand2 and the homeobox transcription factor Bapx1, that mediate dorsoventral (DV) patterning in the anterior pharyngeal arches. First we show that edn1-expressing cells in the first (mandibular) and second (hyoid) pharyngeal arch primordia are located most ventrally and surrounded by hand2-expressing cells. Next we show that along the DV axis of the early first arch primordia, bapx1 is expressed in an intermediate domain, which later marks the jaw joint, and this expression requires edn1 function. bapx1 function is required for formation of the jaw joint, the joint-associated retroarticular process of Meckel's cartilage, and the retroarticular bone. Jaw joint expression of chd and gdf5 also requires bapx1 function. Similar to edn1, hand2 is required for ventral pharyngeal cartilage formation. However, the early ventral arch edn1-dependent expression of five genes (dlx3, EphA3, gsc, msxe and msxb) are all present in hand2 mutants. Further, msxe and msxb are upregulated in hand2 mutant ventral arches. Slightly later, an edn1-dependent ventral first arch expression domain of gsc is absent in hand2 mutants, providing a common downstream target of edn1 and hand2. In hand2 mutants, bapx1 expression is present at the joint region, and expanded ventrally. In addition, expression of eng2, normally restricted to first arch dorsal mesoderm, expands ventrally in hand2 and edn1 mutants. Thus, ventral pharyngeal specification involves repression of dorsal and intermediate (joint region) fates. Together our results reveal two critical edn1 effectors that pattern the vertebrate jaw: hand2 specifies ventral pharyngeal cartilage of the lower jaw and bapx1 specifies the jaw joint.

The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development.

The precursors of several organs reside within the lateral plate mesoderm of vertebrate embryos. Here, we demonstrate that 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.