Beth A. Firulli

Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities

Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage–dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A– and protein phosphatase 2A–regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A–mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.

PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation.

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.

Mutations within Helix I of Twist1 Result in Distinct Limb Defects and Variation of DNA Binding Affinities

Twist1 is a basic helix-loop-helix (bHLH) factor that plays an important role in limb development. Haploinsufficiency of Twist1 results in polydactyly via the inability of Twist1 to antagonistically regulate the related factor Hand2. The mechanism modulating Twist1-Hand2 antagonism is via phosphoregulation of conserved threonine and serine residues in helix I of the bHLH domain. Phosphoregulation alters the dimerization affinities for both proteins. Here we show that the expression of Twist1 and Twist1 phosphoregulation mutants results in distinct limb phenotypes in mice. In addition to dimer regulation, Twist1 phosphoregulation affects the DNA binding affinities of Twist1 in a partner-dependent and cis-element-dependent manner. In order to gain a better understanding of the specific Twist1 transcriptional complexes that function during limb morphogensis, we employ a series of Twist1-tethered dimers that include the known Twist1 partners, E12 and Hand2, as well as a tethered Twist1 homodimer. We show that these dimers behave in a manner similar to monomerically expressed bHLH factors and result in distinct limb phenotypes that correlate well with those observed from the limb expression of Twist1 and Twist1 phosphoregulation mutants. Taken together, this study shows that the Twist1 dimer affinity for a given partner can modulate the DNA binding affinity and that Twist1 dimer choice determines phenotypic outcome during limb development.

An absence of Twist1 results in aberrant cardiac neural crest morphogenesis

The basic helix-loop-helix transcription factor Twist1 plays an essential role in mesenchymal cell populations during embryonic development and in pathological disease. Remodeling of the cardiac outflow tract (OFT) into the functionally separate aortic arch and pulmonary trunk is dependent upon the dynamic, coordinated contribution of multiple mesenchymal cell populations. Here, we report that Twist1(-/-) mice exhibit OFTs that contain amorphic cellular nodules within their OFT endocardial cushions. The nodular mesenchyme expresses the related bHLH factors Hand1 and Hand2, but reduced levels of the normal cushion marker Periostin. Lineage mapping confirms that nodule cells are exclusively of cardiac neural crest origin (cNCC), and are not ectopic cardiomyocytes or smooth muscle cells. These studies also reveal a delay in cNCC colonization of the OFT cushions. Furthermore, these mapping studies uncover nodules in the pharyngeal arches, and identify Twist1(-/-) neural crest cell defects within the dorsal neural tube, which exhibits an expanded domain of Wnt1-Cre-lineage marked cells. Together, these data support a model where Twist1 is required both for proper cNCC delamination, and for emigration from the dorsal neural tube and along cNCC migration pathways. Within the Twist1(-/-) neural crest cell populations that do emigrate to the OFT, a Hand-expressing subpopulation displays defective maturation, tracking, and, presumably, cell-cell adhesion, further compromising cNCC morphogenesis.

Cooperative interaction of Nkx2.5 and Mef2c transcription factors during heart development

The interactions of diverse transcription factors mediate the molecular programs that regulate mammalian heart development. Among these, Nkx2.5 and the Mef2c regulate common downstream targets and exhibit striking phenotypic similarities when disrupted, suggesting a potential interaction during heart development. Co‐immunoprecipitation and mammalian two‐hybrid experiments revealed a direct molecular interaction between Nkx2.5 and Mef2c. Assessment of mRNA expression verified spatiotemporal cardiac coexpression. Finally, genetic interaction studies employing histological and molecular analyses showed that, although Nkx2.5−/− and Mef2c−/− individual mutants both have identifiable ventricles, Nkx2.5−/−;Mef2c−/− double mutants do not, and that mutant cardiomyocytes express only atrial and second heart field markers. Molecular marker and cell death and proliferation analyses provide evidence that ventricular hypoplasia is the result of defective ventricular cell differentiation. Collectively, these data support a hypothesis where physical, functional, and genetic interactions between Nkx2.5 and Mef2c are necessary for ventricle formation. Developmental Dynamics 237:3809–3819, 2008.

Analysis of the Hand1 cell lineage reveals novel contributions to cardiovascular, neural crest, extra‐embryonic, and lateral mesoderm derivatives

The basic Helix‐Loop‐Helix (bHLH) transcription factors Hand1 and Hand2 play critical roles in the development of multiple organ systems during embryogenesis. The dynamic expression patterns of these two factors within developing tissues obfuscate their respective unique and redundant organogenic functions. To define cell lineages potentially dependent upon Hand gene expression, we generated a mutant allele in which the coding region of Hand1 is replaced by Cre recombinase. Subsequent Cre‐mediated activation of β‐galactosidase or eYFP reporter alleles enabled lineage trace analyses that clearly define the fate of Hand1‐expressing cells. Hand1‐driven Cre marks specific lineages within the extra embryonic tissues, placenta, sympathetic nervous system, limbs, jaw, and several cell types within the cardiovascular system. Comparisons between Hand1 expression and Hand1‐lineage greatly refine our understanding of its dynamic spatial‐temporal expression domains and raise the possibility of novel Hand1 functions in structures not thought to be Hand1‐dependent. Developmental Dynamics 239:3086–3097, 2010.

A Phosphomimetic Mutation in the Sall1 Repression Motif Disrupts Recruitment of the Nucleosome Remodeling and Deacetylase Complex and Repression of Gbx2

The multizinc finger transcription factor Sall1 is a critical developmental regulator that mediates repression through the recruitment of the nucleosome remodeling and deacetylase (NuRD) complex. Although a short conserved peptide motif in Sall1 is sufficient to recruit NuRD, its ability to regulate native Sall1 target genes in vivo has not been demonstrated. In this report, we demonstrate an in vivo role for the Sall1 repression motif and describe a novel direct target gene of Sall1, Gbx2, that is directly repressed in a NuRD-dependent fashion. The ability of Sall1 to repress Gbx2 was impaired in Xenopus embryos expressing mutant forms of Sall1 that are defective for NuRD binding. Finally, we demonstrate that protein kinase C phosphorylates serine 2 of the Sall1 repression motif and reveal that a phosphomimetic mutation of serine 2 disrupts the ability of Sall1 to repress Gbx2 in cell culture and Xenopus embryos. Together, these studies establish that Sall1 recruits NuRD via the Sall1 repression motif to mediate repression of a native target gene and suggest a model in which dynamic control of gene expression by Sall1 is modulated by serine phosphorylation of the Sall1 repression motif.

Hand2 Loss-of-Function in Hand1-Expressing Cells Reveals Distinct Roles in Epicardial and Coronary Vessel Development

Rationale: The basic helix–loop–helix (bHLH) transcription factors Hand1 and Hand2 are essential for embryonic development. Given their requirement for cardiogenesis, it is imperative to determine their impact on cardiovascular function. Objective: To deduce the role of Hand2 within the epicardium. Method and Results: We engineered a Hand1 allele expressing Cre recombinase. Cardiac Hand1 expression is largely limited to cells of the primary heart field, overlapping little with Hand2 expression. Hand1 is expressed within the septum transversum, and the Hand1 lineage marks the proepicardial organ and epicardium. To examine Hand factor functional overlap, we conditionally deleted Hand2 from Hand1-expressing cells. Hand2 mutants display defective epicardialization and fail to form coronary arteries, coincident with altered extracellular matrix deposition and Pdgfr expression. Conclusions: These data demonstrate a hierarchal relationship whereby transient Hand1 septum transversum expression defines epicardial precursors that are subsequently dependent on Hand2 function.

Hand2 Is an Essential Regulator for Two Notch-Dependent Functions within the Embryonic Endocardium

The basic-helix-loop-helix (bHLH) transcription factor Hand2 plays critical roles during cardiac morphogenesis via expression and function within myocardial, neural crest, and epicardial cell populations. Here, we show that Hand2 plays two essential Notch-dependent roles within the endocardium. Endocardial ablation of Hand2 results in failure to develop a patent tricuspid valve, intraventricular septum defects, and hypotrabeculated ventricles, which collectively resemble the human congenital defect tricuspid atresia. We show endocardial Hand2 to be an integral downstream component of a Notch endocardium-to-myocardium signaling pathway and a direct transcriptional regulator of Neuregulin1. Additionally, Hand2 participates in endocardium-to-endocardium-based cell signaling, with Hand2 mutant hearts displaying an increased density of coronary lumens. Molecular analyses further reveal dysregulation of several crucial components of Vegf signaling, including VegfA, VegfR2, Nrp1, and VegfR3. Thus, Hand2 functions as a crucial downstream transcriptional effector of endocardial Notch signaling during both cardiogenesis and coronary vasculogenesis.

A Phox2- and Hand2-Dependent Hand1 cis-Regulatory Element Reveals a Unique Gene Dosage Requirement for Hand2 during Sympathetic Neurogenesis

Neural crest cell specification and differentiation to a sympathetic neuronal fate serves as an important model for neurogenesis and depends upon the function of both bHLH transcription factors, notably Hand2, and homeodomain transcription factors, including Phox2b. Here, we define a 1007 bp cis-regulatory element 5′ of the Hand1 gene sufficient to drive reporter expression within the sympathetic chain of transgenic mice. Comparative genomic analyses uncovered evolutionarily conserved consensus-binding sites within this element, which chromatin immunoprecipitation and electrophoretic mobility shift assays confirm are bound by Hand2 and Phox2b. Mutational analyses revealed that the conserved Phox2 and E-box binding sites are necessary for proper cis-regulatory element activity, and expression analyses on both Hand2 conditionally null and hypomorphic backgrounds demonstrate that Hand2 is required for reporter activation in a gene dosage-dependent manner. We demonstrate that Hand2 and Hand1 differentially bind the E-boxes in this cis-regulatory element, establishing molecular differences between these two factors. Finally, we demonstrate that Hand1 is dispensable for normal tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) expression in sympathetic neurons, even when Hand2 gene dosage is concurrently reduced by half. Together, these data define a tissue-specific Hand1 cis-regulatory element controlled by two factors essential for the development of the sympathetic nervous system and provide in vivo regulatory evidence to support previous findings that Hand2, rather than Hand1, is predominantly responsible for regulating TH, DBH, and Hand1 expression in developing sympathetic neurons.