Peter Cserjesi

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.

Dicer is required for survival of differentiating neural crest cells

The neural crest (NC) lineage gives rise to a wide array of cell types ranging from neurons and glia of the peripheral nervous system to skeletal elements of the head. The mechanisms regulating NC differentiation into such a large number of cell types remain largely unknown. MicroRNAs (miRNAs) play key roles in regulating developmental events suggesting they may also play a role during NC differentiation. To determine what roles miRNAs play in differentiation of NC-derived tissues, we deleted the miRNA processing gene Dicer in NC cells using the Wnt1-Cre deleter line. We show that deletion of Dicer soon after NC cells have formed does not affect their migration and colonization of their targets in the embryo. However, the post-migratory NC is dependent on Dicer for survival. In the head, loss of Dicer leads to a loss of NC-derived craniofacial bones while in the trunk, cells of the enteric, sensory and sympathetic nervous systems are lost during development. We found that loss of Dicer does not prevent the initial differentiation of NC but as development progresses, NC derivatives are lost due to apoptotic cell death. When Dicer is deleted, both Caspase-dependent and -independent apoptotic pathways are activated in the sensory ganglia but only the Caspase-dependent apoptotic program was activated in the sympathetic nervous system showing that the specific endogenous apoptotic programs are turned on by loss of Dicer. Our results show that Dicer and miRNAs, are required for survival of NC-derived tissues by preventing apoptosis during differentiation.

BmprIa is required in mesenchymal tissue and has limited redundant function with BmprIb in tooth and palate development.

The BMP signaling plays a pivotal role in the development of craniofacial organs, including the tooth and palate. BmprIa and BmprIb encode two type I BMP receptors that are primarily responsible for BMP signaling transduction. We investigated mesenchymal tissue-specific requirement of BmprIa and its functional redundancy with BmprIb during the development of mouse tooth and palate. BmprIa and BmprIb exhibit partially overlapping and distinct expression patterns in the developing tooth and palatal shelf. Neural crest-specific inactivation of BmprIa leads to formation of an unusual type of anterior clefting of the secondary palate, an arrest of tooth development at the bud/early cap stages, and severe hypoplasia of the mandible. Defective tooth and palate development is accompanied by the down-regulation of BMP-responsive genes and reduced cell proliferation levels in the palatal and dental mesenchyme. To determine if BmprIb could substitute for BmprIa during tooth and palate development, we expressed a constitutively active form of BmprIb (caBmprIb) in the neural crest cells in which BmprIa was simultaneously inactivated. We found that substitution of BmprIa by caBmprIb in neural rest cells rescues the development of molars and maxillary incisor, but the rescued teeth exhibit a delayed odontoblast and ameloblast differentiation. In contrast, caBmprIb fails to rescue the palatal and mandibular defects including the lack of lower incisors. Our results demonstrate an essential role for BmprIa in the mesenchymal component and a limited functional redundancy between BmprIa and BmprIb in a tissue-specific manner during tooth and palate development.

Hand2 is required in the epithelium for palatogenesis in mice

The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in the development of multiple organs, including craniofacial organs. Mice carrying Hand2 hypomorphic alleles (Hand2(LoxP/-)) display a cleft palate phenotype. A specific deletion of the Hand2 branchial arch-specific enhancer also leads to a hypoplastic mandible and cleft palate formation in mice. However, the underlying mechanism of Hand2 regulation of palate development remains unknown. Here we show that Hand2 is expressed in both the epithelium and mesenchyme of the developing palate. While mesenchymal specific inactivation of Hand2 has no impact on palate development, epithelial specific deletion of Hand2 creates a cleft palate phenotype. Hand2 appears to exert distinct roles in the anterior and posterior palate. In the anterior palate of Hand2(LoxP/-) mice, premature death of periderm cells and a down-regulation of Shh are observed in the medial edge epithelium (MEE), accompanied by a decreased level of cell proliferation in the palatal mesenchyme. In the posterior palate, a lower dose of Hand2 causes aberrant periderm cell death on the surface of the epithelium, triggering abnormal fusion between the palatal shelf and mandible and preventing palatal shelf elevation. We further demonstrate that BMP activities are essential for the expression of Hand2 in the palate. We conclude that Hand2 is an intrinsic regulator in the epithelium and is required for palate development.

Cardiac Neural Crest Expression of Hand2 Regulates Outflow and Second Heart Field Development

The cardiac neural crest (cNC) lineage plays key roles in heart development by directly contributing to heart structures and regulating development of other heart lineages. The basic helix–loop–helix factor Hand2 regulates development of cardiovascular structures and NC-derived tissues including those that contribute to face and peripheral nervous system. Although Hand2 is expressed in cNC, its role has not been examined because of an early embryonic lethality when Hand2 is deleted in the NC lineage. We find that the lethality is attributable to loss of norepinephrine synthesis that can be overcome by activating adrenergic receptors. In rescued embryos, loss of Hand2 in the NC lineage leads to the misalignment of the outflow tract and aortic arch arteries. Defects include pulmonary stenosis, interrupted aortic artery, retroesophageal right subclavian artery, and ventricular septum defect, which resemble congenital heart defects attributed to defects in the NC. Hand2 functions in part by regulating signaling from the cNC to other cardiac lineages but not by regulating migration or survival of the cNC. Loss of Hand2 in NC also uncovered a novel role for the cNC in regulating proliferation and differentiation of the second heart field–derived myocardium that persists late into development. These results show that the cNC functions as a major signaling center for heart development and Hand2 plays a pivotal role in regulating both cell-autonomous and -nonautonomous functions of the cNC.

MEF2C is required for the normal allocation of cells between the ventricular and sinoatrial precursors of the primary heart field

Targeted deletion of the mef2c gene results in a small left ventricle and complete loss of the right ventricle (Lin et al. [1997] Science 276:1404–1407). Absence of the right ventricle is from defective differentiation of cells from the secondary heart field. Our studies of the dysmorphogenesis of the left ventricle uncovered morphological and transcriptional abnormalities at the transition from the cardiac crescent to the linear‐tube stage heart. Use of the cgata6LacZ transgene demonstrated that lacZ‐positive cells, which normally mark the precursors to the atrioventricular canal and adjacent regions of the left ventricle and atria, remain in the sinoatrial region of the mutant. This, along with the absence of a morphologically distinct atrioventricular canal, indicates a misapportioning of cells between the inflow and outflow segments. The underlying genetic program was also affected with altered expression of mlc2a, mlc2v, and irx4 in outflow segment precursors of the primary heart field. In addition, the sinoatrial‐enriched transcription factor, tbx5, was ectopically expressed in the primitive ventricle and ventricle‐specific splicing of mef2b was lost, suggesting that the mutant ventricle had acquired atrial‐specific characteristics. Collectively, these results suggest a fundamental role of MEF2C in ventricular cardiomyocyte differentiation and apportioning of cells between inflow and outflow precursors in the primary heart field. Developmental Dynamics 235:1809–1821, 2006.

The basic helix‐loop‐helix factor Hand2 regulates autonomic nervous system development

Mammalian autonomic nervous system (ANS) development requires the combinatorial action of a number of transcription factors, which include Mash1, Phox2b, and GATA3. Here we show that the bHLH transcription factor, Hand2 (dHAND), is expressed concurrently with Mash1 during sympathetic nervous system (SNS) development and that the expression of Hand2 is not dependent on Mash1. This suggests that these two bHLH factors work in parallel during SNS development. We also show that ectopic expression of Hand2 activates the neuronal program and promotes the acquisition of a phenotype corresponding to peripheral neurons including neurons of the SNS lineage in P19 embryonic carcinoma cells. We propose that Hand2 works in parallel with other members of the transcriptional network to regulate ANS developmental but can ectopically activate the program by a cross‐regulatory mechanism that includes the activation of Mash1. We show that this function is dependent on its interaction with the histone acetyltransferase p300/CBP, indicating that Hand2 functions to promote ANS development as part of a larger transcriptional complex. Developmental Dynamics 234:613–621, 2005.

Expression Level of Hand2 Affects Specification of Enteric Neurons and Gastrointestinal Function in Mice

BACKGROUND & AIMS Hand2 is a basic helix-loop-helix transcription factor required for terminal differentiation of enteric neurons. We studied Hand2 haploinsufficient mice, to determine whether reduced expression of Hand2 allows sufficient enteric neurogenesis for survival, but not for development of a normal enteric nervous system (ENS). METHODS Enteric transcripts that encode Hand2 and the neuron-specific embryonic lethal abnormal vision proteins HuB, HuC, and HuD were quantified. Immunocytochemistry was used to identify and quantify neurons. Apoptosis was analyzed with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling procedure. Intracellular microelectrodes were used to record inhibitory junction potentials. Gastrointestinal transit and colonic motility were measured in vivo. RESULTS Levels of of enteric Hand2 transcripts were associated with genotypes of mice, in the following order: Hand2(+/+) > Hand2(LoxP/+) > Hand2(+/-) > Hand2(LoxP/-). Parallel reductions were found in expression of HuD and in regional and phenotypic manners. Numbers of neurons, numbers of neuronal nitric oxide synthase(+) and calretinin(+), but not substance P(+) or vasoactive intestinal peptide(+) neurons, decreased. No effects were observed in stomach or cecum. Apoptosis was not detected, consistent with the concept that Hand2 inhibits neuronal differentiation, rather than regulates survival. The amplitude of inhibitory junction potentials in colonic circular muscle was similar in Hand2 wild-type and haploinsufficient mice, although in haploinsufficient mice, the purinergic component was reduced and a nitrergic component appeared. The abnormal ENS of haploinsufficient mice slowed gastrointestinal motility but protected mice against colitis. CONCLUSIONS Reduced expression of factors required for development of the ENS can cause defects in the ENS that are subtle enough to escape detection yet cause significant abnormalities in bowel function.