Single cell evaluation of endocardial Hand2 gene regulatory networks reveals HAND2-dependent pathways that impact cardiac morphogenesis.

The transcription factor HAND2 plays essential roles during cardiogenesis. Hand2 endocardial deletion (H2CKO) results in tricuspid atresia or double inlet left ventricle with accompanying intraventricular septum defects, hypo-trabeculated ventricles and an increased density of coronary lumens. To understand the regulatory mechanisms of these phenotypes, single cell transcriptome analysis of mouse E11.5 H2CKO hearts was performed revealing a number of disrupted endocardial regulatory pathways. Using HAND2 DNA occupancy data, we identify several HAND2-dependent enhancers, including two endothelial enhancers for the shear-stress master regulator KLF2. A 1.8 kb enhancer located 50 kb upstream of the Klf2 TSS imparts specific endothelial/endocardial expression within the vasculature and endocardium. This enhancer is HAND2-dependent for ventricular endocardium expression but HAND2-independent for Klf2 vascular and valve expression. Deletion of this Klf2 enhancer results in reduced Klf2 expression within ventricular endocardium. These data reveal that HAND2 functions within endocardial gene regulatory networks including shear-stress response.

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

BACKGROUND: 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. RESULTS: 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 potential of these elements using 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. CONCLUSIONS: Together, these results start to uncover the diverse regulatory potential inherent to the Hand2 upstream regulatory interval.

HAND transcription factors cooperatively specify the aorta and pulmonary trunk.

Congenital heart defects (CHDs) affecting the cardiac outflow tract (OFT) constitute a significant cause of morbidity and mortality. The OFT develops from migratory cell populations which include the cardiac neural crest cells (cNCCs) and secondary heart field (SHF) derived myocardium and endocardium. The related transcription factors HAND1 and HAND2 have been implicated in human CHDs involving the OFT. Although Hand1 is expressed within the OFT, Hand1 NCC-specific conditional knockout mice (H1CKOs) are viable. Here we show that these H1CKOs present a low penetrance of OFT phenotypes, whereas SHF-specific Hand1 ablation does not reveal any cardiac phenotypes. Further, HAND1 and HAND2 appear functionally redundant within the cNCCs, as a reduction/ablation of Hand2 on an NCC-specific H1CKO background causes pronounced OFT defects. Double conditional Hand1 and Hand2 NCC knockouts exhibit persistent truncus arteriosus (PTA) with 100% penetrance. NCC lineage-tracing and Sema3c in situ mRNA expression reveal that Sema3c-expressing cells are mis-localized, resulting in a malformed septal bridge within the OFTs of H1CKO;H2CKO embryos. Interestingly, Hand1 and Hand2 also genetically interact within the SHF, as SHF H1CKOs on a heterozygous Hand2 background exhibit Ventricular Septal Defects (VSDs) with incomplete penetrance. Previously, we identified a BMP, HAND2, and GATA-dependent Hand1 OFT enhancer sufficient to drive reporter gene expression within the nascent OFT and aorta. Using these transcription inputs as a probe, we identify a novel Hand2 OFT enhancer, suggesting that a conserved BMP-GATA dependent mechanism transcriptionally regulates both HAND factors. These findings support the hypothesis that HAND factors interpret BMP signaling within the cNCCs to cooperatively coordinate OFT morphogenesis.

Variation in a Left Ventricle-Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function.

RATIONALE: The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r2>0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5' to the gene encoding the basic helix-loop-helix transcription factor HAND1 (heart- and neural crest derivatives-expressed protein 1). OBJECTIVE: Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. METHODS AND RESULTS: We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis-regulatory activity. Two evolutionarily conserved GATA transcription factor cis-binding elements within this enhancer are bound by GATA4 and are necessary for cis-regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis-regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. CONCLUSIONS: Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans.

Defective Hand1 phosphoregulation uncovers essential roles for Hand1 in limb morphogenesis.

The morphogenesis of the vertebrate limbs is a complex process in which cell signaling and transcriptional regulation coordinate diverse structural adaptations in diverse species. In this study, we examine the consequences of altering Hand1 dimer choice regulation within developing vertebrate limbs. Although Hand1 deletion via the limb-specific Prrx1-Cre reveals a non-essential role for Hand1 in mouse limb morphogenesis, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, results in a severe truncation of proximal-anterior limb elements. Molecular analysis reveals a non-cell-autonomous mechanism that causes widespread cell death within the embryonic limb bud. In addition, we observe changes in proximal-anterior gene regulation, including a reduction in the expression of Irx3, Irx5, Gli3 and Alx4, all of which are upregulated in Hand2 limb conditional knockouts. A reduction of Hand2 and Shh gene dosage improves the integrity of anterior limb structures, validating the importance of the Twist-family bHLH dimer pool in limb morphogenesis.

Partially Penetrant Cardiac Neural Crest Defects in Hand1 Phosphomutant Mice: Dimer Choice That Is Not So Critical.

Hand1 is a basic Helix-loop-Helix transcription factor that exhibits post-translationally regulated dimer partner choice that allows for a diverse set of Hand1 transcriptional complexes. Indeed, when Hand1 phosphoregulation is altered, conditionally activated hypophorylation (Hand1PO4-) and phosphorylation mimic (Hand1PO4+) Hand1 alleles disrupt both craniofacial and limb morphogenesis with 100% penetrance. Interestingly, activation of conditional Hand1 Phosphomutant alleles within post-migratory neural crest cells produce heart defects that include ventricular septal defects, double-outlet right ventricle, persistent truncus arteriosus with partial penetrance. Single versus double-lobed thymus is a distinguishing feature between Wnt1-Cre;Hand1PO4-/+ and Wnt1-Cre;Hand1PO4+/+ mice. These data show that although Hand1 dimer regulation plays critical and consistent roles in disrupting craniofacial and limb morphogenesis, Hand1 dimer regulation during cardiac outflow track formation is less critical for normal morphogenesis. This review will present the OFT phenotypes observed in Hand1 Phosphomutant mice, and discuss possible mechanisms of how penetrance differences within the same tissues within the same embryos could be variable.

Nkx2.5 regulates endothelin converting enzyme-1 during pharyngeal arch patterning.

In gnathostomes, dorsoventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches is crucial for the development of hinged jaws. One of the key signals that mediate this process is Endothelin-1 (EDN1). Loss of EDN1 binding to the Endothelin-A receptor (EDNRA) results in loss of EDNRA signaling and subsequent facial birth defects in humans, mice and zebrafish. A rate-limiting step in this crucial signaling pathway is the conversion of immature EDN1 into a mature active form by Endothelin converting enzyme-1 (ECE1). However, surprisingly little is known about how Ece1 transcription is induced or regulated. We show here that Nkx2.5 is required for proper craniofacial development in zebrafish and acts in part by upregulating ece1 expression. Disruption of nkx2.5 in zebrafish embryos results in defects in both ventral and dorsal pharyngeal arch-derived elements, with changes in ventral arch gene expression consistent with a disruption in Ednra signaling. ece1 mRNA rescues the nkx2.5 morphant phenotype, indicating that Nkx2.5 functions through modulating Ece1 expression or function. These studies illustrate a new function for Nkx2.5 in embryonic development and provide new avenues with which to pursue potential mechanisms underlying human facial disorders.

Hand1 phosphoregulation within the distal arch neural crest is essential for craniofacial morphogenesis.

In this study we examine the consequences of altering Hand1 phosphoregulation in the developing neural crest cells (NCCs) of mice. Whereas Hand1 deletion in NCCs reveals a nonessential role for Hand1 in craniofacial development and embryonic survival, altering Hand1 phosphoregulation, and consequently Hand1 dimerization affinities, in NCCs results in severe mid-facial clefting and neonatal death. Hand1 phosphorylation mutants exhibit a non-cell-autonomous increase in pharyngeal arch cell death accompanied by alterations in Fgf8 and Shh pathway expression. Together, our data indicate that the extreme distal pharyngeal arch expression domain of Hand1 defines a novel bHLH-dependent activity, and that disruption of established Hand1 dimer phosphoregulation within this domain disrupts normal craniofacial patterning.

Hand2 elevates cardiomyocyte production during zebrafish heart development and regeneration.

Embryonic heart formation requires the production of an appropriate number of cardiomyocytes; likewise, cardiac regeneration following injury relies upon the recovery of lost cardiomyocytes. The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in promoting cardiomyocyte formation. It is unclear, however, whether Hand2 plays an instructive or permissive role during this process. Here, we find that overexpression of hand2 in the early zebrafish embryo is able to enhance cardiomyocyte production, resulting in an enlarged heart with a striking increase in the size of the outflow tract. Our evidence indicates that these increases are dependent on the interactions of Hand2 in multimeric complexes and are independent of direct DNA binding by Hand2. Proliferation assays reveal that hand2 can impact cardiomyocyte production by promoting division of late-differentiating cardiac progenitors within the second heart field. Additionally, our data suggest that hand2 can influence cardiomyocyte production by altering the patterning of the anterior lateral plate mesoderm, potentially favoring formation of the first heart field at the expense of hematopoietic and vascular lineages. The potency of hand2 during embryonic cardiogenesis suggested that hand2 could also impact cardiac regeneration in adult zebrafish; indeed, we find that overexpression of hand2 can augment the regenerative proliferation of cardiomyocytes in response to injury. Together, our studies demonstrate that hand2 can drive cardiomyocyte production in multiple contexts and through multiple mechanisms. These results contribute to our understanding of the potential origins of congenital heart disease and inform future strategies in regenerative medicine.

Twist1 controls a cell-specification switch governing cell fate decisions within the cardiac neural crest.

Neural crest cells are multipotent progenitor cells that can generate both ectodermal cell types, such as neurons, and mesodermal cell types, such as smooth muscle. The mechanisms controlling this cell fate choice are not known. The basic Helix-loop-Helix (bHLH) transcription factor Twist1 is expressed throughout the migratory and post-migratory cardiac neural crest. Twist1 ablation or mutation of the Twist-box causes differentiation of ectopic neuronal cells, which molecularly resemble sympathetic ganglia, in the cardiac outflow tract. Twist1 interacts with the pro-neural factor Sox10 via its Twist-box domain and binds to the Phox2b promoter to repress transcriptional activity. Mesodermal cardiac neural crest trans-differentiation into ectodermal sympathetic ganglia-like neurons is dependent upon Phox2b function. Ectopic Twist1 expression in neural crest precursors disrupts sympathetic neurogenesis. These data demonstrate that Twist1 functions in post-migratory neural crest cells to repress pro-neural factors and thereby regulate cell fate determination between ectodermal and mesodermal lineages.

Loss of Hand2 in a population of Periostin lineage cells results in pronounced bradycardia and neonatal death.

The Periostin Cre (Postn-Cre) lineage includes endocardial and neural crest derived mesenchymal cells of the cardiac cushions, neural crest-derived components of the sympathetic and enteric nervous systems, and cardiac fibroblasts. In this study, we use the Postn-Cre transgenic allele to conditionally ablate Hand2 (H2CKO). We find that Postn-Cre H2CKOs die shortly after birth despite a lack of obvious cardiac structural defects. To ascertain the cause of death, we performed a detailed comparison of the Postn-Cre lineage and Hand2 expression at mid and late stages of embryonic development. Gene expression analyses demonstrate that Postn-Cre ablates Hand2 from the adrenal medulla as well as the sphenopalatine ganglia of the head. In both cases, Hand2 loss-of-function dramatically reduces expression of Dopamine Beta Hydroxylase (Dbh), a gene encoding a crucial catecholaminergic biosynthetic enzyme. Expression of the genes Tyrosine Hydroxylase (Th) and Phenylethanolamine N-methyltransferase (Pnmt), which also encode essential catecholaminergic enzymes, were severely reduced in postnatal adrenal glands. Electrocardiograms demonstrate that 3-day postnatal Postn-Cre H2CKO pups exhibit sinus bradycardia. In conjunction with the aforementioned gene expression analyses, these results strongly suggest that the observed postnatal lethality occurs due to a catecholamine deficiency and subsequent heart failure.

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.

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 ß-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.

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.

Neonatal Deletion of Hand1 and Hand2 within Murine Cardiac Conduction System Reveals a Novel Role for HAND2 in Rhythm Homeostasis.

The cardiac conduction system, a network of specialized cells, is required for the functioning of the heart. The basic helix loop helix factors Hand1 and Hand2 are required for cardiac morphogenesis and have been implicated in cardiac conduction system development and maintenance. Here we use embryonic and post-natal specific Cre lines to interrogate the role of Hand1 and Hand2 in the function of the murine cardiac conduction system. Results demonstrate that loss of HAND1 in the post-natal conduction system does not result in any change in electrocardiogram parameters or within the ventricular conduction system as determined by optical voltage mapping. Deletion of Hand2 within the post-natal conduction system results in sex-dependent reduction in PR interval duration in these mice, suggesting a novel role for HAND2 in regulating the atrioventricular conduction. Surprisingly, results show that loss of both HAND factors within the post-natal conduction system does not cause any consistent changes in cardiac conduction system function. Deletion of Hand2 in the embryonic left ventricle results in inconsistent prolongation of PR interval and susceptibility to atrial arrhythmias. Thus, these results suggest a novel role for HAND2 in homeostasis of the murine cardiac conduction system and that HAND1 loss potentially rescues the shortened HAND2 PR phenotype.

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 ß-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.

Analysis of a Hand1 hypomorphic allele reveals a critical threshold for embryonic viability.

Loss-of-function analysis of the basic helix-loop-helix (bHLH) transcription factor Hand1 indicates critical roles in development. In an effort to generate a Hand1 cDNA knock-in reporter mouse, we generated two hypomorphic alleles, which extend embryonic survival to between embryonic day (E) 10.5 and E12.5. Heart morphogenesis appears largely normal; however, hypomorphic mice display thin left ventricular myocardium and reduction in pharyngeal mesoderm. Caudal defects, large allantois, and thickened yolk sac are observed and consistent with systemic Hand1 gene deletion. Hand1 mRNA is expressed at 30% of wild-type littermates and known Hand1-dependent genes show intermediate expression compared with wild-type and Hand1 null mice. Interestingly, putative bHLH partners, Hand2 and Twist1, show altered expression in both Hand1 null and hypomorphic backgrounds and intercrossing the Hand1 hypomorphic mice onto the Hand2 systemic null background exacerbates the cardiac and lateral mesoderm phenotypes. Together, these data define a critical threshold of Hand1 expression that is necessary for embryonic survival.

Gene replacement strategies to test the functional redundancy of basic helix-loop-helix transcription factor.

Basic helix-loop-helix (bHLH) transcription factors control developmental decisions for a wide range of embryonic cell types. Hand1 and Hand2 are closely related bHLH proteins that control cardiac, craniofacial, and limb development. Within the developing heart, Hand1 expression becomes restricted predominantly to the left ventricle, whereas Hand2 becomes restricted predominantly to the left ventricle, for which findings have shown each Hand factor to be necessary for normal chamber formation. Forced overexpression of Hand1 throughout the early developing heart induces abnormal interventricular septal development, with resulting pathogenesis of congenital heart defects. To investigate the potential transcriptional mechanisms involved in heart morphogenesis by Hand2, this study used a replacement targeting approach to knock Hand2 into the Hand1 locus and ectopically express one copy of Hand2 within the endogenous Hand1 expression domain in the developing hearts of transgenic mice. The findings show that high-percentage Hand1 ( Hand2 ) chimeras die at birth and exhibit a range of congenital heart defects. These findings suggest that Hand factors may act via unique transcriptional mechanisms mediated by bHLH factor partner choice, supporting the notion that alterations of Hand factor stoichiometry may be as deleterious to normal heart morphogenesis as Hand factor loss of function.

HAND1 loss-of-function within the embryonic myocardium reveals survivable congenital cardiac defects and adult heart failure.

AIMS: To examine the role of the basic Helix-loop-Helix (bHLH) transcription factor HAND1 in embryonic and adult myocardium. METHODS AND RESULTS: Hand1 is expressed within the cardiomyocytes of the left ventricle (LV) and myocardial cuff between embryonic days (E) 9.5-13.5. Hand gene dosage plays an important role in ventricular morphology and the contribution of Hand1 to congenital heart defects requires further interrogation. Conditional ablation of Hand1 was carried out using either Nkx2.5 knockin Cre (Nkx2.5Cre) or α-myosin heavy chain Cre (αMhc-Cre) driver. Interrogation of transcriptome data via ingenuity pathway analysis reveals several gene regulatory pathways disrupted including translation and cardiac hypertrophy-related pathways. Embryo and adult hearts were subjected to histological, functional, and molecular analyses. Myocardial deletion of Hand1 results in morphological defects that include cardiac conduction system defects, survivable interventricular septal defects, and abnormal LV papillary muscles (PMs). Resulting Hand1 conditional mutants are born at Mendelian frequencies; but the morphological alterations acquired during cardiac development result in, the mice developing diastolic heart failure. CONCLUSION: Collectively, these data reveal that HAND1 contributes to the morphogenic patterning and maturation of cardiomyocytes during embryogenesis and although survivable, indicates a role for Hand1 within the developing conduction system and PM 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.