Population Of Neural Crest Cells Research Articles

The SWI/SNF Complex in Neural Crest Cell Development and Disease.

While the neural crest cell population gives rise to an extraordinary array of derivatives, including elements of the craniofacial skeleton, skin pigmentation, and peripheral nervous system, it is today increasingly recognized that Schwann cell precursors are also multipotent. Two mammalian paralogs of the SWI/SNF (switch/sucrose nonfermentable) chromatin-remodeling complexes, BAF (Brg1-associated factors) and PBAF (polybromo-associated BAF), are critical for neural crest specification during normal mammalian development. There is increasing evidence that pathogenic variants in components of the BAF and PBAF complexes play central roles in the pathogenesis of neural crest-derived tumors. Transgenic mouse models demonstrate a temporal window early in development where pathogenic variants in Smarcb1 result in the formation of aggressive, poorly differentiated tumors, such as rhabdoid tumors. By contrast, later in development, homozygous inactivation of Smarcb1 requires additional pathogenic variants in tumor suppressor genes to drive the development of differentiated adult neoplasms derived from the neural crest, which have a comparatively good prognosis in humans.

Folic Acid Attenuates MSG-Induced Teratogenicity during A 2-Month Pregnancy by Preventing Neural Crest Cell Destruction and Malformation in Chick Embryo Models

Monosodium glutamate (MSG), commonly used as a food enhancer, has been reported to have teratogenicity during the first 3 days of development. Furthermore, the neural crest cells (NCCs) are crucial for embryonic development during organogenesis. The present study aimed to investigate the treatment effect of folic acid (FA) on MSG-induced teratogenicity, focusing on the toxicity and teratogenic effects on somatic and neural crest cells in chick embryos as models. Six hundred and fifty fertilized eggs were divided into control, FA, MSG, and MSG with FA groups. The chemicals were administered, and the results were investigated after 3 days of incubation. The morphology and histology were studied using stereomicroscopy and hematoxylin and eosin staining, respectively. The NCC population was confirmed by the presence of HNK-1 using immunohistochemistry. The finding showed that the MSG at 2 mg/kg of egg weight induced retardation, tissue malformation, craniofacial, and heart defects, whereas the FA alleviated those adverse effects and reduced the MSG-induced NCCs destruction in the eyes, heart, stomach, and nerves. In conclusion, although MSG harms embryos, FA effectively diminished its teratogenicity in the chick embryo model. These experimental protocols are beneficial for teratogenic studies on preventing birth defects that are harmful to the embryo. HIGHLIGHTS The 4 mg/kg egg weight of monosodium glutamate exposure leads to chick embryo lethality Folic acid treatment is effective at 2 mg/kg of monosodium glutamate exposure Folic acid is essential for supervised neural crest cell functions following monosodium glutamate-induced teratogenesis These experimental protocols are beneficial for teratogenic studies GRAPHICAL ABSTRACT

Abstract P3087: Single-cell Transcriptomics Reveals Cell-type Specific Transcriptional Responses In Murine Embryonic Hearts Exposed To Maternal Hyperglycemia

Congenital heart disease (CHD) is a significant pediatric health burden in the United States and a leading non-infectious cause of mortality in the infants. According to CDC, ~1 in 4 babies born with cardiac defects develop critical CHD and require medical interventions during first year of their lives. Despite the strong heritable basis of CHD, genetic variations explain nearly 30% of cases. The phenotypic heterogeneity and incomplete penetrance of the disease implies a vital role of environmental factors as non-genetic contributors of CHD. Women with pregestational diabetes mellitus (PGDM) are about four times more likely to have a pregnancy affected by CHD compared to women without diabetes. Previous studies have established that maternal hyperglycemia (matHG) before pregnancy and in first trimester profoundly increases the risk of CHD among the offspring, with a preponderance of conotruncal and septal defects. However, the cell-type-specific sensitivity to matHG responsible for such cardiac malformations remains unclear. Using a chemically induced murine PGDM model, we first demonstrated increased incidence of CHD in matHG-exposed embryos compared to control (CNTRL) embryos. To evaluate the underlying cellular basis of CHD risk in diabetic pregnancies, we performed in vivo single-cell RNA-sequencing in CNTRL and matHG-exposed embryonic hearts. The transcriptomic data revealed cardiac cell-type-specific molecular changes associated matHG. Bioinformatics analysis showed significant gene expression differences in Isl1+ multipotent progenitor cells, Tnnt2+ cardiomyocytes, Tbx18+ epicardial cells, Sox9+ fibromesenchymal cells, Cdh5+ endocardial/endothelial cells, and in Dlx5+ neural crest cell populations. Pseudotime trajectory analysis suggested HG-induced impairments in genes critical for cardiomyocyte differentiation. Furthermore, in vivo Isl1-derived cell fate mapping studies suggested matHG-mediated perturbations in cardiomyocyte proliferation and differentiation at critical stages of cardiac development. In combination with our scRNA-seq and cell fate mapping data, future mechanistic studies will delineate cell-type-specific gene regulatory network perturbed in matHG environment. Early diagnosis and treatment of diabetes in pregnancy is of great clinical significance and more in-depth studies will enable the development of preventative therapeutic measures to ameliorate PGDM associated CHD.

(Fish) tales of congenital diseases: molecularly distinguishing cranial neural crest cell populations

Congenital diseases that affect the anterior segment are typically due to cranial neural crest defects. This transient embryonic stem cell population ultimately gives rise to the bone, cartilage, and connective tissue of the jaw and mid face, and within the eye contributes to the corneal, iris, ciliary body, aqueous outflow pathways, and sclera. Thus, due to their common origin, congenital eye anomalies are often associated with facial defects.

MicroRNAs promote neural crest multipotency

Restriction of developmental potential occurs when pluripotent cells undergo gastrulation and germ layer specification. Although cranial neural crest cells are specified from the ectoderm, they are capable of generating both ectodermal and mesodermal lineages. How mammalian neural crest give rise to multiple lineages is not well understood. Here, we use single‐cell ATAC sequencing to characterize chromatin accessibility of cranial neural crest and identify a transient increase in accessibility during specification. This increase in chromatin accessibility coincides with co‐expression of both pluripotency and neural crest transcriptional programs. Analysis of the differentiation trajectory during neural crest specification using single‐cell mRNA sequencing revealed that pluripotent cells of the anterior epiblast resolve into two populations of premigratory cranial neural crest corresponding to ectoderm and mesoderm fates. Our data identify two populations of premigratory cranial neural crest cells that deploy distinct pluripotency programs and rely upon post‐transcriptional gene regulation to expand developmental potential and direct cell fate.

Expression of Neural Crest Specifiers During the Two Waves of Trunk NCC Migration in Turtle Embryos

The bones of the plastron, the ventral portion of the turtle shell, develop through intramembranous ossification, in the same manner as facial bones, suggesting they are also produced by neural crest cells (NCCs). Previous work has demonstrated the existence of a unique second migration of NCCs away from the neural tube of turtle (Trachemys scripta) embryos; these migrate ventrally, appear to be skeletogenic and may contribute to the bones of the plastron. The goal of this project is to examine the expression of neural crest specifiers in the premigratory NCCs located in the neural tube and in migrating NCCs using immunofluorescence and whole mount in situ hybridization. During the switch‐over period, the neural crest specifiers Sox9 and Sox10 are both expressed in premigratory NCCs. However, their expression diverges in migrating NCCs; the cells that retain Sox10 are located within the major migratory stream in the rostral somite leading to the production of the dorsal root ganglion and other peripheral nerves, consistent with a neurogenic cell fate. By contrast, cells that retain Sox9 appear to take a novel migratory pathway through the caudal portion of the somite, and are excluded from the dorsal root ganglia, consistent with a non‐neurogenic and perhaps skeletogenic cell fate. We are currently examining the expression of transcription factors that have been found to be preferentially expressed in either trunk NCCs or cranial NCCs in the well‐characterized chicken (G. gallus) system. Elucidating the molecular control of NCC specification in the unique population of trunk NCCs would further our understanding of its role in the development of the turtle shell.

AP-2α and AP-2β cooperatively function in the craniofacial surface ectoderm to regulate chromatin and gene expression dynamics during facial development.

The facial surface ectoderm is essential for normal development of the underlying cranial neural crest cell populations, providing signals that direct appropriate growth, patterning, and morphogenesis. Despite the importance of the ectoderm as a signaling center, the molecular cues and genetic programs implemented within this tissue are understudied. Here, we show that removal of two members of the AP-2 transcription factor family, AP-2α and AP-2ß, within the early embryonic ectoderm of the mouse leads to major alterations in the craniofacial complex. Significantly, there are clefts in both the upper face and mandible, accompanied by fusion of the upper and lower jaws in the hinge region. Comparison of ATAC-seq and RNA-seq analyses between controls and mutants revealed significant changes in chromatin accessibility and gene expression centered on multiple AP-2 binding motifs associated with enhancer elements within these ectodermal lineages. In particular, loss of these AP-2 proteins affects both skin differentiation as well as multiple signaling pathways, most notably the WNT pathway. We also determined that the mutant clefting phenotypes that correlated with reduced WNT signaling could be rescued by Wnt1 ligand overexpression in the ectoderm. Collectively, these findings highlight a conserved ancestral function for AP-2 transcription factors in ectodermal development and signaling, and provide a framework from which to understand the gene regulatory network operating within this tissue that directs vertebrate craniofacial development.

BMP3 is a novel locus involved in the causality of ocular coloboma

Coloboma, a congenital disorder characterized by gaps in ocular tissues, is caused when the choroid fissure fails to close during embryonic development. Several loci have been associated with coloboma, but these represent less than 40% of those that are involved with this disease. Here, we describe a novel coloboma-causing locus, BMP3. Whole exome sequencing and Sanger sequencing of patients with coloboma identified three variants in BMP3, two of which are predicted to be disease causing. Consistent with this, bmp3 mutant zebrafish have aberrant fissure closure. bmp3 is expressed in the ventral head mesenchyme and regulates phosphorylated Smad3 in a population of cells adjacent to the choroid fissure. Furthermore, mutations in bmp3 sensitize embryos to Smad3 inhibitor treatment resulting in open choroid fissures. Micro CT scans and Alcian blue staining of zebrafish demonstrate that mutations in bmp3 cause midface hypoplasia, suggesting that bmp3 regulates cranial neural crest cells. Consistent with this, we see active Smad3 in a population of periocular neural crest cells, and bmp3 mutant zebrafish have reduced neural crest cells in the choroid fissure. Taken together, these data suggest that Bmp3 controls Smad3 phosphorylation in neural crest cells to regulate early craniofacial and ocular development.

Identification of regulatory elements for MafB expression in the cardiac neural crest.

Cardiac neural crest cells arise in the caudal hindbrain and then migrate to the heart through the pharyngeal arches. These cells contribute to the formation of the heart, including the outflow tract, and are unique to this neural crest population. MafB is a transcription factor expressed specifically in early migrating cardiac neural crest cells as well as in rhombomeres (r) 5 and 6. Here, we identified the regulatory region in the chicken genome controlling the expression of endogenous MafB transcripts and used these essential elements to express MafB in the cardiac neural crest in reporter assays. A reporter driven by this regulatory region was employed to trace the migration of these cells into the pharyngeal arches. This regulatory region demonstrated transcriptional activity in the cardiac neural crest but not in other neural crest cell subpopulations, such as the cranial and trunk cells. This study provides insights into the gene regulatory mechanisms that specify cardiac neural crest cells among neural crest cell populations.

Earliest migratory cephalic NC cells are potent to differentiate into dental ectomesenchyme of the two lungfish dentitions: tetrapodomorph ancestral condition of unconstrained capability of mesencephalic NC cells to form oral teeth.

Reciprocal interactions between epithelial and neural crest-derived mesenchymal cells have been recognized in the evolutionary modulation of tetrapod odontodes, skeletal structures that include the teeth and tooth-integrated basal tissue. Using cell-tracking experiments, it has been demonstrated that mandibular neural crest cells, labelled during migration, extensively populate dental papillae of all tooth phenotypes of the lobe-finned fish, the Australian lungfish (Neoceratodus forsteri). Here, I report on an extension of this experimental study that earliest migrating NC cells are able to differentiate into odontogenic ectomesenchyme. Using vital dye cell-tracking to mark the mesencephalic neural crest prior to migration, I have found that the corresponding population of earliest migratory cells selectively relocated to dental papillae of both temporary and permanent dentitions of Neoceratodus. I noticed a gradient in distribution of the labelled cells which populated posterior teeth, pterygoid and prearticular (including associated trabecular and Meckelian cartilages; major relocation) much more densely than those in anterior marginal positions, temporary and vomeral permanent teeth (minor relocation). Contrary to mice and zebrafish, the odontogenic potency of mesencephalic neural crest cells is already programmed at the onset of the migration event in lungfish. This may imply that the morphogenic potential of mesencephalic neural crest cells to form teeth has been heterochronically shifted and constrained to later migratory populations of neural crest cells during the developmental evolution of derived tetrapods, or/and arrested in their expression in the oral development of some modern osteichthyans.

Single-cell RNA analysis identifies pre-migratory neural crest cells expressing markers of differentiated derivatives.

The neural crest is a migratory population of stem-like cells that contribute to multiple traits including the bones of the skull, peripheral nervous system, and pigment. How neural crest cells differentiate into diverse cell types is a fundamental question in the study of vertebrate biology. Here, we use single-cell RNA sequencing to characterize transcriptional changes associated with neural crest cell development in the zebrafish trunk during the early stages of migration. We show that neural crest cells are transcriptionally diverse and identify pre-migratory populations already expressing genes associated with differentiated derivatives, specifically in the xanthophore lineage. Further, we identify a population of Rohon-Beard neurons in the data. The data presented identify novel genetic markers for multiple trunk neural crest cell populations and Rohon-Beard neurons providing insight into previously uncharacterized genes critical for vertebrate development.

A UNIQUE MUSCLE; DISSECTING THE ROLE OF SIGNALING PATHWAYS IN DEVELOPMENTAL ANOMALIES OF THE TONGUE

<h3>Background</h3> The vertebrate tongue is a complex muscular organ involved in mastication, taste sensation, and articulation. The tongue is affected in many syndromes, diseases, and malignancies. It has recently been shown that a cascade of signaling interactions between different cell populations during embryogenesis orchestrates the development of the tongue. In humans, a number of congenital abnormalities affect the gross morphology of the tongue and can occur in isolation or as part of a developmental syndrome such as aglossia, microglossia, macroglossia, and bifid tongue. <h3>Objective</h3> We present an overview of the gross anatomy and embryology of mammalian tongue development, review the clinical presentation of tongue anomalies, and briefly look at their genetic etiology. We focused on one of the anomalies in mouse models and utilized multiple genetic approaches to investigate local temporospatial requirements for sonic hedgehog (Shh) signaling during tongue development. <h3>Results</h3> Mice lacking a <i>Shh cis</i> enhancer, <i>MFCS4</i>, with reduced Shh in dorsal tongue epithelium, have perturbed lingual septum tendon formation and disrupted intrinsic muscle patterning, with these defects reproduced following global <i>Shh</i> deletion from E10.5 mouse embryos. Shh responsiveness was diminished in local cranial neural crest cell (CNCC) populations in both mutants. Shh targets these cells through primary cilium. CNCC-specific deletion of orofaciodigital syndrome 1, which encodes a ciliary protein, led to loss of normal myotube arrangement and microglossia. <h3>Conclusions</h3> We demonstrate the cause of microglossia in syndromes affecting Shh signaling and show that Shh signaling is required in the cranial neural crest cells for lingual tendon differentiation and intrinsic muscle patterning.

Онтологія варіантів будови та вад розвитку черепа. Частина I

Annually severe congenital abnormalities occur in 1 of 33 newborns or about 125.000 live births in the United States and are the leading cause of infant mortality. Craniofacial anomalies account for about one-third of all abnormalities. The prevalence of human congenital malformations is an important characteristic of public health. According to the World Health Organization, the birth rate of children with cleft lip and palate is on average 1:750 newborns, which is 20-30% of all human malformations and 86% of maxillofacial malformations. They contribute significantly to infant morbidity and disability as well as millions of dollars in health care costs each year. Development of the human skull begins about 23-26 days after fertilization, when a multipotent population of cranial neural crest cells migrates from the dorsal part of the neuraxis to the embryonic head region, giving rise to the frontal bone and interparietal part of the occipital bone, while the rest of the cranial arch comes mainly from the mesoderm. A malformation is a deviation from the normal development of an organ or tissue. Among the causes are chromosomal abnormalities, single gene defects, teratogenic or multi-factor agents, that is, a combination of genetic and external factors; the smallest number of cases are idiopathic. Exogenous causes of human congenital abnormalities include: physical (mechanical, thermal, radiation); chemical (hypoxia, malnutrition, hormonal discordance, teratogenic poisons); biological (viruses, bacteria and their toxins, protozoa); mental factors. Endogenous causes are heredity (changes in hereditary structures, mutations); biological inadequacy of germ cells (endocrine disorders, “over-ripening” of germ cells); parents’ age. Congenital craniofacial abnormalities are caused by improper growth and/or development of the brain and facial parts of the skull and soft tissue. Craniofacial abnormalities affecting the skull include macrocephaly, microcephaly and unilateral deformities. Sometimes cranial sutures fuse too early and craniosynostosis occurs, which is accompanied by various cranial deformities. This leads to abnormal cranial development, which can cause dysmorphia, and in the most severe cases catastrophically affect brain development. Conclusion. Based on the analysis of the literature sources, an attempt has been made to summarize the existing information and classifications of congenital cranial abnormalities, as well as the interpretation of various anomalies of the brain and facial skull in alphabetic order. The most common stigmas and malformations of the skull bones are briefly characterized

The alx3 gene shapes the zebrafish neurocranium by regulating frontonasal neural crest cell differentiation timing.

During craniofacial development, different populations of cartilage- and bone-forming cells develop in precise locations in the head. Most of these cells are derived from pluripotent cranial neural crest cells and differentiate with distinct developmental timing and cellular morphologies. The mechanisms that divide neural crest cells into discrete populations are not fully understood. Here, we use single-cell RNA sequencing to transcriptomically define different populations of cranial neural crest cells. We discovered that the gene family encoding the Alx transcription factors is enriched in the frontonasal population of neural crest cells. Genetic mutant analyses indicate that alx3 functions to regulate the distinct differentiation timing and cellular morphologies among frontonasal neural crest cell subpopulations. This study furthers our understanding of how genes controlling developmental timing shape craniofacial skeletal elements.

The Ocular Neural Crest: Specification, Migration, and Then What?

During vertebrate embryonic development, a population of dorsal neural tube-derived stem cells, termed the neural crest (NC), undergo a series of morphogenetic changes and extensive migration to become a diverse array of cell types. Around the developing eye, this multipotent ocular NC cell population, called the periocular mesenchyme (POM), comprises migratory mesenchymal cells that eventually give rise to many of the elements in the anterior of the eye, such as the cornea, sclera, trabecular meshwork, and iris. Molecular cell biology and genetic analyses of congenital eye diseases have provided important information on the regulation of NC contributions to this area of the eye. Nevertheless, a complete understanding of the NC as a contributor to ocular development remains elusive. In addition, positional information during ocular NC migration and the molecular pathways that regulate end tissue differentiation have yet to be fully elucidated. Further, the clinical challenges of ocular diseases, such as Axenfeld-Rieger syndrome (ARS), Peters anomaly (PA) and primary congenital glaucoma (PCG), strongly suggest the need for better treatments. While several aspects of NC evolution have recently been reviewed, this discussion will consolidate the most recent current knowledge on the specification, migration, and contributions of the NC to ocular development, highlighting the anterior segment and the knowledge obtained from the clinical manifestations of its associated diseases. Ultimately, this knowledge can inform translational discoveries with potential for sorely needed regenerative therapies.

From head to tail: regionalization of the neural crest.

The neural crest is regionalized along the anteroposterior axis, as demonstrated by foundational lineage-tracing experiments that showed the restricted developmental potential of neural crest cells originating in the head. Here, we explore how recent studies of experimental embryology, genetic circuits and stem cell differentiation have shaped our understanding of the mechanisms that establish axial-specific populations of neural crest cells. Additionally, we evaluate how comparative, anatomical and genomic approaches have informed our current understanding of the evolution of the neural crest and its contribution to the vertebrate body.

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

Establishing Primary Cultures of Trunk Neural Crest Cells.

Neural crest cells constitute a unique population of progenitor cells with extensive stem cell capacities able to navigate throughout various environments in the embryo and are a source of multiple cell types, including neurons, glia, melanocytes, smooth muscles, endocrine cells, cardiac cells, and also skeletal and supportive tissues in the head. Neural crest cells are not restricted to the embryo but persist as well in adult tissues where they provide a reservoir of stem cells with great therapeutic promise. Many fundamental questions in cell, developmental, and stem cell biology can be addressed using this system. During the last decades there has been an increased availability of elaborated techniques, animal models, and molecular tools to tackle neural crest cell development. However, these approaches are often very challenging and difficult to establish and they are not adapted for rapid functional investigations of mechanisms driving cell migration and differentiation. In addition, they are not adequate for collecting pure populations of neural crest cells usable in large scale analyses and for stem cell studies. Transferring and adapting the neural crest system in tissue culture may then represent an attractive alternative, opening up numerous prospects. Here we describe a simple method for establishing primary cultures of neural crest cells derived from trunk neural tubes using the avian embryo as a source of cells. This protocol is suited for producing pure populations of neural crest cells that can be processed for cytological, cellular, and functional approaches aimed at characterizing their phenotype, behavior, and potential. © 2020 Wiley Periodicals LLC. Basic Protocol: Primary cultures of avian trunk neural crest cells Support Protocol: Adaptations for immunofluorescence labeling and videomicroscopy.

Temporal Modulations of NODAL, BMP, and WNT Signals Guide the Spatial Patterning in Self-Organized Human Ectoderm Tissues

Summary Developmental biology studies using model organisms suggest that the emergence of spatial patterning in the ectoderm is mediated by the morphogen gradients. However, it is still unclear whether the morphogen gradient is necessary and dominates the cell spatial patterning, particularly in human genetic background. Here, we demonstrate that human pluripotent stem cells can self-organize to concentric rings of all major cell types in ectoderm when cultured on micropatterned surfaces in a chemically defined condition. We reveal that modulating the dynamics of NODAL, BMP, and WNT signals is sufficient to control the spatial order of different cell types. Our mathematical model suggests that changes in wavelength and phase of signaling patterns formed via reaction-diffusion may be the mechanism by which temporal information is translated into spatial information. Together, our work demonstrates that in vitro human ectoderm microtissues have great potential in understanding the mechanisms of early-stage human development.

Mis-Expression of a Cranial Neural Crest Cell-Specific Gene Program in Cardiac Neural Crest Cells Modulates HAND Factor Expression, Causing Cardiac Outflow Tract Phenotypes.

Congenital heart defects (CHDs) occur with such a frequency that they constitute a significant cause of morbidity and mortality in both children and adults. A significant portion of CHDs can be attributed to aberrant development of the cardiac outflow tract (OFT), and of one of its cellular progenitors known as the cardiac neural crest cells (NCCs). The gene regulatory networks that identify cardiac NCCs as a distinct NCC population are not completely understood. Heart and neural crest derivatives (HAND) bHLH transcription factors play essential roles in NCC morphogenesis. The Hand1PA/OFT enhancer is dependent upon bone morphogenic protein (BMP) signaling in both cranial and cardiac NCCs. The Hand1PA/OFT enhancer is directly repressed by the endothelin-induced transcription factors DLX5 and DLX6 in cranial but not cardiac NCCs. This transcriptional distinction offers the unique opportunity to interrogate NCC specification, and to understand why, despite similarities, cranial NCC fate determination is so diverse. We generated a conditionally active transgene that can ectopically express DLX5 within the developing mouse embryo in a Cre-recombinase-dependent manner. Ectopic DLX5 expression represses cranial NCC Hand1PA/OFT-lacZ reporter expression more effectively than cardiac NCC reporter expression. Ectopic DLX5 expression induces broad domains of NCC cell death within the cranial pharyngeal arches, but minimal cell death in cardiac NCC populations. This study shows that transcription control of NCC gene regulatory programs is influenced by their initial specification at the dorsal neural tube.