Neural Crest Cell Development Research Articles

A Hand for the Epicardium

See related article, pages 940–949 In mammals, formation of the properly septated 4-chambered heart and coronary vasculature is essential for unidirectional blood flow and survival of the organism. This process involves complex cell-cell interactions, the dysregulation of which can profoundly alter organogenesis, leading to a plethora of structural or functional cardiac defects. No wonder that congenital heart disease (CHD) represents the largest class of birth defects in humans. CHD, even the more subtle, subclinical form, is a major risk factor for early onset of serious cardiovascular complications and premature death. Despite important advances in diagnosis and surgical repair, preventive or therapeutic approaches have been hampered by our incomplete understanding of the molecular basis of most CHDs. Over the past 15 years, work in several model systems has identified genes required for normal heart development and started unraveling intricate regulatory relationships among many of them. Importantly, mutations in several of these cardiac regulators were subsequently linked to human CHD.1 Of those, the basic helix-loop-helix transcription factors Hand1 (eHAND) and Hand2 (dHAND) have emerged as important regulators of heart, limb, and neural crest cell development. Within the developing heart, Hand1 and Hand2 have overlapping expression in the cardiac crescent, but Hand1 expression becomes more restricted to the left ventricle after cardiac looping, whereas Hand2 becomes more localized to the right ventricle and derivatives of the secondary heart field (SHF).2 Gene targeting …

A phenotype‐driven ENU mutagenesis screen identifies novel alleles with functional roles in early mouse craniofacial development

Proper craniofacial development begins during gastrulation and requires the coordinated integration of each germ layer tissue (ectoderm, mesoderm, and endoderm) and its derivatives in concert with the precise regulation of cell proliferation, migration, and differentiation. Neural crest cells, which are derived from ectoderm, are a migratory progenitor cell population that generates most of the cartilage, bone, and connective tissue of the head and face. Neural crest cell development is regulated by a combination of intrinsic cell autonomous signals acquired during their formation, balanced with extrinsic signals from tissues with which the neural crest cells interact during their migration and differentiation. Although craniofacial anomalies are typically attributed to defects in neural crest cell development, the cause may be intrinsic or extrinsic. Therefore, we performed a phenotype-driven ENU mutagenesis screen in mice with the aim of identifying novel alleles in an unbiased manner, that are critically required for early craniofacial development. Here we describe 10 new mutant lines, which exhibit phenotypes affecting frontonasal and pharyngeal arch patterning, neural and vascular development as well as sensory organ morphogenesis. Interestingly, our data imply that neural crest cells and endothelial cells may employ similar developmental programs and be interdependent during early embryogenesis, which collectively is critical for normal craniofacial morphogenesis. Furthermore our novel mutants that model human conditions such as exencephaly, craniorachischisis, DiGeorge, and Velocardiofacial sydnromes could be very useful in furthering our understanding of the complexities of specific human diseases.

Making faces: neural crest cells in craniofacial development and congenital birth defects such as Treacher Collins syndrome

Craniofacial anomalies account for approximately one-third of congenital defects. The majority of bone, cartilage and connective tissue in the head and face are derived from neural crest cells. Consequently, defects in neural crest cell patterning are thought to underlie most craniofacial anomalies. Understanding the etiology and pathogenesis of craniofacial anomalies is dependent upon a thorough knowledge of the mechanisms that govern neural crest cell development. Treacher Collins syndrome (TCS) which is characterised by hypoplasia of the facial bones, is caused by mutations in TCOF1 and deficiencies in the number of migrating neural crest cells and their proliferative capacity. We are investigating the function of Tcof1 in neural crest cells in connection with its role in TCS. Haploinsufficiency of Tcof1 affects ribosome biogenesis as well as cell proliferation and survival, however, inhibition of apoptosis is sufficient to prevent the manifestation of craniofacial anomalies characteristic of TCS. Our work therefore is facilitating the development of therapeutic approaches to prevent the pathogenesis of craniofacial malformation syndromes, which has broad implications for other congenital birth defects of similar etiology to TCS. This research was supported by March of Dimes grants FY05-82 and FY08-265 and the National Institute of Dental and Craniofacial Research DE016082-06

Genomically integrated transgenes are stably and conditionally expressed in neural crest cell-specific lineages

Neural crest cells (NCCs) are a transient embryonic structure that gives rise to a variety of cells including peripheral nervous system, melanocytes, and Schwann cells. To understand the molecular mechanisms underlying NCC development, a gene manipulation of NCCs by in ovo electroporation technique is a powerful tool, particularly in chicken embryos, the model animal that has long been used for the NCC research. However, since expression of introduced genes by the conventional electroporation method is transient, the mechanisms of late development of NCCs remain unexplored. We here report novel methods by which late-developing NCCs are successfully manipulated with electroporated genes. Introduced genes can be stably and/or conditionally expressed in a NCC-specific manner by combining 4 different techniques: Tol2 transposon-mediated genomic integration (Sato et al., 2007), a NCC-specific enhancer of the Sox10 gene (identified in this study), Cre/loxP system, and tet-on inducible expression (Watanabe et al., 2007). This is the first demonstration that late-developing NCCs in chickens are gene-manipulated specifically and conditionally. These methods have further allowed us to obtain ex vivo live-images of individual Schwann cells that are associated in axon bundles in peripheral tissues. Cellular activity and morphology dynamically change as development proceeds. This study has opened a new way to understand at the molecular and cellular levels how late NCCs develop in association with other tissues during embryogenesis.

Metabolic syndromes and neural crest development

Aim of this study is to investigate for the possible connection between abnormal neural crest cell (NCC) development and NCC-derived abnormal facial and cerebral structures in 3 children with pyruvate-dehydrogenase (PDH) and in 10 cases with oxidative phosphorylation deficiency diagnosed from the Author by standard laboratory assays [i.e. 3 cases of Kearns-Sayre syndrome (KSS), 2 cases of Leigh syndrome, 1 case of KSS with De Toni-Debre-Fanconi and rachitis (Berio disease), 1 case of KSS with aortic insuffiency and sub-aortic septum hyperthophy, 3 cases of chronic progressive external ophthalmoplegia]. There patients presented with hyperlactacidemia, hyperpyruvicemia and facial abnormalities, similar to those observed in the fetal alcohol syndrome (a typical neurocristopathy) due to PDH deficiency, down-regulating NCC genes. The Author hypothesizes that the metabolic defect of scarce energy production is responsible of abnormal NCC proliferation/migration and consequent facial abnormalities.

Dicer activity in neural crest cells is essential for craniofacial organogenesis and pharyngeal arch artery morphogenesis

MicroRNAs (miRNAs) play important roles in regulating gene expression during numerous biological/pathological processes. Dicer encodes an RNase III endonuclease that is essential for generating most, if not all, functional miRNAs. In this work, we applied a conditional gene inactivation approach to examine the function of Dicer during neural crest cell (NCC) development. Mice with NCC-specific inactivation of Dicer died perinatally. Cranial and cardiac NCC migration into target tissues was not affected by Dicer disruption, but their subsequent development was disturbed. NCC derivatives and their associated mesoderm-derived cells displayed massive apoptosis, leading to severe abnormalities during craniofacial morphogenesis and organogenesis. In addition, the 4th pharyngeal arch artery (PAA) remodeling was affected, resulting in interrupted aortic arch artery type B (IAA-B) in mutant animals. Taken together, our results show that Dicer activity in NCCs is essential for craniofacial development and pharyngeal arch artery morphogenesis.

Disruption of long‐distance highly conserved noncoding elements in neurocristopathies

One of the key discoveries of vertebrate genome sequencing projects has been the identification of highly conserved noncoding elements (CNEs). Some characteristics of CNEs include their high frequency in mammalian genomes, their potential regulatory role in gene expression, and their enrichment in gene deserts nearby master developmental genes. The abnormal development of neural crest cells (NCCs) leads to a broad spectrum of congenital malformation(s), termed neurocristopathies, and/or tumor predisposition. Here we review recent findings that disruptions of CNEs, within or at long distance from the coding sequences of key genes involved in NCC development, result in neurocristopathies via the alteration of tissue- or stage-specific long-distance regulation of gene expression. While most studies on human genetic disorders have focused on protein-coding sequences, these examples suggest that investigation of genomic alterations of CNEs will provide a broader understanding of the molecular etiology of both rare and common human congenital malformations.

The neural crest-enriched microRNA miR-452 regulates epithelial-mesenchymal signaling in the first pharyngeal arch

Neural crest cells (NCCs) are a subset of multipotent, migratory stem cells that populate a large number of tissues during development and are important for craniofacial and cardiac morphogenesis. Although microRNAs (miRNAs) have emerged as important regulators of development and disease, little is known about their role in NCC development. Here, we show that loss of miRNA biogenesis by NCC-specific disruption of murine Dicer results in embryos lacking craniofacial cartilaginous structures, cardiac outflow tract septation and thymic and dorsal root ganglia development. Dicer mutant embryos had reduced expression of Dlx2, a transcriptional regulator of pharyngeal arch development, in the first pharyngeal arch (PA1). miR-452 was enriched in NCCs, was sufficient to rescue Dlx2 expression in Dicer mutant pharyngeal arches, and regulated non-cell-autonomous signaling involving Wnt5a, Shh and Fgf8 that converged on Dlx2 regulation in PA1. Correspondingly, knockdown of miR-452 in vivo decreased Dlx2 expression in the mandibular component of PA1, leading to craniofacial defects. These results suggest that post-transcriptional regulation by miRNAs is required for differentiation of NCC-derived tissues and that miR-452 is involved in epithelial-mesenchymal signaling in the pharyngeal arch.

Prdm1a Regulates sox10 and islet1 in the development of neural crest and Rohon‐Beard sensory neurons

The PR domain containing 1a, with ZNF domain factor, gene (prdm1a) plays an integral role in the development of a number of different cell types during vertebrate embryogenesis, including neural crest cells, Rohon-Beard (RB) sensory neurons and the cranial neural crest-derived craniofacial skeletal elements. To better understand how Prdm1a regulates the development of various cell types in zebrafish, we performed a microarray analysis comparing wild type and prdm1a mutant embryos and identified a number of genes with altered expression in the absence of prdm1a. Rescue analysis determined that two of these, sox10 and islet1, lie downstream of Prdm1a in the development of neural crest cells and RB neurons, respectively. In addition, we identified a number of other novel downstream targets of Prdm1a that may be important for the development of diverse tissues during zebrafish embryogenesis.

P49. Cell reprogramming factors of neural crest cells

The iPS-technology has opened a previously unexplored way to study the mechanisms of cell differentiation. Thus, cells that have already differentiated or committed to a certain cell type can potentially be reprogrammed to either a less differentiated state or a different type of cells. Such cell reprogramming technology has so far been applied to an in vitro situation. Toward the goal of achieving cell reprogramming directly in vivo, we have focused on neural crest cells (NCCs), multipotent precursors of peripheral nervous system. NCCs are highly amenable for in vivo gene manipulation using in ovo electroporation technique, particularly in chicken embryos. In addition, the entire cell lineage derived from NCCs has been described, and many transcriptional factors are available that regulate the development of NCCs and their derivatives. Combining these advantages of NCCs with recently established technologies such as tet-on inducible expression (Watanabe et al., 2007. Dev. Biol.) and transposon-mediated stable integration of electroporated genes (Sato et al., 2007. Dev. Biol.), NCCs have become an excellent model to achieve the in vivo cell reprogramming. We have found that Sox9, Sox 10, and canonical Wnt signal are powerful candidates with which NCCs and their derivatives can be reprogrammed directly in vivo.

Inositol hexakisphosphate kinase-2 acts as an effector of the vertebrate Hedgehog pathway

Inositol phosphate (IP) kinases constitute an emerging class of cellular kinases linked to multiple cellular activities. Here, we report a previously uncharacterized cellular function in Hedgehog (Hh) signaling for the IP kinase designated inositol hexakisphosphate kinase-2 (IP6K2) that produces diphosphoryl inositol phosphates (PP-IPs). In zebrafish embryos, IP6K2 activity was required for normal development of craniofacial structures, somites, and neural crest cells. ip6k2 depletion in both zebrafish and mammalian cells also inhibited Hh target gene expression. Inhibiting IP(6) kinase activity using N(2)-(m-(trifluoromethy)lbenzyl) N(6)-(p-nitrobenzyl)purine (TNP) resulted in altered Hh signal transduction. In zebrafish, restoring IP6K2 levels with exogenous ip6k2 mRNA reversed the effects of IP6K2 depletion. Furthermore, overexpression of ip6k2 in mammalian cells enhanced the Hh pathway response, suggesting IP6K2 is a positive regulator of Hh signaling. Perturbations from IP6K2 depletion or TNP were reversed by overexpressing smoM2, gli1, or ip6k2. Moreover, the inhibitory effect of cyclopamine was reversed by overexpressing ip6k2. This identified roles for the inositol kinase pathway in early vertebrate development and tissue morphogenesis, and in Hh signaling. We propose that IP6K2 activity is required at the level or downstream of Smoothened but upstream of the transcription activator Gli1.

P89. Genomically integrated transgenes are conditionally manipulable to be expressed in the neural crest-specific cell lineage

During vertebrate development, the neural crest (NC) is a transient structure that gives rise to a variety of tissues, including all types of peripheral nervous system in the trunk. Neural crest cells (NCCs) emigrate from the dorsal aspect of the neural tube, and migrate over distances in the body before reaching their targets. Whereas early specification of NCCs has extensively been studied, it remains unclear how NCCs migrate and find their target at later stages in development. Recently, Sato et al., established a Tol2-transposon-mediated gene transfer of electroporated genes in chickens to enable long-term labeling/gene manipulation of electroporated cells (Dev. Biol., 2007). By extending these techniques, we aimed at a long-term gene manipulation of cells in a NCC-specific manner. We started our analyses by electroporating the plasmid Tol2-CAGGS-EGFP (Tol2: sequence required for transposition, CAGGS: ubiquitous promoter) into an early neural tube. This resulted in a long-term expression of EGFP retained until at least to embryonic day 7 (E7) in most of NC-derivatives. However, this method also labels motor neurons (not NC-derived), which extend their axons from the neural tube. Therefore, we next attempted to exploit an enhancer of Sox 10, the gene known to be expressed in a NC-specific manner. By analyzing genomic sequences over ~30 kb, one region of 3.6 kb long was found to drive NC-specific expression when subcloned into the reporter plasmid tk-EGFP (a gift of Prof. H. Kondo, Osaka University). Since Sox10 expression ceases at later stages, we combined Sox10 enhancer with Cre/loxP system, the technique widely used in mouse genetics, and we observed stable expression of EGFP that was specific to NCCs. This technique is expected to enable cell- and tissue type-specific gene manipulation at later stages in development. We further combined this NC-specific gene manipulation with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene in NC-derivatives could be experimentally induced upon tetracycline administration at relatively late stages such as E6, when a variety of NCC developmental processes are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late NCC development, for which chickens are most suitable model animals.

Progress of pregestational diabetes mellitus and congenital heart defects

Pregestational diabetes mellitus may cause fetal heart defects,and it is considered to be an important non-genetic risk factors for congenital heart defects. However, the pathogenesis is unclear. Recent studies have shown that hyperglycemia, which acts as a primary teratogen in pregestational diabetes mellitus, may affect the endocardial cushion formation and neural crest cell development. Cardiac oxidative stress damage, increased myocardial cell apoptosis,increased synthesis of extracellular matrix, as well as the alteration of heart development-related genes expression are important pathogenic mechanism. In this paper, we review the progress in the effect and the pathogenesis of pregestational diabetes mellitus on heart development. Key words: Pregestational diabetes mellitus; Congenital heart defects; Endocardial cushion; Neural crest cell

Examining the evidence for vascular pathogenesis of selected birth defects

Vascular mechanisms have been proposed to be involved in the pathogenesis of a number of defects, including transverse-limb defects, intestinal atresias, gastroschisis, hydranencephaly, porencephaly, oromandibular-limb hypogenesis sequence, and oculoauriculovertebral spectrum (OAVS). Here, we examine the available clinical, epidemiologic, and experimental evidence for four defects (transverse-limb defects, intestinal atresias, gastroschisis, and OAVS) for which vascular pathogenesis has been hypothesized. Based on our review, transverse-limb defects appear to sometimes be due to vascular events related to placental vascular abnormalities, hypoperfusion, abnormal development of blood vessels, intrauterine compression, hemoglobinopathies, or exposure to vasoactive agents, although epidemiological studies have not consistently demonstrated the latter association. However, transverse-limb defects can also be due to abnormal developmental events, such as aberrant molecular signaling in the apical ectodermal ridge. Some intestinal atresias may have a vascular origin, with the hypothesis supported by experiments in canines. However, evidence is accumulating that a more common mechanism might be related to improper molecular signaling related to gut specification early in development. In contrast, evidence to support vascular pathogenesis for gastroschisis and OAVS is less compelling. Instead, these defects probably arise from interference with basic developmental events [e.g., body wall closure (gastroschisis) and neural crest cell development (OAVS)]. These conclusions are important for counseling parents of children with these defects and for guiding the design of future epidemiological studies and experiments to further characterize the causes of these defects.

Hes1 Is Required for the Development of Craniofacial Structures Derived From Ectomesenchymal Neural Crest Cells

The cranial neural crest cells contribute extensively to the formation of skeletogenic mesenchyme in the head and neck. Hes1 functions as a repressor of basic helix-loop-helix transcription factors and is implicated in controlling the maintenance of undifferentiated cells and the timing of cell differentiation. We show here that Hes1 homozygous null mutant mice exhibit multiple craniofacial malformations including calvaria agenesis, defective anterior cranial base, shortened maxilla and mandible, and abnormal palate and tongue. In the null mutant cranium, the calvarial bones, meninges including the dura mater and skin were not formed, and the brain was therefore exposed without the outer cover. The defective anterior cranial base in the mutants was attributable to the lack of presphenoid bone and the flexed cranial base angle, which was in contrast with the flat cranial base of wild-type mice. Furthermore, in the null mutants, palatal shelf growth was impaired because of the early elevation of the palatal shelves, resulting in a narrow palate and oral cavity, which were consistently associated with a small size of the tongue. These craniofacial anomalies could be the result of the defective development of neural crest cells. Taken together, it is supposed that Hes1 signaling plays an essential role in regulating the development of various craniofacial structures derived from the cranial neural crest cells.

Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus

Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. We used Xenopus laevis as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, we demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, we analyzed the function of phospholipase C beta 3 (PLC) in Xenopus development and showed that PLC is the effector of 5-HT2B during craniofacial development. Our results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to our understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch.

Cadherin 6B induces BMP signaling and de-epithelialization during the epithelial mesenchymal transition of the neural crest

The development of neural crest cells involves an epithelial-mesenchymal transition (EMT) associated with the restriction of cadherin 6B expression to the pre-migratory neural crest cells (PMNCCs), as well as a loss of N-cadherin expression. We find that cadherin 6B, which is highly expressed in PMNCCs, persists in early migrating neural crest cells and is required for their emigration from the neural tube. Cadherin 6B-expressing PMNCCs exhibit a general loss of epithelial junctional polarity and acquire motile properties before their delamination from the neuroepithelium. Cadherin 6B selectively induces the de-epithelialization of PMNCCs, which is mediated by stimulation of BMP signaling, whereas N-cadherin inhibits de-epithelialization and BMP signaling. As BMP signaling also induces cadherin 6B expression and represses N-cadherin, cadherin-regulated BMP signaling may create two opposing feedback loops. Thus, the overall EMT of neural crest cells occurs via two distinct steps: a cadherin 6B and BMP signaling-mediated de-epithelialization, and a subsequent delamination through the basement membrane.

Regulation of cadherin expression in the chicken neural crest by the Wnt/ β-catenin signaling pathway

In neural crest cell development, the expression of the cell adhesion proteins cadherin-7 and cadherin-11 commences after delamination of the neural crest cells from the neuroepithelium. The canonical Wnt signaling pathway is known to drive this delamination step and is a candidate for inducing expression of these cadherins at this time. This project was initiated to investigate the role of canonical Wnt signaling in the expression of cadherin-7 and cadherin-11 by treating neural crest cells with Wnt3a ligand. Expression of cadherin-11 was first confirmed in the neural crest cells for the chicken embryo. The changes in the expression level of cadherin-7 and -11 following the treatment with Wnt3a ligand were studied using real-time RT-PCR and immunostaining. Statistically significant up-regulation in the mRNA expression of cadherin-7 and cadherin-11 and in the amount of cadherin-7 and cadherin-11 protein found in cell-cell interfaces between neural crest cells was observed in response to Wnt, demonstrating that cadherin-7 and cadherin-11 expressed by the migrating neural crest cells can be regulated by the canonical Wnt pathway.

Analysis of early human neural crest development

The outstanding migration and differentiation capacities of neural crest cells (NCCs) have fascinated scientists since Wilhelm His described this cell population in 1868. Today, after intense research using vertebrate model organisms, we have gained considerable knowledge regarding the origin, migration and differentiation of NCCs. However, our understanding of NCC development in human embryos remains largely uncharacterized, despite the role the neural crest plays in several human pathologies. Here, we report for the first time the expression of a battery of molecular markers before, during, or following NCC migration in human embryos from Carnegie Stages (CS) 12 to 18. Our work demonstrates the expression of Sox9, Sox10 and Pax3 transcription factors in premigratory NCCs, while actively migrating NCCs display the additional transcription factors Pax7 and AP-2α. Importantly, while HNK-1 labels few migrating NCCs, p75 NTR labels a large proportion of this population. However, the broad expression of p75 NTR – and other markers – beyond the neural crest stresses the need for the identification of additional markers to improve our capacity to investigate human NCC development, and to enable the generation of better diagnostic and therapeutic tools.

Elevated glucose induces congenital heart defects by altering the expression of tbx5, tbx20, and has2 in developing zebrafish embryos

Maternal diabetes increases the risk of congenital heart defects in infants, and hyperglycemia acts as a major teratogen. Multiple steps of cardiac development, including endocardial cushion morphogenesis and development of neural crest cells, are challenged under elevated glucose conditions. However, the direct effect of hyperglycemia on embryo heart organogenesis remains to be investigated. Zebrafish embryos in different stages were exposed to D-glucose for 12 or 24 hr to determine the sensitive window during early heart development. In the subsequent study, 6 hr post-fertilization embryos were treated with either 25 mmol/liter D-glucose or L-glucose for 24 hr. The expression of genes was analyzed by whole-mount in situ hybridization. The highest incidence of cardiac malformations was found during 6-30 hpf exposure periods. After 24 hr exposure, D-glucose-treated embryos exhibited significant developmental delay and diverse cardiac malformations, but embryos exposed to L-glucose showed no apparent phenotype. Further investigation of the origin of heart defects showed that cardiac looping was affected earliest, while the specification of cardiac progenitors and heart tube assembly were complete. Moreover, the expression patterns of tbx5, tbx20, and has2 were altered in the defective hearts. Our data demonstrate that elevated glucose alone induces cardiac defects in zebrafish embryos by altering the expression pattern of tbx5, tbx20, and has2 in the heart. We also show the first evidence that cardiac looping is affected earliest during heart organogenesis. These research results are important for devising preventive and therapeutic strategies aimed at reducing the occurrence of congenital heart defects in diabetic pregnancy.