Sema3E is required for migration of cranial neural crest cells in zebrafish: Implications for the pathogenesis of CHARGE syndrome

CHARGE syndrome is a congenital disorder with multiple malformations in the craniofacial structures, and cardiovascular and genital systems, which are mainly affected by neural crest defects caused by loss‐of‐function mutations within chromodomain helicase DNA‐binding protein 7 (CHD7). However, many patients with CHARGE syndrome test negative for CHD7. Semaphorin 3E (sema3E) is a gene reported to be mutated in patients with CHARGE syndrome. However, its role in the pathogenesis of CHARGE syndrome has not been verified experimentally. Here, we report that the knockdown of sema3E results in severe craniofacial malformations, including small eyes, defective cartilage and an abnormal number of otoliths in zebrafish embryos, which resemble the major features of CHARGE syndrome. Further analysis reveals that the migratory cranial neural crest cells are scattered in the region of the hindbrain, and the postmigratory neural crest cells are reduced in the pharyngeal arches upon sema3E knockdown. Notably, immunostaining and time‐lapse imaging analyses of a neural crest cell‐labelled transgenic fish line, sox10:EGFP, show that the migration of cranial neural crest cells is severely impaired, and many of these cells are misrouted upon sema3E knockdown. Furthermore, the sox10‐expressing cranial neural crest cells are scattered in chd7 homozygous mutants, which phenocopied the phenotype in sema3E morphants. Overexpression of sema3E rescues the phenotype of scattered cranial neural crest cells in chd7 homozygotes, indicating that chd7 may control the expression of sema3E to regulate cranial neural crest cell migration. Collectively, our data demonstrate that sema3E is involved in the pathogenesis of CHARGE syndrome by modulating cranial neural crest cell migration.     

Expression of actin‐binding proteins and requirement for actin‐depolymerizing factor in chick neural crest cells

Background: Neural crest cells are multipotent cells that migrate extensively throughout vertebrate embryos to form diverse lineages. Cell migration requires polarized, organized actin networks that provide the driving force for motility. Actin‐binding proteins that regulate neural crest cell migration are just beginning to be defined. Results: We recently identified a number of actin‐associated factors through proteomic profiling of methylated proteins in migratory neural crest cells. Here, we report the previously undocumented expression pattern of three of these proteins in chick early neural crest development: doublecortin (DCX), tropomyosin‐1 (TPM‐1), and actin depolymerizing factor (ADF). All three genes are expressed with varying degrees of specificity and intensity in premigratory and migratory neural crest cells, and their resulting proteins exhibit distinct subcellular localization in migratory neural crest cells. Morpholino knock down of ADF reveals it is required for Sox10 gene expression, but minimally important during neural crest migration. Conclusions: Neural crest cells express DCX, TPM‐1, and ADF. ADF is necessary during neural crest specification, but largely dispensable for migration. Developmental Dynamics 243:731–739, 2014. © 2013 Wiley Periodicals, Inc.     

Wnt11r is required for cranial neural crest migration

wnt11r is a recently identified member of the Wnt family of genes, which has been proposed to be the true Xenopus homologue to the mammalian wnt11 gene. In this study we have examined the role of wnt11r on neural crest development. Expression analysis of wnt11r and comparison with the neural crest marker snail2 and the noncanonical Wnt, wnt11, shows wnt11r is expressed at the medial or neural plate side of the neural crest while wnt11 is expressed at the lateral or epidermal side. Injection of wnt11r morpholino leads to strong inhibition of neural crest migration with no effect on neural crest induction or maintenance. This effect can be rescued by co‐injection of Wnt11r but not by Wnt11 mRNA, demonstrating the specificity of the loss of function treatment. Finally, neural crest graft experiments show that wnt11r is required in a non–cell‐autonomous manner to control neural crest migration. Developmental Dynamics 237:3404–3409, 2008. © 2008 Wiley‐Liss, Inc.     

Pard3 regulates contact between neural crest cells and the timing of Schwann cell differentiation but is not essential for neural crest migration or myelination

Background : Schwann cells, which arise from the neural crest, are the myelinating glia of the peripheral nervous system. During development neural crest and their Schwann cell derivatives engage in a sequence of events that comprise delamination from the neuroepithelium, directed migration, axon ensheathment, and myelin membrane synthesis. At each step neural crest and Schwann cells are polarized, suggesting important roles for molecules that create cellular asymmetries. In this work we investigated the possibility that one polarity protein, Pard3, contributes to the polarized features of neural crest and Schwann cells that are associated with directed migration and myelination. Results : We analyzed mutant zebrafish embryos deficient for maternal and zygotic pard3 function. Time‐lapse imaging revealed that neural crest delamination was normal but that migrating cells were disorganized with substantial amounts of overlapping membrane. Nevertheless, neural crest cells migrated to appropriate peripheral targets. Schwann cells wrapped motor axons and, although myelin gene expression was delayed, myelination proceeded to completion. Conclusions : Pard3 mediates contact inhibition between neural crest cells and promotes timely myelin gene expression but is not essential for neural crest migration or myelination. Developmental Dynamics 243:1511–1523, 2014. © 2014 Wiley Periodicals, Inc.     

Neuregulin‐1 is a chemoattractant and chemokinetic molecule for trunk neural crest cells

Background: Trunk neural crest cells migrate rapidly along characteristic pathways within the developing vertebrate embryo. Proper trunk neural crest cell migration is necessary for the morphogenesis of much of the peripheral nervous system, melanocytes, and the adrenal medulla. Numerous molecules help guide trunk neural crest cell migration throughout the early embryo. Results: The trophic factor NRG1 is a chemoattractant through in vitro chemotaxis assays and in vivo silencing via a DN‐erbB receptor. Interestingly, we also observed changes in migratory responses consistent with a chemokinetic effect of NRG1 in trunk neural crest velocity. Conclusions: NRG1 is a trunk neural crest cell chemoattractant and chemokinetic molecule. Developmental Dynamics 247:888–902, 2018.. © 2018 Wiley Periodicals, Inc.     

Genetic disruption of CYP26B1 severely affects development of neural crest derived head structures,but does not compromise hindbrain patterning

Cyp26b1 encodes a cytochrome‐P450 enzyme that catabolizes retinoic acid (RA), a vitamin A derived signaling molecule. We have examined Cyp26b1^?/? mice and report that mutants exhibit numerous abnormalities in cranial neural crest cell derived tissues. At embryonic day (E) 18.5 Cyp26b1^?/? animals exhibit a truncated mandible, abnormal tooth buds, reduced ossification of calvaria, and are missing structures of the maxilla and nasal process. Some of these abnormalities may be due to defects in formation of Meckel's cartilage, which is truncated with an unfused distal region at E14.5 in mutant animals. Despite the severe malformations, we did not detect any abnormalities in rhombomere segmentation, or in patterning and migration of anterior hindbrain derived neural crest cells. Abnormal migration of neural crest cells toward the posterior branchial arches was observed, which may underlie defects in larynx and hyoid development. These data suggest different periods of sensitivity of anterior and posterior hindbrain neural crest derivatives to elevated levels of RA in the absence of CYP26B1. Developmental Dynamics 238:732–745, 2009. © 2009 Wiley‐Liss, Inc.     

Pdgfra and Pdgfrb genetically interact during craniofacial development

Background: One of the most prevalent congenital birth defects is cleft palate. The palatal skeleton is derived from the cranial neural crest and platelet‐derived growth factors (Pdgf) are critical in palatogenesis. Of the two Pdgf receptors, pdgfra is required for neural crest migration and palatogenesis. However, the role pdgfrb plays in the neural crest, or whether pdgfra and pdgfrb interact during palatogenesis is unclear. Results: We find that pdgfrb is dispensable for craniofacial development in zebrafish. However, the palatal defect in pdgfra;pdgfrb double mutants is significantly more severe than in pdgfra single mutants. Data in mouse suggest this interaction is conserved and that neural crest requires both genes. In zebrafish, pdgfra and pdgfrb are both expressed by neural crest within the pharyngeal arches, and pharmacological analyses demonstrate Pdgf signaling is required at these times. While neither proliferation nor cell death appears affected, time‐lapsed confocal analysis of pdgfra;pdgfrb mutants shows a failure of proper neural crest condensation during palatogenesis. Conclusions: We provide data showing that pdgfra and pdgfrb interact during palatogenesis in both zebrafish and mouse. In zebrafish, this interaction affects proper condensation of maxillary neural crest cells, revealing a previously unknown interaction between Pdgfra and Pdgfrb during palate formation. Developmental Dynamics 245:641–652, 2016. © 2016 Wiley Periodicals, Inc.     

Antagonistic activities of Rho and Rac GTPases underlie the transition from neural crest delamination to migration

Background: Neural crest progenitors arise as epithelial cells and then undergo a transition into mesenchyme that generates motility. Previously, we showed that active Rho maintains crest cells in the epithelial conformation by keeping stress fibers and membrane‐bound N‐cadherin. Results: While Rho disappears from cell membranes upon delamination, active Rac1 becomes apparent in lamellipodia of mesenchymal cells. Loss of Rac1 function at trunk levels inhibited NC migration but did not prevent cell emigration that is associated with N‐cadherin downregulation and G1/S transition. Furthermore, inhibition of Rho stimulated premature Rac1 activity and consequent formation of lamellipodia, leading to NC migration. To examine whether timely migration influences cell fate, Rac1 activity was transiently inhibited to delay dispersion of early NC cells that generate neural derivatives, and its activity was restored by the time of melanoblast migration. Even if confronted with a melanocytic environment, late‐dispersing progenitors colonized sensory ganglia where they generated neurons and glia. Conclusions: In the context of crest delamination and migration, activities of Rho and Rac are differential, sequential, and antagonistic. Furthermore, transient inhibition of Rac1 that delays the onset of crest dispersion raises the possibility that the fate of trunk neural progenitors might be restricted prior to migration. Developmental Dynamics 241:1155–1168, 2012. © 2012 Wiley Periodicals, Inc.     

Notch pathway regulation of neural crest cell development in vivo

Background : The function of Notch signaling in murine neural crest–derived cell lineages in vivo was examined. Results : Conditional gain (Wnt1Cre;Rosa^Notch) or loss (Wnt1Cre;RBP‐J^f/f) of Notch signaling in neural crest cells (NCCs) in vivo results in craniofacial, cardiac, and trunk abnormalities. Severe craniofacial malformations are apparent in Wnt1Cre;Rosa^Notch embryos, while less severe skull abnormalities are evident in Wnt1Cre;RBP‐J^f/f mice. Deficient cardiac neural crest migration, resulting in cardiac outflow tract malformations, occurs with increased or decreased Notch signaling in NCCs. Smooth muscle cell differentiation also is impaired in pharyngeal NCC derivatives in both Wnt1Cre;Rosa^Notch and Wnt1Cre;RBP‐J^f/f embryos. Neurogenesis is absent and gliogenesis is increased in the dorsal root ganglia of Wnt1Cre;Rosa^Notch embryos, while neurogenesis is increased and gliogenesis is decreased in Wnt1Cre;RBP‐J^f/f embryos. Conclusions : Together, these studies demonstrate essential cell‐autonomous roles for appropriate levels of Notch signaling during NCC migration, proliferation, and differentiation with critical implications in craniofacial, cardiac, and neurogenic development and disease. Developmental Dynamics 241:376–389, 2012. © 2011 Wiley Periodicals, Inc.     

Immunolocalization of N-CAM in the heart of the early developing rat embryo

Background: Neural cell adhesion molecule (N-CAM) is important in the migration of neural crest cells and is expressed in the developing heart. The pattern of expression of N-CAM in the heart of early rat embryos was investigated to shed light on the potential role of N-CAM in cardiac neural crest cell migration. Methods: N-CAM expression was studied by immunohistochemistry in Sprague-Dawley rat hearts between embryonic days 11.5 and 15.5 HNK-1 immunoreactivity was also investigated for comparison with that of N-CAM. Results: A continuity of N-CAM immunoreactivity was transiently detected from the outflow tract to the recurrent nerve. N-CAM was also expressed around the sinus venosus, inferior vena cava, sinotrial septum, and coronary sinus, as well as on mesenchymal cells in the atrioventricular endocardial cushion tissues. Conclusions: The continuous N-CAM immunoreactivity from the outflow tract to the recurrent nerve appeared to represent the pathway along which cardiac neural crest cells migrate. N-CAM-immunoreactive sites around the sinus venosus may correspond to migrating neural crest cells that differentiate into nerve fibers or cardiac ganglia. Results indicate that N-CAM may play an important role in the migration, proliferation, and transformation of neural crest cells, thereby contributing to cardiac morphogenesis and to innervation around the heart and great arteries. © 1995 Wiley-Liss, Inc.     

Late‐emigrating trunk neural crest cells in turtle embryos generate an osteogenic ectomesenchyme in the plastron

Background : The turtle plastron is composed of a keratinized epidermis overlying nine dermal bones. Its developmental origin has been controversial; recent evidence suggests that the plastral bones derive from trunk neural crest cells (NCCs). Results: This study extends the observations that there is a turtle‐specific, second wave of trunk NCC delamination and migration, after the original NCCs have reached their destination and differentiated. This second wave was confirmed by immunohistochemistry in whole‐mounts and serial sections, by injecting DiI (1,1′, di‐octadecyl‐3,3,3′,3′,‐tetramethylindo‐carbocyanine perchlorate) into the lumen of the neural tube and tracing labeled cells into the plastron, and by isolating neural tubes from older turtle embryos and observing delaminating NCCs. This later migration gives rise to a plastral ectomesenchyme that expresses NCC markers and can be induced to initiate bone formation. Conclusions: The NCCs of this second migration have properties similar to those of the earlier NCCs, but also express markers characteristic of cranial NCCs. The majority of the cells of the plastron mesenchyme express neural crest markers, and have osteogenic differentiation capabilities that are similar or identical to craniofacial ectomesenchyme. Our evidence supports the contention that turtle plastron bones are derived from a late emigrating population of cells derived from the trunk neural crest. Developmental Dynamics 242:1223–1235, 2013. © 2013 Wiley Periodicals, Inc.     

A segmented pattern of cell death during development of the chick embryo

Summary During the early development of the chick embryo, specific groups of cells die in characteristic patterns. In this study, Nile Blue sulphate staining was used to reveal a novel pattern of segmentally repeated cell death in the paraxial mesoderm of the chick prior to stage 23. This pattern varies according to the developmental stage of the embryo and shifts rostrocaudally, corresponding to progressing somite differentiation. Initially, during early somite differentiation, cell death is restricted to the rostral half of the somite (the rostral pattern of cell death). After the somite has differentiated into dermomyotome and sclerotome, dead cells appear in superficial tissues in a pyramidal pattern which lies in register (rostrocaudally) with the central part of the sclerotome. Finally, small bands of dying cells are seen between the neural tube and the expanding sclerotome. This third pattern (the ventral path) lies in register with the rostral part of the caudal half of the sclerotome. We show by fluorescent labelling of the migrating neural crest that these patterns of cell death correspond to the routes of neural crest migration. In addition, serial sectioning of stage 23 chick embryos confirms that the position of dying cells correlates with the known routes of neural crest migration and with the sites of development of certain neural crest-derived tissues.     

Nosip functions during vertebrate eye and cranial cartilage development

Background: The nitric oxide synthase interacting protein (Nosip) has been associated with diverse human diseases including psychological disorders. In line, early neurogenesis of mouse and Xenopus is impaired upon Nosip deficiency. Nosip knockout mice show craniofacial defects and the down‐regulation of Nosip in the mouse and Xenopus leads to microcephaly. Until now, the exact underlying molecular mechanisms of these malformations were still unknown. Results: Here, we show that nosip is expressed in the developing ocular system as well as the anterior neural crest cells of Xenopus laevis. Furthermore, Nosip inhibition causes severe defects in eye formation in the mouse and Xenopus. Retinal lamination as well as dorso–ventral patterning of the retina were affected in Nosip‐depleted Xenopus embryos. Marker gene analysis using rax, pax6 and otx2 reveals an interference with the eye field induction and differentiation. A closer look on Nosip‐deficient Xenopus embryos furthermore reveals disrupted cranial cartilage structures and an inhibition of anterior neural crest cell induction and migration shown by twist, snai2, and egr2. Moreover, foxc1 as downstream factor of retinoic acid signalling is affected upon Nosip deficiency. Conclusions: Nosip is a crucial factor for the development of anterior neural tissue such the eyes and neural crest cells. Developmental Dynamics 247:1070‐1082, 2018. © 2018 Wiley Periodicals, Inc.