Neural crest cell genes and the domestication syndrome: A comparative analysis of selection.

Neural crest cell genes control the migration of neural crest cells to multiple parts of developing vertebrate embryos. A recent hypothesis posits that the "domestication syndrome" characteristic of domesticated animals is driven by selection for tameness acting on neural crest cell genes, particularly those affecting cell migration. This is posited to explain why this syndrome involves many disparate phenotypic effects. These effects can be connected to deficits in neural crest cell migration. This hypothesis predicts that patterns of selection on these neural crest cell genes will differ between domesticated species and related wild species. Specifically, it predicts higher levels of positive selection on these genes in domesticated species, relative to closely related wild species. Here we test this prediction in a comparative framework. We obtained DNA sequences from a public database (NCBI) for eleven key neural crest cell genes from a set of thirty domesticated vertebrates and matched close relatives that remain wild. We used the program Contrast-FEL in the software suite HyPhy to compare the number of sites under positive selection (as measured by non-synonymous to synonymous nucleotide substitution rates across codons) between these two types of taxa in a phylogenetic framework. We found that domesticated lineages showed a consistently higher level of positive selection on these key genes, relative to their closely related wild counterparts. In addition, we found support for relaxation of selection and purifying selection. We argue that this result is consistent with an important role for these genes in the domestication syndrome.

Phox2b Immunohistochemical Staining in Detecting Enteric Neural Crest Cells in Hirschsprung Disease.

BACKGROUND: It can be challenging to recognize undifferentiated/immature ganglion cells, especially single forms. Ganglion cells and glia are derived from enteric neural crest cells (ENCCs), a group of autonomic nervous system (ANS)-lineage neural crest progenitors that PHOX2B regulates. Phox2b is an excellent marker for neoplastic and non-neoplastic ANS cells (eg, peripheral neuroblastic tumors [pNTs]). We hypothesized that Phox2b immunohistochemical staining (IHC) would also be useful for detecting ENCCs. METHODS: Hematoxylin and eosin, calretinin IHC, and Phox2b IHC were reviewed on 21 pull-through specimens and on a cohort of 12 rectal biopsies. RESULTS: Phox2b IHC demonstrated nuclear positivity in all of the ganglion cells across the different phases of differentiation without background staining. The Phox2b result correlated with the morphological findings, calretinin IHC results, and diagnoses based on the routine diagnostic method. The intensity was uniformly strong in the undifferentiated/immature forms and became variable in the mature forms; this pattern was similar to that seen in pNTs. CONCLUSION: Phox2b IHC was highly sensitive and specific for detecting ganglion cells. It worked especially well for immature ganglion cells, seen in premature neonates, and scattered single forms in transition zones. In basic research settings, Phox2b can be a useful marker for early differentiation of ENCCs.

Dual labeling of neural crest cells and blood vessels within chicken embryos using Chick(GFP) neural tube grafting and carbocyanine dye DiI injection.

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chick(GFP) embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. Chick(GFP)-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Novel method for isolating human melanoblasts from keratinocyte culture.

The characterization of melanoblasts is important for understanding their in vivo development, melanoma formation, and pigment-related disorders. However, no methods have been reported for the isolation of melanoblasts from human skin. Using a 'calcium-pulse' technique, involving the differentiation of human keratinocytes with high calcium and the subsequent spontaneous separation of the epidermal sheets, we effectively isolated human melanoblasts (keratinocyte-adapted melanoblasts, KaMBs) from keratinocyte culture. The KaMBs expressed early melanogenesis-related genes, including BRN2, which is a known melanoblast marker. Moreover, the KaMBs displayed much higher proliferative and growth capacities than the primary melanocytes. Considering that keratinocytes might provide an in vivo-like environment for KaMBs during isolation and in vitro culture, the 'calcium-pulse' technique offers an unprecedented, easy, and efficient method for the isolation of human melanoblasts, retaining the original characteristics of these cells.

Visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy.

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

GATA factors in human neuroblastoma: distinctive expression patterns in clinical subtypes.

BACKGROUND: The aim of this study is to elucidate the expression patterns of GATA transcription factors in neuroblastoma and the developing sympathetic nervous system (SNS). METHODS: GATA-2, -3 and -4 and their cofactor friend-of-GATA (FOG)-2 were investigated in primary neuroblastoma by immunohistochemistry, real-time RT-PCR (n=73) and microarray analysis (n=251). In addition, GATA-2, -3 and FOG-2 expression was determined by northern-blot hybridisation. In the developing murine SNS, Gata-4 and Fog-2 were examined by immunohistochemistry. RESULTS: Although Gata-2, -3 and Fog-2 are expressed in the developing nervous system, Gata-4 was not detected. In contrast, protein expression of all factors was observed in human neuroblastoma. Northern-blot hybridisation and real-time RT-PCR suggested specific expression patterns of the four genes in primary neuroblastoma, but did not show unequivocal results. In the large cohort examined by microarrays, a significant association of GATA-2, -3 and FOG-2 expression with low-risk features was observed, whereas GATA-4 mRNA levels correlated with MYCN-amplification. CONCLUSION: The transcription factors GATA-2 and -3, which are essential for normal SNS development, and their cofactor FOG-2 are downregulated in aggressive but not in favourable neuroblastoma. In contrast, upregulation of GATA-4 appears to be a common feature of this malignancy and might contribute to neuroblastoma pathogenesis.

Zwilling-A and -B, two related myelin proteins of teleosts, which originate from a single bicistronic transcript.

Myelination, the ensheathment of axons by membranes of highly specialized glial cells, has been a crucial innovation during early vertebrate evolution. It enables high nerve signal conduction velocities, while maintaining nervous system size and energy requirements at moderate levels. Consequently, myelination has been conserved in all extant gnathostome vertebrates. In a genomewide mRNA expression screen, we identified several novel neural crest and myelin-specific transcripts in the zebrafish (Danio rerio). Here, we describe the characterization of two proteins, Zwilling-A and -B (ZwiA and ZwiB), which are exclusively expressed in myelinating glia of teleosts. They are structurally homologous and are translated from a common, bicistronic transcript. No similarities to sequences or domains of other proteins were detected. Analysis of phylogeny, genomic organization, and genomic syntenies suggests that the zwi gene has appeared soon after the teleost-specific genome duplication event and evolved under conservative selective pressure. We hypothesize that ZwiA and ZwiB serve important physiological functions in teleost myelin.

Development of mesenchymal stem cells partially originate from the neural crest.

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells isolated from many adult tissues. Previous studies reported that MSCs can differentiate to both mesodermal and neural lineages by a phenomenon referred to as ''dedifferentiation'' or ''transdifferentiation''. However, since MSCs have only been defined in vitro, much of their development in vivo is still unknown. Here, we prospectively identified MSCs in the bone marrow from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. EGFP-positive MSCs formed spheres that expressed neural crest stem cell genes and differentiated into neurons, glial cells, and myofibroblasts. Interestingly, we observed MSCs both in the GFP(+) and GFP(-) fraction and found that there were no significant differences in the in vitro characteristics between these two populations. Our results suggest that MSCs in adult bone marrow have at least two developmental origins, one of which is the neural crest.

[Odontoblast-like cell phenotype differentiation of cranial neural crest stem cell in vivo].

OBJECTIVE: To investigate the feasibility of tooth regeneration by seeding cranial neural crest stem cell (CNCSC) in vivo. METHODS: Cranial neural tubes, dissected from mouse E9 d, were explanted onto fibronectin-coated dishes. CNCSC emigrated from the explanted neural tubes, and were cultured in a free-serum medium containing modified DMEM/F12. CNCSC, induced by FGF8, BMP2, TGFbeta1 and dentin matrix non-collagen protein (DMNCP), were cultured with collagen/chitosan, and implanted into the subcutaneous part of immunodeficiency mouse. The expression of collagen I/dentin sialophosphoprotein (DSPP) was analyzed by immunocytochemistry. RESULTS: With the scaffolds destroying, columnar cells possessing polarized nuclei and matrix produced by cells were showed in some regions. Immunohistochemical staining demonstrated that collagen type I and DSPP were expressed throughout the cytoplasm and matrix produced by cells. CONCLUSION: By tissue engineering approach, our experiments further verify the odontoblast-like cell phenotype differentiation of CNCSC in vivo.

Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development.

BACKGROUND: Congenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice. METHODS AND RESULTS: We analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos. CONCLUSION: It is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.

Generation of mice deficient for Lbx2, a gene expressed in the urogenital system, nervous system, and Pax3 dependent tissues.

Lbx2 is a member of the ladybird family of homeobox genes. The first murine ortholog identified, Lbx1, is required for hypaxial musculature and dorsal spinal cord neuron development. The second murine ortholog, Lbx2, is expressed in the developing urogenital and nervous systems. To elucidate the function of Lbx2, we generated a gene-targeted allele of Lbx2 in mice. Lbx2 deficiency did not impair mouse development, and Lbx2 null mice appeared healthy and fertile. Replacement of Lbx2 by the lacZ gene provides a valuable histological marker for Lbx2-expressing cells. Given the important role of Pax3 in neural crest, we intercrossed our Lbx2 deficient mice with Splotch Pax3 mutant mice to determine if Pax3 affects Lbx2 expression. There was reduced Lbx2 expression in dorsal root ganglia and cranial nerve ganglia with Pax3 deficiency, but not in the genital tubercle. This suggested that Pax3 is required for Lbx2 expression in affected neural crest-derived tissues.

Neural crest cell deficiency of c-myc causes skull and hearing defects.

The proto-oncogene c-myc has a central role in multiple processes important for embryonic development, including cell proliferation, growth, apoptosis, and differentiation. We have investigated the role of c-myc in neural crest by using Wnt1-Cre-mediated deletion of a conditional mutation of the c-myc gene. c-myc deficiency in neural crest resulted in viable adult mice that have defects in coat color, skull frontal bone, and middle ear ossicle development. Physiological hearing studies demonstrated a significant hearing deficit in the mutant mice. In this report, we focus on the craniofacial and hearing defects. To further examine neural crest cells affected by c-myc deficiency, we fate mapped Wnt1-Cre expressing neural crest cells using the ROSA26 Cre reporter transgene. The phenotype obtained demonstrates the critical role that c-myc has in neural crest during craniofacial development as well as in providing a model for examining human congenital skull defects and deafness.

Semaphorin 3d promotes cell proliferation and neural crest cell development downstream of TCF in the zebrafish hindbrain.

Neural crest cells (NCCs) are pluripotent migratory cells that are crucial to the development of the peripheral nervous system, pigment cells and craniofacial cartilage and bone. NCCs are specified within the dorsal ectoderm and undergo an epithelial to mesenchymal transition (EMT) in order to migrate to target destinations where they differentiate. Here we report a role for a member of the semaphorin family of cell guidance molecules in NCC development. Morpholino-mediated knockdown of Sema3d inhibits the proliferation of hindbrain neuroepithelial cells. In addition, Sema3d knockdown reduces markers of migratory NCCs and disrupts NCC-derived tissues. Similarly, expression of a dominant-repressor form of TCF (DeltaTCF) reduces hindbrain cell proliferation and leads to a disruption of migratory NCC markers. Moreover, expression of DeltaTCF downregulates sema3d RNA expression. Finally, Sema3d overexpression rescues reduced proliferation caused by DeltaTCF expression, suggesting that Sema3d lies downstream of Wnt/TCF signaling in the molecular pathway thought to control cell cycle in NCC precursors.

Hepatic stellate cells do not derive from the neural crest.

BACKGROUND/AIMS: Hepatic stellate cells (HSC) have been hypothesised to derive from the neural crest, based on their expression of multiple neural/neuroendocrine features and their contacts with autonomic nerve endings. METHODS: We studied the emergence of HSC in the liver during embryonic development in a transgenic mouse line expressing yellow fluorescent protein (YFP) in all neural crest cells and their derivatives. Cellular YFP expression in these mice was compared with desmin expression between embryonic day (E) 11.5 and adulthood. RESULTS: YFP was abundantly expressed in neural crest cells delaminating from the neural tube and in all known neural crest-derived structures and cell populations. In particular, YFP expressing cells perfectly mimicked the spatial and temporal pattern of enteric nervous system development from neural crest cells migrating from the postotic region. Cells within the adrenal medulla were also YFP positive. Analysis of the liver showed that desmin-expressing, stellate-shaped, perisinusoidally located HSC were evident from E11.5 onwards. However, no detectable YFP expression was seen in the developing liver or in HSC, from E11.5 until adulthood. CONCLUSIONS: These findings suggest HSC do not descend from the neural crest, and therefore may derive from the septum transversum mesenchyme, from endoderm or from the mesothelial liver capsule.

BMP and FGF-2 regulate neurogenin-2 expression and the differentiation of sensory neurons and glia.

We have examined the effects of signaling molecules and Notch signaling on the mechanisms regulating neurogenin (ngn)-2 expression. This ngn-2 is a transcription factor that is essential for the specification of early differentiating sensory neurons in the dorsal root ganglia. In the presence of bone morphogenetic protein (BMP), anti-ngn-2-positive cells appeared in mouse trunk neural crest cell cultures, and they expressed Brn3, indicating that ngn-2-expressing cells are sensory neurons. These cells did not differentiate after fibroblast growth factor (FGF)-2 treatment or after Notch activation. The suppression of ngn-2 expression by FGF-2 was recovered by treatment with a Notch signaling inhibitor. Thus, FGF-2 may prevent ngn-2 expression through Notch activation. Whereas BMP-4 inhibited glial differentiation, FGF-2 promoted gliogenesis by means of Notch activation. Our data suggest that BMP and FGF-2 act as positive and negative regulators in ngn-2 expression, respectively, and that these signaling molecules regulate the differentiation of sensory neurons and glia.

Abnormalities in neural crest cell migration in laminin alpha5 mutant mice.

Although numerous in vitro experiments suggest that extracellular matrix molecules like laminin can influence neural crest migration, little is known about their function in the embryo. Here, we show that laminin alpha5, a gene up-regulated during neural crest induction, is localized in regions of newly formed cranial and trunk neural folds and adjacent neural crest migratory pathways in a manner largely conserved between chick and mouse. In laminin alpha5 mutant mice, neural crest migratory streams appear expanded in width compared to wild type. Conversely, neural folds exposed to laminin alpha5 in vitro show a reduction by half in the number of migratory neural crest cells. During gangliogenesis, laminin alpha5 mutants exhibit defects in condensing cranial sensory and trunk sympathetic ganglia. However, ganglia apparently recover at later stages. These data suggest that the laminin alpha5 subunit functions as a cue that restricts neural crest cells, focusing their migratory pathways and condensation into ganglia. Thus, it is required for proper migration and timely differentiation of some neural crest populations.

Regulatory targets for transcription factor AP2 in Xenopus embryos.

The transcription factor AP2 (TFAP2) has an important role in regulating gene expression in both epidermis and neural crest cells. In order to further characterize these functions we have used a hormone inducible TFAP2alpha fusion protein in a Xenopus animal cap assay to identify downstream targets of this factor. The most common pattern comprised genes predominantly expressed in the epidermis. A second group was expressed at high levels in the neural crest, but all of these were also expressed in the epidermis as well as in other tissues in which TFAP2alpha has not been detected, suggesting modular control involving both TFAP2-dependent and TFAP2-independent components. In addition, a few strongly induced genes did not overlap at all in expression pattern with that of TFAP2alpha in the early embryo, and were also activated precociously in the experimentally manipulated ectoderm, and thus likely represent inappropriate regulatory interactions. A final group was identified that were repressed by TFAP2alpha and were expressed in the neural plate. These results provide further support for the importance of TFAP2alpha in ectoderm development, and also highlight the molecular linkage between the epidermis and neural crest in the Xenopus embryo.

Large-scale reprogramming of cranial neural crest gene expression by retinoic acid exposure.

Although retinoic acid (RA), the active form of vitamin A, is required for normal embryonic growth and development, it is also a powerful teratogen. Infants born to mothers exposed to retinoids during pregnancy have a 25-fold increased risk for malformations, nearly exclusively of cranial neural crest-derived tissues. To characterize neural crest cell responses to RA, we exposed murine crest cultures to teratogenic levels of RA and subjected their RNA to microarray-based gene expression profile analysis using Affymetrix MG-U74Av2 GeneChips. RNAs were isolated from independent cultures treated with 10(-6) M RA for 6, 12, 24, or 48 h. Statistical analyses of gene expression profile data facilitated identification of the 205 top-ranked differentially regulated genes whose expression was reproducibly changed by RA over time. Cluster analyses of these genes across the independently treated sample series revealed distinctive kinetic patterns of altered gene expression. The largest group was transiently affected within the first 6 h of exposure, representing early responding genes. Group 2 showed sustained induction by RA over all times, whereas group 3 was characterized by the suppression of a time-dependent expression increase normally seen in untreated cells. Additional patterns demonstrated time-dependent increased or decreased expression among genes not normally regulated to a significant extent. Gene function analysis revealed that more than one-third of all RA-regulated genes were associated with developmental regulation, including both canonical and noncanonical Wnt signaling pathways. Multiple genes associated with cell adhesion and cell cycle regulation, recognized targets for the biological effects of RA, were also affected. Taken together, these results support the hypothesis that the teratogenic effects of RA derive from reprogramming gene expression of a host of genes, which play critical roles during embryonic development regulating pathways that determine subsequent differentiation of cranial neural crest cells.

An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries.

BACKGROUND: The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. RESULTS: Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. CONCLUSIONS: Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online.

Roles of HNK-1 carbohydrate epitope and its synthetic glucuronyltransferase genes on migration of rat neural crest cells.

HNK-1 carbohydrate epitope is localized on the surface of avian neural crest cells (NCCs), and is necessary for their migration. However, it is still disputed whether the epitope works in similar ways in mammalian embryos. In this study, we found that HNK-1 carbohydrate epitope was specifically detected in some of the cranial ganglia, migrating trunk NCCs and some non-NCC derivatives in the rat embryo. Two genes encoding glucuronyltransferases that synthesize the HNK-1 epitope in vitro (GlcAT-P and GlcAT-D) were recently identified in the rat. Interestingly, the NCCs in the cranial ganglia expressed the GlcAT-D gene, whereas the migrating trunk NCCs expressed the GlcAT-P gene. To investigate in vivo functions of the GlcATs in the NCC migration further, we overexpressed GlcAT genes by electroporation in the cranial NCCs in cultured rat embryos. Transfection of both GlcAT genes resulted in efficient synthesis of the HNK-1 epitope in the NCCs. GlcAT-P overexpression increased distance of cranial NCC migration, whereas GlcAT-D overexpression did not show this effect. Our data suggest that the HNK-1 epitope synthesized by different GlcATs is involved in migration in the sublineages of the NCCs in the rat embryo, and that GlcAT-P and GlcAT-D mediate different effects on the NCC migration.