Perturbation of Hoxb5 signaling in vagal and trunk neural crest cells causes apoptosis and neurocristopathies in mice

Neural crest cells (NCCs) migrate from different regions along the anterior–posterior axis of the neural tube (NT) to form different structures. Defective NCC development causes congenital neurocristopathies affecting multiple NCC-derived tissues in human. Perturbed Hoxb5 signaling in vagal NCC causes enteric nervous system (ENS) defects. This study aims to further investigate if perturbed Hoxb5 signaling in trunk NCC contributes to defects of other NCC-derived tissues besides the ENS. We perturbed Hoxb5 signaling in NCC from the entire NT, and investigated its impact in the development of tissues derived from these cells in mice. Perturbation of Hoxb5 signaling in these NCC resulted in Sox9 downregulation, NCC apoptosis, hypoplastic sympathetic and dorsal root ganglia, hypopigmentation and ENS defects. Mutant mice with NCC-specific Sox9 deletion also displayed some of these phenotypes. In vitro and in vivo assays indicated that the Sox9 promoter was bound and trans-activated by Hoxb5. In ovo studies further revealed that Sox9 alleviated apoptosis induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Sox9 expression in chick NT. This study demonstrates that Hoxb5 regulates Sox9 expression in NCC and disruption of this signaling causes Sox9 downregulation, NCC apoptosis and multiple NCC-developmental defects. Phenotypes such as ENS deficiency, hypopigmentation and some of the neurological defects are reported in patients with Hirschsprung disease (HSCR). Whether dysregulation of Hoxb5 signaling and early depletion of NCC contribute to ENS defect and other neurocristopathies in HSCR patients deserves further investigation.     

Semaphorin signaling guides cranial neural crest cell migration in zebrafish

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We find that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs.     

Fgf20b is required for the ectomesenchymal fate establishment of cranial neural crest cells in zebrafish

In cranial skeletal development, the establishment of the ectomesenchymal lineage within the cranial neural crest is of great significance. Fgfs are polypeptide growth factors with diverse functions in development and metabolism. Fgf20b knockdown zebrafish embryos showed dysplastic neurocranial and pharyngeal cartilages. Ectomesenchymal cells from cranial neural crest cells were significantly decreased in Fgf20b knockdown embryos, but cranial neural crest cells with a non-ectomesnchymal fate were increased. However, the proliferation and apoptosis of cranial neural crest cells were essentially unchanged. Fgfr1 knockdown embryos also showed dysplastic neurocranial and pharyngeal cartilages. The present findings indicate that Fgf20b is required for ectomesenchymal fate establishment via the activation of Fgfr1 in zebrafish.     

A role for chemokine signaling in neural crest cell migration and craniofacial development

Neural crest cells (NCCs) are a unique population of multipotent cells that migrate along defined pathways throughout the embryo and give rise to many diverse cell types including pigment cells, craniofacial cartilage and the peripheral nervous system (PNS). Aberrant migration of NCCs results in a wide variety of congenital birth defects including craniofacial abnormalities. The chemokine Sdf1 and its receptors, Cxcr4 and Cxcr7, have been identified as key components in the regulation of cell migration in a variety of tissues. Here we describe a novel role for the zebrafish chemokine receptor Cxcr4a in the development and migration of cranial NCCs (CNCCs). We find that loss of Cxcr4a, but not Cxcr7b, results in aberrant CNCC migration defects in the neurocranium, as well as cranial ganglia dysmorphogenesis. Moreover, overexpression of either Sdf1b or Cxcr4a causes aberrant CNCC migration and results in ectopic craniofacial cartilages. We propose a model in which Sdf1b signaling from the pharyngeal arch endoderm and optic stalk to Cxcr4a expressing CNCCs is important for both the proper condensation of the CNCCs into pharyngeal arches and the subsequent patterning and morphogenesis of the neural crest derived tissues.     

Disruption of Smad4 in neural crest cells leads to mid-gestation death with pharyngeal arch, craniofacial and cardiac defects

TGFβ/BMP signaling pathways are essential for normal development of neural crest cells (NCCs). Smad4 encodes the only common Smad protein in mammals, which is a critical nuclear mediator of TGFβ/BMP signaling. In this work, we sought to investigate the roles of Smad4 for development of NCCs. To overcome the early embryonic lethality of Smad4 null mice, we specifically disrupted Smad4 in NCCs using a Cre/loxP system. The mutant mice died at mid-gestation with defects in facial primordia, pharyngeal arches, outflow tract and cardiac ventricles. Further examination revealed that mutant embryos displayed severe molecular defects starting from E9.5. Expression of multiple genes, including Msx1, 2, Ap-2α, Pax3, and Sox9, which play critical roles for NCC development, was downregulated by NCC disruption of Smad4. Moreover, increased cell death was observed in pharyngeal arches from E10.5. However, the cell proliferation rate in these areas was not substantially altered. Taken together, these findings provide compelling genetic evidence that Smad4-mediated activities of TGFβ/BMP signals are essential for appropriate NCC development.     

Crispld2 is required for neural crest cell migration and cell viability during zebrafish craniofacial development

The CAP superfamily member, CRISPLD2, has previously been shown to be associated with nonsyndromic cleft lip and palate (NSCLP) in human populations and to be essential for normal craniofacial development in the zebrafish. Additionally, in rodent models, CRISPLD2 has been shown to play a role in normal lung and kidney development. However, the specific role of CRISPLD2 during these developmental processes has yet to be determined. In this study, it was demonstrated that Crispld2 protein localizes to the orofacial region of the zebrafish embryo and knockdown of crispld2 resulted in abnormal migration of neural crest cells (NCCs) during both early and late time points. An increase in cell death after crispld2 knockdown as well as an increase in apoptotic marker genes was also shown. This data suggests that Crispld2 modulates the migration, differentiation, and/or survival of NCCs during early craniofacial development. These results indicate an important role for Crispld2 in NCC migration during craniofacial development and suggests involvement of Crispld2 in cell viability during formation of the orofacies. genesis 53:660–667, 2015. © 2015 Wiley Periodicals, Inc.     

Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Neural crest cells (NCCs) are a multipotent embryonic cell population that contributes to the formation of various craniofacial structures including teeth. It has been generally believed that dental enamel is an ectodermal derivative, whereas the dentin–pulp complex and the surrounding supporting tissues originate from NCC-derived mesenchyme. These traditional concepts stem mainly from several early studies of fishes and amphibians. Recently, Wnt1-Cre/R26R mice, a mouse model for NCC lineage analysis, revealed the contribution of NCCs to mammalian tooth development. However, the discrepancy of expression patterns between different NCC-specific transgenic mouse lines makes it compulsory to revisit the cell lineage in mammalian tooth development. Here, we reevaluated the NCC lineage during mouse tooth development by using P0-Cre/R26R mice, another NCC-specific transgenic mouse line. Inconsistent with the traditional concepts, we observed the potential contribution of NCCs to developing enamel organ and enamel formation. We also demonstrated that the P0-Cre transgene was specifically expressed in migrating NCC in the hindbrain region, where NCC contributes to tooth, validating their applicability for NCC lineage analysis. Our unanticipated finding may change the general understanding of tooth development and provide new insights into dental stem cell biology.     

Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.     

inka1b expression in the head mesoderm is dispensable for facial cartilage development

Inka box actin regulator 1 (Inka1) is a novel protein identified in Xenopus and is found in vertebrates. While Inka1 is required for facial skeletal development in Xenopus and zebrafish, it is dispensable in mice despite its conserved expression in the cranial neural crest, indicating that Inka1 function in facial skeletal development is not conserved among vertebrates. Zebrafish bears two paralogs of inka1 (inka1a and inka1b) in the genome, with the biological roles of inka1b barely known. Here, we analyzed the expression and function of inka1b during facial skeletal development in zebrafish. inka1b was expressed sequentially in the head mesoderm adjacent to the pharyngeal pouches essential for facial skeletal development at the stage of arch segmentation. However, a loss-of-function mutation in inka1b displayed normal head development, including the pouches and facial cartilages. The normal head of inka1b mutant fish was unlikely a result of the genetic redundancy of inka1b with inka1a, given the distinct expression of inka1a and inka1b in the cranial neural crest and head mesoderm, respectively, during craniofacial development. Our findings suggest that the inka1b expression in the head mesoderm might not be essential for head development in zebrafish.     

Cranial neural crest cell migration in cockatiel Nymphicus hollandicus (Aves: Psittaciformes)

Parrots have developed unique jaw muscles in their evolutionary history. The M. pseudomasseter, which completely covers the lateral side of the jugal bar, is regarded as a jaw muscle unique to parrots. In a previous study, I presented a hypothesis on the relevance of modifications in the regulation of cranial neural crest cell (NCC) development to the generation of this novel jaw muscle based on histological analyses (Tokita [2004] J Morphol 259:69-81). In the present study, I investigated distribution and migration patterns of cranial neural crest cells (NCCs) through parrot embryogenesis with immunohistochemical techniques to further understand the role of cranial NCCs in the evolution of the M. pseudomasseter, and to provide new information on the relative plasticity in cranial NCC migration at early stages of avian development. The basic nature of cranial NCC development was mostly conserved between chick and parrot. In both, cranial NCCs migrated from the dorsal tip of the neural tube in a ventral direction. Three major populations were identified in their cranial NCCs. Migration pathways of these cells were almost identical between chick and parrot. The principal difference was seen in the relative timing of cranial NCC migration. In the parrot, cranial NCC migration into the first pharyngeal arch was more advanced than in the chick at early stages of development. Such a temporal shift in cranial NCC migration might influence architectural patterning of parrot jaw muscles that generates new muscle like M. pseudomasseter.     

Elucidating timing and function of endothelin-A receptor signaling during craniofacial development using neural crest cell-specific gene deletion and receptor antagonism

Cranial neural crest cells (NCCs) play an intimate role in craniofacial development. Multiple signaling cascades participate in patterning cranial NCCs, some of which are regulated by endothelin-A receptor (Ednra) signaling. Ednra^−/− embryos die at birth from severe craniofacial defects resulting from disruption of neural crest cell patterning and differentiation. These defects include homeotic transformation of lower jaw structures into upper jaw-like structures, suggesting that some cephalic NCCs alter their “identity” in the absence of Ednra signaling. To elucidate the temporal necessity for Ednra signaling in vivo, we undertook two strategies. We first used a conditional knockout strategy in which mice containing a conditionally targeted Ednra allele (Ednra^fl) were bred with mice from the Hand2-Cre and Wnt1-Cre transgenic mouse strains, two strains in which Cre expression occurs at different time periods within cranial NCCs. In our second approach, we used an Ednra-specific antagonist to treat wild type pregnant mice between embryonic days E8.0 and E10.0, a time frame encompassing the early migration and proliferation of cranial NCCs. The combined results suggest that Ednra function is crucial for NCC development between E8.25 and E9.0, a time period encompassing the arrival of NCCs in the arches and/or early post-migratory patterning. After this time period, Ednra signaling is dispensable. Interestingly, middle ear structures are enlarged and malformed in a majority of Ednra^fl/fl;Wnt1-Cre embryos, instead resembling structures found in extinct predecessors of mammals. These observations suggest that the advent of Ednra signaling in cranial NCCs may have been a crucial event in the evolution of the mammalian middle ear ossicles.     

In Vivo Expression of EphrinA5-Fc in Mice Results in Cephalic Neural Crest Agenesis and Craniofacial Abnormalities

Eph receptors and their ligands ephrins have been implicated in guiding the directed migration of neural crest cells (NCCs). In this study, we found that Wnt1-Cre-mediated expression of ephrinA5-Fc along the dorsal midline of the dien- and mesencephalon resulted in severe craniofacial malformation of mouse embryo. Interestingly, expression of cephalic NCC markers decreased significantly in the frontonasal process and branchial arches 1 and 2, which are target areas for the migratory cephalic NCCs originating in the dien- and mesencephalon. In addition, these craniofacial tissues were much smaller in mutant embryos expressing ephrinA5-Fc. Importantly, EphA7-positive cephalic NCCs were absent along the dorsal dien- and mesencephalon of mutant embryos expressing ephrinA5-Fc, suggesting that the generation of cephalic NCCs is disrupted due to ephrinA5-Fc expression. NCC explant experiments suggested that ephrinA5-Fc perturbed survival of cephalic NCC precursors in the dorsal midline tissue rather than affecting their migratory capacity, which was consistent with our previous report that expression of ephrinA5-Fc in the dorsal midline is responsible for severe neuroepithelial cell apoptotic death. Taken together, our findings strongly suggest that expression of ephrinA5-Fc decreases a population of cephalic NCC precursors in the dorsal midline of the dien- and mesencephalon, thereby disrupting craniofacial development in the mouse embryos.     

Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch

Morphogenesis of the vertebrate head relies on proper dorsal-ventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches. Endothelin-1 (Edn1)-induced signaling through the endothelin-A receptor (Ednra) is crucial for cranial NCC patterning within the mandibular portion of the first pharyngeal arch, from which the lower jaw arises. Deletion of Edn1, Ednra or endothelin-converting enzyme in mice causes perinatal lethality due to severe craniofacial birth defects. These include homeotic transformation of mandibular arch-derived structures into more maxillary-like structures, indicating a loss of NCC identity. All cranial NCCs express Ednra whereas Edn1 expression is limited to the overlying ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm of the mandibular arch as well as more caudal arches. To define the developmental significance of Edn1 from each of these layers, we used Cre/loxP technology to inactivate Edn1 in a tissue-specific manner. We show that deletion of Edn1 in either the mesoderm or endoderm alone does not result in cellular or molecular changes in craniofacial development. However, ectodermal deletion of Edn1 results in craniofacial defects with concomitant changes in the expression of early mandibular arch patterning genes. Importantly, our results also both define for the first time in mice an intermediate mandibular arch domain similar to the one defined in zebrafish and show that this region is most sensitive to loss of Edn1. Together, our results illustrate an integral role for ectoderm-derived Edn1 in early arch morphogenesis, particularly in the intermediate domain.     

Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest

The Pax3/7 gene family has a fundamental and conserved role during neural crest formation. In people, PAX3 mutation causes Waardenburg syndrome, and murine Pax3 is essential for pigment formation. However, it is unclear exactly how Pax3 functions within the neural crest. Here we show that pax3 is expressed before other pax3/7 members, including duplicated pax3b, pax7 and pax7b genes, early in zebrafish neural crest development. Knockdown of Pax3 protein by antisense morpholino oligonucleotides results in defective fate specification of xanthophores, with complete ablation in the trunk. Other pigment lineages are specified and differentiate. As a consequence of xanthophore loss, expression of pax7, a marker of the xanthophore lineage, is reduced in neural crest. Morpholino knockdown of Pax7 protein shows that Pax7 itself is dispensable for xanthophore fate specification, although yellow pigmentation is reduced. Loss of xanthophores after reduction of Pax3 correlates with a delay in melanoblast differentiation followed by significant increase in melanophores, suggestive of a Pax3-driven fate switch within a chromatophore precursor or stem cell. Analysis of other neural crest derivatives reveals that, in the absence of Pax3, the enteric nervous system is ablated from its inception. Therefore, Pax3 in zebrafish is required for specification of two specific lineages of neural crest, xanthophores and enteric neurons.     

Expression of five frizzleds during zebrafish craniofacial development

Wnt/Planar Cell Polarity (PCP) signaling is critical for proper animal development. While initially identified in Drosophila, this pathway is also essential for the proper development of vertebrates. Zebrafish mutants, defective in the Wnt/PCP pathway, frequently display defects in convergence and extension gastrulation movements and additional later abnormalities including problems with craniofacial cartilage morphogenesis. Although multiple Frizzled (Fzd) homologues, Wnt receptors, were identified in zebrafish, it is unknown which Fzd plays a role in shaping the early larvae head skeleton. In an effort to determine which Frizzleds are involved in this process, we analyzed the expression of five zebrafish frizzled homologues fzd2, 6, 7a, 7b, and 8a from 2–4 days post-fertilization (dpf). During the analyzed developmental time points fzd2 and fzd6 are broadly expressed throughout the head, while the expression of fzd7a, 7b and 8a is much more restricted. Closer examination revealed that fzd7b is expressed in the neural crest and the mesodermal core of the pharyngeal arches and in the chondrocytes of newly stacked craniofacial cartilage elements. However, fzd7a is only expressed in the neural crest of the pharyngeal arches and fzd8a is expressed in the pharyngeal endoderm.     

Neural crest cell-specific deletion of Rac1 results in defective cell-matrix interactions and severe craniofacial and cardiovascular malformations

The small GTP-binding protein Rac1, a member of the Rho family of small GTPases, has been implicated in regulation of many cellular processes including adhesion, migration and cytokinesis. These functions have largely been attributed to its ability to reorganize cytoskeleton. While the function of Rac1 is relatively well known in vitro, its role in vivo has been poorly understood. It has previously been shown that in neural crest cells (NCCs) Rac1 is required in a stage-specific manner to acquire responsiveness to mitogenic EGF signals. Here we demonstrate that mouse embryos lacking Rac1 in neural crest cells (Rac1/Wnt1-Cre) showed abnormal craniofacial development including regional ectodermal detachment associated with mesenchymal acellularity culminating in cleft face at E12. Rac1/Wnt1-Cre mutants also displayed inappropriate remodelling of pharyngeal arch arteries and defective outflow tract septation resulting in the formation of a common arterial trunk (‘persistent truncus arteriosus’ or PTA). The mesenchyme around the aortic sac also developed acellular regions, and the distal aortic sac became grossly dysmorphic, forming a pair of bilateral, highly dilated arterial structures connecting to the dorsal aortas. Smooth muscle cells lacking Rac1 failed to differentiate appropriately, and subpopulations of post-migratory NCCs demonstrated aberrant cell death and attenuated proliferation. These novel data demonstrate that while Rac1 is not required for normal NCC migration in vivo, it plays a critical cell-autonomous role in post-migratory NCCs during craniofacial and cardiac development by regulating the integrity of the craniofacial and pharyngeal mesenchyme.