Yawei Kong

Neural Crest Development and Craniofacial Morphogenesis Is Coordinated by Nitric Oxide and Histone Acetylation

Cranial neural crest (CNC) cells are patterned and coalesce to facial prominences that undergo convergence and extension to generate the craniofacial form. We applied a chemical genetics approach to identify pathways that regulate craniofacial development during embryogenesis. Treatment with the nitric oxide synthase inhibitor 1-(2-[trifluoromethyl] phenyl) imidazole (TRIM) abrogated first pharyngeal arch structures and induced ectopic ceratobranchial formation. TRIM promoted a progenitor CNC fate and inhibited chondrogenic differentiation, which were mediated through impaired nitric oxide (NO) production without appreciable effect on global protein S-nitrosylation. Instead, TRIM perturbed hox gene patterning and caused histone hypoacetylation. Rescue of TRIM phenotype was achieved with overexpression of histone acetyltransferase kat6a, inhibition of histone deacetylase, and complementary NO. These studies demonstrate that NO signaling and histone acetylation are coordinated mechanisms that regulate CNC patterning, differentiation, and convergence during craniofacial morphogenesis.

Requirement for frzb and fzd7a in cranial neural crest convergence and extension mechanisms during zebrafish palate and jaw morphogenesis

Regulation of convergence and extension by wnt-frizzled signaling is a common theme in embryogenesis. This study examines the functional requirements of frzb and fzd7a in convergence and extension mechanisms during craniofacial development. Using a morpholino knockdown approach, we found that frzb and fzd7a are dispensable for directed migration of the bilateral trabeculae, but necessary for the convergence and extension of the palatal elements, where the extension process is mediated by chondrocyte proliferation, morphologic change and intercalation. In contrast, frzb and fzd7a are required for convergence of the mandibular prominences, where knockdown of either frzb or fzd7a resulted in complete loss of lower jaw structures. Further, we found that bapx1 was specifically downregulated in the wnt9a/frzb/fzd7a morphants, while general neural crest markers were unaffected. In addition, expression of wnt9a and frzb was also absent in the edn-/- mutant. Notably, over-expression of bapx1 was sufficient to partially rescue mandibular elements in the wnt9a/frzb/fzd7a morphants, demonstrating genetic epistasis of bapx1 acting downstream of edn1 and wnt9a/frzb/fzd7a in lower jaw development. This study underscores the important role of wnt-frizzled signaling in convergence and extension in palate and craniofacial morphogenesis, distinct regulation of upper vs. lower jaw structures, and integration of wnt-frizzled with endothelin signaling to coordinate shaping of the facial form.

Functional Analysis of SPECC1L in Craniofacial Development and Oblique Facial Cleft Pathogenesis

Background: Oblique facial clefts, also known as Tessier clefts, are severe orofacial clefts, the genetic basis of which is poorly understood. Human genetics studies revealed that disruption in SPECC1L resulted in oblique facial clefts, demonstrating that oblique facial cleft malformation has a genetic basis. An important step toward innovation in treatment of oblique facial clefts would be improved understanding of its genetic pathogenesis. The authors exploit the zebrafish model to elucidate the function of SPECC1L by studying its homolog, specc1lb. Methods: Gene and protein expression analysis was carried out by reverse-transcriptase polymerase chain reaction and immunohistochemistry staining. Morpholino knockdown, mRNA rescue, lineage tracing and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assays were performed for functional analysis. Results: Expression of specc1lb was detected in epithelia juxtaposed to chondrocytes. Knockdown of specc1lb resulted in bilateral clefts between median and lateral elements of the ethmoid plate, structures analogous to the frontonasal process and the paired maxillary processes. Lineage tracing analysis revealed that cranial neural crest cells contributing to the frontonasal prominence failed to integrate with the maxillary prominence populations. Cells contributing to lower jaw structures were able to migrate to their destined pharyngeal segment but failed to converge to form mandibular elements. Conclusions: These results demonstrate that specc1lb is required for integration of frontonasal and maxillary elements and convergence of mandibular prominences. The authors confirm the role of SPECC1L in orofacial cleft pathogenesis in the first animal model of Tessier cleft, providing morphogenetic insight into the mechanisms of normal craniofacial development and oblique facial cleft pathogenesis.

Featured Article: Characterization of cultured multipotent zebrafish neural crest cells

The neural crest is a unique cell population associated with vertebrate evolution. Neural crest cells (NCCs) are characterized by their multipotent and migratory potentials. While zebrafish is a powerful genetic model organism, the isolation and culture of zebrafish NCCs would provide a useful adjunct to fully interrogate the genetic networks that regulate NCC development. Here we report for the first time the isolation, in vitro culture, and characterization of NCCs from zebrafish embryos. NCCs were isolated from transgenic sox10:egfp embryos using fluorescence activated cell sorting and cultured in complex culture medium without feeder layers. NCC multilineage differentiation was determined by immunocytochemistry and real-time qPCR, cell migration was assessed by wound healing assay, and the proliferation index was calculated by immunostaining against the mitosis marker phospho-histone H3. Cultured NCCs expressed major neural crest lineage markers such as sox10, sox9a, hnk1, p75, dlx2a, and pax3, and the pluripotency markers c-myc and klf4. We showed that the cultured NCCs can be differentiated into multiple neural crest lineages, contributing to neurons, glial cells, smooth muscle cells, melanocytes, and chondrocytes. We applied the NCC in vitro model to study the effect of retinoic acid on NCC development. We showed that retinoic acid had a profound effect on NCC morphology and differentiation, significantly inhibited proliferation and enhanced cell migration. The availability of high numbers of NCCs and reproducible functional assays offers new opportunities for mechanistic studies of neural crest development, in genetic and chemical biology applications.

Roles of Wnt pathway genes wls, wnt9a, wnt5b, frzb and gpc4 in regulating convergent-extension during zebrafish palate morphogenesis

The Wnt signaling pathway is crucial for tissue morphogenesis, participating in cellular behavior changes, notably during the process of convergent-extension. Interactions between Wnt-secreting and receiving cells during convergent-extension remain elusive. We investigated the role and genetic interactions of Wnt ligands and their trafficking factors Wls, Gpc4 and Frzb in the context of palate morphogenesis in zebrafish. We describe that the chaperon Wls and its ligands Wnt9a and Wnt5b are expressed in the ectoderm, whereas juxtaposed chondrocytes express Frzb and Gpc4. Using wls, gpc4, frzb, wnt9a and wnt5b mutants, we genetically dissected the Wnt signals operating between secreting ectoderm and receiving chondrocytes. Our analysis delineates that non-canonical Wnt signaling is required for cell intercalation, and that wnt5b and wnt9a are required for palate extension in the anteroposterior and transverse axes, respectively. Summary: Detailed morphogenetic analyses reveal the relative contributions of Wnt pathway genes to the regulation of convergence and extension cell behaviors during zebrafish palate development.

Abstract 13: Functional Analysis of Connective Tissue Growth Factor (ctgf) in Neural Crest And Craniofacial Development.

PurPose: During vertebrate embryogenesis, cranial neural crest cells (CNCCs) contribute extensively to the formation of facial structures, including cartilage, bone and connective tissue. CNCCs are patterned and fated to distinct anatomical derivatives early at the onset of migration from the neural tube, target to pharyngeal segments and undergo multi-lineage differentiation. Several key pathways regulate CNCC migration and differentiation, including connective tissue growth factor (ctgf). ctgf is a member of the CCN family of secreted proteins, exhibits multiple activities in cell adhesion, migration, and differentiation. Previous studies have shown that ctgfnull mice were born with defects including cleft palate and severe chondrodysplasia, suggesting a critical role of ctgf in craniofacial development. However, the mechanistic basis of how ctgf affects CNCCs behavior and how ctgf is coordinated with other signaling pathway such as wnt signaling during development remain unclear. To this end, we took advantage of genetic approaches in zebrafish generating ctgf-mutant cleft palate model to elucidate the potential function of ctgf in CNCC migration, proliferation and/or convergence extension mechanisms.

Abstract 69: analysis of the role of wls in craniofacial development.

BaCkground: Wnt signaling is a critical pathway regulating craniofacial development, where dysregulation leads to orofacial clefts. Intracellular trafficking and secretion of wnt ligands is chaperoned by wntless (wls). In human, the WLS gene is on the short arm of the chromosome 1 (1p31.3) and several deletions of this region associated with craniofacial malformations have been reported. We hypothesize that the wls gene functions to modulate wnt signaling important in morphogenesis of the craniofacial skeleton.

Abstract 78: APPLICATION OF ZEBRAFISH NEURAL CREST CULTURE ASSAYS TO ELUCIDATE STEM CELL PROPERTIES

Purpose: Vertebrate neural crest development depends on pluripotent, migratory neural crest (NC) cells. Isolation and culture of Zebra sh NC cells has not been previously reported. In vitro culture of NC cells allows evaluation of in vivo  ndings in a more controlled environment. Here we report for the  rst time the isolation, in vitro culture and characterization of NC cells from Zebra sh embryos. We apply the NC culture to determine if these cells possess stem cell or progenitor cell properties of multi-lineage differentiation, maintenance and renewal.

Abstract 9: CONDITIONAL NEURAL CREST CELL ABLATION TO INTERROGATE CRANIOFACIAL REGENERATION IN VERTEBRATE MODEL

Background: It has long been hypothesized that neural crest (NC) stem or progenitor cells exist and they are able to undergo self-renewal, proliferation and multi-lineage differentiation. Most of the NC stem-like properties described to date were gleaned from in vitro assays, such as neural tube explants. In order to generate an in vivo regeneration assay, we develped a lineage-restricted inducible transgenic model where NC cells can be selectively ablated and their regenerative potential be assessed.

Abstract 5: NEURAL CREST TRANSCRIPTOME SCREEN IDENTIFIES NOVEL GENETIC REGULATORS IN CRANIOFACIAL DEVELOPMENT

Background: Cranial neural crest (CNC) cells migrate and elaborate into craniofacial structures, dysregulation of which leads to craniofacial malformations. Many of the intrinsic genetic programs regulating CNC fate commitment and morphogenesis remain largely unknown. Using comparative transcriptome analysis between premigration (10 somites) and postmigration (24 hpf) CNC cells, we aimed to segregate the early migrating genes from those in late pharyngeal morphogenesis. We utilized the zebra sh model to carry out high-throughput expression and function analysis and identi ed novel genes that modulate CNC development.