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Functional Analysis Of Connective Tissue Growth Factor (ctgf) In Neural Crest And Craniofacial Development
Yawei Kong, Ph.D., Michael Grimaldi, B.A., Tatiana Hoyos, M.D., Eric C. Liao, M.D., Ph.D..
Massachusetts General Hospital; Shriners Hospitals for Children Boston; Harvard Medical School, Boston, MA, USA.
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 ctgf-null 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.
Gene expression profiling was analyzed by Affymetrix gene-chip. Spatiotemporal expression analysis was performed by wholemount RNA in situ hybridization (WISH). Morpholino-mediated gene knockdown was performed to assess gene function in vivo. Targeted mutagenesis of ctgf locus was achieved by the latest CRISPR/Cas genome editing method.
Expression profiles of two zebrafish homologues for ctgf gene, ctgfa and ctgfb, were characterized by WISH and found to be compatible with a role in craniofacial development. During embryogenesis (24-96 hpf), we observed that the spatiotemporal expression of both ctgfa and ctgfb faithfully delineate the pharyngeal arch region and the derived craniofacial skeleton. Specifically, ctgfa was expressed in the ectoderm that surrounds the palate, the oral ectoderm, as well as the chondrocytes that constitute the palate. Despite their similar expression patterns, in vivo functional analysis by Morpholino-mediated gene knockdown showed that impaired ctgfa leads to craniofacial defects, featured by loss of facial soft tissues, missing lower jaw, and truncated palate with a cleft phenotye at the anterior edge of the seam between median and lateral ethmoid plate. However, ctgfb-knockdown had subtle effects on craniofacial morphogenesis. Collectively, these results suggest an indispensible role for ctgfa in palate and lower jaw development. To gain mechanistic insight into ctgf function during craniofacial development, we have generated both ctgfa and ctgfb mutants by CRISPR/Cas, where targeted short-nucleotide deletion resulted in truncated protein translation. This ctgfa/ctgfb mutant model provides us with an important tool to access the genetic basis of cleft palate malformation.
This study underscores the important role of ctgf in CNCC development, and highlights the utility of the zebrafish model to interrogate palate and craniofacial morphogenesis using reverse genetic approaches. Our ongoing work to elucidate the molecular basis of ctgf-associated cleft palate helps to identify other risk loci and develop potentially preventive measures via pharmacologic manipulation of craniofacial development during early embryogenesis.
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