Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Palate and Mandibular Form are Regulated by Actin Capping Protein CAPZB Function
Kusumika Mukherjee, PhD1, Kana Ishii, MD1, Michael J. Grimaldi, B.A.1, Christina Nguyen, B.S.1, Michael Talkowski, PhD1, James Gusella, PhD1, Richard Maas, MD, PhD2, Cynthia C. Morton, PhD2, Eric C. Liao, MD, PhD1.
1Massachusetts General Hospital, Boston, MA, USA, 2Brigham and Women's Hospital, Boston, MA, USA.

PURPOSE: Craniofacial malformations are among the most common congenital anomalies worldwide with a strong genetic etiology. Utilizing a functional genomics gene discovery pipeline, we have identified a new candidate gene, CAPZB that encodes an actin-capping protein and regulates the growth of polymerizing actin filaments. CAPZB is also important in maintaining the structural integrity of sarcomere, which is the functional unit of muscles. The isolated disruption of CAPZB was first identified in a 6-month old female presenting cleft palate (CP), micrognathia (Robin sequence) and hypotonia. We have exploited the zebrafish to model the phenotypes observed in the human case and to carry out detailed analysis of molecular and developmental processes to determine the function of capzb in craniofacial morphogenesis.
METHODS:The spatiotemporal gene expression of capzb was determined by whole mount in situ hybridization (WISH) during embryogenesis. The capzb mutant line, generated from an insertional mutagenesis screen, was analyzed by quantitative and non-quantitative PCR. Craniofacial cartilaginous structures and muscles were examined by Alcian Blue stain, immunohistochemistry, and lifeact:GFP transgenics, respectively. Multispectral clonal analysis of neural crest cells (NCCs) was performed in zebrabow;sox10:Cre transgenics. Lineage tracing experiments and standard cell proliferation and apoptosis assays were performed in sox10:kaede and sox10:GFP transgenics respectively.
RESULTS:WISH analysis shows that capzb is maternally inherited and ubiquitously expressed, demonstrating its potential requirement in the function of many tissue types. Indeed, we observed that absence of Capzb manifests in defective heart and circulatory system as well as malformed fins. Moreover, analysis of the cartilaginous structures of capzb mutants show that the lower jaw elements are smaller and retrusive (micrognathia) and the palate is only partially fused with a cleft in the anterior palate (CP). The actin cytoskeleton is in disarray and myofibrils are highly disorganized, leading to atrophied muscles (hypotonia). Preliminary results from clonal lineage analysis suggest that defects in NCCs migration may be causing the craniofacial anomalies. There is no observed defect in cell proliferation and no increased cell death in the mutants. Importantly, over-expression of capzb by mRNA injection also perturbs normal craniofacial development, suggesting that a correct stoichiometry of Capzb binding to polymerizing actin is critical to cell morphology and tissue morphogenesis.
CONCLUSION:We have successfully modeled the Robin sequence phenotypes of cleft palate and hypoplastic mandible exhibited by the proband in a capzb animal model. We identified CAPZB to be important in craniofacial and global morphogenesis, disruption of which is pathologic for palate development and lower jaw extension. Ultimately, the capzb mutant has provided us with a molecular example of how form follows function by revealing how disruption of a basic cellular process like regulation of actin cytoskeletal dynamics can cause NCC morphology and migration defects and translate into craniofacial dysmorphism.


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