K. Mukherjee1,2, M. J. Grimaldi1, M. Talkowski2,4, J. F. Gusella2,4, R. Maas2,3, C. C. Morton2,5, E. C. Liao1,2 1Massachusetts General Hospital,Center For Regenerative Medicine,Boston, MA, USA 2Harvard School Of Medicine,Brookline, MA, USA 3Brigham And Women’s Hospital,Division Of Genetics,Boston, MA, USA 4Massachusetts General Hospital,Center For Human Genetic Research,Boston, MA, USA 5Brigham And Women’s Hospital,Department Of Pathology,Boston, MA, USA
Introduction: Orofacial clefts are among the most common congenital anomalies. The genetic basis for cleft palate and other craniofacial malformations is being elucidated. The Developmental Genome Anatomy project (DGAP) has developed whole genome sequencing strategies to identify genes contributing to such human congenital anomalies. The isolated disruption of CAPZB was identified in a 6-month old DGAP patient presenting cleft palate, micrognathia and hypotonia. We exploit the zebrafish model to determine the function of capzb and to understand the role of actin dynamics in craniofacial development and cleft pathogenesis.
Methods: The Meckel’s cartilage and ethmoid plate are analogous to the mammalian mandible and primary palate respectively, making zebrafish an ideal model to study the genetic and developmental basis of palate and lower jaw morphogenesis. The spatiotemporal gene expression of capzb is determined by whole mount in situ hybridization (WISH) during early embryogenesis. Furthermore, craniofacial cartilaginous structures and muscles are examined in the capzb mutant identified from an insertional mutagenesis screen.
Results: WISH analysis shows that capzb is ubiquitously expressed, demonstrating its potential requirement in the function of many tissue types. Preliminary analysis of the capzb mutant show that the lower jaw elements are smaller and retrusive and the palate is only partially fused, leading to a cleft. The actin cytoskeleton is disorganized without capzb, leading to loss of cell morphology in the palate chondrocytes. The capzb mutants also show highly disorganized myofibrils leading to atrophied muscles.
Conclusion: We successfully modeled the phenotypes observed in the DGAP patient, in the zebrafish. We show that the capzb mutants exhibit micrognathia, cleft palate and atrophied muscles. We identify CAPZB to be important in craniofacial and muscle morphogenesis, disruption of which is pathologic for both palate and muscle development. Preliminary results from characterization of the capzb mutant suggest that it plays a role in palate fusion and lower jaw extension, and affects tissue types where actin organization is critical to cell morphology and higher order organ morphogenesis. We show that capzb is maternally transcribed and hence the mutants survive through embryogenesis even with loss of a critical gene involved in basic actin dynamics. Experiments are under way to delineate the mechanisms of capzb function in craniofacial morphogenesis, regulating fundamental cellular mechanisms where actin cytoskeleton and cell signaling pathways intersect. This study illustrates how clinically based studies can uncover fundamental mechanisms that govern cell biology and tissue morphogenesis.
