L. J. Rochard1, K. Mukherjee1, T. J. Hoyos1, E. C. Liao1 1Massachusetts General Hospital,Center For Regenerative Medicine,Boston, MA, USA
Introduction: Orofacial clefts, such as cleft lip and palate (CL/P) are the most common congenital anomalies worldwide. The etiology of non-syndromic CL/P is complex, with interplay of genetic, epigenetic and environmental factors. We investigate naturally occurring human chromosomal aberrations resulting in orofacial clefts to identify the underlying genetic basis and elucidate the role of such genes in craniofacial development. This functional genomics approach identified ATG4C to be important in palate development. ATG4C is a key enzyme that catalyzes the cellular recycling of autophagosome, organelles that are essential for cellular homeostasis and stress response. Autophagy is required to respond to malnutrition and hypoxia states. Environmental stresses such as hypoxia or smoke have been implicated in CL/P but how they interact with the genome during pregnancy to lead to the malformation is not known. Implication of autophagy in cleft pathogenesis is exciting, as it potentially elucidates how environmental stress potentiates cleft malformation.
Methods: We utilize the zebrafish model to investigate the role of autophagy in craniofacial development. Spatiotemporal gene expression analysis of atg4c was performed by wholemount RNA in situ hybridization (WISH). Morpholino-mediated gene knockdown was performed to assess gene function in vivo. Targeted mutagenesis of atg4c locus was achieved by CRISPR/Cas genome editing method.
Results:Genetic analysis of a patient presenting chromosomal translocation identified breakpoint in the ATG4C gene, which encodes a cysteine protease involved in maturation and recycling of LC3, a key protein of the autophagic pathway. Genomic database (DECIPHER) analysis identified additional patients reported with CL/P. Morpholino mediated gene knockdown of atg4c in zebrafish phenocopied cleft palate. CRISPR mediated targeted mutagenesis developed craniofacial anomalies in stress conditions, such as inhibition of mTOR pathway or induction of hypoxia.
Conclusion:The link between environmental stress and craniofacial malformations has been elusive; therefore identification of autophagy as a regulator of embryonic craniofacial development is exciting and leads to novel understanding of CL/P pathogenesis. Importantly, Autophagy is under intense investigation as a pathway important in carcinogenesis and bone homeostasis, and as such, many drugs regulating this pathway have been developed. The zebrafish model affords the opportunity to test these drugs in small molecule screens to identify chemical modifiers that may augment cellular stress response and mitigate craniofacial malformation, to prevent CL/P before they form.
