Cloning and Functional Characterization of the Retinoic Acid-Catabolizing Enzyme CYP26B1 in Mouse Development

Abstract

Retinoic acid (RA) is an active metabolite of vitamin A that is essential for embryonic development, and homeostasis of adult tissues. RA is a ligand for the nuclear retinoic acid receptor, and RA-mediated signaling is critical for regulation of cell proliferation, differentiation and apoptosis. There is a spatio-temporal distribution of RA in the developing embryo such that some tissues are rich in RA, while others are devoid. This patterned distribution of RA is tightly controlled through the coordinated expression of RA-synthesizing (retinaldehyde dehydrogenase) and RA-catabolizing (CYP26) enzymes. In this thesis, I describe the cloning of a mouse gene encoding one of the CYP26 proteins, Cyp26b1. Cyp26b1 was shown to be highly expressed in the embryo, with transcripts localized to the hindbrain, limb buds and branchial arches. We also used homologous recombination to generate a line of transgenic mice with a loss-of-function deletion in Cyp26b1. These mice die shortly after birth with severe malformations affecting the limbs, craniofacial structures and epidermis; phenotypes that are all reminiscent of RA teratogenesis. We present an extensive characterization of the craniofacial and epidermal abnormalities in Cyp26b1-/- animals, and examine several molecular pathways that may be deregulated. CYP26B1 null embryos exhibit a truncated mandible, lack numerous facial bones, and show reduced ossification of the calvaria. Molecular analysis of Cyp26b1-/- embryos indicates hindbrain and branchial arch patterning is largely unaffected in early to mid-gestational mutants. However, there appear to be some subtle abnormalities in neural crest cell migration, which may contribute to the development of some of the observed phenotypes. CYP26B1 null mutants also lack hair follicles, which appears to be due to a downregulation of -catenin mediated signaling. Thus, in addition to cloning and characterizing the expression of murine Cyp26b1, we have demonstrated in vivo, that regulation of RA distribution by CYP26B1 is essential for morphogenesis of the epidermis and craniofacial structures.