Purpose: To examine the embryonic expression of cytochrome P4501b1 (Cyp1b1) gene by whole mount in situ hybridization.
Methods: FVB/NcrlBR mouse embryos staged at 9.5, 10.5, and 11.5 dpc were obtained by timed breeding experiments. Antisense and sense RNA probes labeled with digoxigenin UTP were generated by in vitro transcription of an 848 bp Cyp1b1 cDNA fragment that was subcloned into transcription vector pCRII-TOPO. The digoxigenin labeled RNA was localized using an alkaline phosphatase conjugated anti-digoxigenin Fab fragment. Colorimetric detection of the digoxigenin labeled probe was performed with substrate solution containing 4-nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
Results: During early stages of murine development Cyp1b1 mRNA was detected in the developing eye, hindbrain, branchial arches, forelimb bud, ligaments supporting the liver primordium and developing kidney. In the eye and forelimb bud Cyp1b1 displayed restricted expression along the axes of development. In the developing eye Cyp1b1 exhibited dorsal expression with respect to the dorso-distal/proximo-ventral axis and anterior expression with respect to the anterior-nasal/posterior-temporal axis. In the forelimb bud Cyp1b1 expression was localized posteriorly. The polarity of Cyp1b1 expression was lost at 11.5 dpc, at which time expression was additionally seen in ventral (eye) and anterior (forelimb bud) areas.
Conclusions: The spatio-temporal expression patterns observed in this study suggest that during early stages of murine development, Cyp1b1 participates in establishment and/or maintenance of polarity along the axes of embryonic development. Expression of Cyp1b1 in the dorso-distal end of the optic cup, from which the ciliary body and iris are derived, correlates with the expression patterns seen in adult tissues and the abnormal development of these structures as part of the glaucoma phenotypes resulting from Cyp1b1 mutations.