| In utero inhibition of the Hedgehog (Hh) signaling pathway by the Veratrum alkaloid cyclopamine causes serious birth defects of the brain and face in several animal models. In humans, homologous defects of the forebrain and face (holoprosencephaly) as well as isolated defects of the face (cleft lip/palate) occur in 1/15,000 and 1/600 live births respectively, with poorly understood etiological bases underlying both. Emerging evidence points toward endogenous small molecule modulation as a key Hh pathway regulatory mechanism that is sensitive to inhibition by a structurally diverse group of exogenous small molecules. We found that several dietary alkaloids, structurally similar to cyclopamine, act as relatively weak Hh signaling inhibitors. Additionally, employing a high-throughput small molecule screening assay, we identified three structurally diverse Hh signaling inhibitors; an antifungal, a human dietary supplement, and a human hormone. While each of the Hh signaling inhibitors identified here are variably less potent than cyclopamine, they act additively through a mechanism similar to cyclopamine. Finally, using correlative in vitro whole embryo culture assays and in vivo pharmacokinetic analyses, we established an in vivo cyclopamine administration regimen for induction of cleft lip and palate defects that phenocopy human clinical birth defects. Moving forward, further characterization of the mouse model of cleft lip and palate and of the environmental Hh signaling inhibitors described here, as well as a broadened effort to screen additional relevant environmental compounds may provide important avenues in which to examine the underlying etiological factors of common and morbid human birth defects. |
