Molecular mechanisms that pattern the face are poorly understood. Disruptions to signaling by the protein Sonic hedgehog (SHH) cause facial malformations. During development of the frontonasal process (FNP), which is the precursor of the mid- and upper face, Shh is first expressed in forebrain and subsequently in FNP ectoderm. Targets of SHH, Patched1 and Gli1, were expressed in ectoderm and neural crest-derived mesenchyme of the FNP adjacent to Shh expression domains. We used two approaches to determine if Shh expression in the forebrain is necessary for patterning the FNP. Previously, we filled the neural tube with cells expressing an antibody that prevents SHH from binding its receptor Patched1. These treatments reduced expression of Patched1 and Gli1 in the FNP, and led to anophthalmia, hypotelorism, and upper beak truncations. Objective: We tested if SHH from the forebrain acts directly on FNP tissues. Method: To prevent mesenchymal and ectodermal cells from responding to SHH we used a plasmid encoding a dominant-negative form of the SHH receptor (Patched^Dloop2). For controls we used a plasmid encoding alkaline phosphatase. Premigratory neural crest cells were transfected by injecting the plasmids into the neural tube followed by electroporation. Electrodes were placed on either side of the neural tube and three pulses (40 volts/50 milliseconds) were applied. Results: Seventy-two (n=5/5) and 96 (n=5/5) hours after treatment embryos exhibited unilateral or bilateral anophthalmia and hypotelorism. By 7 (n=2/2) and 10 (n=3/3) days, embryos with bilateral anophthalmia exhibited upper beak truncations. Beaks of embryos with unilateral anophthalmia deviated toward the malformed eye, indicating localized growth defects. Control embryos appeared normal (n=11). Conclusion: Rendering FNP cells incapable of transducing a Shh signal causes anopthalmia, hypotelorism, and truncations in FNP-derived structures, which is a phenotype equivalent to that observed by blocking the ligand. JK supported by NIH T35-DE07103. G. Struhl provided Patched^Dloop2).

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