GPR161-GLI3 repressor signaling at cilia directs apical constriction and cell fate during cranial neural tube closure.

Failure to close the cranial neural tube, known as exencephaly/anencephaly, is a lethal congenital defect. However, the mechanisms driving patterning and reshaping of the broad cranial neural folds are poorly understood. Loss of the primary cilium-localized G protein-coupled receptor GPR161 causes ectopic, excessive hedgehog signaling in the mouse neural tube and fully penetrant exencephaly. GPR161 promotes GLI3 transcriptional repressor (GLI3R) formation while preventing GLI2 transcriptional activator formation. Here, we studied the mechanisms underlying cranial closure in mice using a Gpr161 mutant allelic series, epistasis between Gpr161 knockout and GLI effectors, and in toto imaging of cell behavior. A functional non-ciliary Gpr161 knock-in implicated GPR161 ciliary localization directly in initiation and maintenance of cranial closure. Furthermore, Gli3R expression, but not Gli2 loss, rescued exencephaly in Gpr161 knockout mice. GLI3R specifically restricted forebrain ventral floor plate expansion and mediated apical constriction in the lateral midbrain neural folds prior to closure. These results reveal metamere-specific, cilia-dependent hedgehog repression thresholds in control of spatially restricted gene expression and dynamic cell behavior during cranial closure. Targeted interventions increasing hedgehog repression could ameliorate regional cranial defects.