Context-dependent ciliary regulation of hedgehog pathway repression in tissue morphogenesis.

A fundamental problem in tissue morphogenesis is identifying how subcellular signaling regulates mesoscale organization of tissues. The primary cilium is a paradigmatic organelle for compartmentalized subcellular signaling. How signaling emanating from cilia orchestrates tissue organization-especially, the role of cilia-generated effectors in mediating diverse morpho-phenotypic outcomes-is not well understood. In the hedgehog pathway, bifunctional GLI transcription factors generate both GLI-activators (GLI-A) and GLI-repressors (GLI-R). The formation of GLI-A/GLI-R requires cilia. However, how these counterregulatory effectors coordinate cilia-regulated morphogenetic pathways is unclear. Here we determined GLI-A/GLI-R requirements in phenotypes arising from lack of hedgehog pathway repression (derepression) during mouse neural tube and skeletal development. We studied hedgehog pathway repression by the GPCR GPR161, and the ankyrin repeat protein ANKMY2 that direct cAMP/protein kinase-A signaling by cilia in GLI-R generation. We performed genetic epistasis between Gpr161 or Ankmy2 mutants, and Gli2/Gli3 knockouts, Gli3R knock-in and knockout of Smoothened, the hedgehog pathway transducer. We also tested the role of cilia-generated signaling using a Gpr161 ciliary localization knock-in mutant that is cAMP signaling competent. We found that the cilia-dependent derepression phenotypes arose in three modes: lack of GLI-R only, excess GLI-A formation only, or dual regulation of either lack of GLI-R or excess GLI-A formation. These modes were mostly independent of Smoothened. The cAMP signaling-competent non-ciliary Gpr161 knock-in recapitulated Gpr161 loss-of-function tissue phenotypes solely from lack of GLI-R only. Our results show complex tissue-specific GLI-effector requirements in morphogenesis and point to tissue-specific GLI-R thresholds generated by cilia in hedgehog pathway repression. Broadly, our study sets up a conceptual framework for rationalization of different modes of signaling generated by the primary cilium in mediating morphogenesis in diverse tissues.

Studying Hedgehog Signaling During Mouse Neural Tube Development.

The identity of ventral neural progenitors in the neural tube is largely dependent on Hedgehog (Hh) signaling. Variations in staining patterns are excellent indicators of aberrant Hh signaling. Here we describe the basic protocol to stain for progenitor populations based on transcription factor expression. We also provide an overview of ciliary and centrosomal staining in the neural tube.

Ankmy2 Prevents Smoothened-Independent Hyperactivation of the Hedgehog Pathway via Cilia-Regulated Adenylyl Cyclase Signaling.

The mechanisms underlying subcellular targeting of cAMP-generating adenylyl cyclases and processes regulated by their compartmentalization are poorly understood. Here, we identify Ankmy2 as a repressor of the Hedgehog pathway via adenylyl cyclase targeting. Ankmy2 binds to multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia. Mice lacking Ankmy2 are mid-embryonic lethal. Knockout embryos have increased Hedgehog signaling and completely open neural tubes showing co-expansion of all ventral neuroprogenitor markers, comparable to the loss of the Hedgehog receptor Patched1. Ventralization in Ankmy2 knockout is completely independent of the Hedgehog pathway transducer Smoothened. Instead, ventralization results from the reduced formation of Gli2 and Gli3 repressors and early depletion of adenylyl cyclase III in neuroepithelial cilia, implicating deficient pathway repression. Ventralization in Ankmy2 knockout requires both cilia and Gli2 activation. These findings indicate that cilia-dependent adenylyl cyclase signaling represses the Hedgehog pathway and promotes morphogenetic patterning.