CDON contributes to Hedgehog-dependent patterning and growth of the developing limb.

Hedgehog (HH) signaling is a major driver of tissue patterning during embryonic development through the regulation of a multitude of cell behaviors including cell fate specification, proliferation, migration, and survival. HH ligands signal through the canonical receptor PTCH1 and three co-receptors, GAS1, CDON and BOC. While previous studies demonstrated an overlapping and collective requirement for these co-receptors in early HH-dependent processes, the early embryonic lethality of Gas1;Cdon;Boc mutants precluded an assessment of their collective contribution to later HH-dependent signaling events. Specifically, a collective role for these co-receptors during limb development has yet to be explored. Here, we investigate the combined contribution of these co-receptors to digit specification, limb patterning and long bone growth through limb-specific conditional deletion of Cdon in a Gas1;Boc null background. Combined deletion of Gas1, Cdon and Boc in the limb results in digit loss as well as defects in limb outgrowth and long bone patterning. Taken together, these data demonstrate that GAS1, CDON and BOC are collectively required for HH-dependent patterning and growth of the developing limb.

Unique lingual expression of the Hedgehog pathway antagonist Hedgehog-interacting protein in filiform papillae during homeostasis and ectopic expression in fungiform papillae during Hedgehog signaling inhibition.

BACKGROUND: Hedgehog (HH) signaling is essential for homeostasis in gustatory fungiform papillae (FP) and taste buds. However, activities of HH antagonists in these tissues remain unexplored. We investigated a potential role for HH-interacting protein (HHIP), an endogenous pathway antagonist, in regulating HH signaling during taste organ homeostasis. We found a restricted pattern of Hhip-expressing cells in the anterior epithelium of each nongustatory filiform papilla (FILIF) only. To test for roles in antagonism of HH signaling, we investigated HHIP after pathway inhibition with SMO inhibition via sonidegib and Smo deletion, Gli2 deletion/suppression, or with chorda tympani/lingual nerve cut. RESULTS: In all approaches, the HHIP expression pattern was retained in FILIF suggesting HH-independent regulation of HHIP. Remarkably, after pathway inhibition, HHIP expression was detected also in the conical, FILIF-like atypical FP. We found a close association of de novo expression of HHIP in atypical FP with loss of Gli1+, HH-responding cells. Further, we report that PTCH1 is another potential HH antagonist in FILIF that co-localizes with HHIP. CONCLUSIONS: After HH pathway inhibition the ectopic expression of HHIP correlates with a FILIF-like morphology in atypical FP and we propose that localized expression of the HH antagonist HHIP regulates pathway inhibition to maintain FILIF during tongue homeostasis.

Neuropilin-1 promotes Hedgehog signaling through a novel cytoplasmic motif.

Hedgehog (HH) signaling critically regulates embryonic and postnatal development as well as adult tissue homeostasis, and its perturbation can lead to developmental disorders, birth defects, and cancers. Neuropilins (NRPs), which have well-defined roles in Semaphorin and VEGF signaling, positively regulate HH pathway function, although their mechanism of action in HH signaling remains unclear. Here, using luciferase-based reporter assays, we provide evidence that NRP1 regulates HH signaling specifically at the level of GLI transcriptional activator function. Moreover, we show that NRP1 localization to the primary cilium, a key platform for HH signal transduction, does not correlate with HH signal promotion. Rather, a structure-function analysis suggests that the NRP1 cytoplasmic and transmembrane domains are necessary and sufficient to regulate HH pathway activity. Furthermore, we identify a previously uncharacterized, 12-amino acid region within the NRP1 cytoplasmic domain that mediates HH signal promotion. Overall, our results provide mechanistic insight into NRP1 function within and potentially beyond the HH signaling pathway. These insights have implications for the development of novel modulators of HH-driven developmental disorders and diseases.

Dual and opposing roles for the kinesin-2 motor, KIF17, in Hedgehog-dependent cerebellar development.

While the kinesin-2 motors KIF3A and KIF3B have essential roles in ciliogenesis and Hedgehog (HH) signal transduction, potential role(s) for another kinesin-2 motor, KIF17, in HH signaling have yet to be explored. Here, we investigated the contribution of KIF17 to HH-dependent cerebellar development, where Kif17 is expressed in both HH-producing Purkinje cells and HH-responding cerebellar granule neuron progenitors (CGNPs). Germline Kif17 deletion in mice results in cerebellar hypoplasia due to reduced CGNP proliferation, a consequence of decreased HH pathway activity mediated through decreased Sonic HH (SHH) protein. Notably, Purkinje cell-specific Kif17 deletion partially phenocopies Kif17 germline mutants. Unexpectedly, CGNP-specific Kif17 deletion results in the opposite phenotype-increased CGNP proliferation and HH target gene expression due to altered GLI transcription factor processing. Together, these data identify KIF17 as a key regulator of HH-dependent cerebellar development, with dual and opposing roles in HH-producing Purkinje cells and HH-responding CGNPs.

Distinct expression patterns of Hedgehog signaling components in mouse gustatory system during postnatal tongue development and adult homeostasis.

The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.

Plexins promote Hedgehog signaling through their cytoplasmic GAP activity.

Hedgehog signaling controls tissue patterning during embryonic and postnatal development and continues to play important roles throughout life. Characterizing the full complement of Hedgehog pathway components is essential to understanding its wide-ranging functions. Previous work has identified neuropilins, established semaphorin receptors, as positive regulators of Hedgehog signaling. Neuropilins require plexin co-receptors to mediate semaphorin signaling, but the role of plexins in Hedgehog signaling has not yet been explored. Here, we provide evidence that multiple plexins promote Hedgehog signaling in NIH/3T3 mouse fibroblasts and that plexin loss of function in these cells results in significantly reduced Hedgehog pathway activity. Catalytic activity of the plexin GTPase-activating protein (GAP) domain is required for Hedgehog signal promotion, and constitutive activation of the GAP domain further amplifies Hedgehog signaling. Additionally, we demonstrate that plexins promote Hedgehog signaling at the level of GLI transcription factors and that this promotion requires intact primary cilia. Finally, we find that plexin loss of function significantly reduces the response to Hedgehog pathway activation in the mouse dentate gyrus. Together, these data identify plexins as novel components of the Hedgehog pathway and provide insight into their mechanism of action.

Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment. EXPERIMENTAL DESIGN: We used a combination of pharmacologic inhibition, gain- and loss-of-function genetic experiments, cytometry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC. RESULTS: We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression. CONCLUSIONS: Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.