Probes for GLI1

Inturned (INTU), a cilia and planar polarity effector, performs prominent ciliogenic functions during morphogenesis, such as in the skin. INTU is expressed in adult tissues but its role in tissue maintenance is unknown. Here, we report that the expression of the INTU gene is aberrantly elevated in human basal cell carcinoma (BCC), coinciding with increased primary cilia formation and activated hedgehog (Hh) signaling. Disrupting Intu in an oncogenic mutant Smo (SmoM2)-driven BCC mouse model prevented the formation of BCC through suppressing primary cilia formation and Hh signaling, suggesting that Intu performs a permissive role during BCC formation. INTU is essential for intraflagellar transport A complex assembly during ciliogenesis. To further determine whether Intu is directly involved in the activation of Hh signaling downstream of ciliogenesis, we examined the Hh signaling pathway in mouse embryonic fibroblasts, which readily responds to the Hh pathway activation. Depleting Intu blocked Smo agonist-induced Hh pathway activation, whereas the expression of Gli2ΔN, a constitutively active Gli2, restored Hh pathway activation in Intu-deficient cells, suggesting that INTU functions upstream of Gli2 activation. In contrast, overexpressing Intu did not promote ciliogenesis or Hh signaling. Taken together, data obtained from this study suggest that INTU is indispensable during BCC tumorigenesis and that its aberrant upregulation is likely a prerequisite for primary cilia formation during Hh-dependent tumorigenesis.

Hedgehog (Hh) pathway inhibitors such as vismodegib are highly effective for treating basal cell carcinoma (BCC); however, residual tumor cells frequently persist and regenerate the primary tumor upon drug discontinuation. Here, we show that BCCs are organized into two molecularly and functionally distinct compartments. Whereas interior Hh+/Notch+ suprabasal cells undergo apoptosis in response to vismodegib, peripheral Hh+++/Notch− basal cells survive throughout treatment. Inhibiting Notch specifically promotes tumor persistence without causing drug resistance, while activating Notch is sufficient to regress already established lesions. Altogether, these findings suggest that the three-dimensional architecture of BCCs establishes a natural hierarchy of drug response in the tumor and that this hierarchy can be overcome, for better or worse, by modulating Notch.

The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts.

The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap-null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK-JUN signaling, a well-established tumor-driving cascade. Importantly, in this context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCC pathogenesis and thereby a viable target for drug-resistant BCC.

Although hundreds of cytosolic or transmembrane molecules form the primary cilium, few secreted molecules are known to contribute to ciliogenesis. Here, homologous secreted metalloproteases ADAMTS9 and ADAMTS20 are identified as ciliogenesis regulators that act intracellularly. Secreted and furin-processed ADAMTS9 bound heparan sulfate and was internalized by LRP1, LRP2 and clathrin-mediated endocytosis to be gathered in Rab11 vesicles with a unique periciliary localization defined by super-resolution microscopy. CRISPR-Cas9 inactivation of ADAMTS9 impaired ciliogenesis in RPE-1 cells, which was restored by catalytically active ADAMTS9 or ADAMTS20 acting in trans, but not by their proteolytically inactive mutants. Their mutagenesis in mice impaired neural and yolk sac ciliogenesis, leading to morphogenetic anomalies resulting from impaired hedgehog signaling, which is transduced by primary cilia. In addition to their cognate extracellular proteolytic activity, ADAMTS9 and ADAMTS20 thus have an additional proteolytic role intracellularly, revealing an unexpected regulatory dimension in ciliogenesis.

Gut mesenchyme provides key stem cell niche signals such as Wnt ligands, but how these signals are regulated is unclear. Because Hedgehog (Hh) signaling is critical for gut mesenchymal development and tumorigenesis, we investigated Hh-mediated mechanisms by analyzing mice deleted for key negative regulators of Hh signaling, Sufu and/or Spop, in the gut mesenchyme, and demonstrated their dosage-dependent roles. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction. Consistent with increased epithelial proliferation caused by abnormal Hh activation in development, Sufu reduction promotes intestinal tumorigenesis, whereas Gli2 heterozygosity suppresses it. Our analyses of chromatin and GLI2 binding genomic regions reveal its transcriptional regulation of stem cell niche signals through enhancers, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis