Probes for INS

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.

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.

Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.

Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.