For homeostasis, lingual taste papilla organs require regulation of epithelial cell survival and renewal, with sustained innervation and stromal interactions. To investigate a role for Hedgehog/GLI signaling in adult taste organs we used a panel of conditional mouse models to manipulate GLI activity within epithelial cells of the fungiform and circumvallate papillae. Hedgehog signaling suppression rapidly led to taste bud loss, papilla disruption, and decreased proliferation in domains of papilla epithelium that contribute to taste cells. Hedgehog responding cells were eliminated from the epithelium but retained in the papilla stromal core. Despite papilla disruption and loss of taste buds that are a major source of Hedgehog ligand, innervation to taste papillae was maintained, and not misdirected, even after prolonged GLI blockade. Further, vimentin-positive fibroblasts remained in the papilla core. However, retained innervation and stromal cells were not sufficient to maintain taste bud cells in the context of compromised epithelial Hedgehog signaling. Importantly taste organ disruption after GLI blockade was reversible in papillae that retained some taste bud cell remnants where reactivation of Hedgehog signaling led to regeneration of papilla epithelium and taste buds. Therefore, taste bud progenitors were either retained during epithelial GLI blockade or readily repopulated during recovery, and were poised to regenerate taste buds once Hedgehog signaling was restored, with innervation and papilla connective tissue elements in place. Our data argue that Hedgehog signaling is essential for adult tongue tissue maintenance and that taste papilla epithelial cells represent the key targets for physiologic Hedgehog-dependent regulation of taste organ homeostasis. Because disruption of GLI transcriptional activity in taste papilla epithelium is sufficient to drive taste organ loss, similar to pharmacologic Hedgehog pathway inhibition, the findings suggest that taste alterations in cancer patients using systemic Hedgehog pathway inhibitors result principally from interruption of signaling activity in taste papillae.
Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception1. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders2–4. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88Tg737Rpw mice, also known as ORPK mice)5 have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.
The olfactory epithelium (OE) is one of the few tissues to undergo constitutive neurogenesis throughout the mammalian lifespan. It is composed of multiple cell types including olfactory sensory neurons (OSNs) that are readily replaced by two populations of basal stem cells, frequently dividing globose basal cells and quiescent horizontal basal cells (HBCs). However, the precise mechanisms by which these cells mediate OE regeneration are unclear. Here, we show for the first time that the HBC subpopulation of basal stem cells uniquely possesses primary cilia that are aligned in an apical orientation in direct apposition to sustentacular cell end feet. The positioning of these cilia suggests that they function in the detection of growth signals and/or differentiation cues. To test this idea, we generated an inducible, cell type-specific Ift88 knock-out mouse line (K5rtTA;tetOCre;Ift88 fl/fl ) to disrupt cilia formation and maintenance specifically in HBCs. Surprisingly, the loss of HBC cilia did not affect the maintenance of the adult OE but dramatically impaired the regeneration of OSNs following lesion. Furthermore, the loss of cilia during development resulted in a region-specific decrease in neurogenesis, implicating HBCs in the establishment of the OE. Together, these results suggest a novel role for primary cilia in HBC activation, proliferation, and differentiation.
Oxaliplatin, satraplatin, and picoplatin are cisplatin analogs that interact with DNA forming intrastrand and interstrand DNA cross-links (ICLs). Replicative bypass of cisplatin DNA adducts requires the cooperative actions of at least three translesion DNA synthesis (TLS) polymerases: Pol, REV1, and Pol. Because oxaliplatin, satraplatin, and picoplatin contain bulkier chemical groups attached to the platinum core compared with cisplatin, we hypothesized that these chemical additions may impede replicative bypass by TLS polymerases and reduce tolerance to platinum-containing adducts. We examined multiple responses of cancer cells to oxaliplatin, satraplatin, or picoplatin treatment under conditions where expression of a TLS polymerase was limited. Our studies revealed that, although Pol contributes to the tolerance of cisplatin adducts, it plays a lesser role in promoting replication through oxaliplatin, satraplatin, and picoplatin adducts. REV1 and Pol were necessary for tolerance to all four platinum analogs and prevention of hyperactivation of the DNA damage response after treatment. In addition, REV1 and Pol were important for the resolution of DNA double-stranded breaks created during replication-associated repair of platinum-containing ICLs. Consistent with ICLs being the predominant cytotoxic lesion, depletion of REV1 or Pol rendered two different model cell systems extremely sensitive to all four drugs, whereas Pol depletion had little effect. Together, our data suggest that REV1 and Pol are critical for promoting resistance to all four clinically relevant platinumbased drugs by promoting both translesion DNA synthesis and DNA repair.
Cilia are evolutionarily conserved organelles found on many mammalian cell types, including neuronal populations. Although neuronal cilia, including those on olfactory sensory neurons (OSNs), are often delineated by localization of adenylyl cyclase 3 (AC3, also known as ADCY3), the mechanisms responsible for targeting integral membrane proteins are largely unknown. Post-translational modification by small ubiquitin-like modifier (SUMO) proteins plays an important role in protein localization processes such as nuclear–cytosolic transport. Here, we identified through bioinformatic analysis that adenylyl cyclases harbor conserved SUMOylation motifs, and show that AC3 is a substrate for SUMO modification. Functionally, overexpression of the SUMO protease SENP2 prevented ciliary localization of AC3, without affecting ciliation or cilia maintenance. Furthermore, AC3-SUMO mutants did not localize to cilia. To test whether SUMOylation is sufficient for cilia entry, we compared localization of ANO2, which possesses a SUMO motif, and ANO1, which lacks SUMOylation sites and does not localize to cilia. Introduction of SUMOylation sites into ANO1 was not sufficient for ciliary entry. These data suggest that SUMOylation is necessary but not sufficient for ciliary trafficking of select constituents, further establishing the link between ciliary and nuclear import.
The olfactory epithelium (OE) is one of the few tissues to undergo constitutive neurogenesis throughout the lifespan of an organism. The OE is composed of differentiated olfactory sensory neurons (OSNs), supporting cells, and two populations of basal stem cells, globose basal cells (GBCs) and horizontal basal cells (HBCs). While GBCs frequently divide and differentiate into OSNs and supporting cells to restore the OE, HBCs are more quiescent yet able to restore the OE following severe insult. In the mouse OE, immunohistochemical (IHC) analysis of Arl13b (a cilia‐localized protein) revealed that in addition to being expressed in olfactory cilia of OSNs, Arl13b‐labeled primary cilia are on HBCs. Due to the growing evidence that cilia play a role in stem cell formation and function, we hypothesized that primary cilia on HBCs regulate their stem cell properties. To test this hypothesis, we generated a triple transgenic mouse (K5rtTA;tetOCre;Ift88fl/fl) that incorporates an inducible tetracycline system, the K5 promoter that in the OE is specifically expressed in HBCs, and the Ift88 conditional allele whose deletion results in a loss of cilia. Upon four weeks of doxycycline treatment in these mice, there was a significant loss of HBC cilia with no change in the cell composition of the OE. However, IHC analysis of various OE cell‐specific markers revealed that three weeks following a methimazole‐induced injury to the OE of mice with depleted HBC cilia resulted in an altered recovery of the OE. These data suggest that cilia are critical for HBC differentiation during the restoration of the OE, making HBC cilia important for olfactory tissue maintenance.Supported by R01DC009606 (to J.R.M.) and F31DC013496 (to A.M.J.)
The olfactory epithelium (OE) is a specialized neuroepithelium that is replenished by two stem cell populations: globose basal cells (GBCs) and horizontal basal cells (HBCs). Previous work indicated that HBCs contain primary cilia, organelles that mediate Hedgehog (HH) pathway activity. However, a role for HH signaling in HBCs has not been investigated. We find that GLI2 and GLI3, transcriptional effectors of the HH pathway, are expressed in HBCs in the adult OE and that their expression expands following injury. Further, Gli2-expressing descendants contribute to all major OE cell types during OE regeneration. HBC-specific expression of constitutively active GLI2 drives inappropriate HBC proliferation, alters HBC identity, and culminates in a failure of HBCs to differentiate into olfactory sensory neurons (OSNs) following injury. HBC-specific deletion of endogenous Gli2 and Gli3 results in decreased HBCs and OSNs following OE injury. These data identify GLI2 and GLI3 as key regulators of HBC-mediated OE regeneration.
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