Renal cystic diseases are a leading cause of renal failure. Mutations associated with renal cystic diseases reside in genes encoding proteins that localize to primary cilia. These cystoproteins can disrupt ciliary structure or cilia-mediated signaling, although molecular mechanisms connecting cilia function to renal cystogenesis remain unclear. The ciliary gene, Thm1(Ttc21b), negatively regulates Hedgehog signaling and is most commonly mutated in ciliopathies. We report that loss of murine Thm1 causes cystic kidney disease, with persistent proliferation of renal cells, elevated cAMP levels, and enhanced expression of Hedgehog signaling genes. Notably, the cAMP-mediated cystogenic potential of Thm1-null kidney explants was reduced by genetically deleting Gli2, a major transcriptional activator of the Hedgehog pathway, or by culturing with small molecule Hedgehog inhibitors. These Hedgehog inhibitors acted independently of protein kinase A and Wnt inhibitors. Furthermore, simultaneous deletion of Gli2 attenuated the renal cystic disease associated with deletion of Thm1. Finally, transcripts of Hedgehog target genes increased in cystic kidneys of two other orthologous mouse mutants, jck and Pkd1, and Hedgehog inhibitors reduced cystogenesis in jck and Pkd1 cultured kidneys. Thus, enhanced Hedgehog activity may have a general role in renal cystogenesis and thereby present a novel therapeutic target.

Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutation of PKD1 or PKD2, which encode polycystin 1 and 2, respectively. The polycystins localize to primary cilia and the functional loss of the polycystin complex leads to the formation and progressive growth of fluid-filled cysts in the kidney. The pathogenesis of ADPKD is complex and molecular mechanisms connecting ciliary dysfunction to renal cystogenesis are unclear. Primary cilia mediate Hedgehog signaling, which modulates cell proliferation and differentiation in a tissue-dependent manner. Previously, we showed that Hedgehog signaling was increased in cystic kidneys of several PKD mouse models and that Hedgehog inhibition prevented cyst formation in embryonic PKD mouse kidneys treated with cAMP. Here, we show that in human ADPKD tissue, Hedgehog target and activator, Glioma 1, was elevated and localized to cyst-lining epithelial cells and to interstitial cells, suggesting increased autocrine and paracrine Hedgehog signaling in ADPKD, respectively. Further, Hedgehog inhibitors reduced basal and cAMP-induced proliferation of ADPKD cells and cyst formation in vitro. These data suggest that Hedgehog signaling is increased in human ADPKD and that suppression of Hedgehog signaling can counter cellular processes that promote cyst growth in vitro.

Primary cilia are sensory organelles that are essential for eukaryotic development and health. These antenna-like structures are synthesized by intraflagellar transport protein complexes, IFT-B and IFT-A, which mediate bidirectional protein trafficking along the ciliary axoneme. Here using mouse embryonic fibroblasts (MEF), we investigate the ciliary roles of two mammalian orthologues of Chlamydomonas IFT-A gene, IFT139, namely Thm1 (also known as Ttc21b) and Thm2 (Ttc21a). Thm1 loss causes perinatal lethality, and Thm2 loss allows survival into adulthood. At E14.5, the number of Thm1;Thm2 double mutant embryos is lower than that for a Mendelian ratio, indicating deletion of Thm1 and Thm2 causes mid-gestational lethality. We examined the ciliary phenotypes of mutant MEF. Thm1-mutant MEF show decreased cilia assembly, increased cilia disassembly, shortened primary cilia, a retrograde IFT defect for IFT and BBS proteins, and reduced ciliary entry of membrane-associated proteins. Thm1-mutant cilia also show a retrograde transport defect for the Hedgehog transducer, Smoothened, and an impaired response to Smoothened agonist, SAG. Thm2-null MEF show normal ciliary dynamics and Hedgehog signaling, but additional loss of a Thm1 allele impairs response to SAG. Further, Thm1;Thm2 doublemutant MEF show enhanced cilia disassembly, and increased impairment of INPP5E ciliary import. Thus, Thm1 and Thm2 have unique and redundant roles in MEF. Thm1 regulates cilia assembly, and alone and together with Thm2, regulates cilia disassembly, ciliary entry of membrane-associated protein, Hedgehog signaling, and embryogenesis. These findings shed light on mechanisms underlying Thm1-, Thm2or IFT-A-mediated ciliopathies. K E Y W O R D S development, mouse embryonic fibroblasts, mouse model, primary cilia 6370 | WANG et Al.

Ciliopathies are genetic syndromes that link osteochondrodysplasias to dysfunction of primary cilia. Primary cilia extend from the surface of bone and cartilage cells, to receive extracellular cues and mediate signaling pathways. Mutations in several genes that encode components of the intraflagellar transport-A ciliary protein complex have been identified in skeletal ciliopathies, including THM1. Here, we report a role for genetic interaction between Thm1 and its paralog, Thm2, in skeletogenesis. THM2 localizes to the ciliary axoneme, but unlike its paralog, Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood.Since paralogs often have redundant functions, we crossed a Thm1 null (aln) allele into the Thm2 colony. After 5 generations of backcrossing the colony onto a C57BL6/J background, we observed that by postnatal day 14, Thm2 -/-; Thm1 aln/+ mice are smaller than control littermates. Thm2 -/-; Thm1 aln/+ mice exhibit shortened long bones, narrow ribcage, shortened cranium and mandibular defects. Mutant mice also show aberrant architecture of the tibial growth plate, with an expanded proliferation zone and diminished hypertrophic zone, indicating impaired chondrocyte differentiation. Using microcomputed tomography, Thm2 -/-; Thm1 aln/+ tibia were revealed to have reduced cortical and trabecular bone mineral density. Deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog (Hh) pathway, exacerbated the small phenotype of Thm2 -/-; Thm1 aln/+ mice and caused small stature in Thm2-null mice. Together, these data reveal Thm2 as a novel locus that sensitizes to Hh signaling in skeletal development. Further, Thm2 -/-; Thm1 aln/+ mice present a new postnatal ciliopathy model of osteochondrodysplasia.