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Mutations in the intraflagellar transport-A (IFT-A) gene, THM1, have been identified in skeletal ciliopathies. Here, we report a genetic interaction between Thm1, and its paralog, Thm2, in postnatal skeletogenesis. THM2 localizes to primary cilia, but Thm2 deficiency does not affect ciliogenesis and Thm2-null mice survive into adulthood. However, by postnatal day 14, Thm2(−/−); Thm1(aln/+) mice exhibit small stature and small mandible. Radiography and microcomputed tomography reveal Thm2(−/−); Thm1(aln/+) tibia are less opaque and have reduced cortical and trabecular bone mineral density. In the mutant tibial growth plate, the proliferation zone is expanded and the hypertrophic zone is diminished, indicating impaired chondrocyte differentiation. Additionally, mutant growth plate chondrocytes show increased Hedgehog signaling. Yet deletion of one allele of Gli2, a major transcriptional activator of the Hedgehog pathway, exacerbated the Thm2(−/−); Thm1(aln/+) small phenotype, and further revealed that Thm2(−/−); Gli2(+/−) mice have small stature. In Thm2(−/−); Thm1(aln/+) primary osteoblasts, a Hedgehog signaling defect was not detected, but bone nodule formation was markedly impaired. This indicates a signaling pathway is altered, and we propose that this pathway may potentially interact with Gli2. Together, our data reveal that loss of Thm2 with one allele of Thm1, Gli2, or both, present new IFT mouse models of osteochondrodysplasia. Our data also suggest Thm2 as a modifier of Hedgehog signaling in postnatal skeletal development.

Polycystic liver disease (PLD) is characterized by the growth of numerous biliary cysts and presents in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD), causing significant morbidity. Interestingly, deletion of intraflagellar transport-B (IFT-B) genes in adult mouse models of ADPKD attenuates severity of PKD and PLD. Here we examine the role of deletion of IFT-A gene, Thm1, in PLD of juvenile and adult Pkd2 conditional knock-out mice. Perinatal deletion of Thm1 results in disorganized and expanded biliary regions, biliary fibrosis, shortened primary cilia on CK19+ biliary epithelial cells, and reduced Notch signaling. In contrast, perinatal deletion of Pkd2 causes PLD, with multiple CK19+ biliary epithelial cell-lined cysts, fibrosis, lengthened primary cilia, and increased Notch and ERK signaling. Perinatal deletion of Thm1 in Pkd2 conditional knock-out mice increased hepatomegaly and liver necrosis, indicating enhanced liver disease severity. In contrast to effects in the developing liver, deletion of Thm1 in adult mice, alone and together with Pkd2, did not cause a biliary phenotype nor affect Pkd2-mutant PLD, respectively. However, similar to juvenile PLD, Notch and ERK signaling were increased in adult Pkd2-mutant cyst-lining cholangiocytes. Taken together, Thm1 is required for biliary tract development, likely by enabling Notch signaling, and proper biliary development restricts PLD severity. Unlike IFT-B genes, Thm1 does not affect hepatic cystogenesis, suggesting divergent regulation of signaling and cystogenic processes in the liver by IFT-B and –A. Notably, increased Notch signaling in cyst-lining cholangiocytes may indicate that aberrant activation of this pathway promotes hepatic cystogenesis, presenting as a novel potential therapeutic target.

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multi-protein complexes. Deletion of different IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult Autosomal Dominant (AD) PKD mouse models, while deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, here we investigate the impact of global deletion of an IFT-A gene, Thm1, in juvenile and adult ADPKD mouse models. Both juvenile and adult models exhibited increased kidney weight:body weight (KW/BW) ratios, renal cysts, inflammation, lengthened renal cilia, and increased levels of the nutrient sensor, O-linked β-N-acetylglucosamine (O-GlcNAc). Thm1 deletion in juvenile ADPKD mice reduced KW/BW ratios and cortical collecting duct cystogenesis, but increased proximal tubular and glomerular dilations and did not reduce inflammation, cilia lengths, and O-GlcNAc signaling. In contrast, Thm1 deletion in adult ADPKD mice markedly attenuated renal cystogenesis, inflammation, cilia lengths, and O-GlcNAc. Thus, unlike IFT-B genes, the role of Thm1 deletion in ADPKD mouse models is development-specific. Unlike an IFT-A adaptor gene, deleting Thm1 in juvenile ADPKD mice is partially ameliorative. Our studies suggest that different microenvironmental factors found in distinct nephron segments and between developing and mature kidneys modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a novel feature and critical regulator of certain key ADPKD pathological processes.

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 bi-directional 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 double mutant 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-, Thm2- or IFT-A-mediated ciliopathies.

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, includingTHM1. Here, we report a role for genetic interaction betweenThm1and its paralog,Thm2,in skeletogenesis. THM2 localizes to the ciliary axoneme, but unlike its paralog,Thm2deficiency does not affect ciliogenesis andThm2-null mice survive into adulthood. Since paralogs often have redundant functions, we crossed aThm1null (aln) allele into theThm2colony. After 5 generations of backcrossing the colony onto a C57BL6/J background, we observed that by postnatal day 14,Thm2-/-; Thm1aln/+mice are smaller than control littermates.Thm2-/-; Thm1aln/+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-/-; Thm1aln/+tibia were revealed to have reduced cortical and trabecular bone mineral density. Deletion of one allele ofGli2, a major transcriptional activator of the Hedgehog (Hh) pathway, exacerbated the small phenotype ofThm2-/-; Thm1aln/+mice and caused small stature inThm2-null mice. Together, these data revealThm2as a novel locus that sensitizes to Hh signaling in skeletal development. Further,Thm2-/-; Thm1aln/+mice present a new postnatal ciliopathy model of osteochondrodysplasia.

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 bi-directional protein trafficking along the ciliary axoneme. Here using mouse embryonic fibroblasts (MEF), we investigate the ciliary roles of two mammalian orthologues ofChlamydomonasIFT-A gene,IFT139, namelyThm1(also known asTtc21b) andThm2 (Ttc21a). Thm1loss causes perinatal lethality, andThm2loss allows survival into adulthood. At E14.5, the number ofThm1;Thm2double mutant embryos is lower than that for a Mendelian ratio, indicating deletion ofThm1andThm2causes mid-gestational lethality. We examined the ciliary phenotypes of mutant MEF.Thm1-mutant MEF show decreased cilia assembly, 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 aThm1allele impairs response to SAG. Further,Thm1;Thm2double mutant MEF show enhanced cilia disassembly, and relative toThm1-null MEF, increased impairment of IFT81 retrograde transport and of INPP5E ciliary import. Thus,Thm1andThm2have unique and redundant roles in MEF.Thm1regulates cilia assembly, and together withThm2, cilia disassembly. Moreover,Thm1alone and together withThm2, regulates ciliary protein trafficking, Hedgehog signaling, and embryogenesis. These findings shed light on mechanisms underlyingThm1-,Thm2- or IFT-A-mediated ciliopathies.

Primary cilia are singular, sensory organelles that extend from the plasma membrane of most quiescent mammalian cells. These slender, microtubule-based organelles receive and transduce extracellular cues and regulate signaling pathways. Primary cilia are critical to the development and function of many tissue types, and mutation of ciliary genes causes multi-system disorders, termed ciliopathies. Notably, renal cystic disease is one of the most common clinical features of ciliopathies, highlighting a central role for primary cilia in the kidney. Additionally, acute kidney injury and chronic kidney disease are associated with altered primary cilia lengths on renal epithelial cells, suggesting ciliary dynamics and renal physiology are linked. Here we describe methods to examine primary cilia in kidney tissue and in cultured renal cells. We include immunofluorescence and scanning electron microscopy to determine ciliary localization of proteins and cilia structure. Further, we detail cellular assays to measure cilia assembly and disassembly, which regulate cilia length.