RSG1 is required for cilia-dependent neural tube closure.

Cilia play a key role in the regulation of signaling pathways required for embryonic development, including the proper formation of the neural tube, the precursor to the brain and spinal cord. Forward genetic screens were used to generate mouse lines that display neural tube defects (NTD) and secondary phenotypes useful in interrogating function. We describe here the L3P mutant line that displays phenotypes of disrupted Sonic hedgehog signaling and affects the initiation of cilia formation. A point mutation was mapped in the L3P line to the gene Rsg1, which encodes a GTPase-like protein. The mutation lies within the GTP-binding pocket and disrupts the highly conserved G1 domain. The mutant protein and other centrosomal and IFT proteins still localize appropriately to the basal body of cilia, suggesting that RSG1 GTPase activity is not required for basal body maturation but is needed for a downstream step in axonemal elongation.

NEKL-4 regulates microtubule stability and mitochondrial health in ciliated neurons.

Ciliopathies are often caused by defects in the ciliary microtubule core. Glutamylation is abundant in cilia, and its dysregulation may contribute to ciliopathies and neurodegeneration. Mutation of the deglutamylase CCP1 causes infantile-onset neurodegeneration. In C. elegans, ccpp-1 loss causes age-related ciliary degradation that is suppressed by a mutation in the conserved NEK10 homolog nekl-4. NEKL-4 is absent from cilia, yet it negatively regulates ciliary stability via an unknown, glutamylation-independent mechanism. We show that NEKL-4 was mitochondria-associated. Additionally, nekl-4 mutants had longer mitochondria, a higher baseline mitochondrial oxidation state, and suppressed ccpp-1∆ mutant lifespan extension in response to oxidative stress. A kinase-dead nekl-4(KD) mutant ectopically localized to ccpp-1∆ cilia and rescued degenerating microtubule doublet B-tubules. A nondegradable nekl-4(PEST∆) mutant resembled the ccpp-1∆ mutant with dye-filling defects and B-tubule breaks. The nekl-4(PEST∆) Dyf phenotype was suppressed by mutation in the depolymerizing kinesin-8 KLP-13/KIF19A. We conclude that NEKL-4 influences ciliary stability by activating ciliary kinesins and promoting mitochondrial homeostasis.

Syndromic ciliopathy: a taiwanese single-center study.

BACKGROUND: Syndromic ciliopathies are a group of congenital disorders characterized by broad clinical and genetic overlap, including obesity, visual problems, skeletal anomalies, mental retardation, and renal diseases. The hallmark of the pathophysiology among these disorders is defective ciliary functions or formation. Many different genes have been implicated in the pathogenesis of these diseases, but some patients still remain unclear about their genotypes. METHODS: The aim of this study was to identify the genetic causes in patients with syndromic ciliopathy. Patients suspected of or meeting clinical diagnostic criteria for any type of syndromic ciliopathy were recruited at a single diagnostic medical center in Southern Taiwan. Whole exome sequencing (WES) was employed to identify their genotypes and elucidate the mutation spectrum in Taiwanese patients with syndromic ciliopathy. Clinical information was collected at the time of patient enrollment. RESULTS: A total of 14 cases were molecularly diagnosed with syndromic ciliopathy. Among these cases, 10 had Bardet-Biedl syndrome (BBS), comprising eight BBS2 patients and two BBS7 patients. Additionally, two cases were diagnosed with Alström syndrome, one with Oral-facial-digital syndrome type 14, and another with Joubert syndrome type 10. A total of 4 novel variants were identified. A recurrent splice site mutation, BBS2: c.534 + 1G > T, was present in all eight BBS2 patients, suggesting a founder effect. One BBS2 patient with homozygous c.534 + 1G > T mutations carried a third ciliopathic allele, TTC21B: c.264_267dupTAGA, a nonsense mutation resulting in a premature stop codon and protein truncation. CONCLUSIONS: Whole exome sequencing (WES) assists in identifying molecular pathogenic variants in ciliopathic patients, as well as the genetic hotspot mutations in specific populations. It should be considered as the first-line genetic testing for heterogeneous disorders characterized by the involvement of multiple genes and diverse clinical manifestations.

Polyploidy Promotes Hypertranscription, Apoptosis Resistance, and Ciliogenesis in Cancer Cells and Mesenchymal Stem Cells of Various Origins: Comparative Transcriptome In Silico Study.

Mesenchymal stem cells (MSC) attract an increasing amount of attention due to their unique therapeutic properties. Yet, MSC can undergo undesirable genetic and epigenetic changes during their propagation in vitro. In this study, we investigated whether polyploidy can compromise MSC oncological safety and therapeutic properties. For this purpose, we compared the impact of polyploidy on the transcriptome of cancer cells and MSC of various origins (bone marrow, placenta, and heart). First, we identified genes that are consistently ploidy-induced or ploidy-repressed through all comparisons. Then, we selected the master regulators using the protein interaction enrichment analysis (PIEA). The obtained ploidy-related gene signatures were verified using the data gained from polyploid and diploid populations of early cardiomyocytes (CARD) originating from iPSC. The multistep bioinformatic analysis applied to the cancer cells, MSC, and CARD indicated that polyploidy plays a pivotal role in driving the cell into hypertranscription. It was evident from the upregulation of gene modules implicated in housekeeping functions, stemness, unicellularity, DNA repair, and chromatin opening by means of histone acetylation operating via DNA damage associated with the NUA4/TIP60 complex. These features were complemented by the activation of the pathways implicated in centrosome maintenance and ciliogenesis and by the impairment of the pathways related to apoptosis, the circadian clock, and immunity. Overall, our findings suggest that, although polyploidy does not induce oncologic transformation of MSC, it might compromise their therapeutic properties because of global epigenetic changes and alterations in fundamental biological processes. The obtained results can contribute to the development and implementation of approaches enhancing the therapeutic properties of MSC by removing polyploid cells from the cell population.

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii.

Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.

A neurodevelopmental disorder associated with a loss-of-function missense mutation in RAB35.

Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.

Cep131-Cep162 and Cby-Fam92 complexes cooperatively maintain Cep290 at the basal body and contribute to ciliogenesis initiation.

Cilia play critical roles in cell signal transduction and organ development. Defects in cilia function result in a variety of genetic disorders. Cep290 is an evolutionarily conserved ciliopathy protein that bridges the ciliary membrane and axoneme at the basal body (BB) and plays critical roles in the initiation of ciliogenesis and TZ assembly. How Cep290 is maintained at BB and whether axonemal and ciliary membrane localized cues converge to determine the localization of Cep290 remain unknown. Here, we report that the Cep131-Cep162 module near the axoneme and the Cby-Fam92 module close to the membrane synergistically control the BB localization of Cep290 and the subsequent initiation of ciliogenesis in Drosophila. Concurrent deletion of any protein of the Cep131-Cep162 module and of the Cby-Fam92 module leads to a complete loss of Cep290 from BB and blocks ciliogenesis at its initiation stage. Our results reveal that the first step of ciliogenesis strictly depends on cooperative and retroactive interactions between Cep131-Cep162, Cby-Fam92 and Cep290, which may contribute to the complex pathogenesis of Cep290-related ciliopathies.

Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3T31A and ARL3T31A/C118F mutations.

ARL3 is essential for cilia development, and mutations in ARL3 are closely associated with ciliopathies. In a previous study, we observed distinct phenotypes of retinal dystrophy in patients with heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated transformed immortal fibroblast cells from patients carrying heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE-19 cells. Results showed that both ARL3T31A and ARL3T31A/C118F mutations led to a decrease in cilium formation. The ARL3T31A/C118F mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3T31A mutation did not induce significant changes in fibroblasts. RNA-sequencing results indicated that compared to ARL3T31A , ARL3T31A/C118F fibroblasts exhibited a higher enrichment of biological processes related to neuron projection development, tissue morphogenesis, and extracellular matrix (ECM) organization, with noticeable alterations in pathways such as ECM-receptor interaction, focal adhesion, and TGF-ß signaling. Similar changes were observed in the proteomic results in ARPE-19 cells. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3T31A/C118F group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3T31A/C118F fibroblasts. Our results not only enhance our understanding of ARL3-related diseases but also provide new insights into the analysis of heterozygous and compound heterozygous mutations in genetics.

IFT27 regulates the long-term maintenance of photoreceptor outer segments in zebrafish.

Approximately a quarter of Retinitis Pigmentosa (RP) is caused by mutations in transport-related genes in cilia. IFT27 (Intraflagellar Transport 27), a core component of the ciliary intraflagellar transport (IFT) system, has been implicated as a significant pathogenic gene in RP. The pathogenic mechanisms and subsequent pathology related to IFT27 mutations in RP are largely obscure. Here, we utilized TALEN technology to create an ift27 knockout (ift27-/-) zebrafish model. Electroretinography (ERG) detection showed impaired vision in this model. Histopathological examinations disclosed that ift27 mutations cause progressive degeneration of photoreceptors in zebrafish, and this degeneration was late-onset. Immunofluorescence labeling of outer segments showed that rods degenerated before cones, aligning with the conventional characterization of RP. In cultured human retinal pigment epithelial cells, we found that IFT27 was involved in maintaining ciliary morphology. Furthermore, decreased IFT27 expression resulted in the inhibition of the Hedgehog (Hh) signaling pathway, including decreased expression of key factors in the Hh pathway and abnormal localization of the ciliary mediator Gli2. In summary, we generated an ift27-/- zebrafish line with retinal degeneration which mimicked the symptoms of RP patients, highlighting IFT27's integral role in the long-term maintenance of cilia via the Hh signaling pathway. This work may furnish new insights into the treatment or delay of RP caused by IFT27 mutations.

Skewed X-chromosome inactivation drives the proportion of DNAAF6-defective airway motile cilia and variable expressivity in primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the absence of ciliary dynein arms, whereas their heterozygous mothers are supposedly healthy. Our objective was to assess the possible clinical and ciliary consequences of X-chromosome inactivation (XCI) in these mothers. METHODS: XCI patterns of six mothers of male patients with DNAAF6-related PCD were determined by DNA-methylation studies and compared with their clinical phenotype (6/6 mothers), as well as their ciliary phenotype (4/6 mothers), as assessed by immunofluorescence and high-speed videomicroscopy analyses. The mutated X chromosome was tracked to assess the percentage of cells with a normal inactivated DNAAF6 allele. RESULTS: The mothers' phenotypes ranged from absence of symptoms to mild/moderate or severe airway phenotypes, closely reflecting their XCI pattern. Analyses of the symptomatic mothers' airway ciliated cells revealed the coexistence of normal cells and cells with immotile cilia lacking dynein arms, whose ratio closely mirrored their XCI pattern. CONCLUSION: This study highlights the importance of searching for heterozygous pathogenic DNAAF6 mutations in all female relatives of male PCD patients with a DNAAF6 defect, as well as in females consulting for mild chronic respiratory symptoms. Our results also demonstrate that about one-third-ranging from 20% to 50%-normal ciliated airway cells sufficed to avoid severe PCD, a result paving the way for gene therapy.

Genetic variants in primary cilia-related genes associated with the prognosis of first-line chemotherapy in colorectal cancer.

BACKGROUND: Primary cilia are antenna-like organelles that conduct physical and chemical signals, which affect cell proliferation, migration, and differentiation. Some researchers have reported the correlation between primary cilia-related genes and prognosis of colorectal cancer (CRC). METHODS: The association between single nucleotide polymorphisms (SNPs) of primary cilia-related genes and outcome after the first-line chemotherapy was explored by the Cox regression model. Expression qualitative trait locus (eQTL) analysis was performed to explore the impact of SNPs on gene expression. Tumor Immune Estimation Resource and TISIDB databases were used for investigating the relevance between ODF2L and tumor infiltration immune cells and immunomodulators. RESULTS: We identified that rs4288473 C allele of ODF2L had poor progression-free survival (PFS) and overall survival (OS) of CRC patients in the additive model (adjusted HRPFS = 1.39, 95% CI = 1.14-1.70, p = 1.36 × 10-3 , and adjusted HROS = 1.31, 95% CI = 1.03-1.65, p = 2.62 × 10-2 ). The stratified analysis indicated that rs4288573 CC/CT genotype was involved with poor prognosis in the irinotecan-treated subgroup (PPFS = 1.03 × 10-2 , POS = 3.29 × 10-3 ). Besides, ODF2L mRNA expression level was notably up-regrated in CRC tissues. The C allele of rs4288573 was notably related to higher ODF2L mRNA expression levels based on eQTL analysis. Functionally, knockdown of ODF2L inhibited cell proliferation and decrease the chemoresistance of HCT-116 and DLD-1 cells to irinotecan. CONCLUSION: Our study indicates that rs4288573 in ODF2L is a potential predictor of the chemotherapy prognosis of CRC.

Novel compound heterozygous variants in ARL13B lead to Joubert syndrome.

Joubert syndrome (JBTS) is a systematic developmental disorder mainly characterized by a pathognomonic mid-hindbrain malformation. All known JBTS-associated genes encode proteins involved in the function of antenna-like cellular organelle, primary cilium, which plays essential roles in cellular signal transduction and development. Here, we identified four unreported variants in ARL13B in two patients with the classical features of JBTS. ARL13B is a member of the Ras GTPase family and functions in ciliogenesis and cilia-related signaling. The two missense variants in ARL13B harbored the substitutions of amino acids at evolutionarily conserved positions. Using model cell lines, we found that the accumulations of the missense variants in cilia were impaired and the variants showed attenuated functions in ciliogenesis or the trafficking of INPP5E. Overall, these findings expanded the ARL13B pathogenetic variant spectrum of JBTS.

POMC Neuron BBSome Regulation of Body Weight is Independent of its Ciliary Function.

The BBSome, a complex of several Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as a critical regulator of energy homeostasis. Although the BBSome is best known for its involvement in cilia trafficking, through a process that involve BBS3, it also regulates the localization of cell membrane receptors underlying metabolic regulation. Here, we show that inducible Bbs1 gene deletion selectively in proopiomelanocortin (POMC) neurons cause a gradual increase in body weight, which was associated with higher fat mass. In contrast, inducible deletion of Bbs3 gene in POMC neurons failed to affect body weight and adiposity. Interestingly, loss of BBS1 in POMC neurons led to glucose intolerance and insulin insensitivity, whereas BBS3 deficiency in these neurons is associated with slight impairment in glucose handling, but normal insulin sensitivity. BBS1 deficiency altered the plasma membrane localization of serotonin 5-HT2C receptor (5-HT2CR) and ciliary trafficking of neuropeptide Y2 receptor (NPY2R).In contrast, BBS3 deficiency, which disrupted the ciliary localization of the BBSome, did not interfere with plasma membrane expression of 5-HT2CR, but reduced the trafficking of NPY2R to cilia. We also show that deficiency in BBS1, but not BBS3, alters mitochondria dynamics and decreased total and phosphorylated levels of dynamin-like protein 1 (DRP1) protein. Importantly, rescuing DRP1 activity restored mitochondria dynamics and localization of 5-HT2CR and NPY2R in BBS1-deficient cells. The contrasting effects on energy and glucose homeostasis evoked by POMC neuron deletion of BBS1 versus BBS3 indicate that BBSome regulation of metabolism is not related to its ciliary function in these neurons.

The genetic framework of primary ciliary dyskinesia assessed by soft computing analysis.

BACKGROUND: International guidelines disagree on how best to diagnose primary ciliary dyskinesia (PCD), not least because many tests rely on pattern recognition. We hypothesized that quantitative distribution of ciliary ultrastructural and motion abnormalities would detect most frequent PCD-causing groups of genes by soft computing analysis. METHODS: Archived data on transmission electron microscopy and high-speed video analysis from 212 PCD patients were re-examined to quantitate distribution of ultrastructural (10 parameters) and functional ciliary features (4 beat pattern and 2 frequency parameters). The correlation between ultrastructural and motion features was evaluated by blinded clustering analysis of the first two principal components, obtained from ultrastructural variables for each patient. Soft computing was applied to ultrastructure to predict ciliary beat frequency (CBF) and motion patterns by a regression model. Another model classified the patients into the five most frequent PCD-causing gene groups, from their ultrastructure, CBF and beat patterns. RESULTS: The patients were subdivided into six clusters with similar values to homologous ultrastructural phenotype, motion patterns, and CBF, except for clusters 1 and 4, attributable to normal ultrastructure. The regression model confirmed the ability to predict functional ciliary features from ultrastructural parameters. The genetic classification model identified most of the different groups of genes, starting from all quantitative parameters. CONCLUSIONS: Applying soft computing methodologies to PCD diagnostic tests optimizes their value by moving from pattern recognition to quantification. The approach may also be useful to evaluate atypical PCD, and novel genetic abnormalities of unclear disease-producing potential in the future.

Joubert syndrome causing mutation in C2 domain of CC2D2A affects structural integrity of cilia and cellular signaling molecules.

Cilia are organelles extend from cells to sense external signals for tuning intracellular signaling for optimal cellular functioning. They have evolved sensory and motor roles in various cells for tissue organization and homeostasis in development and post-development. More than a thousand genes are required for cilia function. Mutations in them cause multisystem disorders termed ciliopathies. The null mutations in CC2D2A result in Meckel syndrome (MKS), which is embryonic lethal, whereas patients who have missense mutations in the C2 domain of CC2D2A display Joubert syndrome (JBTS). They survive with blindness and mental retardation. How C2 domain defects cause disease conditions is not understood. To answer this question, C2 domain of Cc2d2a (mice gene) was knocked down (KD) in IMCD-3 cells by shRNA. This resulted in defective cilia morphology observed by immunofluorescence analysis. To further probe the cellular signaling alteration in affected cells, gene expression profiling was done by RNAseq and compared with the controls. Bioinformatics analysis revealed that the differentially expressed genes (DEGs) have functions in cilia. Among the 61 cilia DEGs identified, 50 genes were downregulated and 11 genes were upregulated. These cilia genes are involved in cilium assembly, protein trafficking to the cilium, intraflagellar transport (IFT), cellular signaling like polarity patterning, and Hedgehog signaling pathway. This suggests that the C2 domain of CC2D2A plays a critical role in cilia assembly and molecular signaling hosted in cilia for cellular homeostasis. Taken together, the missense mutations in the C2 domain of CC2D2A seen in JBTS might have affected cilia-mediated signaling in neurons of the retina and brain.

Racgap1 knockdown results in cells with multiple cilia due to cytokinesis failure.

Most mammalian cells have a single primary cilium that acts as a signalling hub in mediating cellular functions. However, little is known about the mechanisms that result in aberrant supernumerary primary cilia per cell. In this study, we re-analysed a previously published whole-genome siRNA-based reverse genetic screen for genes mediating ciliogenesis to identify knockdowns that permit multi-ciliation. We identified siRNA knockdowns that caused significant formation of supernumerary cilia, validated candidate hits in different cell-lines and confirmed that RACGAP1, a component of the centralspindlin complex, was the strongest candidate hit at the whole-genome level. Following loss of RACGAP1, mother centrioles were specified correctly prior to ciliogenesis and the cilia appeared normal. Live cell imaging revealed that increased cilia incidence was caused by cytokinesis failure which led to the formation of multinucleate cells with supernumerary cilia. This suggests that the signalling mechanisms for ciliogenesis are unable to identify supernumerary centrosomes and therefore allow ciliation of duplicated centrosomes as if they were in a new diploid daughter cell. These results, demonstrating that aberrant ciliogenesis is de-coupled from cell cycle regulation, have functional implications in diseases marked by centrosomal amplification.

Functions of cilia in cardiac development and disease.

Errors in embryonic cardiac development are a leading cause of congenital heart defects (CHDs), including morphological abnormalities of the heart that are often detected after birth. In the past few decades, an emerging role for cilia in the pathogenesis of CHD has been identified, but this topic still largely remains an unexplored area. Mouse forward genetic screens and whole exome sequencing analysis of CHD patients have identified enrichment for de novo mutations in ciliary genes or non-ciliary genes, which regulate cilia-related pathways, linking cilia function to aberrant cardiac development. Key events in cardiac morphogenesis, including left-right asymmetric development of the heart, are dependent upon cilia function. Cilia dysfunction during left-right axis formation contributes to CHD as evidenced by the substantial proportion of heterotaxy patients displaying complex CHD. Cilia-transduced signaling also regulates later events during heart development such as cardiac valve formation, outflow tract septation, ventricle development, and atrioventricular septa formation. In this review, we summarize the role of motile and non-motile (primary cilia) in cardiac asymmetry establishment and later events during heart development.

Deletion of IFT20 exclusively in the RPE ablates primary cilia and leads to retinal degeneration.

Vision impairment places a serious burden on the aging society, affecting the lives of millions of people. Many retinal diseases are of genetic origin, of which over 50% are due to mutations in cilia-associated genes. Most research on retinal degeneration has focused on the ciliated photoreceptor cells of the retina. However, the contribution of primary cilia in other ocular cell types has largely been ignored. The retinal pigment epithelium (RPE) is a monolayer epithelium at the back of the eye intricately associated with photoreceptors and essential for visual function. It is already known that primary cilia in the RPE are critical for its development and maturation; however, it remains unclear whether this affects RPE function and retinal tissue homeostasis. We generated a conditional knockout mouse model, in which IFT20 is exclusively deleted in the RPE, ablating primary cilia. This leads to defective RPE function, followed by photoreceptor degeneration and, ultimately, vision impairment. Transcriptomic analysis offers insights into mechanisms underlying pathogenic changes, which include transcripts related to epithelial homeostasis, the visual cycle, and phagocytosis. Due to the loss of cilia exclusively in the RPE, this mouse model enables us to tease out the functional role of RPE cilia and their contribution to retinal degeneration, providing a powerful tool for basic and translational research in syndromic and non-syndromic retinal degeneration. Non-ciliary mechanisms of IFT20 in the RPE may also contribute to pathogenesis and cannot be excluded, especially considering the increasing evidence of non-ciliary functions of ciliary proteins.

Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function.

Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their requirement for muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Depletion of MuSC after regeneration suggests impaired self-renewal, also consistent with an exacerbated phenotype in TA3iSC-KO mice after repeat injury. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data show that TA3 and primary cilia are important for MuSC self-renewal and pharmacological treatment can efficiently restore muscle regeneration.

The G protein alpha chaperone and guanine-nucleotide exchange factor RIC-8 regulates cilia morphogenesis in Caenorhabditis elegans sensory neurons.

Heterotrimeric G (αßγ) proteins are canonical transducers of G-protein-coupled receptor (GPCR) signaling and play critical roles in communication between cells and their environment. Many GPCRs and heterotrimeric G proteins localize to primary cilia and modulate cilia morphology via mechanisms that are not well understood. Here, we show that RIC-8, a cytosolic guanine nucleotide exchange factor (GEF) and chaperone for Gα protein subunits, shapes cilia membrane morphology in a subset of Caenorhabditis elegans sensory neurons. Consistent with its role in ciliogenesis, C. elegans RIC-8 localizes to cilia in different sensory neuron types. Using domain mutagenesis, we demonstrate that while the GEF function alone is not sufficient, both the GEF and Gα-interacting chaperone motifs of RIC-8 are required for its role in cilia morphogenesis. We identify ODR-3 as the RIC-8 Gα client and demonstrate that RIC-8 functions in the same genetic pathway with another component of the non-canonical G protein signaling AGS-3 to shape cilia morphology. Notably, despite defects in AWC cilia morphology, ags-3 null mutants exhibit normal chemotaxis toward benzaldehyde unlike odr-3 mutant animals. Collectively, our findings describe a novel function for the evolutionarily conserved protein RIC-8 and non-canonical RIC-8-AGS-3-ODR-3 signaling in cilia morphogenesis and uncouple Gα ODR-3 functions in ciliogenesis and olfaction.