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Primary cilia are microtubule-based organelles that act as cellular antennae to mediate vertebrate development and growth factor signalling. Defects in primary cilia result in a group of inherited developmental conditions known as ciliopathies. Ciliopathies often present with cystic kidney disease, a major cause of early renal failure that requires renal replacement therapies. Currently, only one drug, Tolvaptan, is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions for these conditions remain a pressing clinical need.We screened clinical development compounds for positive effects on cilia formation and function and identified fasudil hydrochloride as the top hit. Fasudil is a generic, off-patent drug that is a potent but broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In a parallel whole genome siRNA-based reverse genetics phenotypic screen of positive modulators of cilia formation, we identified ROCK2 as the target molecule. We demonstrate that ROCK2 is a key mediator of cilium formation and function through effects on actin cytoskeleton remodelling. Our results indicate that specific ROCK2 inhibitors such as belumosudil (KD-025) could be repurposed for pharmacological intervention in cystic kidney disease. We propose that ROCK2 inhibition represents a novel, disease-modifying therapeutic approach for heterogeneous ciliopathies.

5-HT2A, a G-protein coupled receptor, is widely expressed in the human body, including in the gastrointestinal tract, platelets and the nervous system. It mediates various functions, for e.g. learning, memory, mood regulation, platelet aggregation and vasoconstriction, but its involvement in cell-adhesion remains largely unknown. Here we report a novel role for 5-HT2A in cell–matrix adhesion. In HEK293 cells, which are loosely adherent, expression and stimulation of human or rat 5-HT2A receptor by agonists such as serotonin or 2,5-dimethoxy-4-iodoamphetamine (DOI) led to a significant increase in adhesion, while inhibition of 5-HT2A by antipsychotics, such as risperidone, olanzapine or chlorpromazine prevented it. 5-HT2A activation gave rise to stress fibers in these cells and was also required for their maintenance. Mechanistically, the 5-HT2A-mediated adhesion was mediated by downstream PKC and Rho signaling. Since 5-HT2A is associated with many disorders such as dementia, depression and schizophrenia, its role in cell–matrix adhesion could have implications for neural circuits.

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.

SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes

Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/β-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with β-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.

Bar graph showing the percentage of the main features for each distinct ciliopathy syndrome. (PPTX 70 kb)

Vertebrate left-right (LR) asymmetry originates at a transient left-right organizer (LRO), where cilia play a crucial role in breaking symmetry. The LRO is a ciliated structure, however, much remains unknown about the choreography of cilia biogenesis and resorption at this organ. We recently identified a mutation affecting NEK2, a member of the NIMA-like serine-threonine kinase family, in a patient with congenital heart disease associated with abnormal LR development. Here, we report how Nek2 acts through cilia to influence LR patterning. Both overexpression and knockdown of nek2 in Xenopus result in abnormal LR development and reduction of LRO cilia count and motility, phenotypes that are modified by interaction with the Hippo signaling pathway. nek2 knockdown leads to a centriole defect at the LRO, consistent with the known role of Nek2 in centriole separation. Nek2 overexpression results in premature ciliary resorption in cultured cells dependent on function of the tubulin deacetylase HDAC6. Finally, we provide evidence that the known interaction between Nek2 and Nup98, a nucleoporin that localizes to the ciliary base, is important for regulating cilium resorption. Together these data show that Nek2 is a switch balancing ciliogenesis and resorption in the development of LR asymmetry.

Although fibroblast growth factor (FGF) signaling is critical for many developmental processes, the cellular mechanism(s) by which FGF exerts its effects remains obscure. In recent papers, Neugebauer et al. and Hong and Dawid focus on the role of FGF signaling on left-right axis patterning, showing that FGF functions at least in part via an effect on ciliogenesis.

Cilia establish the vertebrate left-right (LR) axis and are integral to the development and function of the kidney, liver, and brain. Left-right asymmetry is established in the ciliated ventral node cells of the mouse. The chiral structure of the cilium provides a reference asymmetry to impose handed LR asymmetric development on the bilaterally symmetric vertebrate embryo. A ciliary mechanism of LR development is evolutionarily conserved, as ciliated organs essential to LR axis formation, called LR organizers, are found in other vertebrates, including rabbit, fish, and Xenopus. Mice with mutations affecting ciliary biogenesis, motility, or sensory function have abnormal LR development and abnormal development of the heart. The axonemal dynein heavy chain left-right dynein (lrd) localizes to the LR organizer and drives counterclockwise movement of node primary cilia. Node primary cilia are an admixture of 9 + 2 and 9 + 0 cilia. Mutations in lrd result in structurally normal, immotile node monocilia. In the mouse, coordinated, directional beating of motile node monocilia at the neural fold stage generates leftward flow of extraembryonic fluid surrounding the node (nodal flow). Nodal flow triggers a rise in intracellular calcium in cells at the left side of the node. The perinodal asymmetric rise in intracellular calcium generated by nodal flow subsequently leads to asymmetric gene expression and morphogenesis.

Cilia establish the vertebrate left-right (LR) axis and are integral to the development and function of the kidney, liver, and brain. Left-right asymmetry is established in the ciliated ventral node cells of the mouse. The chiral structure of the cilium provides a reference asymmetry to impose handed LR asymmetric development on the bilaterally symmetric vertebrate embryo. A ciliary mechanism of LR development is evolutionarily conserved, as ciliated organs essential to LR axis formation, called LR organizers, are found in other vertebrates, including rabbit, fish, and Xenopus. Mice with mutations affecting ciliary biogenesis, motility, or sensory function have abnormal LR development and abnormal development of the heart. The axonemal dynein heavy chain left-right dynein (lrd) localizes to the LR organizer and drives counterclockwise movement of node primary cilia. Node primary cilia are an admixture of 9 + 2 and 9 + 0 cilia. Mutations in lrd result in structurally normal, immotile node monocilia. In the mouse, coordinated, directional beating of motile node monocilia at the neural fold stage generates leftward flow of extraembryonic fluid surrounding the node (nodal flow). Nodal flow triggers a rise in intracellular calcium in cells at the left side of the node. The perinodal asymmetric rise in intracellular calcium generated by nodal flow subsequently leads to asymmetric gene expression and morphogenesis.

Checklist used to cover all previously reported ciliopathy features. (DOCX 36 kb)

Supplemental clinical data: clinical details for the affected cases with mutations in novel candidate genes. (DOCX 35 kb)

Checklist used to cover all previously reported ciliopathy features. (DOCX 36 kb)

Supplemental clinical data: clinical details for the affected cases with mutations in novel candidate genes. (DOCX 35 kb)