Haploinsufficiency of intraflagellar transport protein 172 causes autism-like behavioral phenotypes in mice through BDNF.

INTRODUCTION: Primary cilia are hair-like solitary organelles growing on most mammalian cells that play fundamental roles in embryonic patterning and organogenesis. Defective cilia often cause a suite of inherited diseases called ciliopathies with multifaceted manifestations. Intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium, actively facilitates the formation and absorption of primary cilia. IFT172 is the largest component of the IFT-B complex, and its roles in Bardet-Biedl Syndrome (BBS) have been appreciated with unclear mechanisms. OBJECTIVES: We performed a battery of behavioral tests with Ift172 haploinsufficiency (Ift172+/-) and WT littermates. We use RNA sequencing to identify the genes and signaling pathways that are differentially expressed and enriched in the hippocampus of Ift172+/- mice. Using AAV-mediated sparse labeling, electron microscopic examination, patch clamp and local field potential recording, western blot, luciferase reporter assay, chromatin immunoprecipitation, and neuropharmacological approach, we investigated the underlying mechanisms for the aberrant phenotypes presented by Ift172+/- mice. RESULTS: Ift172+/- mice displayed excessive self-grooming, elevated anxiety, and impaired cognition. RNA sequencing revealed enrichment of differentially expressed genes in pathways relevant to axonogenesis and synaptic plasticity, which were further confirmed by less spine density and synaptic number. Ift172+/- mice demonstrated fewer parvalbumin-expressing neurons, decreased inhibitory synaptic transmission, augmented theta oscillation, and sharp-wave ripples in the CA1 region. Moreover, Ift172 haploinsufficiency caused less BDNF production and less activated BDNF-TrkB signaling pathway through transcription factor Gli3. Application of 7,8-Dihydroxyflavone, a potent small molecular TrkB agonist, fully restored BDNF-TrkB signaling activity and abnormal behavioral phenotypes presented by Ift172+/- mice. With luciferase and chip assays, we provided further evidence that Gli3 may physically interact with BDNF promoter I and regulate BDNF expression. CONCLUSIONS: Our data suggest that Ift172 per se drives neurotrophic effects and, when defective, could cause neurodevelopmental disorders reminiscent of autism-like disorders.

mTOR potentiates senescent phenotypes and primary cilia formation after cisplatin-induced G2 arrest in retinal pigment epithelial cells.

Cisplatin, a platinum-based anticancer drug, is used to treat several types of cancer. Despite its effectiveness, cisplatin-induced side effects have often been reported. Although cisplatin-induced toxicities, such as apoptosis and/or necrosis, have been well studied, the fate of cells after exposure to sublethal doses of cisplatin needs further elucidation. Treatment with a sublethal dose of cisplatin induced cell cycle arrest at the G2 phase in retinal pigment epithelial cells. Following cisplatin withdrawal, the cells irreversibly exited the cell cycle and became senescent. Notably, the progression from the G2 to the G1 phase occurred without mitotic entry, a phenomenon referred to as mitotic bypass, resulting in the accumulation of cells containing 4N DNA content. Cisplatin-exposed cells exhibited morphological changes associated with senescence, including an enlarged size of cell and nucleus and increased granularity. In addition, the senescent cells possessed primary cilia and persistent DNA lesions. Senescence induced by transient exposure to cisplatin involves mTOR activation. Although transient co-exposure with an mTORC1 inhibitor rapamycin did not prevent mitotic bypass and entry into senescence, it delayed the progression of senescence and attenuated senescent phenotypes, resulting in shorter primary cilia formation. Conclusively, cisplatin induces senescence in retinal pigment epithelial cells by promoting mTOR activation.

The effects of primary cilia-mediated mechanical stimulation on nestin+-BMSCs during bone-tendon healing.

INTRODUCTION: Mechanical stimulation has been proven to promote bone-tendon interface (BTI) healing, but the mechanism remains unclear. OBJECTIVE: To investigate the effects of mechanical stimulation on the biological behavior of nestin+-bone mesenchymal stem cells (BMSCs) during the BTI healing, and to reveal the mechanisms of mechanical stimulation affecting BTI healing by primary cilia on the nestin+-BMSCs. METHODS: Transgenic tracing mice (nestin creERT2:: IFT88fl/fl/ROSA26 YFP) with primary cilia on nestin+-BMSCs conditioned knocked out were constructed, and the littermates (nestin creERT2:: ROSA26 YFP) with normal cilia on nestin+-BMSCs were the control. After establishing mouse supraspinatus insertion injury models, samples were collected at week-2 (n = 5 per group), 4 and 8 (n = 15 per group, respectively). In vivo, the repair efficiency was evaluated by histology, imaging, biomechanics, and the migration of nestin+-BMSCs, detected by immunofluorescence staining. In vitro, nestin+ BMSCs were sorted and stimulated by tensile force to study the mechanisms of primary cilium-mediated mechanosensitive basis. RESULTS: Mechanical stimulation (MS) accelerated the recruitment of nestin+-BMSCs and promoted osteogenic and chondrogenic capacity. Histological, imaging and biomechanical results showed that the BTI healing quality of the IFT88+/+, MS group was better than that of the other groups. After the conditionally knockout IFT88 in nestin+-BMSCs, the repair ability of the BTI was obviously deteriorated, even though mechanical stimulation did not increase significantly (IFT88-/-, MS group). In vitro results showed the tensile loading enhanced the proliferation, migration and osteogenic or chondrogenic gene expression of nestin+-BMSCs with normal cilia. On the other hand, osteogenesis and chondrogenic expression were significantly decreased after inhibiting actin- Hippo/YAP pathway components. CONCLUSION: The primary cilia mediated mechanical stimulation regulated osteogenic and chondrogenic differentiation potential of nestin+-BMSCs through the actin- Hippo/YAP pathway, and then promoted the BTI healing process.

Identifying the roles of miR-17 in ciliogenesis and cell cycle.

Emerging evidence suggests a significant contribution of primary cilia to cell division and proliferation. MicroRNAs, especially miR-17, contribute to cell cycle regulation and proliferation. Recent investigations have highlighted the dysregulated expression of miR-17 in various malignancies, underlining its potential role in cancer. However, the correlation between primary cilia and miR-17 has yet to be fully elucidated. The present study examines the presence of miR-17 in primary cilia. The miR-17 expression is studied in selected ciliary protein knockdown cells. Using in situ hybridization (ISH), we identified the subcellular localization of miR-17 in both cilium and cell body. We confirmed the importance of miR-17, progesterone receptor membrane component-2 (PGRMC2), and monosialodihexosylganglioside (GM3S) in cilia formation, as shown by the significant reduction in cilia and cilia length in knockdown cells compared to control. We also demonstrated the involvement of PGRMC2, GM3S, polycystin-2 (PKD2), and miR-17 in cellular proliferation and cell growth. Our studies revealed a hyperproliferative effect in the knockdown cells compared to control cells, suggesting the regulatory roles of PGRMC2/GM3S/PKD2/miR-17 in promoting cell proliferation. Overall, our studies conclude that ciliary proteins are involved in cell division and proliferation. We further hypothesize that primary cilia can serve as compartments to store and control genetic materials, further implicating their complex involvement in cellular processes.

Constructing condylar cartilage organoid to explore primary cilia functions.

An organoid culture system better recapitulates the cellular structure, function, and interaction between cells and the extracellular matrix (ECM) than a two-dimensional (2D) culture system. We here constructed a condylar cartilage organoid to explore the regulatory role of primary cilia. Similar to the natural condylar cartilage, the condylar cartilage organoid exhibited abundant ECM and comprised superficial, proliferative, and hypertrophic zones. Primary cilia in the condylar cartilage organoid were shorter on average than those in the 2D culture chondrocytes, but their average length was equivalent to those in the natural condylar cartilage. Notably, primary cilia in each zone of the condylar cartilage organoid had an average length similar to that of primary cilia in the natural condylar cartilage. According to transcriptomic and biochemical analyses, the expression of cilia-related genes and cilia-related Hedgehog (HH) signaling differed between the condylar cartilage organoid and 2D culture systems. IFT88 knockdown promoted the protein levels of COL-Ⅹ, TRPV4, and HH signaling molecules in the condylar cartilage organoid, but decreased them in the 2D culture system. Notably, the protein levels of COL-Ⅹ, TRPV4, and HH signaling molecules increased in the superficial zone of the si IFT88 condylar cartilage organoid compared with the condylar cartilage organoid. However, the protein levels of aforementioned molecules were not significantly different in proliferative and hypertrophic zones. Collectively, we successfully constructed the condylar cartilage organoid with a better tissue structure and abundant ECM. Moreover, the condylar cartilage organoid is more suitable for exploring primary cilia functions.

BET Inhibition Rescues Acinar-Ductal-Metaplasia and Ciliogenesis and Ameliorates Chronic Pancreatitis-Driven Changes in Mice with Loss of the Polarity Protein Par3.

BACKGROUND & AIMS: The apical-basal polarity of pancreatic acinar cells is essential for maintaining tissue architecture. However, the mechanisms by which polarity proteins regulate acinar pancreas injury and regeneration are poorly understood. METHODS: Cerulein-induced pancreatitis was induced in mice with conditional deletion of the polarity protein Par3 in the pancreas. The impact of Par3 loss on pancreas injury and regeneration was assessed by histologic analyses and transcriptional profiling by RNA sequencing. Mice were pretreated with the bromodomain and extraterminal domain (BET) inhibitor JQ1 before cotreatment with cerulein to determine the effect of BET inhibition on pancreas injury and regeneration. RESULTS: Initially, we show that Par3 is increased in acinar-ductal metaplasia (ADM) lesions present in human and mouse chronic pancreatitis specimens. Although Par3 loss disrupts tight junctions, Par3 is dispensable for pancreatogenesis. However, with aging, Par3 loss results in low-grade inflammation, acinar degeneration, and pancreatic lipomatosis. Par3 loss exacerbates acute pancreatitis-induced injury and chronic pancreatitis-induced acinar cell loss, promotes pancreatic lipomatosis, and prevents regeneration. Par3 loss also results in suppression of chronic pancreatitis-induced ADM and primary ciliogenesis. Notably, targeting BET proteins attenuates chronic pancreatitis-induced loss of primary cilia and promotes ADM in mice lacking pancreatic Par3. Targeting BET proteins also attenuates cerulein-induced acinar cell loss and enhances recovery of acinar cell mass and body weight of mice lacking pancreatic Par3. CONCLUSIONS: Combined, this study demonstrates how Par3 restrains chronic pancreatitis-induced changes in the pancreas and identifies a potential role for BET inhibitors to attenuate pancreas injury and facilitate regeneration.

Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease.

Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's.

Primary Cilia Elongation in Early-Onset Polycystic Kidney Disease with 2 Hypomorphic PKD1 Alleles: A Case Report.

Recent studies have described several children with very early-onset polycystic kidney disease (PKD) that mimicked autosomal recessive polycystic kidney disease because of 2 hypomorphic PKD1 gene variants. However, no reports have described pathological changes in the primary cilia in these cases. We analyzed the primary cilia in the kidney tubules of an early elementary school child who had very early-onset PKD and a history of large, echogenic kidneys in utero. There was no family history of autosomal dominant PKD. The patient developed kidney failure and received a living-donor kidney transplant from his father. Genetic analysis revealed compound heterozygous variants in the PKD1 gene: c.3876C>A (p. Phe1292Leu) and c.5957C>T (p. Thr1986Met). These variants were likely pathogenic based on in silico analysis. The absence of kidney cysts in the parents suggested that these variants were hypomorphic alleles. Pathological examination of the patient's excised kidney showed prominent dilatation of the proximal and distal tubules. Immunofluorescence staining for α-tubulin showed pronounced elongation of the primary cilia. These findings suggest that the hypomorphic PKD1 variants expressed in this patient with very early-onset PKD were pathogenic.

Ciliary length variations impact cilia-mediated signaling and biological responses.

Primary cilia are thin hair-like organelles that protrude from the surface of most mammalian cells. They act as specialized cell antennas that can vary widely in response to specific stimuli. However, the effect of changes in cilia length on cellular signaling and behavior remains unclear. Therefore, we aimed to characterize the elongated primary cilia induced by different chemical agents, lithium chloride (LiCl), cobalt chloride (CoCl2), and rotenone, using human retinal pigmented epithelial 1 (hRPE1) cells expressing ciliary G protein-coupled receptor (GPCR), melanin-concentrating hormone (MCH) receptor 1 (MCHR1). MCH induces cilia shortening mainly via MCHR1-mediated Akt phosphorylation. Therefore, we verified the proper functioning of the MCH-MCHR1 axis in elongated cilia. Although MCH shortened cilia that were elongated by LiCl and rotenone, it did not shorten CoCl2-induced elongated cilia, which exhibited lesser Akt phosphorylation. Furthermore, serum readdition was found to delay cilia shortening in CoCl2-induced elongated cilia. In contrast, rotenone-induced elongated cilia rapidly shortened via a chopping mechanism at the tip of the cilia. Conclusively, we found that each chemical exerted different effects on ciliary GPCR signaling and serum-mediated ciliary structure dynamics in cells with elongated cilia. These results provide a basis for understanding the functional consequences of changes in ciliary length.

An Epilepsy-Associated CILK1 Variant Compromises KATNIP Regulation and Impairs Primary Cilia and Hedgehog Signaling.

Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEFs) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia, and upregulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild-type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. The CILK1 A615T mutant protein was not increased to the same level as the wild-type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single-residue variant of CILK1 is compromised, and this impairs the maintenance of primary cilia and Hedgehog signaling.

Human efferent ductules and epididymis display unique cell lineages with motile and primary cilia.

BACKGROUND: Previous research has illustrated the role of cilia as mechanical and sensory antennae in various organs within the mammalian male reproductive system across different developmental stages. Despite their significance in both organ development and homeostasis, primary cilia in the human male reproductive excurrent duct have been overlooked due to limited access to human specimens. OBJECTIVE: This study aimed to characterize the unique cellular composition of human efferent and epididymal ducts, with a focus on their association with primary cilia. MATERIALS AND METHODS: Human efferent ductules/epididymides from five donors aged 32-47 years, were obtained through our local organ transplant program. Cell lineage specificity and primary cilia features were examined by immunofluorescent staining and confocal microscopy in the efferent ductules and the distinct segments of the epididymis. RESULTS: The epithelium of the human efferent duct exhibited estrogen receptor-positive cells with primary cilia, FoxJ1-positive multiciliated cells with numerous motile cilia, and non-ciliated intraepithelial immune cells. Notably, intraluminal macrophages, identified by CD163/CD68 positivity, were observed to engage in sperm phagocytosis. In all three segments of the human epididymis, primary cilia were found on the surface of principal and basal cells. DISCUSSION AND CONCLUSIONS: Our research indicates that the human efferent ductules create a distinct environment, characterized by the presence of two types of ciliated cells that are in contact with immune cells. The discovery of sensory primary cilia exposed on the surface of reabsorptive cells in the efferent ductules, as well as on basal and principal cells in the epididymis, lays the foundation for complementary functional studies. This research uncovers novel characteristics exclusive to human efferent ductules and epididymides, providing a basis for exploring innovative approaches to male contraception and infertility treatment.

Pleiotropy in FOXC1-attributable phenotypes involves altered ciliation and cilia-dependent signaling.

Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. In this study we investigated whether altered cilia-mediated signaling contributes to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld-Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1 protein, via shRNA mediated inhibition, CRISPR/Cas9 mutagenesis and overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRα], and altered ciliary compartmentalization of the Hh pathway transcription factor, Gli2. Consistent with these data, in primary cultures of murine embryonic meninges, cilia length was significantly reduced in heterozygous and homozygous Foxc1 mutants compared to controls. Meningeal expression of the core Hh signaling components Gli1, Gli3 and Sufu was dysregulated, with comparable dysregulation of Pdgfrα signaling evident from significantly altered Pdgfrα and phosphorylated Pdgfrα expression. On the basis of these clinical and experimental findings, we propose a model that altered cilia-mediated signaling contributes to some FOXC1-induced phenotypes.

Lateral expansion of the mammalian cerebral cortex is related to anchorage of centrosomes in apical neural progenitors.

The centrosome is the main microtubule organizing center in stem cells, and its mother centriole, anchored to the cell membrane, serves as the basal body of the primary cilium. Prolonged anchorage of centrosomes and primary cilia to the apical segment of the membrane of apical neural progenitor cells is considered vital for interkinetic nuclear translocation and repetitive cycling in the ventricular zone. In contrast, the basolateral anchorage of primary cilia has been regarded as the first step in delamination and conversion of apical to basal neural progenitor cells or neurons. Using electron microscopy analysis of serial sections, we show that centrosomes, in a fraction of cells, anchor to the basolateral cell membrane immediately after cell division and before development of cilia. In other cells, centrosomes situate freely in the cytoplasm, increasing their probability of subsequent apical anchorage. In mice, anchored centrosomes in the cells shortly after mitosis predominate during the entire cerebral neurogenesis, whereas in macaque monkeys, cytoplasmic centrosomes are more numerous. Species-specific differences in the ratio of anchored and free cytoplasmic centrosomes appear to be related to prolonged neurogenesis in the ventricular zone that is essential for lateral expansion of the cerebral cortex in primates.

Excess microtubule and F-actin formation mediates shortening and loss of primary cilia in response to a hyperosmotic milieu.

The primary cilium is a small organelle protruding from the cell surface that receives signals from the extracellular milieu. Although dozens of studies have reported that several genetic factors can impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock induced reversible shortening and disassembly of the primary cilia of murine intramedullary collecting duct cells. The shortening of primary cilia caused by hyperosmotic shock followed delocalization of the pericentriolar material (PCM). Excessive microtubule and F-actin formation in the cytoplasm coincided with the hyperosmotic shock-induced changes to primary cilia and the PCM. Treatment with a microtubule-disrupting agent, nocodazole, partially prevented the hyperosmotic shock-induced disassembly of primary cilia and almost completely prevented delocalization of the PCM. An actin polymerization inhibitor, latrunculin A, also partially prevented the hyperosmotic shock-induced shortening and disassembly of primary cilia and almost completely prevented delocalization of the PCM. We demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and the PCM in a manner dependent on excessive formation of microtubule and F-actin.

A review of CDKL: An underestimated protein kinase family.

Cyclin-dependent kinase-like (CDKL) family proteins are serine/threonine protein kinases and is a specific branch of CMGC (including CDK, MAPK, GSK). Its name is due to the sequence similarity with CDK and it consists of 5 members. Their function in protein phosphorylation underpins their important role in cellular activities, including cell cycle, apoptosis, autophagy and microtubule dynamics. CDKL proteins have been demonstrated to regulate the length of primary cilium, which is a dynamic and diverse signaling hub and closely associated with multiple diseases. Furthermore, CDKL proteins have been shown to be involved in the development and progression of several diseases, including cancer, neurodegenerative diseases and kidney disease. In this review, we summarize the structural characteristics and discovered functions of CDKL proteins and their role in diseases, which might be helpful for the development of innovative therapeutic strategies for disease.

A Short Sequence Targets Transmembrane Proteins to Primary Cilia.

Primary cilia are finger-like sensory organelles that extend from the bodies of most cell types and have a distinct lipid and protein composition from the plasma membrane. This partitioning is maintained by a diffusion barrier that restricts the entry of non-ciliary proteins, and allows the selective entry of proteins harboring a ciliary targeting sequence (CTS). However, CTSs are not stereotyped and previously reported sequences are insufficient to drive efficient ciliary localisation across diverse cell types. Here, we describe a short peptide sequence that efficiently targets transmembrane proteins to primary cilia in all tested cell types, including human neurons. We generate human-induced pluripotent stem cell (hiPSC) lines stably expressing a transmembrane construct bearing an extracellular HaloTag and intracellular fluorescent protein, which enables the bright, specific labeling of primary cilia in neurons and other cell types to facilitate studies of cilia in health and disease. We demonstrate the utility of this resource by developing an image analysis pipeline for the automated measurement of primary cilia to detect changes in their length associated with altered signaling or disease state.

Distraction force promotes the osteogenic differentiation of Gli1+ cells in facial sutures via primary cilia-mediated Hedgehog signaling pathway.

BACKGROUND: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown. METHODS: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling. RESULTS: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force. CONCLUSIONS: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.

Patient-derived and gene-edited pluripotent stem cells lacking NPHP1 recapitulate juvenile nephronophthisis in abnormalities of primary cilia and renal cyst formation.

Juvenile nephronophthisis is an inherited renal ciliopathy with cystic kidney disease, renal fibrosis, and end-stage renal failure in children and young adults. Mutations in the NPHP1 gene encoding nephrocystin-1 protein have been identified as the most frequently responsible gene and cause the formation of cysts in the renal medulla. The molecular pathogenesis of juvenile nephronophthisis remains elusive, and no effective medicines to prevent end-stage renal failure exist even today. No human cellular models have been available yet. Here, we report a first disease model of juvenile nephronophthisis using patient-derived and gene-edited human induced pluripotent stem cells (hiPSCs) and kidney organoids derived from these hiPSCs. We established NPHP1-overexpressing hiPSCs from patient-derived hiPSCs and NPHP1-deficient hiPSCs from healthy donor hiPSCs. Comparing these series of hiPSCs, we found abnormalities in primary cilia associated with NPHP1 deficiency in hiPSCs. Kidney organoids generated from the hiPSCs lacking NPHP1 formed renal cysts frequently in suspension culture with constant rotation. This cyst formation in patient-derived kidney organoids was rescued by overexpression of NPHP1. Transcriptome analysis on these kidney organoids revealed that loss of NPHP1 caused lower expression of genes related to primary cilia in epithelial cells and higher expression of genes related to the cell cycle. These findings suggested the relationship between abnormality in primary cilia induced by NPHP1 loss and abnormal proliferative characteristics in the formation of renal cysts. These findings demonstrated that hiPSC-based systematic disease modeling of juvenile nephronophthisis contributed to elucidating the molecular pathogenesis and developing new therapies.

Positive regulation of Hedgehog signaling via phosphorylation of GLI2/GLI3 by DYRK2 kinase.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

Primary Cilia are Required for Cell-Type Determination and Angiogenesis in Pituitary Development.

The functional maturation of the pituitary gland requires adequate cell differentiation and vascular network formation. Although spatiotemporal signaling and transcription factors are known to govern pituitary development, the involvement of primary cilia, nonmoving hair-like organelles, remains unclear. In this study, we uncovered the contribution of primary cilia to cell-type determination and vascular network formation during pituitary development. Homozygous knockout mice lacking a ciliary kinase, Dyrk2-/-, exhibit abnormalities in ciliary structure and pituitary hypoplasia, accompanied by varying degrees of failure in differentiation among all types of hormone-producing cells in the anterior lobe. Aberrations in cell differentiation in Dyrk2-/- mice arise from a decrease in the expression of crucial transcription factors, Lhx4, Lhx3, and Prop1, resulting from the inactivity of Hedgehog (Hh) signaling during the early stages of development. Furthermore, the loss of Dyrk2 results in vascular system abnormalities during the middle to late stages of development. Mechanistically, transcriptome analyses revealed the downregulation of vitronectin-integrin αvß3-VEGFR2 signaling, essential for orchestrating vascular development. Collectively, our findings demonstrate that primary cilia play a pivotal role as critical regulators of cell survival, cell determination, and angiogenesis during pituitary gland development through the activation of Hh signaling. These findings expand our understanding of the potential link between pituitary dysfunction in human disorders and ciliopathies.