Balancing the length of the distal tip by septins is key for stability and signalling function of primary cilia.

Primary cilia are antenna-like organelles required for signalling transduction. How cilia structure is mechanistically maintained at steady-state to promote signalling is largely unknown. Here, we define that mammalian primary cilia axonemes are formed by proximal segment (PS) and distal segment (DS) delineated by tubulin polyglutamylation-rich and -poor regions, respectively. The analysis of proximal/distal segmentation indicated that perturbations leading to cilia over-elongation influenced PS or DS length with a different impact on cilia behaviour. We identified septins as novel repressors of DS growth. We show that septins control the localisation of MKS3 and CEP290 required for a functional transition zone (TZ), and the cilia tip accumulation of the microtubule-capping kinesin KIF7, a cilia-growth inhibitor. Live-cell imaging and analysis of sonic-hedgehog (SHH) signalling activation established that DS over-extension increased cilia ectocytosis events and decreased SHH activation. Our data underlines the importance of understanding cilia segmentation for length control and cilia-dependent signalling.

Non-apoptotic caspase activation ensures the homeostasis of ovarian somatic stem cells.

Current evidence has associated caspase activation with the regulation of basic cellular functions without causing apoptosis. Malfunction of non-apoptotic caspase activities may contribute to specific neurological disorders, metabolic diseases, autoimmune conditions and cancers. However, our understanding of non-apoptotic caspase functions remains limited. Here, we show that non-apoptotic caspase activation prevents the intracellular accumulation of the Patched receptor in autophagosomes and the subsequent Patched-dependent induction of autophagy in Drosophila follicular stem cells. These events ultimately sustain Hedgehog signalling and the physiological properties of ovarian somatic stem cells and their progeny under moderate thermal stress. Importantly, our key findings are partially conserved in ovarian somatic cells of human origin. These observations attribute to caspases a pro-survival role under certain cellular conditions.

KIAA0753 enhances osteoblast differentiation suppressed by diabetes.

Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.

Glycolysis regulates Hedgehog signalling via the plasma membrane potential.

Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator-the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady-state levels of ATP and stabilizes Smoothened (Smo), the 7-pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full-length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N-acylethanolamides-lipoprotein-borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo-a key step in pathway activation-in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.

Feedback control of the Gpr161-Gαs-PKA axis contributes to basal Hedgehog repression in zebrafish.

Hedgehog (Hh) ligands act as morphogens to direct patterning and proliferation during embryonic development. Protein kinase A (PKA) is a central negative regulator of Hh signalling, and in the absence of Hh ligands, PKA activity prevents inappropriate expression of Hh target genes. The orphan G-protein-coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA. Gpr161 acts as an A-kinase-anchoring protein, and is itself phosphorylated by PKA, but the functional significance of PKA phosphorylation of Gpr161 in the context of Hh signalling remains unknown. Here, we show that loss of Gpr161 in zebrafish leads to constitutive activation of medium and low, but not maximal, levels of Hh target gene expression. Furthermore, we find that PKA phosphorylation-deficient forms of Gpr161, which we show directly couple to Gαs, display an increased sensitivity to Shh, resulting in excess high-level Hh signalling. Our results suggest that PKA feedback-mediated phosphorylation of Gpr161 may provide a mechanism for fine-tuning Gpr161 ciliary localisation and PKA activity.

Extensive crosstalk of G protein-coupled receptors with the Hedgehog signalling pathway.

Hedgehog (Hh) ligands orchestrate tissue patterning and growth by acting as morphogens, dictating different cellular responses depending on ligand concentration. Cellular sensitivity to Hh ligands is influenced by heterotrimeric G protein activity, which controls production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). cAMP in turn activates Protein kinase A (PKA), which functions as an inhibitor and (uniquely in Drosophila) as an activator of Hh signalling. A few mammalian Gαi- and Gαs-coupled G protein-coupled receptors (GPCRs) have been shown to influence Sonic hedgehog (Shh) responses in this way. To determine whether this is a more-general phenomenon, we carried out an RNAi screen targeting GPCRs in Drosophila. RNAi-mediated depletion of more than 40% of GPCRs tested either decreased or increased Hh responsiveness in the developing Drosophila wing, closely matching the effects of Gαs and Gαi depletion, respectively. Genetic analysis indicated that the orphan GPCR Mthl5 lowers cAMP levels to attenuate Hh responsiveness. Our results identify Mthl5 as a new Hh signalling pathway modulator in Drosophila and suggest that many GPCRs may crosstalk with the Hh pathway in mammals.

Dysembryogenetic Pathogenesis of Basal Cell Carcinoma: The Evidence to Date.

The Basal Cell Carcinoma (BCC) is a sort of unique tumour due to its combined peculiar histological features and clinical behaviour, such as the constant binary involvement of the epithelium and the stroma, the virtual absence of metastases and the predilection of specific anatomical sites for both onset and spread. A potential correlation between the onset of BCC and a dysembryogenetic process has long been hypothesised. A selective investigation of PubMed-indexed publications supporting this theory retrieved 64 selected articles published between 1901 and 2024. From our analysis of the literature review, five main research domains on the dysembryogenetic pathogenesis of BCC were identified: (1) The correlation between the topographic distribution of BCC and the macroscopic embryology, (2) the correlation between BCC and the microscopic embryology, (3) the genetic BCC, (4) the correlation between BCC and the hair follicle and (5) the correlation between BCC and the molecular embryology with a specific focus on the Hedgehog signalling pathway. A large amount of data from microscopic and molecular research consistently supports the hypothesis of a dysembryogenetic pathogenesis of BCC. Such evidence is promoting advances in the clinical management of this disease, with innovative targeted molecular therapies on an immune modulating basis being developed.

Brain mosaicism of hedgehog signalling and other cilia genes in hypothalamic hamartoma.

Hypothalamic hamartoma (HH) is a rare benign developmental brain lesion commonly associated with a well characterized epilepsy phenotype. Most individuals with HH are non-syndromic without additional developmental anomalies nor a family history of disease. Nonetheless, HH is a feature of Pallister-Hall (PHS) and Oro-Facial-Digital Type VI (OFD VI) syndromes, both characterized by additional developmental anomalies. Initial genetic of analysis HH began with syndromic HH, where germline inherited or de novo variants in GLI3, encoding a central transcription factor in the sonic hedgehog (Shh) signalling pathway, were identified in most individuals with PHS. Following these discoveries in syndromic HH, the hypothesis that post-zygotic mosaicism in related genes may underly non-syndromic HH was tested. We discuss the identified mosaic variants within individuals with non-syndromic HH, review the analytical methodologies and diagnostic yields, and explore understanding of the functional role of the implicated genes with respect to Shh signalling, and cilia development and function. We also outline future challenges in studying non-syndromic HH and suggest potential novel strategies to interrogate brain mosaicism in HH.

Understanding the role of lipids and lipoproteins in development.

Lipids exert diverse functions in living organisms. They form cellular membranes, store and transport energy and play signalling roles. Some lipid species function in all of these processes, making them ideal candidates to coordinate metabolism with cellular homeostasis and animal development. This theme was central to Suzanne Eaton's research in the fruit fly, Drosophila Here, we discuss her work on membrane lipid homeostasis in changing environments and on functions for lipids in the Hedgehog signalling pathway. We further highlight lipoproteins as inter-organ carriers of lipids and lipid-linked morphogens, which communicate dietary and developmental signals throughout the organism.

Suppressor of fused controls cerebellum granule cell proliferation by suppressing Fgf8 and spatially regulating Gli proteins.

Cerebellar granule cell (GC) development relies on precise regulation of sonic hedgehog (Shh)-Gli signalling activity, failure of which is associated with motor disorders and medulloblastoma. Mutations in the pathway regulator suppressor of fused (Sufu), which modulates Gli activators and repressors, are linked to cerebellar dysfunction and tumourigenesis. The mechanism by which Sufu calibrates Shh signalling in GCs is unknown. Math1-Cre-mediated deletion of Sufu in mouse GC progenitors (GCPs) demonstrated that Sufu restricts GCP proliferation and promotes cell cycle exit, by promoting expression of Gli3R and suppressing Gli2 levels. Sufu is also required to promote a high threshold of pathway activity in GCPs. Remarkably, central cerebellar lobules are more deleteriously impacted by Sufu deletion, but are less sensitive to downstream genetic manipulations to reduce Gli2 expression or overexpress a Gli3R mimic, compared with anterior lobules. Transcriptome sequencing uncovered new Sufu targets, especially Fgf8, which is upregulated in Sufu-mutant GCPs. We demonstrate that Fgf8 is necessary and sufficient to drive Sufu-mutant GCP proliferation. This study reveals new insights into the spatial and temporal regulation of cerebellar Shh-Gli signalling, while uncovering new targets, such as Fgf8.

The Shh receptor Boc is important for myelin formation and repair.

Myelination leads to the formation of myelin sheaths surrounding neuronal axons and is crucial for function, plasticity and repair of the central nervous system (CNS). It relies on the interaction of the axons and the oligodendrocytes: the glial cells producing CNS myelin. Here, we have investigated the role of a crucial component of the Sonic hedgehog (Shh) signalling pathway, the co-receptor Boc, in developmental and repairing myelination. During development, Boc mutant mice display a transient decrease in oligodendroglial cell density together with delayed myelination. Despite recovery of oligodendroglial cells at later stages, adult mutants still exhibit a lower production of myelin basic protein correlated with a significant decrease in the calibre of callosal axons and a reduced amount of the neurofilament NF-M. During myelin repair, the altered OPC differentiation observed in the mutant is reminiscent of the phenotype observed after blockade of Shh signalling. In addition, Boc mutant microglia/macrophages unexpectedly exhibit the apparent inability to transition from a highly to a faintly ramified morphology in vivo Altogether, these results identify Boc as an important component of myelin formation and repair.

Utility of sonic hedgehog and keratin 8/18 immunohistochemistry for detecting ballooned hepatocytes.

AIMS: Ballooned hepatocytes represent liver cell degeneration and are histological hallmarks in the diagnosis of non-alcoholic steatohepatitis, a severe form of non-alcoholic fatty liver disease. However, the identification of ballooned hepatocytes is often difficult, especially in the clinical setting of patients with other chronic liver diseases. In this study, we investigated the utility of immunostaining for positive sonic hedgehog (SHh) protein and negative Keratin 8/18 (K8/18) expression on ballooned hepatocytes. METHODS AND RESULTS: Immunohistochemistry for SHh and K8/18 was evaluated independently by two experienced liver pathologists in non-tumorous liver tissue from 100 cases of resected hepatocellular carcinoma of various aetiology. The degree of hepatocyte ballooning was scored as follows: 0, none; 1, few; 2, many ballooned hepatocytes. These evaluations were performed using routine haematoxylin and eosin (H&E) staining, followed by immunostaining for SHh or K8/18. Using SHh or K8/18 immunostaining combined with H&E staining, the score of ballooned hepatocytes was upgraded in 20 and 19 cases, and downgraded in none and 2 cases, respectively. The percentage of observed agreement for ballooned hepatocytes scoring was 85% and 92%, and the weighted kappa value was 0.806 and 0.893 with SHh or K8/18 immunohistochemistry. Considering the immunohistochemistry results, background liver disease diagnosis was changed in 15 out of 100 cases (15%) evaluated. CONCLUSIONS: SHh and K8/18 immunohistochemistry are useful in detecting ballooned hepatocytes, regardless of background liver disease, and improving pathological diagnosis accuracy.

Identification and characterization of primary cilia-positive salivary gland tumours exhibiting basaloid/myoepithelial differentiation.

Primary cilia (PC) are non-motile, antenna-like structures on the cell surface. Many types of neoplasms exhibit PC loss, whereas in some neoplasms PC are retained and involved in tumourigenesis. To elucidate the PC status and characteristics of major salivary gland tumours (SGTs), we examined 100 major SGTs encompassing eight histopathological types by immunohistochemical analysis. PC were present in all (100%) of the pleomorphic adenomas (PAs), basal cell adenomas (BCAs), adenoid cystic carcinomas (AdCCs), and basal cell adenocarcinomas (BCAcs) examined, but absent in all (0%) of the Warthin tumours, salivary duct carcinomas, mucoepidermoid carcinomas, and acinic cell carcinomas examined. PC were also detected by electron-microscopic analysis using the NanoSuit method. It is worthy of note that the former category and latter category of tumours contained and did not contain a basaloid/myoepithelial differentiation component, respectively. The four types of PC-positive SGTs showed longer PC than normal and exhibited a characteristic distribution pattern of the PC in the ductal and basaloid/neoplastic myoepithelial components. Two PC-positive carcinomas (AdCC and BCAc) still possessed PC in their recurrent/metastatic sites. Interestingly, activation of the Hedgehog signalling pathway, shown by predominantly nuclear GLI1 expression, was significantly more frequently observed in PC-positive SGTs. Finally, we identified tau tubulin kinase 2 (TTBK2) as being possibly involved in the production of PC in SGTs. Taken together, our findings indicate that SGTs that exhibit basaloid/myoepithelial differentiation (PA, BCA, AdCC, and BCAc) are ciliated, and their PC exhibit tumour-specific characteristics, are involved in activation of the Hedgehog pathway, and are associated with TTBK2 upregulation, providing a significant and important link between SGT tumourigenesis and PC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Complex crosstalk of Notch and Hedgehog signalling during the development of the central nervous system.

The development of the vertebrate central nervous system (CNS) is tightly regulated by many highly conserved cell signalling pathways. These pathways ensure that differentiation and migration events occur in a specific and spatiotemporally restricted manner. Two of these pathways, Notch and Hedgehog (Hh) signalling, have been shown to form a complex web of interaction throughout different stages of CNS development. Strikingly, some processes employ Notch signalling to regulate Hh response, while others utilise Hh signalling to modulate Notch response. Notch signalling functions upstream of Hh response through controlling the trafficking of integral pathway components as well as through modulating protein levels and transcription of downstream transcriptional factors. In contrast, Hh signalling regulates Notch response by either indirectly controlling expression of key Notch ligands and regulatory proteins or directly through transcriptional control of canonical Notch target genes. Here, we review these interactions and demonstrate the level of interconnectivity between the pathways, highlighting context-dependent modes of crosstalk. Since many other developmental signalling pathways are active in these tissues, it is likely that the interplay between Notch and Hh signalling is not only an example of signalling crosstalk but also functions as a component of a wider, multi-pathway signalling network.

Aberrant expression of the extracellular matrix component Biglycan regulated by Hedgehog signalling promotes colorectal cancer cell proliferation.

Hedgehog (Hh) signalling plays essential roles in regulating embryonic development and contributes to tumour initiation, growth and progression in multiple cancers. The detailed mechanism by which Hh signalling participates in tumour growth warrants thorough study, although several downstream target genes have been identified. Herein, a set of novel targets of Hh signalling was identified in multiple types of tumour cells via RNA-Seq analysis. Among these targets, the expression regulation and oncogenic function of the extracellular matrix component biglycan (BGN) were investigated. Further investigation verified that Hh signalling activates the expression of BGN via the transcription factor Gli2, which directly binds to the promoter region of BGN. Functional assays revealed that BGN facilitates tumour cell growth and proliferation in colorectal cancer (CRC) cells, and xenograft assays confirmed that BGN also promotes tumour growth . Moreover, analysis of clinical CRC samples showed that both the protein and mRNA levels of BGN are increased in CRC tissues compared to those in adjacent tissues, and higher expression of BGN is correlated with poorer prognosis of CRC patients, further confirming the function of BGN in CRC. Taken together, aberrantly activated Hh signalling increases the expression of BGN, possibly regulates the extracellular matrix, and thereby promotes tumour growth in CRC.

Dilated cardiomyopathy associated with a mutation in the dispatched RND transporter family member 1 gene.

Dilated cardiomyopathy is the most common presentation of cardiomyopathy in children with 20-35% of patients having an identified genetic component. There are more than 30 genes implicated in the pathogenesis of dilated cardiomyopathy. We present the first report of a female infant with dilated cardiomyopathy with a genetic variant in the dispatched RND transporter family member 1 gene.

Gankyrin activates the hedgehog signalling to drive metastasis in osteosarcoma.

Gankyrin is a regulatory subunit of the 26-kD proteasome complex and promotes the occurrence and progression of many malignancies. However, the role of gankyrin in osteosarcoma (OS) metastasis remains unclear. Hedgehog signalling has been shown to regulate stem cell homeostasis and cancer metastasis, but the mechanisms that activate this pathway in OS are still poorly understood. Here, a series of in vitro and in vivo assays were carried out to explore the function and mechanism of gankyrin regulating Hedgehog signalling in OS. We demonstrated that gankyrin promotes migration, invasion and regulates the expression of some stemness factors by up-regulating Gli1 in OS. Importantly, our data showed an interaction between gankyrin and Gli1. Moreover, gankyrin suppresses the ubiquitin-mediated degradation of Gli1 protein in OS. Gankyrin also significantly promotes the lung metastasis of OS in vivo. Our findings suggest that gankyrin drives metastasis and regulates the expression of some stemness factors in osteosarcoma by activating Hedgehog signalling, indicating that drug screening for compounds targeting gankyrin may contribute to the development of novel and effective therapies for OS.

Polysaccharides extracted from balanophora polyandra Griff (BPP) ameliorate renal Fibrosis and EMT via inhibiting the Hedgehog pathway.

Renal interstitial fibrosis (RIF) is a crucial pathological change leading to chronic kidney disease (CKD). Currently, no effective medicines have been available for treating it. In our research, we examined the effects of polysaccharides extracted from Balanophora polyandra Griff (BPPs) on kidney fibrosis and epithelial to mesenchymal transition (EMT) in vivo and in vitro, aiming to explore the underlying mechanisms. By using the mice with unilateral urethral obstruction (UUO) as experimental subjects, we examined the medicinal values of BPPs on alleviating RIF. The effects of BPPs were evaluated by examining the histological staining and relative mRNA and protein expression levels of the related genes. The possible underlying mechanisms were further explored with human normal renal proximal tubular epithelia (HK-2 cells) as in vitro model. In UUO mice, BPP treatment could significantly alleviate interstitial fibrosis through reducing the components (Collagens I, III and IV) of extracellular matrix (ECM), and reducing the activation of fibroblasts producing these components, as revealed by inhibiting the hallmarks (fibronectin and α-SMA) of fibroblast activation. Furthermore, BPP administration increased the expression levels of matrix metalloproteinases (MMPs) and declined those of tissue inhibitors of metalloproteinases (TIMPs). BPPs markedly ameliorated EMT in both the kidneys of UUO mice and TGF-ß1 treated HK-2 cells. Moreover, BPP treatment decreased the expression levels of several transcriptional factors involved in regulating E-cadherin expression, including snail, twist and ZEB1. Additionally, the Hedgehog pathway was found to be closely correlated with renal fibrosis and EMT. Altogether, our results clearly demonstrated that BPP treatment effectively inhibited the Hedgehog pathway both in renal tissues of UUO mice and TGF-ß1-treated HK-2 cells. Thus, BPPs ameliorated RIF and EMT in vivo and in vitro via suppressing Hedgehog signalling pathway.

miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis.

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification.

MTOR promotes basal cell carcinoma growth through atypical PKC.

Advanced basal cell carcinomas (BCCs) are driven by the Hedgehog (HH) pathway and often possess inherent resistance to SMO inhibitors. Identifying and targeting pathways that bypass SMO could provide alternative treatments for patients with advanced or metastatic BCC. Here, we use a combination of RNA-sequencing analysis of advanced human BCC tumor-normal pairs and immunostaining of human and mouse BCC samples to identify an MTOR expression signature in BCC. Pharmacological inhibition of MTOR activity in BCC cells significantly reduces cell proliferation without affecting HH signalling. Similarly, treatment of the Ptch1 fl/fl ; Gli1-CreERT2 mouse BCC tumor model with everolimus reduces tumor growth. aPKC, a downstream target of MTOR, shows reduced activity, suggesting that MTOR promotes tumor growth by activating aPKC and demonstrating that suppressing MTOR could be a promising target for BCC patients.