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Understanding the developmental processes and signaling pathways involved in larval myogenesis and metamorphosis is crucial for comprehending the life history and adaptive strategies of marine organisms. In this study, we investigated the temporal and spatial patterns of myogenesis in the mussel Mytilus coruscus (Mc), focusing on the emergence and transformation of major muscle groups during different larval stages. We also explored the role of the Hedgehog (Hh) signaling pathway in regulating myogenesis and larval metamorphosis. The results revealed distinct developmental stages characterized by the emergence of specific muscular components, such as velum retractor muscles and anterior adductor muscles, in D-veliger and umbo larvae, which are responsible for the planktonic stage. In the pediveliger stage, posterior ventral, posterior adductor, and foot muscles appeared. After larval metamorphosis, the velum structure and its corresponding retractor muscles degenerate, indicating the transition from planktonic to benthic life. We observed a conserved pattern of larval musculature development and revealed a high degree of conservation across bivalve species, with comparable emergence times during myogenesis. Furthermore, exposure to the Hh signaling inhibitor cyclopamine impaired larval muscle development, reduced larval swimming activity, and inhibited larval metamorphosis in M. coruscus. Cyclopamine-mediated inhibition of Hh signaling led to reduced expression of four key genes within the Hh signaling pathway (McHh , McPtc , McSmo , and McGli) and the striated myosin heavy chain gene (McMHC). It is hypothesised that the abnormal larval muscle development in cyclopamine-treated groups may be an indirect effect due to disrupted McMHC expression. We provide evidence for the first time that cyclopamine treatment inhibited larval metamorphosis in bivalves, highlighting the potential involvement of Hh signaling in mediating larval muscle development and metamorphosis in M. coruscus. The present study provides insights into the dynamic nature of myogenesis and the regulatory role of the Hh signaling pathway during larval development and metamorphosis in M. coruscus. The results obtained in this study contribute to a better understanding of the evolutionary significance of Hh signaling in bivalves and shed light on the mechanisms underlying larval muscle development and metamorphosis in marine invertebrates. [Display omitted] • Studied larval myogenesis with specific muscle emergence and degeneration in Mytilus coruscus. • Identified conserved patterns of larval musculature across bivalve species. • Hh signaling inhibitor cyclopamine inhibited larval muscle development and larval metamorphosis. • Cyclopamine inhibited the expression of key genes within the Hh signaling and a myosin gene. [ABSTRACT FROM AUTHOR]

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates. Constituent cells of the amphioxus notochord with gene expression. (A) A transverse section of the middle-part of the body showing the notochord (not) at the mid-dorsal line, which is sandwiched by the dorsal-side nerve cord (nc) and the ventral-side pharynx (ph). Note that Brachyury (blue) is expressed in the notochord and in the hepatic cecum (hc), hedgehog (green) in the notochord and in the endostyle (en), and MRLC (myosin regulatory light chain, brown) in the central region of the notochord and body-wall muscle (bwm), but not in muscle (mus). (B) Notochord expression of Brachyury (blue), hedgehog (green), and MRLC (brown). M, Müller cells; DM, dorsal Müller cells; LM, lateral Müller cells; VM, ventral Müller cells; MC, myofibrillar cells. DM and LM express both Brachyury and hedgehog, while VM express low level of hedgehog (broken circle). (C) An image of a serially transverse-sectioned notochord showing the complex arrangement of its constituent cells with expression of the foregoing genes gene. gd, gonad; nh, notochordal horn; v, vacuoles. [Display omitted] • Notochord of adult amphioxus was examined by scRNA-seq. • The notochord is composed of myofibrillar and non-myofibrillar cells. • Some non-myofibrillar cells express Brachyury and hedgehog. • These correspond to Müller cells and likely interact with nerve cord or somites. • Amphioxus notochord has ancestral properties of vertebrate notochord. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) signaling is involved in multiple biological events including development and cancers. It is processed through primary cilia, which are assembled from the mother centriole in most mammalian cells. Pancreatic ductal adenocarcinoma (PDAC) cells generally lose their primary cilia; thus, the Hh signaling pathway is postulated to be independent of the organelle in PDAC. We previously reported that the mother centriole-specific protein, centrosomal protein 164 (CEP164), is required for centriolar localization of the GLI2 transcription factor in Hh signaling and for suppressing the expression of Hh-target genes. In this study, we demonstrated the physical interaction between CEP164 and GLI2, and delineated their binding modes at the mother centriole. The ectopically expressed GLI2-binding region of CEP164 reduced the centriolar GLI2 localization and enhanced the expression of Hh-target genes in PDAC cells. Furthermore, similar phenotypes were observed in PDAC cells lacking primary cilia. These results suggest that the CEP164-GLI2 association at the mother centriole is responsible for controlling Hh signaling, independent of primary cilia in PDAC cells. • The Hedgehog (Hh) signaling is usually processed through primary cilia in normal cells. In this study, we demonstrate the novel mechanism of the Hh singling in non-ciliated PDAC cells. [ABSTRACT FROM AUTHOR]

Di (2-ethylhexyl) phthalate (DEHP), an artificially synthetic plasticizer, is a widespread environmental endocrine disruptor, which has raised substantial concern among the public about its potential reproductive toxicity effects. Taking large amounts of DEHP disrupts the normal functioning of the ovaries, however, the toxicological effects and the mechanisms by which DEHP impairs fetal folliculogenesis remain poorly understood. Our research aims to elucidate the associations between utero exposure to DEHP and fetal folliculogenesis in offspring. In this research, we monitored the spatiotemporal and expression levels of GDF9-Hedgehog (Hh) pathway-related genes during postnatal days 3–14, confirming initially the potential associations between defects in theca cell development and the downregulation of GDF9-Hh signaling. Moreover, utilizing an ovarian organ in vitro culture model, rescue validation experiments demonstrated that the addition of recombinant GDF9 protein effectively alleviate the theca cell damage caused by DEHP, thus supporting the aforementioned associations. In conclusion, our findings validate the significant role of the GDF9-Hh pathway in the enduring reproductive toxicity resulting from prenatal exposure to DEHP. [Display omitted] • Maternal DEHP exposure affects fetal folliculogenesis development. • Prenatal DEHP exposure causes the deficiency of theca cell development in mice. • Prenatal DEHP exposure reduced gene expression related to GDF9-Hh signaling pathway. [ABSTRACT FROM AUTHOR]

The primary cilium is a hair-like projection that controls cell development and tissue homeostasis. Although accumulated studies identify the molecular link between cilia and cilia-related diseases, the underlying etiology of ciliopathies has not been fully understood. In this paper, we determine the function of Rab34, a small GTPase, as a key regulator for controlling ciliogenesis and type I collagen trafficking in craniofacial development. Mechanistically, Rab34 is required to form cilia that control osteogenic proliferation, survival, and differentiation via cilia-mediated Hedgehog signaling. In addition, Rab34 is indispensable for regulating type I collagen trafficking from the ER to the Golgi. These results demonstrate that Rab34 has both ciliary and non-ciliary functions to regulate osteogenesis. Our study highlights the critical function of Rab34, which may contribute to understanding the novel etiology of ciliopathies that are associated with the dysfunction of RAB34 in humans. • Deletion of Rab34 results in multiple ciliopathies-like phenotypes such as craniofacial defects. • Rab34 is required to form cilia that control osteogenesis via cilia-mediated Hedgehog signaling. • Rab34 is critical for regulating type I collagen trafficking from the ER to the Golgi in osteogenesis. [ABSTRACT FROM AUTHOR]

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via β-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/β-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function. [ABSTRACT FROM AUTHOR]

Histone acetylation and deacetylation are associated with diverse biological phenomena via gene transcription, and histone deacetylases (HDACs) regulate protein deacetylation. HDAC8 is associated with childhood neurological disorders that develop in the uterus and may contribute to neurodevelopment. In our previous studies, we found that HDAC8 regulates neuronal differentiation in P19 pluripotent embryonic carcinoma cells (P19EC cells) by regulating embryoid body (EB) formation. However, the mechanism through which HDAC8 is involved in EB formation and neuronal differentiation remains unclear. Here, we show that HDAC8 regulates EB formation and neuronal differentiation by regulating the canonical Hedgehog (Hh) signaling pathway in P19EC cells. We found that HDAC8 is possibly involved in regulating the expression of the Smoothened receptor (Smo), an important receptor in canonical Hh signaling, and treatment with a Smo agonist restored EB formation ability, which was reduced in HDAC8 knockout P19EC cells. Our results demonstrate that HDAC8 functions in EB formation, which is involved in the Hh signaling pathway that is important for embryonic development. • HDAC8 regulates neuronal differentiation by regulating embryoid body (EB) formation. • HDAC8 regulates EB formation by regulating the hedgehog (Hh) pathway. • Smoothened receptor (Smo) is a regulator of the canonical Hh pathway. • The Smo gene might be epigenetically regulated by HDAC8 through its 5′-UTR. • Gli2 might be regulated by HDAC8 through Smo. [ABSTRACT FROM AUTHOR]

The vertebrate embryonic midline vasculature forms in close proximity to the developing skeletal muscle, which originates in the somites. Angioblasts migrate from bilateral positions along the ventral edge of the somites until they meet at the midline, where they sort and differentiate into the dorsal aorta and the cardinal vein. This migration occurs at the same time that myoblasts in the somites are beginning to differentiate into skeletal muscle, a process which requires the activity of the basic helix loop helix (bHLH) transcription factors Myod and Myf5. Here we examined vasculature formation in myod and myf5 mutant zebrafish. In the absence of skeletal myogenesis, angioblasts migrate normally to the midline but form only the cardinal vein and not the dorsal aorta. The phenotype is due to the failure to activate vascular endothelial growth factor ligand vegfaa expression in the somites, which in turn is required in the adjacent angioblasts for dorsal aorta specification. Myod and Myf5 cooperate with Hedgehog signaling to activate and later maintain vegfaa expression in the medial somites, which is required for angiogenic sprouting from the dorsal aorta. Our work reveals that the early embryonic skeletal musculature in teleosts evolved to organize the midline vasculature during development. [Display omitted] • Myod and Myf5 are required for dorsal aorta and intersegmental vessel formation. • Myod and Myf5 are required for somitic expression of the VEGF ligand vegfaa. • Restoring vegfaa expression in Myod/Myf5 double mutants rescues aortic fate. • Myod and Myf5 function cooperatively with Hedgehog signaling to induce vegfaa. • vegfaa maintenance requires continued bHLH activity in muscle cells. Summary statement: The myogenic transcription factors Myod and Myf5 have a novel function in inducing the artery through regulation of VEGF. [ABSTRACT FROM AUTHOR]

Cell-cell communication through evolutionarily conserved signaling pathways governs embryonic development and adult tissue homeostasis. Deregulation of these signaling pathways has been implicated in a wide range of human diseases including cancer. One such pathway is the Hedgehog (Hh) pathway, which was originally discovered in Drosophila and later found to play a fundamental role in human development and diseases. Abnormal Hh pathway activation is a major driver of basal cell carcinomas (BCC) and medulloblastoma. Hh exerts it biological influence through a largely conserved signal transduction pathway from the activation of the GPCR family transmembrane protein Smoothened (Smo) to the conversion of latent Zn-finger transcription factors Gli/Ci proteins from their repressor (GliR/CiR) to activator (GliA/CiA) forms. Studies from model organisms and human patients have provided deep insight into the Hh signal transduction mechanisms, revealed roles of Hh signaling in a wide range of human cancers, and suggested multiple strategies for targeting this pathway in cancer treatment. [ABSTRACT FROM AUTHOR]

The primary cilium is a hair-like sensory compartment that protrudes from the cellular surface. The primary cilium is enriched in a variety of signaling molecules that regulate cellular activities. Stem cells have primary cilia. They reside in a specialized environment, called the stem cell niche. This niche contains a variety of secreted factors, and some of their receptors are localized in the primary cilia of stem cells. Here, we summarize the current understanding of the function of cilia in compartmentalized signaling in stem cells. We describe how ciliary signaling regulates stem cells and progenitor cells during development, tissue homeostasis and tumorigenesis. We summarize our understanding of cilia regulated signaling -primary involving the hedgehog pathway- in stem cells in diverse settings that include neuroepithelial cells, radial glia, cerebellar granule neuron precursors, hematopoietic stem cells, hair follicle stem cells, bone marrow mesenchymal stem cells and mammary gland stem cells. Overall, our review highlights a variety of roles that ciliary signaling plays in regulating stem cells throughout life. [ABSTRACT FROM AUTHOR]

Tendons are cable-like connective tissues that transfer both active and passive forces generated by skeletal muscle to bone. In the mature skeleton, the tendon-bone enthesis is an interfacial zone of transitional tissue located between two mechanically dissimilar tissues: compliant, fibrous tendon to rigid, dense mineralized bone. In this review, we focus on emerging areas in enthesis development related to its structure, function, and mechanobiology, as well as highlight established and emerging signaling pathways and physiological processes that influence the formation and adaptation of this important transitional tissue. • The enthesis is an interfacial zone of transitional tissue located between tendon to bone. • Entheses are found on the periosteal surface of bone on ridges, tuberosities, and tubercles. • Progenitor enthesis cells are "bi-fated" and express factors of both chondrocytes and tenocytes. • Factors that promote enthesis development include mechanical loading, TGF-beta, FGF signaling, and hedgehog signaling. • Extracellular matrix remodeling, hypoxia, and metabolism are emerging mediators of enthesis development. [ABSTRACT FROM AUTHOR]

Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, the unique functions and roles of each IFT during endochondral ossification remain unclear. Here, we show that IFT20 is required for endochondral ossification in mice. Utilizing osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre and Col2-Cre), we deleted Ift20 to examine its function. Although chondrocyte-specific Ift20 deletion with Col2-Cre mice did not cause any overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) mice resulted in shortened limb outgrowth. Primary cilia were absent on chondrocytes of Ift20:Prx1-Cre mice, and ciliary-mediated Hedgehog signaling was attenuated in Ift20:Prx1-Cre mice. Interestingly, loss of Ift20 also increased Fgf18 expression in the perichondrium that sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate that IFT20 is a critical regulator of temporospatial FGF signaling that is required for endochondral ossification. • Mesoderm-specific disruption of Ift20 results in defective limb growth. • IFT20 plays a pivotal role in the perichondrium via Fgf18 expression. • IFT20 is important for the regulation of temporospatial FGF signaling during endochondral ossification. [ABSTRACT FROM AUTHOR]

Cardiac trabeculae are muscular ridge-like structures within the ventricular wall that are crucial for cardiac function. In zebrafish, these structures first form primarily through the delamination of compact wall cardiomyocytes (CMs). Although defects in proteasomal degradation have been associated with decreased cardiac function, whether they also affect cardiac development has not been extensively analyzed. Here we report a role during cardiac wall morphogenesis in zebrafish for the E3 ubiquitin-protein ligase Rbx1, which has been shown to regulate the degradation of key signaling molecules. Although development is largely unperturbed in zebrafish rbx1 mutant larvae, they exhibit CM multi-layering. This phenotype is not affected by blocking ErbB signaling, but fails to manifest itself in the absence of blood flow/cardiac contractility. Surprisingly, rbx1 mutants display ErbB independent Notch reporter expression in the myocardium. We generated tissue-specific rbx1 overexpression lines and found that endothelial, but not myocardial, specific rbx1 expression normalizes the cardiac wall morphogenesis phenotype. In addition, we found that pharmacological activation of Hedgehog signaling ameliorates the multi-layered myocardial wall phenotype in rbx1 mutants. Collectively, our data indicate that endocardial activity of Rbx1 is essential for cardiac wall morphogenesis. [Display omitted] • rbx1 mutants exhibit a multi-layered myocardial wall phenotype. • rbx1 mutants exhibit reduced cardiomyocyte proliferation in their ventricle. • rbx1 regulates endocardial morphology. • Overexpression of rbx1 in endothelial cells can rescue multi-layered myocardial wall phenotype in rbx1 mutants. • Activation of Hedgehog signaling normalizes the multi-layered myocardial wall phenotype in rbx1 mutants. [ABSTRACT FROM AUTHOR]

GPRASP2 is implicated in nervous system diseases, tumors and immune inflammation. In our previous study, G protein-coupled receptor associated sorting protein 2 (GPRASP2) was identified as a novel causal gene for X-linked recessive syndromic hearing loss (SHL). However, the role of GPRASP2 in auditory function has not been elucidated. The Gprasp2 -knockout (KO) mouse HEI-OC1 auditory cells were constructed using CRISPR/Cas9-mediated gene editing. RNA-sequencing (RNA-seq) was used to investigate the differentially expressed genes (DEGs) and DEGs-enriched signaling pathways, which was verified by Western blot. Flow cytometry assay was used to examine cell apoptosis. The cytological pathology was evaluated by laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). Mitochondrial damage was observed in Gprasp2 -KO HEI-OC1 cells. RNA-seq analysis suggested that Gprasp2 -KO was implicated in the apoptosis process, which could be mediated by Hedgehog (Hh) signaling pathway. The key molecules in Hh signaling pathway (Smo, Gli1, Gli2) were detected to be down-regulated in Gprasp2 -KO HEI-OC1 cells. The differential expression of apoptosis molecules (Bcl2, Bax, Caspase-3/cleaved-Caspase-3) indicated that Gprasp2 -KO induced apoptosis in HEI-OC1 cells. The treatment of smoothened agonist (Purmorphamine, PUR) activated the Hh-Gli signaling pathway and reduced apoptosis in Gprasp2 -KO HEI-OC1 cells. This study revealed that Gprasp2 -disruption inhibited Hh signaling pathway and led to cell apoptosis in HEI-OC1 cells, which might provide the potential molecular mechanism of GPRASP2 mutation associated with human SHL. • Gprasp2 disruption induced mitochondrial damage and apoptosis in HEI-OC1 cells. • Gprasp2 disruption inhibited Hedgehog signaling pathway in HEI-OC1 cells. • The smoothened agonist could reduce apoptosis in Gprasp2 -knockout HEI-OC1 cells. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) signaling plays a significant role in embryogenesis and several physiological processes, such as wound healing and organ homeostasis. In a pathological setting, it is associated with oncogenesis and is responsible for disease progression and poor clinical outcomes. Hedgehog signaling mediates downstream actions via Glioma Associated Oncogene Homolog (GLI) transcription factors. Inhibiting Hh signaling is an important oncological strategy in which inhibitors of the ligands SMO or GLI have been looked at. This review briefly narrates the Hh ligands, signal transduction, the target genes involved and comprehensively describes the numerous inhibitors that have been evaluated for use in various neoplastic settings. [Display omitted] • Hedgehog signaling pathway is a key regulator during development. • Dysregulation of Hedgehog signaling is implicated in numerous cancer. • In cancer contexts, Hedgehog signaling leads to disease progression and drug resistance. • Inhibiting Hedgehog signaling can yield clinical benefit in cancer patients. • Inhibitors can act at various targets across the signal transduction pathway. [ABSTRACT FROM AUTHOR]

Medulloblastoma (MB) is the most common malignant brain tumor in children. Transcriptional profiling has so far delineated four major MB subgroups of which one is driven by uncontrolled Hedgehog (Hh) signaling (SHH-MB). This pathway is amenable to drug targeting, yet clinically approved compounds exclusively target the transmembrane component Smoothened (SMO). Unfortunately, drug resistance against SMO inhibitors is encountered frequently, making the identification of novel Hh pathway components mandatory, which could serve as novel drug targets in the future. Here, we have used MB as a tool to delineate novel modulators of Hh signaling and have identified the Acidic Nuclear Phosphoprotein 32 (ANP32) family of proteins as novel regulators. The expression of all three family members (ANP32A, ANP32B, ANP32E) is increased in Hh-induced MB and their expression level is negatively associated with overall survival in SHH-MB patients. Mechanistically, we could find that ANP32 proteins function as positive modulators of mammalian Hh signaling upstream of GLI transcription factors. These findings add hitherto unknown regulators to the mammalian Hh signaling cascade and might spur future translational efforts to combat Hh-driven malignancies. • ANP32 family members are overexpressed in the pediatric malignancy medulloblastoma. • ANP32 proteins represent positive modulators of mammalian Hedgehog (Hh) signaling, both in mouse and human cells. • ANP32 functions are integrated into the Hh pathway upstream of the GLI transcription factors. [ABSTRACT FROM AUTHOR]

• Primary cilia are required for both repressing and activating Hh signaling. • Active repression is as important as the activation arm of Hh pathway. • Ratio sensing or threshold detection of activator/repressor drive morphogenesis. • Cilia regulate differentiation, patterning or proliferation by Hh pathway. • Hh signaling is reactivated and repurposed during adult tissue regeneration. Primary cilia are immotile appendages that have evolved to receive and interpret a variety of different extracellular cues. Cilia play crucial roles in intercellular communication during development and defects in cilia affect multiple tissues accounting for a heterogeneous group of human diseases called ciliopathies. The Hedgehog (Hh) signaling pathway is one of these cues and displays a unique and symbiotic relationship with cilia. Not only does Hh signaling require cilia for its function but the majority of the Hh signaling machinery is physically located within the cilium-centrosome complex. More specifically, cilia are required for both repressing and activating Hh signaling by modifying bifunctional Gli transcription factors into repressors or activators. Defects in balancing, interpreting or establishing these repressor/activator gradients in Hh signaling either require cilia or phenocopy disruption of cilia. Here, we will summarize the current knowledge on how spatiotemporal control of the molecular machinery of the cilium allows for a tight control of basal repression and activation states of the Hh pathway. We will then discuss several paradigms on how cilia influence Hh pathway activity in tissue morphogenesis during development. Last, we will touch on how cilia and Hh signaling are being reactivated and repurposed during adult tissue regeneration. More specifically, we will focus on mesenchymal stem cells within the connective tissue and discuss the similarities and differences of how cilia and ciliary Hh signaling control the formation of fibrotic scar and adipose tissue during fatty fibrosis of several tissues. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) has been identified as a leading cause of tumor-associated death around the world. Presently, it is necessary to find effective and safe therapy for its treatment in clinic. Jervine (Jer), a sterodial alkaloid from rhizomes of Veratrum album, exhibits anti-inflammatory and anti-cancer effects. However, its effects on lung cancer progression are still unknown. In this study, we explored if Jer showed any influences on NSCLC development, as well as the underlying molecular mechanisms. The results showed that Jer time- and dose-dependently reduced the proliferation of NSCLC cells, along with inhibited colony formation capacity. Apoptosis was highly induced by Jer in NSCLC cells through promoting the expression of cleaved Caspase-3. Furthermore, Jer treatment led to autophagy in cancer cells, as evidenced by the fluorescence microscopy results and increases of LC3II. Autophagy inhibitor bafilomycinA1 (BafA1) abrogated the inhibitory effects of Jer on cell proliferation and apoptosis induction, showing that Jer triggered autophagy-mediated apoptosis in NSCLC cells. Additionally, AKT and mammalian target of Rapamycin (mTOR) signaling pathway was highly repressed in cancer cells. Importantly, promoting AKT activation greatly rescued the cell survival, while attenuated autophagy and apoptosis in Jer-incubated NSCLC cells, revealing that Jer-modulated autophagic cell death was through the blockage of AKT signaling. Hedgehog signaling pathway was then found to be suppressed by Jer, as proved by the decreased expression of Sonic Hedgehog (Shh), Hedgehog receptor protein patched homolog 1 (PTCH1), smoothened (SMO) and glioma-associated oncogene homolog 1 (Gli1) in NSCLC cells. Of note, enhancing Shh signaling dramatically diminished the stimulative effects of Jer on autophagy-mediated apoptosis in vitro , demonstrating the importance of Hedgehog signaling in Jer-regulated cell death. Moreover, Jer treatment effectively reduced tumor growth in A549-bearing mice with few toxicity. Together, Jer may be a promising and effective therapeutic strategy for NSCLC treatment. • Jervine induces apoptosis and autophagy in NSCLC cells. • Jervine suppresses Hedgehog signaling and thereafter influences cell proliferation in NSCLC cells. • Jervine inhibits the growth of NSCLC tumor xenograft in vivo. [ABSTRACT FROM AUTHOR]

Aberrant activation of Hedgehog signaling is considered as the key player in hepatic stellate cell (HSC) activation involved in liver fibrosis (LF). The glioma-associated protein gene (GLI) has a predicted paired box 6 (PAX6)-binding site within its transcribed region. Therefore, this study aimed to investigate the relationship between PAX6 and GLI and their contribution to HSC activation and proliferation. PAX6 expression was upregulated in platelet-derived growth factor-BB (PDGF-BB)-induced LX-2 cells. The activation and proliferation of HSC were inhibited by interference of PAX6 with short hairpin RNA (sh PAX6) via curbing Hedgehog signaling. Notably, PAX6 directly bound to the promoter sequence of GLI1 independent of the PTCH/SMO axis. Therefore, we propose that PAX6 upregulation induces HSC activation and proliferation through crosstalk with GLI1 signaling. Thus, these novel mechanistic insights involving the PAX6-mediated regulation of the activation and proliferation of HSC may provide a new therapeutic target for LF. Diagram representing the potential mechanism by which PAX6 regulates Hedgehog signalling activation in HSCs. Based on our findings, PAX6 expression is upregulated in PDGF-BB-induced LX-2 cells, and PAX6 promotes the activation and proliferation of HSCs. Moreover, PAX6 directly binds to the proximal promoter region of GLI1. Thereafter, the Hedgehog signalling pathway is activated, which promotes HSCs activation and proliferation. Image 1 • PAX6 is up-regulated in activated hepatic stellate cells (HSCs). • Silencing PAX6 can inhibit HSC activation and proliferation. • PAX6 directly binds to GLI1 promoter sequence independent of PTCH/SMO axis. • PAX6 promotes HSC activation and proliferation by inducing Hedgehog/GLI1 signaling. [ABSTRACT FROM AUTHOR]

4-Methylbenzylidene camphor (4-MBC) is a widely used organic UV filter in personal care products. Extensive use of 4-MBC and its frequent detection in aquatic ecosystems defile the biota with muscular and neuronal impairments. This study investigates the neurobehavioral toxicity of 4-MBC using Danio rerio as a model organism. Embryos were exposed semi-statically to 4-MBC at 5, 50, and 500 μg/L concentrations for 10-day post fertilization (dpf). Embryos exhibited a significant thigmotaxis and decreased startle touch response with altered expression of nervous system mRNA transcripts on 5 & 10 dpf. Compared to the sham-exposed group, 4-MBC treated larvae exhibited changes in the expression of shha, ngn1, mbp, elavl3, α1-tubulin, syn2a, and gap43 genes. Since ngn1 induction is mediated by shh signaling during sensory neuron specification, the elevated protein expression of NGN1 indicates 4-MBC interference in the sonic hedgehog signaling pathway. This leads to sensory neuron impairment and function such as 'sense' as evident from reduced touch response. In addition, larval brain histology with a reduced number of cells in the Purkinje layer emblazing the defunct motor coordination. Predictive toxicity study also showed a higher affinity of 4-MBC to modeled Shh protein. Thus, the findings of the present work highlighted that 4-MBC is potential to induce developmental neurotoxicity at both behavioral and molecular functional perspectives, and developing D. rerio larvae could be considered as a suitable alternate animal model to assess the neurological dysfunction of organic UV filters. • 4-Methylbenzylidene (4-MBC) camphor alters D. rerio motor behavior. • Decreased touch response due to motor function deficit. • Brain histology reveals loss of neuronal cell population. • Altered expression of neurodevelopmental mRNA transcripts. • Docking studies confirm 4-MBC affinity and modulation with Sonic Hedgehog protein. [ABSTRACT FROM AUTHOR]

Lenvatinib is a frontline tyrosine kinase inhibitor for patients with advanced hepatocellular carcinoma (HCC). However, just 25% of patients benefit from the treatment, and acquired resistance always develops. To date, there are neither effective medications to combat lenvatinib resistance nor accurate markers that might predict how well a patient would respond to the lenvatinib treatment. Thus, novel strategies to recognize and deal with lenvatinib resistance are desperately needed. In the current study, a robust Lenvatinib Resistance index (LRi) model to predict lenvatinib response status in HCC was first established. Subsequently, five candidate drugs (Mercaptopurine, AACOCF3, NU1025, Fasudil, and Exisulind) that were capable of reversing lenvatinib resistance signature were initially selected by performing the connectivity map (CMap) analysis, and fasudil finally stood out by conducting a series of cellular functional assays in vitro and xenograft mouse model. Transcriptomics revealed that the co-administration of lenvatinib and fasudil overcame lenvatinib resistance by remodeling the hedgehog signaling pathway. Mechanistically, the feedback activation of EGFR by lenvatinib led to the activation of the GLI2-ABCC1 pathway, which supported the HCC cell's survival and proliferation. Notably, co-administration of lenvatinib and fasudil significantly inhibited IHH, the upstream switch of the hedgehog pathway, to counteract GLI2 activation and finally enhance the effectiveness of lenvatinib. These findings elucidated a novel EGFR-mediated mechanism of lenvatinib resistance and provided a practical approach to overcoming drug resistance in HCC through meaningful drug repurposing strategies. [Display omitted] [ABSTRACT FROM AUTHOR]

Emerging evidence has uncovered the extremely important roles of long noncoding RNAs (lncRNAs) in the progression of malignant tumors which includes numerous processes of gene regulation. LncRNA NR2F1-AS1 has been confirmed to have close correlation with the tumorigenesis of diverse cancers. However, the underlying regulatory function of it on esophageal squamous cell carcinoma (ESCC) progression is poorly known and thus needs more elucidations. In this study, a markedly elevated expression of NR2F1-AS1 was discovered in ESCC cells. Functional assays demonstrated that NR2F1-AS1 deficiency repressed ESCC progression. Molecular mechanism tests verified that knockdown of NR2F1-AS1 could lower the expression of GLI2 (a key protein molecule of Hedgehog signaling pathway) in ESCC. Additionally, NR2F1-AS1 was confirmed to facilitate ESCC progression via activation of Hedgehog signaling pathway. NR2F1-AS1 activated Hedgehog signaling pathway by regulating GLI2 to upregulate NR2F1 expression in ESCC. Besides, NR2F1 was testified to activate NR2F1-AS1 transcription in ESCC. Final rescue assays further demonstrated that NR2F1 upregulation could reverse the NR2F1-AS1 knockdown-mediated function on ESCC progression. Briefly, NR2F1-induced NR2F1-AS1 promotes ESCC progression through activation of Hedgehog signaling pathway. • NR2F1-AS1 expression is upregulated in ESCC cells and NR2F1-AS1 depletion suppresses ESCC progression. • NR2F1-AS1 facilitates ESCC progression through activation of Hedgehog signaling pathway. • NR2F1-AS1 upregulates NR2F1 expression via regulation of GLI2. • NR2F1-induced NR2F1-AS1 promotes ESCC progression via activating Hedgehog signaling pathway. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that hepatic stellate cells (HSCs) adopt aerobic glycolysis during activation. Hedgehog (Hh) pathway plays a vital role in the process of HSCs activation by regulating metabolism, and activation of the Hh pathway promotes transdifferentiation of HSCs into myofibroblasts. Deoxyelephantopin (DET), a naturally occurring sesquiterpene lactone from Elephantopus scaber, has been shown to exert hepatoprotective as well as anticancer effects. However, the effect of DET on hepatic fibrosis and glycolysis in HSCs have never been elucidated. Here, we studied the function of the DET on HSCs activation and investigated the anti-fibrogenic effects of DET was associated with interfering with glycolysis in HSCs. Our results first demonstrated that DET reduced the expression of a-smooth muscle actin (a-SMA) and a1(I)procollagen at both mRNA and protein levels, and restore lipogenesis in HSCs. Furthermore, DET decreased the expression of hexokinase (HK), phosphofructokinase-2 (PFK2), Glucose transporter 4 (Glut4), and reduced lactate production dose-dependently in HSCs. Moreover, we further revealed that DET reduced fibrotic gene expression, restored lipid accumulation in HSCs. However, the Hh pathway agonist SAG could reverse the above effect of DET. Together, these results indicate DET inhibits aerobic glycolysis in HSCs associated with inhibition of Hh pathway. Our results provided a novel mechanism for DET suppression of HSC activation implicated in antifibrotic therapy. [ABSTRACT FROM AUTHOR]

Aberrant activity of the hedgehog (Hh) pathway is prevalent in pathologies such as cancer. Improved understanding of Hh activity in the aggressive tumor cell phenotype is being pursued for development of targeted therapies. Recently, we described a link between Hh activity and carbonic anhydrase XII (CAXII) expression. Extracellular facing CAs (IX/XII) are highly expressed in hypoxia, contribute to tumor pH regulation and are thus of clinical interest. Here we have extended the investigation of potential interactions between Hh activity and CAXII utilizing genomic disruption/knockout of either GLI1 (the main transcriptional factor induced with Hh activity) or CAXII in the triple negative breast cancer cell lines MDA-MB-231 and BT-549. Knockout of GLI1 and CAXII significantly decreased hallmarks of tumor aggressiveness including proliferation and migration. Most intriguingly, CAXII knockout caused a massive induction of the Sonic hedgehog (Shh) ligand expression (gene and protein). This novel finding indicates that CAXII plays a potential role in suppression of Shh and may act in a feedback loop to regulate overall Hh activity. Enhanced knowledge of these CA-Hh interactions in future studies may be of value in understanding this currently 'incurable' subclass of breast cancer. • Disruption of GLI1 expression alters tumor cell CAXII expression. • GLI1 and CAXII knockout compromise TNBC cell proliferation and migration. • CAXII removal strongly induces expression and maturation of the Sonic Hedgehog ligand. [ABSTRACT FROM AUTHOR]

Human papilloma virus oncogenes and estradiol are major etiologic factors associated with cervical cancer. In order to understand the mechanism by which these two factors promote carcinogenesis, the role of the Hedgehog (Hh) signaling pathway was evaluated during the normal growth of cervical epithelium and in the presence of E6/E7 oncogenes and exogenous estradiol. Hh signaling activity was determined in live animals (i.e., Gli-Luc reporter levels) during the estrous cycle and was found to be higher in the cervical area during the major growth phases, proestrus-estrus, in comparison to the diestrus phase. The same pattern was observed in transgenic mice expressing the E6/E7 oncogenes, though with notably higher levels than in control mice. Adding estradiol also markedly increased Gli activity in the cervix and the skin. In agreement with the correlation between high bioluminescence and tissue growth in different context, cervical cell proliferation was reduced upon Hh signaling inhibition in mice. Treatment with itraconazole, a putative novel Hh inhibitor, at an early stage of cervical carcinogenesis, did not decrease Hh signaling but it did reduce growth. Therefore, Hh signaling likely contributes to cervical carcinogenesis and itraconazole is effective to reduce growth but by a mechanism involving additional signaling pathways. Image 1 • Hedgehog activity occurs during the growth phases of estrous cycle. • E6/E7 oncogenes increase Gli activity in the cervix and the skin. • Estradiol increases Gli activity in the cervix and the skin. • Itraconazole, a hedgehog inhibitor, reduces growth in a model of cervical cancer. [ABSTRACT FROM AUTHOR]

Adipogenesis, which directly control body fat mass, plays a crucial role in lipid metabolism and obesity-related diseases. Hedgehog interacting protein (Hhip) belongs to Hedgehog (Hh) signaling pathway. The Hh signaling pathway was already linked with adipogenesis in previous reports, however, the physiological functions of Hhip on lipid deposition are still poorly understood. In this study, the level of Hhip was down-regulated during the development of porcine adipose tissues. Recombinant Hedgehog interacting protein (rHhip) could down-regulate cell cycle related genes and cell numbers in S phage to inhibit cell proliferation. Moreover, rHhip could increase adipocytes differentiation by targeting canonical Hh signaling, indicated by the increase of lipid accumulation and up-regulation of Glut4 and PPARγ expression. Collectively, these findings illustrated the essential role of Hhip in the proliferation and differentiation of adipocytes, and provided a potential novel target for preventing obesity. • Hhip moderately expresses in porcine adipose tissues and its expression gradually decreases along with the growth of pigs. • Hhip could inhibit preadipocytes proliferation by down-regulating expression of Cyclin B, Cyclin D and Cyclin E. • Hhip increases lipid droplets by suppressing canonical Hh-Gli signaling and increasing the expression of Glut4 and PPARγ. [ABSTRACT FROM AUTHOR]

Abstract Inverse gradients of transcriptional repressors antagonize the transcriptional effector response to morphogens. However, the role of such inverse regulation might not manifest solely from lack of repressors. Sonic hedgehog (Shh) patterns the forebrain by being expressed ventrally; however, absence of antagonizing Gli3 repressor paradoxically cause insufficient pathway activation. Interestingly, lack of the primary cilia-localized G-protein-coupled receptor, Gpr161 increases Shh signaling in the mouse neural tube from coordinated lack of Gli3 repressor and Smoothened-independent activation. Here, by deleting Gpr161 in mouse neuroepithelial cells and radial glia at early mid-gestation we detected derepression of Shh signaling throughout forebrain, allowing determination of the pathophysiological consequences. Accumulation of cerebrospinal fluid (hydrocephalus) was apparent by birth, although usual causative defects in multiciliated ependymal cells or aqueduct were not seen. Rather, the ventricular surface was expanded (ventriculomegaly) during embryogenesis from radial glial overproliferation. Cortical phenotypes included polymicrogyria in the medial cingulate cortex, increased proliferation of intermediate progenitors and basal radial glia, and altered neocortical cytoarchitectonic structure with increased upper layer and decreased deep layer neurons. Finally, periventricular nodular heterotopia resulted from disrupted neuronal migration, while the radial glial scaffold was unaffected. Overall, suppression of Shh pathway during early mid-gestation prevents ventricular overgrowth, and regulates cortical gyration and neocortical/periventricular cytoarchitecture. Graphical abstract Image 1 Highlights • Deleting Gpr161 in mid-gestation causes derepression of Shh signaling in forebrain. • Hydrocephalus was apparent by birth without defects in ependymal cells or aqueduct. • Hydrocephalus was caused by ventriculomegaly from radial glial overproliferation. • Polymicrogyria, increased intermediate progenitors and basal radial glia were seen. • Periventricular nodular heterotopia resulted from neuronal migration defects. [ABSTRACT FROM AUTHOR]

Abstract The Hedgehog pathway is essential for embryonic development but also for tissue and organ homeostasis in adult organisms. Activation of this pathway leads to the expression of target genes involved in proliferation, angiogenesis and stem cell self-renewal. Moreover, abnormal persistence of Hedgehog signaling is directly involved in a wide range of human cancers. Development of novel strategies targeting the Hedgehog pathway has become a subject of increased interest in anticancer therapy. These data are sustained by pre-clinical studies demonstrating that Hedgehog pathway inhibitors could represent an effective strategy against a heterogeneous panel of malignancies. Limited activity in other tumor types could be explained by the existence of crosstalk between the Hedgehog pathway and other signaling pathways that can compensate for its function. This review describes the Hedgehog pathway in detail, with its physiological roles during embryogenesis and adult tissues, and summarizing the preclinical evidence on its inhibition, the crosstalk between Hedgehog and other cancer-related pathways and finally the potential therapeutic effects of emerging compounds. [ABSTRACT FROM AUTHOR]

Abstract Nicotine, the main toxic substance in cigarette smoke, significantly reduced the differentiation and maturation ratio of Leydig cell in murine testes. To investigate the underlying mechanism, C57BL/6J mice were divided into control (CT) and nicotine treated (NT) groups. Next generation RNA sequencing and bio-informatics analysis were carried out to analysis the effects of nicotine on the RNA profile of Leydig cells. Expression level of 7 pathways remarkably changed after nicotine treatment. As the positive regulating pathway of Leydig cell differentiation, Hedgehog signaling pathway was found among these pathways. PTCH1 and β-TrCP were down-regulated in nicotine treated mice Leydig cells, while GSK3β, Gli2 and Gli2 fragments increased significantly. Nicotine stimulated the destabilization of Gli2 via β-TrCP induced ubiquitination and degradation. Gli2 was phosphorylated by up-expressed GSK3β during this process. Destabilization of Gli2 reduced the activation rate of target genes of Hedgehog signaling pathway such as Ptch1. The differentiation of Leydig cell positively regulated by Hh pathway was thus inhibited by nicotine exposure. Consequently, the male reproduction process powered by Leydic cell-mediated androgen secretion was thus influenced. In conclusion, we find that nicotine inhibits murine Leydig cell differentiation and maturation via regulating Hedgehog signal pathway. Highlights • Nicotine inhibits Leydig cell differentiation and maturation of male mice. • Nicotine changes the expression of Hedgehog signal pathway. • Nicotine down regulates Hedgehog signal pathway via Gli2 degradation. [ABSTRACT FROM AUTHOR]

Abstract Lung cancer is a leading lethal disease with a 5-year survival rate of only 16%. Inadequate potent anti-cancer drugs appear to be a bottleneck in the treatment of lung cancer; hence, how to develop effective anti-lung cancer therapeutics is an urgent problem. In this study, we aim to explore a novel compound with potent anti-lung cancer effect and study its anti-cancer mechanisms. We found that triptonide at very low concentrations of 5–10 nM caused a marked suppression of cell proliferation and colony formation of lung cancer cells. More interestingly, triptonide also robustly inhibited the lung cancer cell formation of tumor spheres, and reduced the stemness and tumorigenicity of the sphere-forming cells. In vivo studies showed that administration of triptonide significantly inhibited the tumor growth with low toxicity. Molecular mechanistic studies revealed that triptonide significantly decreased expression of the Gli1 at both mRNA and protein levels by repressing Gli1 gene promoter activity. Additionally, triptonide reduced the levels of cancer stem cell key signaling protein sonic hedgehog (Shh), but increased the amount of Ptch1, a protein binding to SMO to diminish the Shh signal transduction, thus inhibition of the Shh–Gli1 signaling pathway. Together, our findings show that triptonide effectively inhibits lung cancer cell growth, stemness, and tumorigenicity, and support the notion that triptonide is a new Shh–Gli1 signaling inhibitor and a novel anti-lung cancer drug candidate for further developing effective lung cancer therapeutics. [ABSTRACT FROM AUTHOR]

Abstract Introduction Erectile dysfunction (ED) is a significant health concern that greatly impacts quality of life, and is common in men as they age, impacting 52% of men between the ages of 40 and 70. A significant underlying cause of ED development is injury to the cavernous nerve (CN), a peripheral nerve that innervates the penis. CN injury also occurs in up to 82% of prostatectomy patients. We recently showed that Sonic hedgehog (SHH) protein delivered by peptide amphiphile (PA) nanofiber hydrogel to the CN and penis of a prostatectomy model of CN injury, is neuroprotective, accelerates CN regeneration, improves erectile function ~60%, preserves penile smooth muscle 56% and suppresses collagen deposition 30%. This regenerative potential is substantial in an adult prostatectomy model (P120). However prostatectomy patients are typically older (61.5 ± 9.6 years) and our models should mimic patient conditions more effectively when considering translation. In this study we examine regenerative potential in an aged prostatectomy model (P200–329). Methods The caudal portion of the pelvic ganglia (MPG) and CN were dissected from adult (n = 11), and aged (n = 13) Sprague Dawley rats, and were grown in organ culture 3 days. Uninjured and 2 day CN crushed MPG/CN were exposed to Affi-Gel beads containing SHH protein, PBS (control), or 5e1 SHH inhibitor. Neurites were quantified by counting the number of growth cones normalized by tissue perimeter (mm) and immunohistochemistry for SHH, patched1 (PTCH1), smoothened (SMO), GLI1-3, and GAP43 were performed. Results SHH treatment increased neurites 3.5-fold, in uninjured adult, and 5.7-fold in aged rats. Two days after CN crush, SHH treatment increased neurites 1.8-fold in adult rats and 2.5-fold in aged rats. SHH inhibition inhibited neurite formation in uninjured MPG/CN but not in 2 day CN crushed MPG/CN. PTCH1 and SMO (SHH receptors), and SHH transcriptional activators/repressors, GLI1-3, were abundant in aged MPG/CN with unaltered localization. ROCK1 was induced with SHH treatment. Conclusions Reintroduction of SHH protein in an aged prostatectomy model is even more effective in promoting neurite formation/CN regeneration than in the adult. The first 48 h after CN injury are a critical window when growth factors are released, that impact later neurite formation. These studies are significant because most prostatectomy patients are not young and healthy, as with adult rats, so the aged prostatectomy model will more accurately simulate ED patient response. Understanding how neurite formation changes with age is critical for clinical translation of SHH PA to prostatectomy patients. Highlights • SHH protein is more effective in promoting neurite formation/CN regeneration in aged rats than in the adult. • The first 48 h after CN injury are a critical window when growth factors are released, that impact later neurite formation. • SHH pathway signaling machinery remains intact in aged MPG/CN and are able to respond to exogenous SHH protein. • These studies are significant because the aged prostatectomy model will more accurately simulate ED patient response. • Understanding how neurite formation changes with age is critical for clinical translation of SHH PA to prostatectomy patients [ABSTRACT FROM AUTHOR]

Abstract Hedgehog (Hh) pathway hyperactivation has been observed in various tumors, including breast cancer, and Hh pathway inhibitors have demonstrated antitumor activity in breast cancer. The tumor microenvironment (TME) has been shown to play an important role in modulating cancer cell drug sensitivity, but the TME response to Hh pathway inhibitors is unclear. In the current study, we observed increased TME infiltration of macrophages in breast cancer tissue, and specifically, M2 polarized macrophages after neoadjuvant chemotherapy. Furthermore, we observed an enhanced tolerance to Hh pathway inhibitors in MDA-MB-231 cells after co-culturing with M2 macrophages. In addition, we demonstrated that Hh pathway inhibition significantly induced IL6 expression, and validated that the tolerance to Hh pathway inhibitors was IL6-dependent. This study demonstrates a role of macrophages in Hh pathway inhibition resistance and a role of macrophage-derived IL6 in this resistance of breast cancer cells to Hh inhibition. These data indicate that antagonizing IL6 together with Hh pathway inhibitors may be a novel therapeutic strategy for breast cancer. Highlights • Breast cancer tissue has higher M1 and M2 macrophage infiltration. • Neoadjuvant chemotherapy increases breast cancer tissue M2 macrophage infiltration. • M2 macrophages attenuate the efficacy of a Hh inhibitor in breast cancer cells. • Hh pathway inhibition induces polarization of macrophages toward an M2 phenotype. • M2 macrophage-derived IL6 enhances cyclopamine resistance in breast cancer cells. [ABSTRACT FROM AUTHOR]

Abstract The Sonic Hedgehog signaling (Shh) pathway has been implicated in both proliferation of myoblast cells and terminal differentiation of muscle fibers, and contradictory results of these effects have been described. To clarify the role of Shh during myogenesis, we decided to study the effects of recombinant Shh and the distribution of Gli-1 during in vitro and in situ embryonic chick skeletal muscle differentiation at later stages of development. Gli-1 was found in small aggregates near the nucleus in mononucleated myoblasts and in multinucleated myotubes both in vitro and in situ chick muscle cells. Some Gli-1 aggregates colocalized with gamma-tubulin positive-centrosomes. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle fibers in situ. Recombinant Shh added to in vitro grown muscle cells induced the nuclear translocation of Gli-1, as well as an increase in the number of myoblasts and in the number of nuclei within myotubes. We suggest that Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the rapid cellular redistribution of Gli-1 upon specific signals during muscle differentiation. Highlights • Gli-1 is present in aggregates near the nuclei of chick myoblasts and myotubes. • Gli-1 aggregates colocalized with gamma-tubulin positive-centrosomes. • Recombinant Shh added to muscle cells induced the nuclear translocation of Gli-1. • Gli-1 aggregates are storage sites for its rapid cellular redistribution. [ABSTRACT FROM AUTHOR]

Tissue autotomy is a unique adaptive response to environmental stress, followed by regeneration process compensating for the loss of body parts. The crustaceans present remarkable activity of appendage autotomy and regeneration, however, the molecular mechanism is still unclear. In this study, the Eriocheir sinensis Hedgehog (EsHH) and Smoothened (EsSMO) were identified in the regenerative limbs, and the function of Hedgehog signaling pathway on limb regeneration was evaluated. At the blastema growth stage of limb regeneration, the expression of EsHH and EsSMO was up-regulated in response to limb autotomy stress, and down-regulated at blastema differentiation stage. To clarify the effect of Hedgehog pathway during limb regeneration, the regenerative efficiency was evaluated with Smoothened inhibitor cyclopamine or RNAi (ds-HH) injection. We observed that the regenerative efficiency was significantly repressed with blockage of Hedgehog pathway at both the basal growth stage and the proecdysial growth stage, which was indicated by the delay of wound healing and blastema growth, as well as a decrease in the size of newly formed limbs. In addition, gene expression and BrdU incorporation assay showed that the proliferation and myogenic differentiation of blastema cells were suppressed with either cyclopamine or ds- HH injection. Thus, these results suggest that Hedgehog signaling pathway is essential for the establishment of limb regeneration in E. sinensis through promoting the proliferation and myogenic differentiation of blastema cells. • Hedgehog (EsHH) and Smoothened (EsSMO) homolog were identified from Chinese mitten crab. • Expression of EsHH was up-regulated at the basal growth phase, and down-regulated at the proecdysial growth stage. • Blockage of Hedgehog pathway with Smoothened inhibitor or RNAi (ds-HH) repressed limb regeneration in E. sinensis. • Hedgehog pathway regulates limb regeneration in through promoting proliferation and myogenic differentiation of blastema cells. [ABSTRACT FROM AUTHOR]

[Display omitted] The Hedgehog/Glioma-associated oncogene (Hh/Gli) signaling pathway plays an essential role in embryonic development and tissue homeostasis. Aberrant regulation of this pathway has been linked to various human malignancies. Gli1, the downstream transcription factor of the Hh pathway, is the ultimate effector of the canonical Hh pathway and has been identified as a common regulator of several tumorigenic pathways prevalent in Hh-independent cancers. Thus Gli1 represents a unique and promising drug target for a wide range of cancers. However, the identification and development of small molecules that directly target Gli1 protein have progressed slowly, due to an insufficient efficacy and selectivity. Herein, we developed novel small-molecule Gli1 degraders based on the hydrophobic tagging (HyT) strategy. The Gli1 HyT degrader 8e potently inhibited the proliferation of Gli1-overexpressed HT29 colorectal cancer cells, induced Gli1 degradation with a DC 50 value of 5.4 μM in HT29 and achieved 70% degradation at 7.5 μM in MEF PTCH1−/− and MEF SUFU−/− cell lines, via proteasome pathway. Compared to the canonical Hh antagonist Vismodegib, 8e exhibited much stronger potency in suppressing the mRNA expression of Hh target genes in Hh-overactivated MEF PTCH1−/− and Vismodegib resistant MEF SUFU−/− cells. Our study provides small molecule Gli1 degraders effectively interfering with both canonical and noncanonical Hh signaling and overcoming current Smoothened (SMO) antagonists resistance, which might pave a new avenue for developing therapeutic modalities targeting Hh/Gli1 signaling pathway. [ABSTRACT FROM AUTHOR]

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. • Hypothalamic hamartoma is a benign brain lesion associated with epilepsy. • Somatic and germline variants contribute to pathogenesis of hypothalamic hamartoma. • Associated genes function within the sonic hedgehog pathway or primary cilia. • This review summarizes and contextualizes genetic findings, and future directions. [ABSTRACT FROM AUTHOR]

The blood-nerve and myelin barrier shield peripheral neurons and their axons. These barriers are sealed by tight junction proteins, which control the passage of potentially noxious molecules including proinflammatory cytokines via paracellular pathways. Peripheral nerve barrier breakdown occurs in various neuropathies, such as chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and traumatic neuropathy. Here, we studied the functional role of the tight junction protein claudin-12 in regulating peripheral nerve barrier integrity and CIDP pathogenesis. Sections from sural nerve biopsies from 23 patients with CIDP and non-inflammatory idiopathic polyneuropathy (PNP) were analyzed for claudin-12 and -19 immunoreactivity. Cldn12 -KO mice were generated and subjected to the chronic constriction injury (CCI) model of neuropathy. These mice were then characterized using a battery of barrier and behavioral tests, histology, immunohistochemistry, and mRNA/protein expression. In phenotype rescue experiments, the proinflammatory cytokine TNFα was neutralized with the anti-TNFα antibody etanercept; the peripheral nerve barrier was stabilized with the sonic hedgehog agonist smoothened (SAG). Compared to those without pain, patients with painful neuropathy exhibited reduced claudin-12 expression independently of fiber loss. Accordingly, global Cldn12 -KO in male mice, but not fertile female mice, selectively caused mechanical allodynia associated with a leaky myelin barrier, increased TNFα, decreased sonic hedgehog (SHH), and loss of small axons accompanied by reduced peripheral myelin protein 22 (Pmp22). Other barriers and neurological functions remained intact. The Cldn12 -KO phenotype could be rescued either by neutralizing TNFα with etanercept or stabilizing the barrier with SAG, which both also upregulated the Schwann cell barrier proteins Cldn19 and Pmp22. These results point to a critical role for claudin-12 in maintaining the myelin barrier presumably via Pmp22 and highlight restoration of the hedgehog pathway as a potential treatment strategy for painful inflammatory neuropathy. [Display omitted] • Patients with painful polyneuropathy express less claudin-12 (CLDN12) then patients with painless polyneuropathy. • Cldn12 -KO mice are characterized by mechanical allodynia, increased endoneurial TNFα and a leaky myelin barrier. • TNFα trapping normalizes mechanical nociceptive thresholds and restores the phenotype via neutralization of the endoneurial proalgesic mediator. • Treatment with a sonic hedgehog agonist reverses mechanical allodynia and restores the barrier via Cldn19 and Pmp22. • Cldn12 has a novel role in the PNS stabilizing the myelin barrier. [ABSTRACT FROM AUTHOR]

Abstract Osteoporosis is widely viewed as a major public health concern, but the exact magnitude of the problem is uncertain. MicroRNAs play a key role in maintaining bone development and metabolism. This study aims to investigate the effects that microRNA-874 (miR-874) has on osteoblast proliferation and differentiation in osteoporosis rats by targeting SUFU through the Hedgehog signaling pathway. Twenty Wistar female rats were selected for following experiment, and another 20 rats were served as the normal group. Their osteogenic tissues were obtained and the positive expression of SUFU in tissues was determined. Rat osteoblasts were isolated and. The targeting relationship between SUFU and miR-874 was verified and the expression of miR-874, SUFU, Sonic Hedgehog (Shh), Ptch, Smoothened (Smo), bone morphogenetic protein (BMP2), Runx2, proliferating cell nuclear antigen (PCNA) and Bcl-2 associated X protein (Bax) were identified. Besides, cell viability apoptosis, and differentiation were confirmed respectively. Moreover, calcium nodules were observed. Overexpression of SUFU and Bax but lower expression of miR-874, Shh, Ptch, Smo, BMP2, Runx2, and PCNA were found in osteoporosis mice. Besides, elevated expression of miR-874, Shh, Ptch, Smo, BMP2, Runx2 and PCNA, as well as increased cell viability, ALP activity and calcium nodules but decreased expression of SUFU and Bax, and reduced cell apoptosis were confirmed when treated with miR-874 mimic. And it is reciprocal when miR-874 was inhibited. Our study demonstrated that through targeted inhibition of SUFU and activation of Hedgehog signaling pathway, miR-874 could promote the proliferation and differentiation of osteoblasts in osteoporosis rats. [ABSTRACT FROM AUTHOR]

Abstract Primary cilia represent small, yet distinct compartments of the plasma membrane. They are speculated to exercise chemo- and mechanosensory functions and to serve as signaling hubs for crucial pathways such as the Wnt and hedgehog cascades. It is therefore necessary that specific integral membrane proteins, in particular sensors and receptors, are sorted to the cilium and not to the surrounding somatic plasma membrane upon being synthesized at the rough endoplasmic reticulum. Apparently no singular "zip code" for the primary cilium exists but rather several ciliary targeting signals whose biochemical and cell biological implications are just about being unravelled. Among the better understood proteins residing in the primary cilium is polycystin-2 which is mutated in patients suffering from autosomal-dominant polycystic kidney disease. A special case in the context of this review concerns the connecting cilium which serves as the trafficking pathway for proteins involved in visual sensation of retinal photoreceptor cells. In order to efficiently capture photons, the photopigments are organized in discs or membrane invaginations. Mutations in certain proteins involved in these processes lead to retinal degeneration and ultimately to blindness. One example is peripherin/rds which is mutated in the rds (r etinal d egeneration s low) mouse. The trafficking of peripherin/rds from the inner to the outer segment of photoreceptor cells by way of the connecting cilium also seems to diverge at the Golgi apparatus, and the routes of polycystin-2 and peripherin/rds may represent paradigms of ciliary proteins for the type IV pathway of unconventional protein "secretion". This review is part of a special issue of Seminars in Cell and Developmental Biology edited by Walter Nickel and Catherine Rabouille. [ABSTRACT FROM AUTHOR]

Abstract The transcription factor Gli2 plays crucial roles in the transduction of Hedgehog (Hh) signals, yet the mechanisms that control Gli2 degradation remain unclear. Here we have identified the eubiquitinating enzyme otubain2 (OTUB2) as a regulator of Gli2 protein degradation. We found that OTUB2 was coimmunoprecipitated with Gli2. Knockdown of OTUB2 decreased Gli2 protein level while the proteasome inhibitor MG-132 treatment restored Gli2 expression. Additionally, OTUB2 overexpression stabilized Gli2 protein in U2OS cells and extended the half-life of Gli2. We also found that knockdown of OTUB2 reduced deubiquitination of Gli2 in vivo. In vitro deubiquitination assay showed that ubiquitinated Gli2 was decreased by wild-type OTUB2 but not OTUB2 mutations. We also found that OTUB2 knockdown suppressed the ALP activity and the expression of the common markers BMP2 and RUNX2 during osteogenesis of MSCs in response to Shh and Smo agonists, which indicated OTUB2 may have effect on osteogenic differentiation by regulating Hh signaling. Highlights • OTUB2 stabilizes Gli2 protein. • OTUB2 deubiquitinates Gli2. • OTUB2 promotes osteogenesis of MSCs. [ABSTRACT FROM AUTHOR]

Abstract Primary cilia are appendages observed in most types of cells, and serve as cellular antennae for sensing environmental signals. Evidence is accumulating that correct ciliogenesis and ciliary functions are indispensable for normal skeletal development by regulating signaling pathways important for bone development. However, whether ciliogenesis is regulated by bone-related factors in osteoblasts is largely unknown. Here we show that Kruppel-Like Factor 4 (KLF4), which is known to repress osteoblast differentiation, supports the formation and maintenance of cilia in cultured osteoblasts; however, the length of the cilia observed in KLF4-induced cells were significantly shorter compared to the control cells. Basal Hedgehog signaling was repressed by KLF4. Significantly, activating Hedgehog signaling using a Smoothened agonist significantly rescued osteoblast mineralization and osteoblastic gene expressions. Global gene expression analysis showed that KLF4 induced number of genes including the nuclear receptor, Pregnane X receptor (PXR), and PXR repressed calvarial osteoblast mineralization and repressed Gli1 expression similar as the effect observed by inducing KLF4. Our results implicate that KLF4 plays important roles for maintaining osteoblasts in an immature state by repressing basal activation of the Hedgehog signaling. [ABSTRACT FROM AUTHOR]

Abstract Icaritin is a compound extracted from herb, recent study have found it is able to influence the activity of various types of cancer. Our aim was to investigate the effects of Icaritin on the physiological activities of esophageal cancer stem cells (CSCs). In this study, esophageal cancer cells were cultured and CD133 positive esophageal CSCs were sorted by flow cytometry. Changes in the physiological activity of esophageal CSCs following treatment with different concentrations of Icaritin (0, 12.5, 25, 50, and 100 μmol/L) were evaluated. The CCK-8 method and Transwell assay were used to determine the effects of Icaritin on the proliferation, migration, and invasion of esophageal CSCs. Flow cytometery was used to investigate its effect on the apoptosis of CSCs. The effect of Icaritin on the expression of proteins in Wnt and Hedgehog signaling pathways were determined using western blot test. Consequently, Icaritin inhibited the proliferation, migration, and invasion of esophageal CSCs in a dose-dependent manner. It promoted cell apoptosis, and influenced the levels of proteins in Wnt and Hedgehog signaling pathways. It may act as a promising drug in the therapy of esophageal cancer. Highlights • CSCs were used instead of using cancer cells. • The effect of Icaritin on the Hedgehog and Wnt signaling pathway was investigated. • Our study found that Icaritin down-regulated the level of Hedgehog, Smo and Gli in Hedgehog pathway, up-regulated GSK3β and down-regulated Wnt and β-catenin in Wnt pathway, indicating that Icaritin may inhibit the activity of esophageal CSCs through regulating the expression of proteins in important pathways related to esophageal cancer. [ABSTRACT FROM AUTHOR]

Cleft palate is one of the most common craniofacial congenital defects in humans. It is associated with multiple genetic and environmental risk factors, including mutations in the genes encoding signaling molecules in the sonic hedgehog (Shh) pathway, which are risk factors for cleft palate in both humans and mice. However, the function of Shh signaling in the palatal epithelium during palatal fusion remains largely unknown. Although components of the Shh pathway are localized in the palatal epithelium, specific inhibition of Shh signaling in palatal epithelium does not affect palatogenesis. We therefore utilized a hedgehog (Hh) signaling gain-of-function mouse model, K14-Cre;R26SmoM2 , to uncover the role of Shh signaling in the palatal epithelium during palatal fusion. In this study, we discovered that constitutive activation of Hh signaling in the palatal epithelium results in submucous cleft palate and persistence of the medial edge epithelium (MEE). Further investigation revealed that precise downregulation of Shh signaling is required at a specific time point in the MEE during palatal fusion. Upregulation of Hh signaling in the palatal epithelium maintains the proliferation of MEE cells. This may be due to a dysfunctional p63/Irf6 regulatory loop. The resistance of MEE cells to apoptosis is likely conferred by enhancement of a cell adhesion network through the maintenance of p63 expression. Collectively, our data illustrate that persistent Hh signaling in the palatal epithelium contributes to the etiology and pathogenesis of submucous cleft palate through its interaction with a p63/Irf6-dependent biological regulatory loop and through a p63-induced cell adhesion network. [ABSTRACT FROM AUTHOR]

Hematopoietic progenitor choice between multipotency and differentiation is tightly regulated by intrinsic factors and extrinsic signals from the surrounding microenvironment. The Drosophila melanogaster hematopoietic lymph gland has emerged as a powerful tool to investigate mechanisms that regulate hematopoietic progenitor choice in vivo . The lymph gland contains progenitor cells, which share key characteristics with mammalian hematopoietic progenitors such as quiescence, multipotency and niche-dependence. The lymph gland is zonally arranged, with progenitors located in medullary zone, differentiating cells in the cortical zone, and the stem cell niche or Posterior Signaling Center (PSC) residing at the base of the medullary zone (MZ). This arrangement facilitates investigations into how signaling from the microenvironment controls progenitor choice. The Drosophila Friend of GATA transcriptional regulator, U-shaped, is a conserved hematopoietic regulator. To identify additional novel intrinsic and extrinsic regulators that interface with U-shaped to control hematopoiesis, we conducted an in vivo screen for factors that genetically interact with u-shaped . Smoothened, a downstream effector of Hedgehog signaling, was one of the factors identified in the screen. Here we report our studies that characterized the relationship between Smoothened and U-shaped. We showed that the PSC and Hedgehog signaling are required for U-shaped expression and that U-shaped is an important intrinsic progenitor regulator. These observations identify a potential link between the progenitor regulatory machinery and extrinsic signals from the PSC. Furthermore, we showed that both Hedgehog signaling and the PSC are required to maintain a subpopulation of progenitors. This led to a delineation of PSC-dependent versus PSC-independent progenitors and provided further evidence that the MZ progenitor population is heterogeneous. Overall, we have identified a connection between a conserved hematopoietic master regulator and a putative stem cell niche, which adds to our understanding of how signals from the microenvironment regulate progenitor multipotency. [ABSTRACT FROM AUTHOR]

Neuronal-glial relationships play a critical role in the maintenance of central nervous system architecture and neuronal specification. A deeper understanding of these relationships can elucidate cellular cross-talk capable of sustaining proper development of neural tissues. In the cerebellum, cerebellar granule neuron precursors (CGNPs) proliferate in response to Purkinje neuron-derived Sonic hedgehog (Shh) before ultimately exiting the cell cycle and migrating radially along Bergmann glial fibers. However, the function of Bergmann glia in CGNP proliferation remains not well defined. Interestingly, the Hh pathway is also activated in Bergmann glia, but the role of Shh signaling in these cells is unknown. In this study, we show that specific ablation of Shh signaling using the tamoxifen-inducible TNC YFP-CreER line to eliminate Shh pathway activator Smoothened in Bergmann glia is sufficient to cause severe cerebellar hypoplasia and a significant reduction in CGNP proliferation. TNC YFP-CreER ; Smo F/- ( Smo CKO ) mice demonstrate an obvious reduction in cerebellar size within two days of ablation of Shh signaling. Mutant cerebella have severely reduced proliferation and increased differentiation of CGNPs due to a significant decrease in Shh activity and concomitant activation of Wnt signaling in Smo CKO CGNPs, suggesting that this pathway is involved in cross-talk with the Shh pathway in regulating CGNP proliferation. In addition, Purkinje cells are ectopically located, their dendrites stunted, and the Bergmann glial network disorganized. Collectively, these data demonstrate a previously unappreciated role for Bergmann glial Shh signaling activity in the proliferation of CGNPs and proper maintenance of cerebellar architecture. [ABSTRACT FROM AUTHOR]

The tight control of reactive oxygen species (ROS) levels is required during regeneration. H 2 O 2 in particular assumes clear signalling functions at different steps in this process. Injured nerves induce high levels of H 2 O 2 through the activation of the Hedgehog (Shh) pathway, providing an environment that promotes cell plasticity, progenitor recruitment and blastema formation. In turn, high H 2 O 2 levels contribute to growing axon attraction. Once re-innervation is completed, nerves subsequently downregulate H 2 O 2 levels to their original state. A similar regulatory loop between H 2 O 2 levels and nerves also exists during development. This suggests that redox signalling is a major actor in cell plasticity. [ABSTRACT FROM AUTHOR]

Forelimbs (FLs) and hindlimbs (HLs) develop under the instructive and integrated guidance of signaling centers and transcription factor (TF) action. The development of structures specific to each limb type depends on the limb-specific modulation of these integrated components. Pitx1 is a transcription factor gene expressed in HL, absent in FL, and required for HL-specific patterning and development, in particular for formation of anterior HL skeletal elements. Pitx1 achieves this function by direct TF action on the core limb program, which is largely shared between FL and HL. Shh signaling plays a crucial role in anterior-posterior (AP) patterning in both FL and HL. The present work assessed the relationship between Shh signaling and Pitx1 action for AP patterning. We found that reducing the gene dosage of Shh in the context of the Pitx1-/- HL decreases the severity of the Pitx1-/- phenotype, in particular, the loss of anterior limb structures and the shortening of femur length. However, this did not rescue HL-specific patterning features. Thus, Pitx1 action integrates Shh signaling but not for limb-type-specific patterning. [ABSTRACT FROM AUTHOR]

Background and objective The nature of pulmonary fibrosis involves inadequate repair of the epithelial cell barrier accompanied by impaired regulation of the fibroblast. Moreover, pulmonary fibrosis currently lacks an effective therapeutic drug. This study targets the protection of the epithelial cell and fibroblast to identify a novel, potentially therapeutic drug (i.e., astilbin). Methods In this study, the cytotoxicity of astilbin was firstly detected using CCK-8. A real-time proliferation/migration analysis system was used to test the inhibitory proliferation and migration of astilbin in vitro . The expression of mesenchymal markers and the loss of epithelial cell markers were analyzed to evaluate the antifibrotic activity of astilbin on TGF-β1-treated AEC-II and L929 cells and bleomycin-treated mice. Then, in fibrosis-associated signaling pathways, the regulation of astilbin was tested using RNA sequencing and Cignal Finder 45-Pathway system. Rescue and other experiments were used to confirm this pathway regulation further. Results The data showed that astilbin inhibited proliferation and migration of cell samples. Its treatment resulted in the reduction of pathological score and collagen deposition, with a decrease in α-SMA and Snail and an increase in E-cadherin and SP-C in vivo and in vitro . The fibrosis-associated aberrant genes are some of the most notable components of the Hedgehog signaling pathway. Conclusions Astilbin ameliorates pulmonary fibrosis via blockade of Hedgehog signaling pathway and has potential therapeutic value for lung fibrosis treatment. [ABSTRACT FROM AUTHOR]

Smoothened (Smo) is the essential transducer of Sonic hedgehog (Shh) signaling, which regulates cell fate and proliferation during embryogenesis. We identified a novel mouse mutant, cabbie ( cbb ), and found that its cause is a missense mutation in Smo . We showed the Smo cbb mutation is insensitive to the Shh agonist SAG, perhaps due to the disruption of SAG binding. We characterized Smo cbb for defects in craniofacial and skeletal development, as well as neural tube patterning, and revealed Smo cbb affected processes that require the highest levels of Shh activity. Smo is normally enriched in cilia upon Shh stimulation; however, we detected inefficient enrichment of Smo in Smo cbb mutants whether we stimulated with Shh or SAG. Taken together, our data suggest that the highest levels of vertebrate Hedgehog signaling activity require efficient Smo ciliary enrichment. [ABSTRACT FROM AUTHOR]

Bone is a connective tissue composed of many cell types, including osteoblasts. How bones acquire their unique size and shape during development remains poorly understood. Herein we investigated the molecular and cellular mechanisms of bone morphogenesis in the zebrafish scale by using transgenic lines to enable visualization of specific types of osteoblasts. We demonstrate that the zebrafish scale contains three distinct types of osteoblasts: (i) a monolayer of central osteoblasts along the inner surface of scales; (ii) marginal osteoblasts elongated along the scale edge; and (iii) submarginal osteoblasts located between the central and marginal osteoblast populations. The size of the central osteoblasts increases progressively during development, suggesting that scale growth is mediated primarily by cell growth rather than the recruitment of new osteoblasts. In addition, the total number of central osteoblasts increases in regenerated scales and is correlated with scale size, possibly allowing for the rapid growth of regenerating scales. Moreover, osteoblast proliferation is not detected during regeneration, suggesting that the osteoblasts originate from post-mitotic precursor cells. Sonic hedgehog a (shha) is expressed in the epidermal cells that make contact with the marginal osteoblasts. Pharmacological inhibition of Hedgehog (Hh) signaling during regeneration reduces the number of marginal osteoblasts and interferes with scale growth, indicating that epidermis-derived Shh regulates scale regeneration. Finally, genetic inhibition of Wnt/planar cell polarity (PCP) signaling in the epidermis results in misorientation of scales with regard to the body axis. These results reveal a novel role for the epidermis in the regulation of bone patterning, namely the regeneration of osteoblasts and directional bone growth. [ABSTRACT FROM AUTHOR]