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Objective: Sarcopenia manifests as a chronic, low-level inflammation along with multiple inflammatory cells infiltration. Interleukin (IL)-25 can regulate the function of macrophages. However, the specific role and mechanisms through which IL-25 functions in sarcopenia are still not fully understood and require further investigation., Methods: Aged mice were utilized as sarcopenia models and examined the expression of inflammatory factors. To investigate the effects of IL-25 on sarcopenia, the model mice received IL-25 treatment and underwent in vivo adoptive transfer of IL-25-induced macrophages. Meanwhile, RAW264.7 cells, bone marrow-derived macrophages, satellite cells and C2C12 cells were used in vitro. Shh insufficiency was induced through intramuscular administration of SHH-shRNA adenoviruses. Then, various assays including scratch wound, cell counting kit-8 and Transwell assays, as well as histological staining and molecular biological methods, were conducted., Results: Aged mice exhibited an accelerated decline in muscle strength and mass, along with an increased muscle lipid droplets and macrophage infiltration, and decreased IL-25 levels compared to the young group. IL-25 therapy and the transfer of IL-25-preconditioned macrophages could improve these conditions by promoting M2 macrophage polarization in vivo as well as in vitro. M2 macrophage conditioned medium enhanced satellite cell proliferation and migration, as well as the vitality, migration, and differentiation of C2C12 cells in vitro. Furthermore, IL-25 enhanced Shh expression in macrophages in vitro, and activated the Shh signaling pathway in muscle tissue of aged mice, which could be suppressed by either the inhibitor cyclopamine or Shh knockdown. Mechanistic studies showed that Shh insufficiency suppressed the activation of Akt/mTOR signaling pathway in muscle tissue of aged mice., Conclusion: IL-25 promotes the secretion of Shh by M2 macrophages and activates the Shh/Akt/mTOR signaling pathway, which improves symptoms and function in sarcopenia mice. This suggests that IL-25 has potential as a therapeutic agent for treating sarcopenia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sonic Hedgehog (SHH) is a driver of embryonic patterning that, when corrupted, triggers developmental disorders and cancers. SHH effector responses are organized through primary cilia (PC) that grow and retract with the cell cycle and in response to extracellular cues. Disruption of PC homeostasis corrupts SHH regulation, placing significant pressure on the pathway to maintain ciliary fitness. Mechanisms by which ciliary robustness is ensured in SHH-stimulated cells are not yet known. Herein, we reveal a crosstalk circuit induced by SHH activation of Phospholipase A2α that drives ciliary E-type prostanoid receptor 4 (EP4) signaling to ensure PC function and stabilize ciliary length. We demonstrate that blockade of SHH-EP4 crosstalk destabilizes PC cyclic AMP (cAMP) equilibrium, slows ciliary transport, reduces ciliary length, and attenuates SHH pathway induction. Accordingly, Ep4-/- mice display shortened neuroepithelial PC and altered SHH-dependent neuronal cell fate specification. Thus, SHH initiates coordination between distinct ciliary receptors to maintain PC function and length homeostasis for robust downstream signaling., (© 2024 Ansari et al.)

Background/aim: Glioblastoma is the most frequent type of adult-onset malignant brain tumor and has a very poor prognosis. Glioblastoma stem cells have been shown to be one of the mechanisms by which glioblastoma acquires therapy resistance. Therefore, there is a need to establish novel therapeutic strategies useful for inhibiting this cell population. γ-Glutamylcyclotransferase (GGCT) is an enzyme involved in the synthesis and metabolism of glutathione, which is highly expressed in a wide range of cancer types, including glioblastoma, and inhibition of its expression has been reported to have antitumor effects on various cancer types. The aim of this study was to clarify the function of GGCT in glioblastoma stem cells., Materials and Methods: We searched for pathways affected by GGCT overexpression in mouse embryonic fibroblasts NIH-3T3 by comprehensive gene expression analysis. Knockdown of GGCT and overexpression of desert hedgehog (DHH), a representative ligand of the pathway, were performed in glioblastoma stem cells derived from a mouse glioblastoma model., Results: GGCT overexpression activated the hedgehog pathway. Knockdown of GGCT inhibited proliferation of glioblastoma stem cells and reduced expression of DHH and the downstream target GLI family zinc finger 1 (GLI1). DHH overexpression significantly restored the growth-suppressive effect of GGCT knockdown., Conclusion: High GGCT expression is important for expression of DHH and activation of the hedgehog pathway, which is required to maintain glioblastoma stem cell proliferation. Therefore, inhibition of GGCT function may be useful in suppressing stemness of glioblastoma stem cells accompanied by activation of the hedgehog pathway., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aims: Colon cancer poses a major threat to human health and a heavy burden on the national economy. As a member of the SOX transcription factor family, SRY-box transcription factor 21 (SOX21) is associated with various cancers, but its mechanism of action in colon cancer remains unclear. This study focused on the molecular mechanisms of transcription factor SOX21 in proliferation and metastasis of colon cancer cells., Main Methods: We analyzed SOX21 expression level and its impact on survival in colon cancer patients by bioinformatics analysis. We used public databases for gene correlation, GSEA enrichment analysis. Cell function experiments (colony formation assay, wound healing assay, Transwell migration and invasion assay) were utilized to determine the impact of SOX21 silencing and over-expression on cell proliferation and metastasis. The luciferase reporter assay, CUT&RUN-qPCR assay and Methylation Specific PCR were used to explore SOX21-POU class 4 homeobox 2 (POU4F2) molecular interactions. The molecular mechanisms were verified by Quantitative real-time PCR and Western blot analysis., Key Findings: SOX21 is highly expressed and affects the overall survival of colon cancer patients. SOX21 can attenuates POU4F2 methylation state by binding with it. In addition, this interaction facilitate its transcriptional activation of Hedgehog pathway, mediates epithelial-mesenchymal transition (EMT), consequently promoting the proliferation and metastasis of colon cancer cells., Significance: Our study reveals that SOX21 is an oncogenic molecule and suggests its regulatory role in colon carcinogenesis and progression, providing new insights into the treatment of this disease., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

In years of research on classical pathways, the composition, information transmission mechanism, crosstalk with other pathways, and physiological and pathological effects of hedgehog (HH) pathway have been gradually clarified. HH also plays a critical role in tumor formation and development. According to the update of interpretation of tumor phenotypes, the latest relevant studies have been sorted out, to explore the specific mechanism of HH pathway in regulating different tumor phenotypes through gene mutation and signal regulation. The drugs and natural ingredients involved in regulating HH pathway were also reviewed; five approved drugs and drugs under research exert efficacy by blocking HH pathway, and at least 22 natural components have potential to treat tumors by HH pathway. Nevertheless, there is a deficiency of existing studies. The present review confirmed the great potential of HH pathway in future cancer treatment with factual basis.

Background: Glioma stem cells (GSCs), which are known for their therapy resistance, play a substantial role in treatment inefficacy for glioblastoma multiforme (GBM). TRIM37, a member of the tripartite motif (TRIM) protein family initially linked to a rare growth disorder, has been recognized for its oncogenic role. However, the mechanism by which TRIM37 regulates tumor growth in glioma and GSCs is unclear., Methods: For the in vitro experiments, gene expression was measured by western blotting, RT-qPCR, and immunofluorescence. Cell viability was detected by CCK-8, and cell apoptosis was detected by flow cytometry. The interaction between Enhancer of Zeste Homolog 2 (EZH2) and TRIM37 was verified by co-immunoprecipitation (Co-IP). The interaction between EZH2 and the PTCH1 promoter was verified using dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP). For the in vivo experiments, an orthotopically implanted glioma mouse model was used to validate tumor growth., Results: The expression of TRIM37 is higher in GSCs compared with matched non-GSCs. TRIM37 knockdown promotes apoptosis, decreased stemness in GSCs, and reduces tumor growth in GSCs xenografts of nude mice. TRIM37 and EZH2 co-localize in the nucleus and interact with each other. TRIM37 knockdown or EZH2 inhibition downregulates the protein expressions associated with the Sonic Hedgehog (SHH) pathway. EZH2 epigenetically downregulates PTCH1 to activate SHH pathway in GSCs., Conclusions: TRIM37 maintains the cell growth and stemness in GSCs through the interaction with EZH2. EZH2 activates SHH stem cell signaling pathway by downregulating the expression of SHH pathway suppressor PTCH1. Our findings suggest that TRIM37 may be a potential therapeutic target for GBM., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Alterations to cilia are responsible for a wide range of severe disease; however, understanding of the transcriptional control of ciliogenesis remains incomplete. In this study we investigated whether altered cilia-mediated signaling contributes to the pleiotropic phenotypes caused by the Forkhead transcription factor FOXC1. Here, we show that patients with FOXC1-attributable Axenfeld-Rieger Syndrome (ARS) have a prevalence of ciliopathy-associated phenotypes comparable to syndromic ciliopathies. We demonstrate that altering the level of Foxc1 protein, via shRNA mediated inhibition, CRISPR/Cas9 mutagenesis and overexpression, modifies cilia length in vitro. These structural changes were associated with substantially perturbed cilia-dependent signaling [Hedgehog (Hh) and PDGFRα], and altered ciliary compartmentalization of the Hh pathway transcription factor, Gli2. Consistent with these data, in primary cultures of murine embryonic meninges, cilia length was significantly reduced in heterozygous and homozygous Foxc1 mutants compared to controls. Meningeal expression of the core Hh signaling components Gli1, Gli3 and Sufu was dysregulated, with comparable dysregulation of Pdgfrα signaling evident from significantly altered Pdgfrα and phosphorylated Pdgfrα expression. On the basis of these clinical and experimental findings, we propose a model that altered cilia-mediated signaling contributes to some FOXC1-induced phenotypes., (© 2024. The Author(s).)

Medulloblastoma (MB) is the most common malignant brain tumor in children and is stratified into three major subgroups. The Sonic hedgehog (SHH) subgroup represents ∼30% of all MB cases and has significant survival disparity depending upon TP53 status. Here, we describe a zebrafish model of SHH MB using CRISPR to create mutant ptch1, the primary genetic driver of human SHH MB. In these animals, tumors rapidly arise in the cerebellum and resemble human SHH MB by histology and comparative onco-genomics. Similar to human patients, MB tumors with loss of both ptch1 and tp53 have aggressive tumor histology and significantly worse survival outcomes. The simplicity and scalability of the ptch1-crispant MB model makes it highly amenable to CRISPR-based genome-editing screens to identify genes required for SHH MB tumor formation in vivo, and here we identify the gene encoding Grk3 kinase as one such target., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mTORC1-complex is negatively regulated by TSC1 and TSC2. Activation of Hedgehog signaling is strictly dependent on communication between Smoothened and the Hedgehog-signaling effector and transcription factor, GLI2, in the primary cilium. Details about this communication are not known, and we wanted to explore this further. Here we report that in Tsc2 -/- MEFs constitutively activated mTORC1 led to mis-localization of Smoothened to the plasma membrane, combined with increased concentration of GLI2 in the cilia and reduced Hedgehog signaling, measured by reduced expression of the Hedgehog target gene, Gli1 Inhibition of mTORC1 rescued the cellular localization of Smoothened to the cilia, reduced the cilia concentration of GLI2, and restored Hedgehog signaling. Our results reveal evidence for a two-step activation process of GLI2. The first step includes GLI2 stabilization and cilium localization, whereas the second step includes communication with cilia-localized Smoothened. We found that mTORC1 inhibits the second step. This is the first demonstration that mTORC1 is involved in the regulation of Hedgehog signaling., (© 2024 Larsen et al.)

Liver cancers, including hepatocellular carcinoma (HCC), are the sixth most common cancer and the third leading cause of cancer-related death worldwide, representing a global public health problem. This study evaluated nine patients with HCC. Six of the cases involved hepatic explants, and three involved hepatic segmentectomy for tumor resection. Eight out of nine tumors were HCC, with one being a combined hepatocellular-cholangiocarcinoma tumor. Conventional markers of hepatocellular differentiation (Hep Par-1, arginase, pCEA, and glutamine synthetase) were positive in all patients, while markers of hepatic precursor cells (CK19, CK7, EpCAM, and CD56) were negative in most patients, and when positive, they were detected in small, isolated foci. Based on in silico analysis of HCC tumors from The Cancer Genome Atlas database, we found that Hedgehog (HH) pathway components (GLI1, GLI2, GLI3 and GAS1) have high connectivity values (module membership > 0.7) and are strongly correlated with each other and with other genes in biologically relevant modules for HCC. We further validated this finding by analyzing the gene expression of HH components (PTCH1, GLI1, GLI2 and GLI3) in our samples through qPCR, as well as by immunohistochemical analysis. Additionally, we conducted a chemosensitivity analysis using primary HCC cultures treated with a panel of 18 drugs that affect the HH pathway and/or HCC. Most HCC samples were sensitive to sunitinib. Our results offer a comprehensive view of the molecular landscape of HCC, highlighting the significance of the HH pathway and providing insight into focused treatments for HCC., (© 2024. The Author(s).)

During development, unique combinations of transcription factors and signaling pathways carve the nascent eye-antennal disc of the fruit fly Drosophila melanogaster into several territories that will eventually develop into the compound eye, ocelli, head epidermis, bristles, antenna and maxillary palpus of the adult head. Juxtaposed patterns of Hedgehog (Hh) and Decapentaplegic (Dpp) initiate compound eye development, while reciprocal domains of Dpp and Wingless (Wg) induce formation of the antennal and maxillary palp fields. Hh and Wg signaling, but not Dpp, contribute to the patterning of the dorsal head vertex. Here, we show that combinatorial reductions of the Pax6 transcription factor Twin of Eyeless and either the Wg pathway or the Mirror (Mirr) transcription factor trigger a transformation of the ocelli into a compound eye and the neighboring head epidermis into an antenna. These changes in fate are accompanied by the ectopic expression of Dpp, which might be expected to trigger these changes in fate. However, the transformation of the field cannot be replicated by increasing Dpp levels alone despite the recreation of adjacent Hh-Dpp and Wg-Dpp domains. As such, the emergence of these ectopic organs occurs through a unique regulatory path., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional-targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the insulin-like growth factor (IGF) pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers. Significance: Dissecting transcriptional dependencies driven by super-enhancers uncovers therapeutic targets in Hedgehog-driven cancers and identifies strategies for overcoming resistance to smoothened inhibitors., (©2024 American Association for Cancer Research.)

Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs., (© 2024. The Author(s).)

In Caenorhabditis elegans, expanded families of divergent Hedgehog-related and patched-related proteins promote numerous processes ranging from epithelial and sense organ development to pathogen responses to cuticle shedding during the molt cycle. The molecular functions of these proteins have been mysterious since nematodes lack a canonical Hedgehog signaling pathway. Here we show that Hedgehog-related proteins are components of the cuticle and precuticle apical extracellular matrices that coat, shape, and protect external epithelia. Of four Hedgehog-related proteins imaged, two (GRL-2 and GRL-18) stably associated with the cuticles of specific tubes and two (GRL-7 and WRT-10) labeled precuticle substructures such as furrows or alae. We found that wrt-10 mutations disrupt cuticle alae ridges, consistent with a structural role in matrix organization. We hypothesize that most nematode Hedgehog-related proteins are apical extracellular matrix components, a model that could explain many of the reported functions for this family. These results highlight ancient connections between Hedgehog proteins and the extracellular matrix and suggest that any signaling roles of C. elegans Hedgehog-related proteins will be intimately related to their matrix association., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning" floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2 + clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering., Competing Interests: Declaration of interests A.H. and R.A.N. are full-time employees of Boehringer Ingelheim RCV., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Aims: Current evidence suggests that there is no completely effective method for endometriosis (EMS) without trauma due to diverse adverse effects. Reliable evidence illustrates that inhibiting ferroptosis is a potential strategy for EMS. We sufficiently verified that the expression of endogenous protein PDZ and LIM domain 3 (PDLIM3) was significantly increased in EMS., Methods: PDLIM3 knockdown reduced primary ectopic endometrial stromal cells' (EESCs) viability and migration, and elevated ferroptosis signaling indicators including Fe 2+ , malondialdehyde (MDA), and reactive oxygen species (ROS) in EESCs., Results: Mechanistic studies revealed that inhibition of PDLIM3 accelerated glioma-associated oncogene-1 (Gli1) degradation and further deactivated Hedgehog signaling. Gli1 inhibitor, GANT61, abrogated the impact of PDLIM3 deletion on EESC growth, migration, and ferroptosis. In vivo experiments suggested that PDLIM3 reduction repressed the growth of endometrial lesions. Likewise, repression of PDLIM3 promoted ferroptosis and attenuated Hedgehog signaling in endometrial lesions., Conclusions: Collectively, silencing of PDLIM3 facilitates ferroptosis in EMS by inducing Gli1 degradation and blocking Hedgehog signaling. It may provide an alternative strategy for developing therapeutic agents of EMS in the future., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Sonic hedgehog signaling molecule (SHH) is a key molecule in the cilia-mediated signaling pathway and a critical morphogen in embryogenesis. The association between loss-of-function variants of SHH and holoprosencephaly is well established. In mice experiments, reduced or increased signaling of SHH have been shown to be associated with narrowing or excessive expansion of the facial midline, respectively. Herein, we report two unrelated patients with de novo truncating variants of SHH presenting with hypertelorism rather than hypotelorism. The first patient was a 13-year-old girl. Her facial features included hypertelorism, strabismus, telecanthus, malocclusion, frontal bossing, and wide widow's peak. She had borderline developmental delay and agenesis of the corpus callosum. She had a nonsense variant of SHH: Chr7(GRCh38):g.155802987C > T, NM_000193.4:c.1302G > A, p.(Trp434*). The second patient was a 25-year-old girl. Her facial features included hypertelorism and wide widow's peak. She had developmental delay and agenesis of the corpus callosum. She had a frameshift variant of SHH: Chr7(GRCh38):g.155803072_155803074delCGGinsT, NM_000193.4:c.1215_1217delCCGinsA, p.(Asp405Glufs*92). The hypertelorism phenotype contrasts sharply with the prototypical hypotelorism-holoprosencephaly phenotype associated with loss-of-function of SHH. We concluded that a subset of truncating variants of SHH could be associated with hypertelorism rather than hypotelorism., (© 2024 Wiley Periodicals LLC.)

Purpose: The hyperactivation of epidermal growth factor receptor (EGFR) plays a crucial role in non-small cell lung cancer (NSCLC). Hedgehog (Hh) signaling has been implicated in the tumorigenesis and progression of various cancers, however, its function in NSCLC cells remains controversial. Herein, we present a novel finding that challenges the current understanding of Hh signaling in tumor growth., Methods: Expression of Hh ligands and receptor were assessed using TCGA datasets, immunoblotting and immunohistochemical. Biological function of Hh ligands and receptor in NSCLC were tested using colony formation, cell count kit-8 (CCK-8) and xenograft assays. Biochemical effect of Hh ligands and receptor on regulating EGFR stability and activity were checked via immunoblotting., Results: Expression of Hh ligands and receptor was suppressed in NSCLC tissues, and the lower expression levels of these genes were associated with poor prognosis. Ptch1 binds to EGFR and facilitates its poly-ubiquitylation and degradation independent of downstream transcriptional signaling. Moreover, Hh ligands cooperate with Ptch1 to regulate the protein stability and activity of EGFR. This unique mechanism leads to a suppressive effect on NSCLC tumor growth., Conclusion: Non-canonical Hh signaling pathway, involving cooperation between Hh ligands and their receptor Ptch1, facilitates the degradation of EGFR and attenuates its activity in NSCLC. These findings provide novel insights into the regulation of EGFR protein stability and activity, offer new diagnostic indicators for molecular typing of NSCLC and identify potential targets for targeted therapy of this challenging disease., (© 2024. Springer Nature Switzerland AG.)

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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Given the extensive application of dexamethasone in both clinical settings and the livestock industry, human exposure to this drug can occur through various sources and pathways. Prior research has indicated that prenatal exposure to dexamethasone (PDE) heightens the risk of cognitive and emotional disorders in offspring. Axonal development impairment is a frequent pathological underpinning for neuronal dysfunction in these disorders, yet it remains unclear if it plays a role in the neural damage induced by PDE in the offspring. Through RNA-seq and bioinformatics analysis, we found that various signaling pathways related to nervous system development, including axonal development, were altered in the hippocampus of PDE offspring. Among them, the Sonic Hedgehog (SHH) signaling pathway was the most significantly altered and crucial for axonal development. By using miRNA-seq and targeting miRNAs and glucocorticoid receptor (GR) expression, we identified miR-210-3p and miR-362-5p, which can target and suppress SHH expression. Their abnormal high expression was associated with GR activation in PDE fetal rats. Further testing of PDE offspring rats and infant peripheral blood samples exposed to dexamethasone in utero showed that SHH expression was significantly decreased in peripheral blood mononuclear cells (PBMCs) and was positively correlated with SHH expression in the hippocampus and the expression of the axonal development marker growth-associated protein-43. In summary, PDE-induced hippocampal GR-miR-210-3p/miR-362-5p-SHH signaling axis changes lead to axonal developmental damage. SHH expression in PBMCs may reflect axonal developmental damage in PDE offspring and could serve as a warning marker for fetal axonal developmental damage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here, we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's vesicle (the teleost left-right organizer) is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist Dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1 + cells in the cranial sutures play an important role in bone growth. However, whether Gli1 + cells in facial sutures differentiate into bone under distraction force is unknown., Methods: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1 + cell lineage tracing model was used to observe the distribution of Gli1 + cells and explore the role of Gli1 + cells in facial bone remodeling., Results: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1 + cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2 + cells. Hedgehog signaling was upregulated in Gli1 + cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1 + cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1 + cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1 + cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1 + cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force., Conclusions: Our study indicates that the primary cilia of Gli1 + cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1 + cells in zygomaticomaxillary sutures., (© 2024. The Author(s).)

Background: Heterozygous Indian Hedgehog gene (IHH) variants are associated with brachydactyly type A1 (BDA1). However, in recent years, numerous variants have been identified in patients with short stature and more variable forms of brachydactyly. Many are located in the C-terminal domain of IHH (IHH-C), which lacks signaling activity but is critical for auto-cleavage and activation of the N-terminal (IHH-N) peptide. The absence of functional studies of IHH variants, particularly for those located in IHH-C, has led to these variants being classified as variants of uncertain significance (VUS)., Objective: To establish a simple functional assay to determine the pathogenicity of IHH VUS and confirm that variants in the C-terminal domain affect protein function., Design/methods: In vitro studies were performed for 9 IHH heterozygous variants, to test their effect on secretion and IHH intracellular processing by western blot of cells expressing each variant., Results: IHH secretion was significantly reduced in all mutants, regardless of the location. Similarly, intracellular levels of N-terminal and C-terminal IHH peptides were severely reduced in comparison with the control. Two variants present at a relatively high frequency in the general population also reduced secretion but to a lesser degree in the heterozygous state., Conclusions: These studies provide the first evidence that variants in the C-terminal domain affect the secretion capacity of IHH and thus, reduce availability of IHH ligand, resulting in short stature and mild skeletal defects. The secretion assay permits a relatively easy test to determine the pathogenicity of IHH variants. All studied variants affected secretion and interestingly, more frequent population variants appear to have a deleterious effect and thus contribute to height variation., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Purpose: Microphthalmia is a rare developmental eye disease that affects 1 in 7000 births. Currently, there is no cure for this condition. This study aimed to construct a stable mouse model of microphthalmia, thus providing a new tool for the study of the etiology of microphthalmia., Methods: The Hedgehog signaling pathway plays a crucial role in eye development. One of the key mechanisms of the Sonic Hedgehog signaling is the strong transcriptional activation ability of GLI3, a major mediator of this pathway. This study used CRISPR/Cas9 system to construct a novel TgGli3Ki/Ki lens-specific over-expression mouse line. To identify the ocular characteristics of this line, quantitative PCR, Western blot, hematoxylin and eosin staining, immunofluorescent staining, and RNA-seq were performed on the ocular tissues of this line and normal mice., Results: The TgGli3Ki/Ki lens-specific over-expression mouse model exhibits the ocular phenotype of microphthalmia. In the TgGli3Ki/Ki mouse, Gli3 is over-expressed in the lens, and the size of the eyeball and lens is significantly smaller than the normal one. RNA-seq analysis using the lens and the retina samples from TgGli3Ki/Ki and normal mice indicates that the phototransduction pathway is ectopically activated in the lens. Immunofluorescent staining of the lens samples confirmed this activation., Conclusions: The TgGli3Ki/Ki mouse model consistently manifests the stereotypical microphthalmia phenotype across generations, making it an excellent tool for studying this severe eye disease., Translational Relevance: This study developed a novel animal model to facilitate clinical research on microphthalmia.

The functional maturation of the pituitary gland requires adequate cell differentiation and vascular network formation. Although spatiotemporal signaling and transcription factors are known to govern pituitary development, the involvement of primary cilia, nonmoving hair-like organelles, remains unclear. In this study, we uncovered the contribution of primary cilia to cell-type determination and vascular network formation during pituitary development. Homozygous knockout mice lacking a ciliary kinase, Dyrk2-/-, exhibit abnormalities in ciliary structure and pituitary hypoplasia, accompanied by varying degrees of failure in differentiation among all types of hormone-producing cells in the anterior lobe. Aberrations in cell differentiation in Dyrk2-/- mice arise from a decrease in the expression of crucial transcription factors, Lhx4, Lhx3, and Prop1, resulting from the inactivity of Hedgehog (Hh) signaling during the early stages of development. Furthermore, the loss of Dyrk2 results in vascular system abnormalities during the middle to late stages of development. Mechanistically, transcriptome analyses revealed the downregulation of vitronectin-integrin αvβ3-VEGFR2 signaling, essential for orchestrating vascular development. Collectively, our findings demonstrate that primary cilia play a pivotal role as critical regulators of cell survival, cell determination, and angiogenesis during pituitary gland development through the activation of Hh signaling. These findings expand our understanding of the potential link between pituitary dysfunction in human disorders and ciliopathies., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

The cellular and genetic networks that contribute to the development of the zeugopod (radius and ulna of the forearm, tibia and fibula of the leg) are not well understood, although these bones are susceptible to loss in congenital human syndromes and to the action of teratogens such as thalidomide. Using a new fate-mapping approach with the Chameleon transgenic chicken line, we show that there is a small contribution of SHH-expressing cells to the posterior ulna, posterior carpals and digit 3. We establish that although the majority of the ulna develops in response to paracrine SHH signalling in both the chicken and mouse, there are differences in the contribution of SHH-expressing cells between mouse and chicken as well as between the chicken ulna and fibula. This is evidence that, although zeugopod bones are clearly homologous according to the fossil record, the gene regulatory networks that contribute to their development and evolution are not fixed., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Depleted uranium (DU) retains the radiological toxicities, which accumulates preferentially in the kidneys. Hedgehog (Hh) pathway plays a critical role in tissue injury. However, the role of Hh in DU-induced nephrotoxicity was still unclear. This study was carried out to investigate the effect of Gli2, which was an important transcription effector of Hh signaling, on DU induced nephrotoxicity. To clarify it, CK19 positive tubular epithelial cells specific Gli2 conditional knockout (KO) mice model was exposed to DU, and then histopathological damage and Hh signaling pathway activation was analyzed. Moreover, HEK-293 T cells were exposed to DU with Gant61 or Gli2 overexpression, and cytotoxicity of DU as analyzed. Results showed that DU caused nephrotoxicity accompanied by activation of Hh signaling pathway. Meanwhile, genetic KO of Gli2 reduced DU-induced nephrotoxicity by normalizing biochemical indicators and reducing Hh pathway activation. Pharmacologic inhibition of Gli1/2 by Gant61 reduced DU induced cytotoxicity by inhibiting apoptosis, ROS formation and Hh pathway activation. However, overexpression of Gli2 aggravated DU-induced cytotoxicity by increasing the levels of apoptosis and ROS formation. Taken together, these results revealed that Hh signaling negatively regulated DU-inducted nephrotoxicity, and that inhibition of Gli2 might serve as a promising nephroprotective target for DU-induced kidney injury., (© 2024 Wiley Periodicals LLC.)

The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 μg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The thyroid gland is an important endocrine gland in animals, which mainly secretes thyroid hormones and acts on various organs of the body. Long-chain non-coding RNA (lncRNA) plays an important role in animal reproduction. However, there is still a lack of understanding of their expression patterns and potential roles in the thyroid of Small Tail Han (STH) sheep. In this study, RNA-seq was used to examine the transcriptome expression patterns of lncRNAs and mRNAs in the follicular phase (ww_FT) and luteal phase (ww_LT) in FecB ++ genotype STH Sheep. A total of 17,217 lncRNAs and 39,112 mRNAs were identified including 96 differentially expressed lncRNAs (DELs) and 1054 differentially expressed mRNAs (DEGs). Functional analysis of genes with significant differences in expression level showed that these genes could be enriched in Ras signalling pathway, hedgehog (HH) signalling pathway, ATP-binding cassette (ABC) transporters and other signalling pathways related to animal reproduction. In addition, through correlation analysis for lncRNA-mRNA co-expression and network construction, we found that LNC_009115 and LNC_005796 trans target NIK-related kinase ( NRK ) and poly(A)-specific ribonuclease ( PARN ). LNC_007189 and LNC_002045 trans target progesterone-induced blocking factor 1 ( PIBF1 ), LNC_009013 trans targets small mothers against decapentaplegic ( SMAD1 ) are related to animal reproduction. These genes add new resources for elucidating the regulatory mechanisms of reproduction in sheep with different reproductive cycles of the FecB ++ genotype STH sheep.

Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While granule cell cilia are essential during early developmental stages, they become infrequent upon maturation. Here, we provide nanoscopic resolution of cilia in situ using large-scale electron microscopy volumes and immunostaining of mouse cerebella. In many granule cells, we found intracellular cilia, concealed from the external environment. Cilia were disassembled in differentiating granule cell neurons-in a process we call cilia deconstruction-distinct from premitotic cilia resorption in proliferating progenitors. In differentiating granule cells, cilia deconstruction involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Unlike ciliated neurons in other brain regions, our results show the deconstruction of concealed cilia in differentiating granule cells, which might prevent mitogenic hedgehog responsiveness. Ciliary deconstruction could be paradigmatic of cilia removal during differentiation in other tissues., (© 2024 Ott et al.)

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice ( Osr2-cre KI ; Rosa26R-Fgf8 ) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-cre KI ; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-cre KI ; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-cre KI ; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin , Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-cre KI ; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-cre KI ; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.

The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we show that Spinal Cord Organoids (SCOs) can be applied to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. We validate this system by measuring the effects of mutating the HH receptor PTCH1 and the impact of HH agonists and antagonists on NPC specification. SCOs represent an accessible and reliable in-vitro tool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Holzner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Non-obese diabetes (NOD) mice are an established, spontaneous model of type 1 diabetes in which diabetes develops through insulitis. Using next-generation sequencing, coupled with pathway analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from 4 to 12 weeks of age. The resulting dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6 and 8 weeks that increased to a regulatory plateau between 10 and 12 weeks. The inflammatory signature is identified by the activation of central immunogenic factors such as Infg, Il1b, and Tnfa, and activation of canonical inflammatory signaling. Analysis of the regulatory landscape revealed the transcription factor Atf3 as a potential novel modulator of inflammatory signaling in the NOD islets. Furthermore, the Hedgehog signaling pathway correlated with Atf3 regulation, suggesting that the two play a role in regulating islet inflammation; however, further studies are needed to establish the nature of this connection., (© 2024. The Author(s).)

This study examined the risk factors for short-term outcomes, focusing particularly on the associations among molecular subgroups. The analysis focused on the data of pediatric patients with medulloblastoma between 2013 and 2023, as well as operative complications, length of stay from surgery to adjuvant treatment, 30-day unplanned reoperation, unplanned readmission, and mortality. 148 patients were included. Patients with the SHH TP53-wildtype exhibited a lower incidence of complications (45.2% vs. 66.0%, odds ratio [OR] 0.358, 95% confidence interval [CI] 0.160 - 0.802). Female sex (0.437, 0.207 - 0.919) was identified as an independent protective factor for complications, and brainstem involvement (1.900, 1.297 - 2.784) was identified as a risk factor. Surgical time was associated with an increased risk of complications (1.004, 1.001 - 1.008), duration of hospitalization (1.006, 1.003 - 1.010), and reoperation (1.003, 1.001 - 1.006). Age was found to be a predictor of improved outcomes, as each additional year was associated with a 14.1% decrease in the likelihood of experiencing a prolonged length of stay (0.859, 0.772 - 0.956). Patients without metastasis exhibited a reduced risk of reoperation (0.322, 0.133 - 0.784) and readmission (0.208, 0.074 - 0.581). There is a significant degree of variability in the occurrence of operative complications in pediatric patients with medulloblastoma. SHH TP53-wildtype medulloblastoma is commonly correlated with a decreased incidence of complications. The short-term outcomes of patients are influenced by various unmodifiable endogenous factors. These findings could enhance the knowledge of onconeurosurgeons and alleviate the challenges associated with patient/parent education through personalized risk communication. However, the importance of a dedicated center with expertise surgical team and experienced neurosurgeon in improving neurosurgical outcomes appears self-evident., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, GLI1 , and PTCH1 expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the GLI1 and PTCH1 gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.

In the mammalian auditory system, frequency discrimination depends on numerous morphological and physiological properties of the organ of Corti, which gradually change along the apex-to-base (tonotopic) axis of the organ. For example, the basilar membrane stiffness changes tonotopically, thus affecting the tuning properties of individual hair cells. At the molecular level, those frequency-specific characteristics are mirrored by gene expression gradients; however, the molecular mechanisms controlling tonotopic gene expression in the mouse cochlea remain elusive. Through analyzing single-cell RNA sequencing (scRNA-seq) data from E12.5 and E14.5 time points, we predicted that morphogens, rather than a cell division-associated mechanism, confer spatial identity in the extending cochlea. Subsequently, we reconstructed the developing cochlea in 3D space from scRNA-seq data to investigate the molecular pathways mediating positional information. The retinoic acid (RA) and hedgehog pathways were found to form opposing apex-to-base gradients, and functional interrogation using mouse cochlear explants suggested that both pathways jointly specify the longitudinal axis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Sonic Hedgehog (SHH) is a fundamental signaling pathway that controls tissue reconstruction, stem cell biology, and differentiation and has a role in gut tissue homeostasis and development. Dysregulation of SHH leads to the development of HCC., Methods, and Results: The present study was conducted to compare the effects of mesenchymal stem cells (MSCs) and curcumin on SHH molecular targets in an experimental model of HCC in rats. One hundred rats were divided equally into the following groups: control group, HCC group, HCC group received MSCs, HCC group received curcumin, and HCC group received MSCs and curcumin. Histopathological examinations were performed, and gene expression of SHH signaling target genes (SHH, PTCH1, SMOH, and GLI1) was assessed by real-time PCR in rat liver tissue. Results showed that SHH target genes were significantly upregulated in HCC-untreated rat groups and in MSC-treated groups, with no significant difference between them. Administration of curcumin with or without combined administration of MSCs led to a significant down-regulation of SHH target genes, with no significant differences between both groups. As regards the histopathological examination of liver tissues, both curcumin and MSCs, either through separate use or their combined use, led to a significant restoration of normal liver pathology., Conclusions: In conclusion, SHH signaling is upregulated in the HCC experimental model. MSCs do not inhibit the upregulated SHH target genes in HCC. Curcumin use with or without MSCs administration led to a significant down-regulation of SHH signaling in HCC and a significant restoration of normal liver pathology., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)-a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein - a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas., (© 2024. The Author(s).)

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Du et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kumari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.

We describe a 46-year-old patient with an IDH-wildtype diffusely infiltrating atypical teratoid/rhabdoid tumour (AT/RT), SHH-1B molecular subtype. The unusual histology and subsequent diagnosis in an adult patient will be discussed., (© 2024 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)