Shh Expression Research Articles

Retracted: miR-135a Inhibits the Invasion and Migration of Esophageal Cancer Stem Cells through the Hedgehog Signaling Pathway by Targeting Smo

Cancer stem cells (CSCs) have been reported to be involved in esophageal cancer (EC) development. Hence, we aim to explore whether microRNA-135a (miR-135a) affects EC and its associated mechanism. Cancerous and adjacent tissues from 138 EC patients were collected. The dual-luciferase reporter gene assay and bioinformatics analysis were used to confirm the interaction between nucleotides. A series of mimics or inhibitors of miR-135a or small interfering RNA (siRNA) against Smo were introduced into EC cells. After that, the expression of miR-135a and Hedgehog (Hh) signaling pathway-related genes (Smo, Gli1, Shh, and Gli2) in tissues and cells was measured, accompanied by evaluation of cell viability, apoptosis, invasion, and migration. High expression of Smo, Gli1, Shh, and Gli2 and low expression of miR-135a were observed in EC. Smo was verified to be a target gene of miR-135a. In addition, overexpression of miR-135a or silencing of Smo decreased the expression of Gli1, Gli2, and Shh, thus inhibiting EC cell proliferation, migration, and invasion and promoting apoptosis. Silencing of miR-135a was observed to reverse the inhibitory role of miR-135a in EC. These results suggest that miR-135a inhibited the migration and invasion of EC cells through inhibition of the Smo/Hh axis.

SHH signaling mediated by a prechordal and brain enhancer controls forebrain organization

Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.

SOX2-induced upregulation of lncRNA LINC01510 promotes papillary thyroid carcinoma progression by modulating miR-335/SHH and activating Hedgehog pathway

LncRNA LINC01510 (LINC01510) was a newly identified tumor-related lncRNA whose dysregulation and potential function have been reported in several tumors. However, the expression, clinical significances, and action mechanisms of LINC01510 in papillary thyroid carcinoma (PTC) are still unclear. In this study, we firstly reported that LINC01510 was highly expressed in both PTC tissues and cell lines. Additionally, we used dual-luciferase reporter assay and confirmed that SOX2 could bind directly to the LINC01510 promoter region, activating its transcription. Functional assays with a series of cell experiments indicated that knockdown of LINC01510 suppressed the proliferation, migration and invasion of SW1736 and TPC-1 cells. Moreover, down-regulation of LINC01510 resulted in accelerated apoptosis by promoting the expression of Caspase3/9. In particular, LINC01510 acted as an endogenous sponge by directly binding miR-335, resulting in the suppression of miR-335 expressions. Besides, we confirmed that SHH was a target of miR-335 and miR-335 over-expression distinctly reduced SHH expression in PTC cells. Finally, in the cytoplasm, we provided evidenced that LINC01510 acted as a sponge for miR-335, reducing its ability to promote SHH expression. In addition, the results of Western blot indicated that knockdown of LINC01510 inhibited the expression of SHH and GLI1, suggesting that Hedgehog pathway was suppressed. Taken together, our findings revealed that the newly identified LINC01510/miR-335/SHH axis could be a therapeutic target for PTC.

Perturbed development of cranial neural crest cells in association with reduced sonic hedgehog signaling underlies the pathogenesis of retinoic-acid-induced cleft palate.

ABSTRACTCleft palate (CP) is one of the most common congenital craniofacial anomalies in humans and can be caused by either single or multiple genetic and environmental factor(s). With respect to environmental factors, excessive intake of vitamin A during early pregnancy is associated with increased incidence of CP in offspring both in humans and in animal models. Vitamin A is metabolized to retinoic acid (RA); however, the pathogenetic mechanism of CP caused by altered RA signaling during early embryogenesis is not fully understood. To investigate the detailed cellular and molecular mechanism of RA-induced CP, we administered all-trans RA to pregnant mice at embryonic day (E)8.5. In the RA-treated group, we observed altered expression of Sox10, which marks cranial neural crest cells (CNCCs). Disruption of Sox10 expression was also observed at E10.5 in the maxillary component of the first branchial arch, which gives rise to secondary palatal shelves. Moreover, we found significant elevation of CNCC apoptosis in RA-treated embryos. RNA-sequencing comparisons of RA-treated embryos compared to controls revealed alterations in Sonic hedgehog (Shh) signaling. More specifically, the expression of Shh and its downstream genes Ptch1 and Gli1 was spatiotemporally downregulated in the developing face of RA-treated embryos. Consistent with these findings, the incidence of CP in association with excessive RA signaling was reduced by administration of the Shh signaling agonist SAG (Smoothened agonist). Altogether, our results uncovered a novel mechanistic association between RA-induced CP with decreased Shh signaling and elevated CNCC apoptosis.

Mesenchyme-specific deletion of Tgf-β1 in the embryonic lung disrupts branching morphogenesis and induces lung hypoplasia

Proper lung development depends on the precise temporal and spatial expression of several morphogenic factors, including Fgf10, Fgf9, Shh, Bmp4, and Tgf-β. Over- or under-expression of these molecules often leads to aberrant embryonic or postnatal lung development. Herein, we deleted the Tgf-β1 gene specifically within the lung embryonic mesenchymal compartment at specific gestational stages to determine the contribution of this cytokine to lung development. Mutant embryos developed severe lung hypoplasia and died at birth due to the inability to breathe. Despite the markedly reduced lung size, proliferation and differentiation of the lung epithelium was not affected by the lack of mesenchymal expression of the Tgf-β1 gene, while apoptosis was significantly increased in the mutant lung parenchyma. Lack of mesenchymal expression of the Tgf-β1 gene was also associated with reduced lung branching morphogenesis, with accompanying inhibition of the local FGF10 signaling pathway as well as abnormal development of the vascular system. To shed light on the mechanism of lung hypoplasia, we quantified the phosphorylation of 226 proteins in the mutant E12.5 lung compared with control. We identified five proteins, Hrs, Vav2, c-Kit, the regulatory subunit of Pi3k (P85), and Fgfr1, that were over- or under-phosphorylated in the mutant lung, suggesting that they could be indispensable effectors of the TGF-β signaling program during embryonic lung development. In conclusion, we have uncovered novel roles of the mesenchyme-specific Tgf-β1 ligand in embryonic mouse lung development and generated a mouse model that may prove helpful to identify some of the key pathogenic mechanisms underlying lung hypoplasia in humans.

Potential role of Hedgehog signaling pathway and myofibroblastic differentiation in central giant cell granuloma-A preliminary study.

This study investigated components of the Hedgehog (HH) signaling pathway (SHH, GLI1), cyclin D1, and smooth muscle actin (SMA) in central giant cell granulomas (CGCG). The relationship between these proteins and myofibroblasts was also studied. Twelve cases of non-aggressive CGCG and 11 cases of aggressive CGCG were studied using immunohistochemistry for SHH, GLI1, Cyclin D1, and SMA. Associations between all proteins in non-aggressive and aggressive CGCG were not significant (P>.05). All cases of CGCG showed significantly higher expression of SMA compared with the other proteins (P<.01). A positive correlation (P=.04) was only observed between SHH and GLI1 for all cases of CGCG. Furthermore, a positive correlation between SHH and GLI1 in non-aggressive CGCG (P=.04) and between GLI1 and cyclin D1 in aggressive CGCG (P=.03) were observed. There was also a negative correlation between the expression of SHH and SMA in non-aggressive CGCG (P=.031). This study provided insights into the activation of the HH signaling pathway in CGCG. In addition, the activation of this pathway (SHH and GLI1) might play some role in the differentiation of stromal myofibroblasts, although these markers including Cyclin D1 and SMA do not indicate aggressiveness of the CGCG. Furthermore, this myofibroblastic differentiation process would occur at the expense of maturation of these lesions.

MicroRNA-124 Enhances Dopamine Receptor Expression and Neuronal Proliferation in Mouse Models of Parkinson’s Disease via the Hedgehog Signaling Pathway by Targeting EDN2

Recent studies have elaborated on the concept that a number of microRNAs (miRNAs) have a potential effect on the pathogenesis and development of Parkinson’s disease (PD). PD is recognized as a common progressive bradykinetic disorder that results from the death of dopaminergic neurons in the substantia nigra. The purpose of this study was to explore whether microRNA-124 (miR-124) affected dopamine receptor (DAR) expression and neuronal proliferation and apoptosis in the 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse models of PD. The targeting relationship of miR-124 and EDN2 was confirmed through bioinformatic predictions and dual-luciferase reporter assay. The expression of miR-124 and EDN2 was altered to assess their effect on the expression of DAR in the substantia nigra and isolated neurons, as well as the neuronal proliferation and apoptosis rate. The obtained results implied that the treatments of miR-124 mimic and si-EDN2 resulted in elevated expressions of Glil, SHH, PTCH1, DAT, DRD1, and DRD2. However, these treatments facilitated neuronal proliferation and suppressed neuronal apoptosis, corresponding to reduced expression of caspase-3 and Bax, as well as increased levels of Bcl-2 expression. These results were discovered to be achieved through the activation of the Hedgehog signaling pathway. With this taken into account, our study demonstrated that miR-124 overexpression promoted DAR expression and neuronal proliferation and suppressed neuronal apoptosis by downregulating EDN2 via activating the Hedgehog signaling pathway.

GATA-6 transcriptionally inhibits Shh to repress cell proliferation and migration in lung squamous cell carcinoma.

GATA-6 is a transcription factor that participates in cell lineage differentiation and organogenesis in many tissue types. The abnormal expression of GATA-6 is associated with the development of diverse cancers. GATA-6 acts as an oncogene or tumor suppressor based on tumor origin. Here, we investigated the effects of GATA-6 on lung squamous cell carcinoma (LSCC). We found that GATA-6 was significantly reduced in LSCC tissues compared with the paired normal tissues. The overexpression of GATA-6 inhibited LSCC cell proliferation and migration. Importantly, a luciferase reporter assay and chromatin immunoprecipitation assay demonstrated that GATA-6 negatively regulated the expression of sonic hedgehog (Shh) by directly binding to its promoter region. Furthermore, N-Shh stimulation rescued the inhibition of LSCC cell proliferation and migration upon GATA-6 overexpression. Thus, GATA-6 inhibited the proliferation and migration of LSCC cells by transcriptionally inhibiting the expression of Shh, indicating that targeting GATA-6 may be a potential approach for LSCC therapy.

Developmental plasticity of epithelial stem cells in tooth and taste bud renewal

In Lake Malawi cichlids, each tooth is replaced in one-for-one fashion every ∼20 to 50 d, and taste buds (TBs) are continuously renewed as in mammals. These structures are colocalized in the fish mouth and throat, from the point of initiation through adulthood. Here, we found that replacement teeth (RT) share a continuous band of epithelium with adjacent TBs and that both organs coexpress stem cell factors in subsets of label-retaining cells. We used RNA-seq to characterize transcriptomes of RT germs and TB-bearing oral epithelium. Analysis revealed differential usage of developmental pathways in RT compared to TB oral epithelia, as well as a repertoire of genome paralogues expressed complimentarily in each organ. Notably, BMP ligands were expressed in RT but excluded from TBs. Morphant fishes bathed in a BMP chemical antagonist exhibited RT with abrogated shh expression in the inner dental epithelium (IDE) and ectopic expression of calb2 (a TB marker) in these very cells. In the mouse, teeth are located on the jaw margin while TBs and other oral papillae are located on the tongue. Previous study reported that tongue intermolar eminence (IE) oral papillae of Follistatin (a BMP antagonist) mouse mutants exhibited dysmorphic invagination. We used these mutants to demonstrate altered transcriptomes and ectopic expression of dental markers in tongue IE. Our results suggest that vertebrate oral epithelium retains inherent plasticity to form tooth and taste-like cell types, mediated by BMP specification of progenitor cells. These findings indicate underappreciated epithelial cell populations with promising potential in bioengineering and dental therapeutics.

Lentiviral‑mediated Shh reverses the adverse effects of high glucose on osteoblast function and promotes bone formation via Sonic hedgehog signaling.

Patients with diabetes tend to have an increased incidence of osteoporosis, which may be associated with hyperglycemia; however, the pathogenic mechanisms governing this interaction remain unknown. The present study sought to investigate whether elevated extracellular glucose levels of bone mesenchymal stem cells (BMSCs) could influence osteoblastic differentiation and whether the intracellular Sonic hedgehog (Shh) pathway could adjust the effects. Furthermore, to verify the results in vivo, a rat tooth extraction model was constructed. BMSCs were incubated in eight types of culture medium, including low glucose (LG), LG + lentivirus (Lenti), LG + Lenti-small interfering RNA (Lenti-siRNA), LG + Lenti-Shh, high glucose (HG), HG + Lenti, HG + Lenti-siRNA and HG + Lenti-Shh. The lentiviral transfection efficiency was observed using a fluorescence microscope; protein and mRNA expression was detected by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The matrix mineralization and alkaline phosphatase (ALP) activity of BMSCs were examined by Alizarin red staining and ALP activity assays, respectively. The expression of osteogenesis-related genes in BMSCs were quantified by RT-qPCR. The alveolar ridge reduction was measured and histological sections were used to evaluate new bone formation in the tooth socket. With high concentrations of glucose, Shh expression, matrix mineralization nodules formation, ALP activity and the levels of bone morphogenic protein 4 (BMP4), bone sialoprotein (BSP) and osteopontin (OPN) expression were greatly reduced compared with LG and corresponding control groups. Whereas activated Shh signaling via Lenti-Shh could increase the number of matrix mineralization nodules, ALP activity, and the expression levels of BMP4, BSP and OPN in BMSCs. Additionally, in vivo assays demonstrated that Lenti-Shh induced additional bone formation. Collectively, the results of the present study indicated that HG inhibited the Shh pathway in osteoblasts and resulted in patterning defects during osteoblastic differentiation and bone formation, while the activation of Shh signaling could suppress these deleterious effects.

Sonic Hedgehog signaling limits atopic dermatitis via Gli2-driven immune regulation.

Hedgehog (Hh) proteins regulate development and tissue homeostasis, but their role in atopic dermatitis (AD) remains unknown. We found that on induction of mouse AD, Sonic Hedgehog (Shh) expression in skin and Hh pathway action in skin T cells were increased. Shh signaling reduced AD pathology and the levels of Shh expression determined disease severity. Hh-mediated transcription in skin T cells in AD-induced mice increased Treg populations and their suppressive function through increased active transforming growth factor–β (TGF-β) in Treg signaling to skin T effector populations to reduce disease progression and pathology. RNA sequencing of skin CD4+ T cells from AD-induced mice demonstrated that Hh signaling increased expression of immunoregulatory genes and reduced expression of inflammatory and chemokine genes. Addition of recombinant Shh to cultures of naive human CD4+ T cells in iTreg culture conditions increased FOXP3 expression. Our findings establish an important role for Shh upregulation in preventing AD, by increased Gli-driven, Treg cell–mediated immune suppression, paving the way for a potential new therapeutic strategy.

Abstract 4679: NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. The long-term prognosis of HCC remains unsatisfactory due to high recurrence rates and chemoresistance. Solid evidence shows that tumor-initiating cells (T-ICs) are the root of tumor relapse and drug resistance, which led to poor prognosis of patients with hepatocellular carcinoma (HCC). Therefore, identification of crucial transcriptional regulator in liver T-IC may improve the prognosis of these patients. Through in vitro liver T-IC enrichment approach, we identified Nuclear Factor (Erythroid-Derived 2)-Like 2 (NRF2) as transcriptions regulator be significantly upregulated in enriched liver T-IC populations. NRF2 regulated liver T-IC properties including self-renewal, tumorigenicity, and liver T-IC marker expression and significantly affected patients’ clinical outcome. Furthermore, NRF2 repression led to sensitization to doxorubicin and 5-FU treatment. Furthermore, we found that ROS-induced NRF2 activation regulates sorafenib resistance of HCC cells. Mechanistically, NRF2 was found to physically bind to promoter SHH, which triggers activation of sonic hedgehog pathway. NRF2 was significantly correlated with SHH expression, which was associated with poor patient’s survival. The effect of NRF2 knockdown was eliminated upon administration of recombinant SHH, demonstrating that NRF2 mediated the TIC function via upregulation of SHH expression. Our study suggested a novel regulatory mechanism for the canonical sonic hedgehog pathway that may function through NRF2/SHH/GLI signaling axis, thus mediating T-IC phenotypes. Targeting NRF2 mediated signaling cascade alone or in combination with other treatment modalities may be a new a potential therapeutic approach for treatment of HCC. Citation Format: Hoi Wing Leung, Eunice Yuen Ting Lau, Kin Wah Lee. NRF2/SHH signaling cascade promotes tumor-initiating cell lineage and drug resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4679.

Neuroprotective effects of a Smoothened receptor agonist against postoperative cognitive dysfunction by promoting autophagy in the dentate gyrus of aged rats

ABSTRACTObjectives: To investigate the effect of purmorphamine (PUR), a Shh co-receptor Smoothened (Smo) agonist, on postoperative cognitive dysfunction (POCD) rat models.Methods: Eighteen-month-old male Sprague–Dawley rats were subjected to intramedullary fixation of a tibial fracture with 7% chloral hydrate anesthesia to mimic human clinical surgery. PUR was administered via an intraperitoneal injection at a dose of 15mg/kg/day for 3 consecutive days at 6 h after surgery. The aged rats were sacrificed after performing a Morris water maze test 1, 3, and 7 days postoperatively to evaluate the expression of related proteins at the appointed time.Results: Compared to the POCD + vehicle group and sham + PUR group, the POCD + PUR group restored neurological deficit (P = 0.01). PUR administration induced upregulation of Shh expression on postoperative day 1 (P = 0.02), which continued on the third day (P = 0.008) but dropped by the 7th day (P = 0.03). Immunofluorescent analysis, similar to western blot analysis, showed a significant increase in the autophagy-marker LC3 (P = 0.006) as well as p62 degradation (P = 0.000) in the dentate gyrus of the aged rats (P = 0.000) after PUR treatment. Importantly, LC3 was mainly found in the presynaptic and postsynaptic membranes of the hippocampus.Conclusions: These results indicate a link between Shh and autophagy in the rat model of POCD, providing new insights into Shh signaling pathway-mediated mechanisms of neuroprotection and cognitive repair after POCD. It also provides a potential entry point for the development of clinical drugs.

Sinusoidal and pericellular fibrosis in adult post-transplant liver biopsies: association with hepatic stellate cell activation and patient outcome

Post-transplant sinusoidal fibrosis (SF) and pericellular fibrosis (PCF) have not been extensively investigated in adults. Fifty-two post-transplant liver biopsies from 28 consented patients (12 men, mean age 49, range 33–67 years) were studied. Tissue morphology, including an arbitrary summative fibrosis score was assessed in detail. Collagen proportionate area (CPA) and alpha-smooth muscle actin (α-SMA) immunostain were evaluated by digital image analysis (DIA). Anti-keratin 7, anti-C4d and anti-sonic hedgehog (Shh) immunostains were scored semi-quantitatively. SF was observed in 36/52 (69.2%) biopsies and most of these (20/36, 55.6%) had centrilobular fibrosis (CLF). PCF was seen in 7/52 (13.5%) biopsies exclusively in cases with CLF. CPA was significantly correlated with time since liver transplantation (p = 0.043), summative fibrosis score and its main components but not with α-SMA. α-SMA-positive area significantly correlated with the Banff rejection score (p = 0.022) and centrilobular inflammatory changes were more severe in cases with CLF (p = 0.003). Hepatocyte ballooning of cholestatic type was associated with PCF (p = 0.016) and Shh expression (p < 0.001). Sinusoidal fibrosis is a frequent occurrence in post-transplant adult livers, with predilection toward centrilobular areas. Graft age and oxidative stress may contribute to SF development, while hepatocyte ballooning may be implicated in PCF development. Hepatic stellate cell (HSC) activation is likely affected by centrilobular inflammation.

Widespread Expression of Sonic Hedgehog (Shh) and Nrf2 in Patient Treated with Cisplatin Predicts Outcome in Resected Tumors and Potential Therapeutic Target for Head and Neck Cancer

Background: Sonic hedgehog (Shh) and Nrf2 play an immense role in chemotherapeutic resistance. These genes have been found to be deregulated in head and neck squamous cell carcinoma (HNSCC). The purpose of this study is to analyze the expression, function and clinical prognostic relationship of Shh and Nrf2 in HNSCC in the context of therapeutic resistance and cancer stem cells (CSCs). Methods: We analyzed a cohort of HNSCC patients to identify potential candidate therapeutic biomarkers correlating with overall survival (OS) and disease-free survival (DFS) from our own data and validated the results using data from TCGA. Expression of Shh and Nrf2 was silenced by siRNA and cell growth, sphere growth and chemotherapeutic resistance were evaluated. Findings: Widespread abundant expression of Shh and Nrf2 protein was associated with shorter OS and DFS. The combination of Shh and Nrf2 expression levels was found to be a mostly significant predictor of patient DFS. The tumor stromal index (SI) was correlated with Shh expression and inversely associated with shorter DFS. Inhibition of Shh by siRNA or cyclopamine resulted in the attenuation of resistant CSC self-renewal, invasion, clonogenic growth and re-sensitization to chemotherapeutic agents. Interpretation: Concomitant high expression of Shh and Nrf2 proved to be an independent predictor of poor OS and DFS in HNSCC patients. These findings suggest that Shh and Nrf2 might serve as therapeutic targets as well as promising dual prognostic therapeutic biomarkers for HNSCC. Funding Statement: Grant-in-Aid from Bangladesh Medical Research Council (BMRC), and University Grant Commission (UGC), Ministry of Education of Bangladesh with the assistance of the World Bank under Higher Education Quality Enhancement Project (HEQEP)– Window -4 (Grant Id. CP4023). Declaration of Interests: The authors declare that they have no conflict of interests. Ethics Approval Statement: This study was approved by the ethics committee of the University of Chittagong and Chittagong Medical Hospital, Chittagong, Bangladesh, and was performed in accordance with the ethical standards laid down in the 1964 declaration of Helsinki and all subsequent revisions. Written informed consent was obtained from all patients used in this study.

Early taste buds are from Shh+ epithelial cells of tongue primordium in distinction from mature taste bud cells which arise from surrounding tissue compartments

Mammalian taste buds emerge perinatally and most become mature 3–4 weeks after birth. Mature taste bud cells in rodents are known to be renewed by the surrounding K14+ basal epithelial cells and potentially other progenitor source(s), but the dynamics between initially developed taste buds and surrounding tissue compartments are unclear. Using the K14-Cre and Dermo1-Cre mouse lines to trace epithelial and mesenchymal cell lineages, we found that early taste buds in E18.5 and newborn mouse tongues are not derived from either lineage. At E11.5 when the tongue primordia (i.e., lingual swellings) emerge, the relatively homogeneous sonic hedgehog-expressing (Shh+) epithelial cells express Keratin (K) 8, a marker that is widely used to label taste buds. Mapping lineage of E11.0 Shh+ epithelium of the tongue rudiment with Shh-CreERT2/RFP mice demonstrated that both the early taste buds and the surrounding lingual epithelium are from the same population of progenitors – Shh+ epithelial cells of the tongue primordium. In combination with previous reports, we propose that Shh+K8+ cells in the homogeneous epithelium of tongue primordium at early embryonic stages are programmed to become taste papilla and taste bud cells. Switching off Shh and K8 expression in the Shh+ epithelial cells of the tongue primordium transforms the cells to non-gustatory cells surrounding papillae, including K14+ basal epithelial cells which will eventually contribute to the cell renewal of mature taste buds.

Sonic Hedgehog Ligand: A Role in Formation of a Mesenchymal Niche in Human Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterised by desmoplasia, thought to support progression and chemotherapeutic resistance. The Hedgehog pathway is known to play an important role in this cancer. While the upregulation of Sonic hedgehog (Shh) in the epithelium of PDAC is known, we investigated its expression in the tumour microenvironment in order to find new targets for new chemotherapeutical approaches. Immunohistochemistry was used for the investigation of Shh and Vimentin in primary human pancreatic tissues. Gene (qRT-PCR) and protein (immunofluorescence) expression of Shh, αSMA (a marker of the mesenchymal phenotype) and periostin (a marker of mesenchymal cells within a mixed population) were investigated in in vitro cell models. Shh expression was significantly upregulated in the stromal and epithelial compartments of poorly-differentiated PDAC samples, with a strong correlation with the amount of stroma present. Characterisation of stromal cells showed that there was expression of Shh ligand in a mixed population comprising αSMA+ myofibroblasts and αSMA− mesenchymal stem cells. Moreover, we demonstrated the interaction between these cell lines by showing a higher rate of mesenchymal cell proliferation and the upregulation of periostin. Therefore, targeting stromal Shh could affect the equilibrium of the tumour microenvironment and its contribution to tumour growth.

Epigenetic regulation of mammalian Hedgehog signaling to the stroma determines the molecular subtype of bladder cancer.

In bladder, loss of mammalian Sonic Hedgehog (Shh) accompanies progression to invasive urothelial carcinoma, but the molecular mechanisms underlying this cancer-initiating event are poorly defined. Here, we show that loss of Shh results from hypermethylation of the CpG shore of the Shh gene, and that inhibition of DNA methylation increases Shh expression to halt the initiation of murine urothelial carcinoma at the early stage of progression. In full-fledged tumors, pharmacologic augmentation of Hedgehog (Hh) pathway activity impedes tumor growth, and this cancer-restraining effect of Hh signaling is mediated by the stromal response to Shh signals, which stimulates subtype conversion of basal to luminal-like urothelial carcinoma. Our findings thus provide a basis to develop subtype-specific strategies for the management of human bladder cancer.

FGF, BMP, and RA signaling are sufficient for the induction of complete limb regeneration from non-regenerating wounds on Ambystoma mexicanum limbs

Some organisms, such as the Mexican axolotl, have the capacity to regenerate complicated biological structures throughout their lives. Which molecular pathways are sufficient to induce a complete endogenous regenerative response in injured tissue is an important question that remains unanswered. Using a gain-of-function regeneration assay, known as the Accessory Limb Model (ALM), we and others have begun to identify the molecular underpinnings of the three essential requirements for limb regeneration; wounding, neurotrophic signaling, and the induction of pattern from cells that retain positional memory. We have previously shown that treatment of Mexican axolotls with exogenous retinoic acid (RA) is sufficient to induce the formation of complete limb structures from blastemas that were generated by deviating a nerve bundle into an anterior-located wound site on the limb. Here we show that these ectopic structures are capable of regenerating and inducing new pattern to form when grafted into new anterior-located wounds. We additionally found that the expression of Alx4 decreases, and Shh expression increases in these anterior located blastemas, but not in the mature anterior tissues, supporting the hypothesis that RA treatment posteriorizes blastema tissue. Based on these and previous observations, we used the ALM assay to test the hypothesis that a complete regenerative response can be generated by treating anterior-located superficial limb wounds with a specific combination of growth factors at defined developmental stages. Our data shows that limb wounds that are first treated with a combination of FGF-2, FGF-8, and BMP-2, followed by RA treatment of the resultant mid-bud stage blastema, will result in the generation of limbs with complete proximal/distal and anterior/posterior limb axes. Thus, the minimal signaling requirements from the nerve and a positional disparity are achieved with the application of this specific combination of signaling molecules.

Dysregulation of sonic hedgehog pathway and pericytes in the brain after lentiviral infection

BackgroundImpairment of the blood–brain barrier (BBB) has been associated with cognitive decline in many CNS diseases, including HIV-associated neurocognitive disorders (HAND). Recent research suggests an important role for the Sonic hedgehog (Shh) signaling pathway in the maintenance of BBB integrity under both physiological and pathological conditions.MethodsIn the present study, we sought to examine the expression of Shh and its downstream effectors in relation to brain pericytes and BBB integrity in HIV-infected humans and rhesus macaques infected with simian immunodeficiency virus (SIV), an animal model of HIV infection and CNS disease. Cortical brain tissues from uninfected (n = 4) and SIV-infected macaques with (SIVE, n = 6) or without encephalitis (SIVnoE, n = 4) were examined using multi-label, semi-quantitative immunofluorescence microscopy of Shh, netrin-1, tight junction protein zona occludens 1 (ZO1), glial fibrillary acidic protein, CD163, platelet-derived growth factor receptor b (PDGFRB), glucose transporter 1, fibrinogen, and SIV Gag p28.ResultsWhile Shh presence in the brain persisted during HIV/SIV infection, both netrin-1 immunoreactivity and the size of PDGFRB+ pericytes, a cellular source of netrin-1, were increased around non-lesion-associated vessels in encephalitis compared to uninfected brain or brain without encephalitis, but were completely absent in encephalitic lesions. Hypertrophied pericytes were strongly localized in areas of fibrinogen extravasation and showed the presence of intracellular SIVp28 and HIVp24 by immunofluorescence in all SIV and HIV encephalitis cases examined, respectively.ConclusionsThe lack of pericytes and netrin-1 in encephalitic lesions, in line with downregulation of ZO1 on the fenestrated endothelium, suggests that pericyte loss, despite the strong presence of Shh, contributes to HIV/SIV-induced BBB disruption and neuropathogenesis in HAND.