Shh Activity Research Articles

Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination center

BackgroundSonic Hedgehog (Shh) has a catalytic cleft characteristic for zinc metallopeptidases and has significant sequence similarities with some bacterial peptidoglycan metallopeptidases defining a subgroup within the M15A family that, besides having the characteristic zinc coordination motif, can bind two calcium ions. Extracellular matrix (ECM) components in animals include heparan-sulfate proteoglycans, which are analogs of bacterial peptidoglycan and are involved in the extracellular distribution of Shh.ResultsWe found that the zinc-coordination center of Shh is required for its association to the ECM as well as for non-cell autonomous signaling. Association with the ECM requires the presence of at least 0.1 μM zinc and is prevented by mutations affecting critical conserved catalytical residues. Consistent with the presence of a conserved calcium binding domain, we find that extracellular calcium inhibits ECM association of Shh.ConclusionsOur results indicate that the putative intrinsic peptidase activity of Shh is required for non-cell autonomous signaling, possibly by enzymatically altering ECM characteristics.

Advanced MRI Findings in Medulloblastomas: Relationship to Genetic Subtypes, Histopathology, and Immunohistochemistry.

For diagnosis of medulloblastoma, the updated World Health Organization classification now demands for genetic typing, defining more precisely the tumor biology, therapy, and prognosis. We investigated potential associations between magnetic resonance imaging (MRI) parameters including apparent diffusion coefficient (ADC) and neuropathologic features of medulloblastoma, focusing on genetic subtypes. This study was a retrospective single-center analysis of 32 patients (eight females, median age = 9 years [range, 1-57], mean 12.6±11.3) from 2012 to 2019. Genetic subtypes (wingless [WNT]; sonic hedgehog [SHH]; non-WNT/non-SHH), histopathology, immunohistochemistry (p53, Ki67), and the following MRI parameters were correlated: tumor volume, location (midline, pontocerebellar, and cerebellar hemisphere), edema, hydrocephalus, metastatic disease (presence/absence and each), contrast-enhancement (minor, moderate, and distinct), cysts (none, small, and large), hemorrhage (none, minor, and major), and ADCmean . The ADCmean was calculated using manually set regions of interest within the solid tumor. Statistics comprised univariate and multivariate testing. Out of 32 tumors, three tumors were WNT activated (9.4%), 13 (40.6%) SHH activated, and 16 (50.0%) non-WNT/non-SHH. Hemispherical location (n = 7/8, P = .003) and presence of edema (8/8; P < .001, specificity 100%, positive predictive value 100%) were significantly associated with SHH activation. The combined parameter "no edema + no metastatic disease + cysts" significantly discriminated WNT-activated from SHH-activated medulloblastoma (P = .036). ADCmean (10-6 mm2 /s) was 484 for WNT-activated, 566 for SHH-activated, and 624 for non-WNT/non-SHH subtypes (P = .080). A significant negative correlation was found between ADCmean and Ki67 (r = -.364, P = .040). MRI analysis enabled noninvasive differentiation of SHH-activated medulloblastoma. ADC alone was not reliable for genetic characterization, but associated with tumor proliferation rate.

Development and validation of a hedgehog heparin-binding assay for high-throughput screening

Sonic hedgehog (Shh) is a morphogenic protein with critical roles in embryogenesis and the development of some cancers. Hence, identifying inhibitors of the Shh pathway is of great therapeutic value. Heparin and HSPGs act as crucial modulators of Shh activity. To identify molecules that antagonize Shh binding to heparin we have developed a solid-phase plate-based assay. The N-terminal domain of Shh (ShhN) protein is first coated in 384-well plates and the direct binding of fluorescein-labeled heparin (flu-heparin) assessed by measuring the fluorescence signal after incubation and wash steps. Binding of ShhN protein to the 384-well plates was confirmed and optimized by a standard ELISA using a monoclonal antibody recognizing folded ShhN. The assay was validated using whole plate minimum and maximum signal wells with a Z’ of 0.68–0.75 determined. Herein, we describe the development and validation of a high throughput screen to identify small molecule antagonists of Shh heparin binding.Overall, this method•Results in an optimized and validated assay for hedgehog heparin binding.•Delivers a cost effective high-throughput screen format for hedgehog heparin antagonist screening

Gpr37l1/prosaposin receptor regulates Ptch1 trafficking, Shh production, and cell proliferation in cerebellar primary astrocytes.

Mammalian cerebellar astrocytes critically regulate the differentiation and maturation of neuronal Purkinje cells and granule precursors. The G protein-coupled receptor 37-like 1 (Gpr37l1) is expressed by Bergmann astrocytes and interacts with patched 1 (Ptch1) at peri-ciliary membranes. Cerebellar primary astrocyte cultures from wild-type and Gpr37l1 null mutant mouse pups were established and studied. Primary cilia were produced by cultures of both genotypes, as well as Ptch1 and smoothened (Smo) components of the sonic hedgehog (Shh) mitogenic pathway. Compared to wild-type cells, Gpr37l1-/- astrocytes displayed striking increases in proliferative activity, Ptch1 protein expression and internalization, intracellular cholesterol content, ciliary localization of Smo, as well as a marked production of active Shh. Similar effects were reproduced by treating wild-type astrocytes with a putative prosaptide ligand of Gpr37l1. These findings indicate that Gpr37l1-Ptch1 interactions specifically regulate Ptch1 internalization and trafficking, with consequent stimulation of Shh production and activation of proliferative signaling.

Shh-Yap signaling controls hepatic ductular reactions in CCl4 -induced liver injury.

Carbon tetrachloride (CCl4 ) exposure can induce hepatic ductular reactions. To date, however, the related mechanism remains largely unknown. Sonic hedgehog (Shh) and Yes-associated protein (Yap) signaling are correlated with liver injury and regeneration. Herein, we investigated the role of Shh and Yap signaling in the fate of ductular reaction cells in CCl4 -treated livers and the possible mechanisms. Wild-type and Shh-EGFP-Cre male mice were exposed to CCl4 (2 mL/kg), and then treated with or without the Shh signaling inhibitor Gant61. The level of liver injury, proliferation of ductular reaction cells, and expression levels of mRNA and protein related to the Shh and Yap signaling components were assessed. Results showed that CCl4 treatment induced liver injury and promoted activation and proliferation of ductular reaction cells. In addition, CCl4 induced the expression of Shh ligands in hepatocytes, accompanied by activation of Shh and Yap1 signaling in the liver. Furthermore, administration of Gant61 ameliorated liver regeneration, inhibited hepatic ductular reactions, and decreased Shh and Yap1 signaling activity. Thus, Shh-Yap1 signaling appears to play an integral role in the proliferation of ductular reaction cells in CCl4 -induced liver injury. This study should improve our understanding of the mechanism of CCl4 -induced liver injury and ductular reactions and provide support for future investigations on liver disease therapy.

Bidirectional Wnt signaling between endoderm and mesoderm confers tracheal identity in mouse and human cells

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. At the initiation of tracheal development, endoderm begins to express Nkx2.1, and then mesoderm expresses the Tbx4 gene. Loss of β-catenin in fetal mouse mesoderm causes loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The mesenchymal Tbx4 expression relies on endodermal Wnt activation and Wnt ligand secretion but is independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generates tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation is required along with WNT to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.

Therapeutic Targeting of Signaling Pathways Related to Cancer Stemness.

The theory of cancer stem cells (CSCs) proposes that the different cells within a tumor, as well as metastasis deriving from it, are originated from a single subpopulation of cells with self-renewal and differentiation capacities. These cancer stem cells are supposed to be critical for tumor expansion and metastasis, tumor relapse and resistance to conventional therapies, such as chemo- and radiotherapy. The acquisition of these abilities has been attributed to the activation of alternative pathways, for instance, WNT, NOTCH, SHH, PI3K, Hippo, or NF-κB pathways, that regulate detoxification mechanisms; increase the metabolic rate; induce resistance to apoptotic, autophagic, and senescence pathways; promote the overexpression of drug transporter proteins; and activate specific stem cell transcription factors. The elimination of CSCs is an important goal in cancer therapeutic approaches because it could decrease relapses and metastatic dissemination, which are main causes of mortality in oncology patients. In this work, we discuss the role of these signaling pathways in CSCs along with their therapeutic potential.

Sonic Hedgehog Signaling Contributes to Chronic Post-Thoracotomy Pain via Activating BDNF/TrkB Pathway in Rats.

PurposeSome patients undergoing thoracotomy may suffer from chronic post-thoracotomy pain (CPTP). Treatment of CPTP has been a clinical challenge and the underlying mechanisms of CPTP remain elusive. Recently, sonic hedgehog (Shh) signaling has been shown to be associated with various pain states but its role in the pathogenesis of CPTP is still unclear.MethodsCPTP was induced in rats by thoracotomy. Rats were divided into CPTP group and non-CPTP group based on the mechanical withdrawal threshold (MWT). Rats were administered with Shh signaling inhibitor cyclopamine and activator smoothened agonist (SAG), and then evaluated by MWT and cold allodynia testing. The expressions of Shh signaling (Shh ligand, patched and smoothened receptor, Gli transcription factors), brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase receptor B (Trk-B), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) in rat T4-5 spinal cord dorsal horn (SDH) were detected by Western blotting and immunohistochemistry.ResultsThe expression of Shh signaling significantly increased and the BDNF/TrkB pathway was activated in T4-5 SDH of CPTP rats. Cyclopamine attenuated hyperalgesia and down-regulated the expressions of Gil1, BDNF, p-TrkB, p-PI3K and p-Akt in CPTP rats. SAG induced hyperalgesia in non-CPTP rats and elevated the expressions of Gil1, BDNF, p-TrkB, p-PI3K and p-Akt.ConclusionShh signaling may contribute to CPTP via activating BDNF/TrkB signaling pathway, and inhibition of Shh signaling may effectively alleviate CPTP.

Osteoblasts are inherently programmed to repel sensory innervation

Tissue innervation is a complex process controlled by the expression profile of signaling molecules secreted by tissue-resident cells that dictate the growth and guidance of axons. Sensory innervation is part of the neuronal network of the bone tissue with a defined spatiotemporal occurrence during bone development. Yet, the current understanding of the mechanisms regulating the map of sensory innervation in the bone tissue is still limited. Here, we demonstrated that differentiation of human mesenchymal stem cells to osteoblasts leads to a marked impairment of their ability to promote axonal growth, evidenced under sensory neurons and osteoblastic-lineage cells crosstalk. The mechanisms by which osteoblast lineage cells provide this nonpermissive environment for axons include paracrine-induced repulsion and loss of neurotrophic factors expression. We identified a drastic reduction of NGF and BDNF production and stimulation of Sema3A, Wnt4, and Shh expression culminating at late stage of OB differentiation. We noted a correlation between Shh expression profile, OB differentiation stages, and OB-mediated axonal repulsion. Blockade of Shh activity and signaling reversed the repulsive action of osteoblasts on sensory axons. Finally, to strengthen our model, we localized the expression of Shh by osteoblasts in bone tissue. Overall, our findings provide evidence that the signaling profile associated with osteoblast phenotype differentiating program can regulate the patterning of sensory innervation, and highlight osteoblast-derived Shh as an essential player in this cue-induced regulation.

Primary cilia-dependent signaling is involved in regulating mesenchymal stem cell proliferation and pluripotency maintenance

Using a large-scale quantitative mesenchymal stem cells (MSCs) membrane proteomics analysis, we identified a large group of ciliary proteins in the MSCs membrane fraction, which prompted us to examine the cilia, intricate organelles that were originally discovered approximately 100 years ago. Here we characterize their primary structure and function in MSCs. We first characterized the primary cilia on undifferentiated human MSCs by immunostaining and verified these observation with scanning and 3D electronic microscopy. To investigate the function of the primary cilia of the MSCs we induced loss of function by means of siRNA knockdown (targeted to two known ciliary proteins: IFT172 and KIF3A). After either of these two proteins was knocked down by the respective siRNA, the MSCs showed fewer and shortened primary cilia. The MSCs proliferation assays showed increased cell proliferative activity under confluent conditions after the siRNA knockdown of IFT172 or KIF3A; among these MSCs, the proportion in S phase was increased in the IFT172 siRNA knockdown group. The expression of stem cell markers on the MSCs, namely, Oct4, Nanog and Sox2, were downregulated after the siRNA-induced knockdown of IFT172 or KIF3A, and the gene expression upregulation of ectoderm lineage markers was notable. Furthermore, manipulation of the primary cilia-dependent Shh pathway, using the Shh activator SAG (smoothened agonist), upregulated the gene expression of pluripotency markers, including Nanog and Oct4, and transcriptional target genes in the Shh pathway. This study confirms that MSCs have primary cilia and provides evidence that primary cilia-dependent signaling pathways play functional roles in MSCs proliferation and stemness maintenance.

Investigation of endoplasmic reticulum stress and sonic hedgehog pathway in diabetic liver injury in mice

AimsDiabetes is a common metabolic disease which damages many organs including the liver and causes endoplasmic reticulum (ER) stress, which originates from non-folded proteins. Sonic hedgehog (Shh) pathway plays a role in liver regeneration and repair. To our knowledge, there is no study showing the relation between ER stress and Shh pathway in the liver in diabetes. Thus, the aim of this study was to investigate the interaction between ER stress and Shh pathway in the liver of diabetic mice. Main methodsSix groups of male mice were formed as control, diabetes (streptozotocine-treated), Shh activator (SAG-treated), Shh inhibitor (SANT1-treated), diabetes + SAG and diabetes + SANT1. At the end of the experiment, mice were weighed, anaesthetized and euthanized. Blood samples were collected, livers were excised and weighed. Thereafter, blood glucose, serum ALT and AST levels, TOS and TAC levels in liver tissue were measured. ER stress marker (GRP78) and Shh pathway molecules (Gli1 and Smo) were evaluated by immunohistochemistry, H-score and western blot analyses. Besides, histopathological examination was performed. Key findingsResults showed that GRP78, Gli1 and Smo were increased in liver due to Type 1 diabetes. The SAG agent decreased GRP78 and increased Gli1 and Smo, leading to liver repair, while the inhibitor SANT1 increased GRP78 and decreased Gli1and Smo, causing progression of the liver stress induced by diabetes. SignificanceIn conclusion, the Shh pathway is related to ER stress and may provide a new strategy for its treatment, especially liver stress induced by diabetes.

Prdm8 regulates pMN progenitor specification for motor neuron and oligodendrocyte fates by modulating the Shh signaling response.

Spinal cord pMN progenitors sequentially produce motor neurons and oligodendrocyte precursor cells (OPCs). Some OPCs differentiate rapidly as myelinating oligodendrocytes, whereas others remain into adulthood. How pMN progenitors switch from producing motor neurons to OPCs with distinct fates is poorly understood. pMN progenitors express prdm8, which encodes a transcriptional repressor, during motor neuron and OPC formation. To determine whether prdm8 controls pMN cell fate specification, we used zebrafish as a model system to investigate prdm8 function. Our analysis revealed that prdm8 mutant embryos have fewer motor neurons resulting from a premature switch from motor neuron to OPC production. Additionally, prdm8 mutant larvae have excess oligodendrocytes and a concomitant deficit of OPCs. Notably, pMN cells of mutant embryos have elevated Shh signaling, coincident with the motor neuron to OPC switch. Inhibition of Shh signaling restored the number of motor neurons to normal but did not rescue the proportion of oligodendrocytes. These data suggest that Prdm8 regulates the motor neuron-OPC switch by controlling the level of Shh activity in pMN progenitors, and also regulates the allocation of oligodendrocyte lineage cell fates.This article has an associated 'The people behind the papers' interview.

TMOD-05. IN VIVO REPROGRAMMING OF POSTMITOTIC NEURONS INDUCES MEDULLOBLASTOMA

Abstract It is widely accepted that the “cell of origin” of tumors has to possess a proliferative capacity. Particularly for brain cancer, the transition of neural progenitors to differentiated postmitotic neurons is considered irreversible in physiological and pathological conditions. Therefore, postmitotic neurons have not been considered as suitable cell-of-origin for brain cancer. Here, we show that neurons reprograming may occur upon Shh activation and it leads to medulloblastoma (MB) formation in vivo. Shh MB is a cerebellar tumor, found in infants and adults that is thought to originate from cerebellar granule neuron progenitors. More recently, it was discovered that the two different forms of SHH MB are distinguished by different transcriptome/methylome levels suggesting that the adult SHH MB may originate from a different cell-of-origin. Relying on these data, we take advantage of a conditional Cre-Lox recombination system to recapitulate the human adult medulloblastoma pathogenesis in mice and demonstrate that post-migratory mature granule neurons can be reprogrammed in vivo. Furthermore, to define the contribution of chromatin changes in granule neurons dedifferentiation in response to Shh activation, we profiled changes in chromatin accessibility by ATAC-seq both in mouse tissue samples and also in human neurons. Upon Shh pathway activation we detected the presence of hyper-accessible chromatin regions corresponding to cis-regulatory elements specifically favouring activation of enhancers and super-enhancers. Our novel model of cancer development could explain the human SHH medulloblastoma onset in adult individuals where granule neuron progenitors are no more present. We strongly believe that our model represents an important starting point to study other tissues where postmitotic cells might originate cancer and therefore open a new field in cancer and stem cell biology.

Sonic Hedgehog repression underlies gigaxonin mutation-induced motor deficits in giant axonal neuropathy.

Growing evidence shows that alterations occurring at early developmental stages contribute to symptoms manifested in adulthood in the setting of neurodegenerative diseases. Here, we studied the molecular mechanisms causing giant axonal neuropathy (GAN), a severe neurodegenerative disease due to loss-of-function of the gigaxonin-E3 ligase. We showed that gigaxonin governs Sonic Hedgehog (Shh) induction, the developmental pathway patterning the dorso-ventral axis of the neural tube and muscles, by controlling the degradation of the Shh-bound Patched receptor. Similar to Shh inhibition, repression of gigaxonin in zebrafish impaired motor neuron specification and somitogenesis and abolished neuromuscular junction formation and locomotion. Shh signaling was impaired in gigaxonin-null zebrafish and was corrected by both pharmacological activation of the Shh pathway and human gigaxonin, pointing to an evolutionary-conserved mechanism regulating Shh signaling. Gigaxonin-dependent inhibition of Shh activation was also demonstrated in primary fibroblasts from patients with GAN and in a Shh activity reporter line depleted in gigaxonin. Our findings establish gigaxonin as a key E3 ligase that positively controls the initiation of Shh transduction, and reveal the causal role of Shh dysfunction in motor deficits, thus highlighting the developmental origin of GAN.

Long non-coding RNAs identify a subset of luminal muscle-invasive bladder cancer patients with favorable prognosis

BackgroundMuscle-invasive bladder cancer (MIBC) is a heterogeneous disease, and gene expression profiling has identified several molecular subtypes with distinct biological and clinicopathological characteristics. While MIBC subtyping has primarily been based on messenger RNA (mRNA), long non-coding RNAs (lncRNAs) may provide additional resolution.MethodsLncRNA expression was quantified from microarray data of a MIBC cohort treated with neoadjuvant chemotherapy (NAC) and radical cystectomy (RC) (n = 223). Unsupervised consensus clustering of highly variant lncRNAs identified a four-cluster solution, which was characterized using a panel of MIBC biomarkers, regulon activity profiles, gene signatures, and survival analysis. The four-cluster solution was confirmed in The Cancer Genome Atlas (TCGA) cohort (n = 405). A single-sample genomic classifier (GC) was trained using ridge-penalized logistic regression and validated in two independent cohorts (n = 255 and n = 94).ResultsNAC and TCGA cohorts both contained an lncRNA cluster (LC3) with favorable prognosis that was enriched with tumors of the luminal-papillary (LP) subtype. In both cohorts, patients with LP tumors in LC3 (LPL-C3) were younger and had organ-confined, node-negative disease. The LPL-C3 tumors had enhanced FGFR3, SHH, and wild-type p53 pathway activity. In the TCGA cohort, LPL-C3 tumors were enriched for FGFR3 mutations and depleted for TP53 and RB1 mutations. A GC trained to identify these LPL-C3 patients showed robust performance in two validation cohorts.ConclusionsUsing lncRNA expression profiles, we identified a biologically distinct subgroup of luminal-papillary MIBC with a favorable prognosis. These data suggest that lncRNAs provide additional information for higher-resolution subtyping, potentially improving precision patient management.

Sonic hedgehog promotes chondrogenesis of rabbit bone marrow stem cells in a rotary cell culture system

BackgroundSonic hedgehog (Shh) is an important signalling protein involved in the induction of early cartilaginous differentiation. Herein, we demonstrate that Shh markedly induces chondrogenesis of rabbit bone marrow stromal cells (BMSCs) under microgravity conditions, and promotes cartilage regeneration.ResultsIn the rotary cell culture system (RCCS), chondrogenic differentiation was revealed by stronger Toluidine Blue and collagen II immunohistochemical staining in the Shh transfection group, and chondroinductive activity of Shh was equivalent to that of TGF-β. Western blotting and qRT-PCR analysis results verified the stronger expression of Sox9, aggrecan (ACAN), and collagen II in rabbit BMSCs treated with Shh or TGF-β in a microgravity environment. Low levels of chondrogenic hypertrophy, osteogenesis, and adipogenesis-related factors were detected in all groups. After transplantation in vivo, histological analysis revealed a significant improvement in cartilage and subchondral repair in the Shh transfection group.ConclusionsThese results suggested that Shh signalling promoted chondrogenesis in rabbit BMSCs under microgravity conditions equivalent to TGF-β, and improved the early stages of the repair of cartilage and subchondral defects. Furthermore, RCCS provided a dynamic culture microenvironment conducive for cell proliferation, aggregation and differentiation.

Critical roles of ARHGAP36 as a signal transduction mediator of Shh pathway in lateral motor columnar specification.

During spinal cord development, Sonic hedgehog (Shh), secreted from the floor plate, plays an important role in the production of motor neurons by patterning the ventral neural tube, which establishes MN progenitor identity. It remains unknown, however, if Shh signaling plays a role in generating columnar diversity of MNs that connect distinct target muscles. Here, we report that Shh, expressed in MNs, is essential for the formation of lateral motor column (LMC) neurons in vertebrate spinal cord. This novel activity of Shh is mediated by its downstream effector ARHGAP36, whose expression is directly induced by the MN-specific transcription factor complex Isl1-Lhx3. Furthermore, we found that AKT stimulates the Shh activity to induce LMC MNs through the stabilization of ARHGAP36 proteins. Taken together, our data reveal that Shh, secreted from MNs, plays a crucial role in generating MN diversity via a regulatory axis of Shh-AKT-ARHGAP36 in the developing mouse spinal cord.

Soluble Heparin and Heparan Sulfate Glycosaminoglycans Interfere with Sonic Hedgehog Solubilization and Receptor Binding.

Sonic hedgehog (Shh) signaling plays a tumor-promoting role in many epithelial cancers. Cancer cells produce soluble a Shh that signals to distant stromal cells that express the receptor Patched (Ptc). These receiving cells respond by producing other soluble factors that promote cancer cell growth, generating a positive feedback loop. To interfere with reinforced Shh signaling, we examined the potential of defined heparin and heparan sulfate (HS) polysaccharides to block Shh solubilization and Ptc receptor binding. We confirm in vitro and in vivo that proteolytic cleavage of the N-terminal Cardin–Weintraub (CW) amino acid motif is a prerequisite for Shh solubilization and function. Consistent with the established binding of soluble heparin or HS to the Shh CW target motif, both polysaccharides impaired proteolytic Shh processing and release from source cells. We also show that HS and heparin bind to, and block, another set of basic amino acids required for unimpaired Shh binding to Ptc receptors on receiving cells. Both modes of Shh activity downregulation depend more on HS size and overall charge than on specific HS sulfation modifications. We conclude that heparin oligosaccharide interference in the physiological roles of HS in Shh release and reception may be used to expand the field of investigation to pharmaceutical intervention of tumor-promoting Shh functions.

Sonic hedgehog signaling is negatively regulated in reactive astrocytes after forebrain stab injury

Following injury to the central nervous system, astrocytes perform critical and complex functions that both promote and antagonize neural repair. Understanding the molecular signaling pathways that coordinate their diverse functional properties is key to developing effective therapeutic strategies. In the healthy, adult CNS, Sonic hedgehog (Shh) signaling is active in mature, differentiated astrocytes. Shh has been shown to undergo injury-induced upregulation and promote neural repair. Here, we investigated whether Shh signaling mediates astrocyte response to injury. Surprisingly, we found that following an acute, focal injury, reactive astrocytes exhibit a pronounced reduction in Shh activity in a spatiotemporally-defined manner. Shh signaling is lost in reactive astrocytes at the lesion site, but persists in mild to moderately reactive astrocytes in distal tissues. Nevertheless, local pharmacological activation of the Shh pathway in astrocytes mitigates inflammation, consistent with a neuroprotective role for Shh signaling after injury. Interestingly, we find that Shh signaling is restored to baseline levels two weeks after injury, a time during which acute inflammation has largely subsided and lesions have matured. Taken together, these data suggest that endogenous Shh signaling in astrocytes is dynamically regulated in a context dependent manner. In addition, exogenous activation of the Shh pathway promotes neuroprotection mediated by reactive astrocytes.

Hedgehog signaling dynamics in mouse embryos determined by a bioluminiscent reporter.

Determination of cellular signaling in live embryos is key to understand the molecular processes that drive development. Here, we show that a transgenic mouse line carrying a luciferase-based gene reporter of Gli-mediated transcriptional activation (Gli-Luc) displays sonic hedgehog (Shh) signaling in discrete developmental processes during short-term cultures of whole embryos or embryo explants. The bioluminescence in E9.5 embryos was detected in regions in which Shh activity has been demonstrated. Later, in E10.5 embryos, bioluminescence intensity markedly increased, mostly corresponding to the high Shh activity of the developing midbrain and limb. Notably, the dynamic range of the Gli-Luc reporter in the developing limb revealed the progressive emergence of bioluminescence in the zone of polarizing activity, where reporter activity locally increased and spatially spread in agreement with the signaling gradient expected for Shh. In the midbrain of E9.5 mouse embryos, bioluminescence was not detected along the ventral region as expected but, instead, Shh-dependent anterior and posterior bioluminescence foci emerged by E10.5 indicating that the Gli-Luc reporter can only respond transcriptionally to relatively high levels of GliA and/or without the interaction with other transcription factors. The present work supports the use of bioluminescence to identify and study the dynamics of centers of morphogen signaling during mouse embryogenesis.