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The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report bi-allelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans., (Copyright © 2020 American Society of Human Genetics. All rights reserved.)

Hedgehog (Hh) signaling has been shown to be involved in regulating both intact and injured peripheral nerves. Therefore, it is critical to understand how Hh signaling is regulated in the peripheral nerve. One of the transcription factors of the Hh signaling pathway, Gli3, functions as both a repressor and an activator of Hh signaling activity. However, it remains unclear whether Gli3 is involved in controlling the intact and/or injured peripheral nerves. We found that Gli3 act as a repressor in the Schwann cells (SCs) of intact sciatic nerves. Although Dhh and Ptch1 expression were present, Hh signaling was not activated in these SCs. Moreover, heterozygous Gli3 mutation (Gli3 -/+ ) induced ectopic Hh signaling activity in SCs. Hh signaling was thus suppressed by Gli3 in the SCs of intact sciatic nerves. Minor morphological changes were observed in the intact nerves from Gli3 -/+ mice. Gli3 expression was significantly decreased following injury and ligand expression switched from Dhh to Shh, which activated Hh signaling in SCs from wild-type mice. Changes of these ligands was found to be important for nerve regeneration in which the downregulation of Gli3 was also involved. In fact, Gli3 -/+ mice exhibited accelerated ligand switching and subsequent nerve regeneration. Both suppression of Hh signaling with Gli3 in the intact nerves and activation of Hh signaling without Gli3 in the injured nerve were observed in the SCs in an autocrine manner. Thus, Gli3 is a key factor in the control of intact peripheral nerve homeostasis and nerve regeneration., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

The Sonic Hedgehog (SHH) pathway is a key signaling pathway orchestrating embryonic development, mainly of the CNS and limbs. In vertebrates, SHH signaling is mediated by the primary cilium, and genetic defects affecting either SHH pathway members or ciliary proteins cause a spectrum of developmental disorders. SUFU is the main negative regulator of the SHH pathway and is essential during development. Indeed, Sufu knock-out is lethal in mice, and recessive pathogenic variants of this gene have never been reported in humans. Through whole-exome sequencing in subjects with Joubert syndrome, we identified four children from two unrelated families carrying homozygous missense variants in SUFU. The children presented congenital ataxia and cerebellar vermis hypoplasia with elongated superior cerebellar peduncles (mild "molar tooth sign"), typical cranio-facial dysmorphisms (hypertelorism, depressed nasal bridge, frontal bossing), and postaxial polydactyly. Two siblings also showed polymicrogyria. Molecular dynamics simulation predicted random movements of the mutated residues, with loss of the native enveloping movement of the binding site around its ligand GLI3. Functional studies on cellular models and fibroblasts showed that both variants significantly reduced SUFU stability and its capacity to bind GLI3 and promote its cleavage into the repressor form GLI3R. In turn, this impaired SUFU-mediated repression of the SHH pathway, as shown by altered expression levels of several target genes. We demonstrate that germline hypomorphic variants of SUFU cause deregulation of SHH signaling, resulting in recessive developmental defects of the CNS and limbs which share features with both SHH-related disorders and ciliopathies., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Tctn1, Tctn2, and Tctn3 are membrane proteins that localize at the transition zone of primary cilia. Tctn1 and Tctn2 mutations have been reported in both humans and mice, but Tctn3 mutations have been reported only in humans. It is also not clear whether the three Tctn proteins are functionally conserved with respect to ciliogenesis and Hedgehog (Hh) signaling. In the present study, we report that loss of Tctn3 gene function in mice results in a decrease in ciliogenesis and Hh signaling. Consistent with this, Tctn3 mutant mice exhibit holoprosencephaly and randomized heart looping and lack the floor plate in the neural tube, the phenotypes similar to those of Tctn1 and Tctn2 mutants. We also show that overexpression of Tctn3, but not Tctn1 or Tctn2, can rescue ciliogenesis in Tctn3 mutant cells. Similarly, replacement of Tctn3 with Tctn1 or Tctn2 in the Tctn3 gene locus results in reduced ciliogenesis and Hh signaling, holoprosencephaly, and randomized heart looping. Surprisingly, however, the neural tube patterning and the proteolytic processing of Gli3 (a transcription regulator for Hh signaling) into a repressor, both of which are usually impaired in ciliary gene mutants, are normal. These results suggest that Tctn1, Tctn2, and Tctn3 are functionally divergent with respect to their role in ciliogenesis and Hh signaling but conserved in neural tube patterning and Gli3 processing., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hedgehog signaling plays crucial roles in the development of calvarial bone, relying on the activation of Gli transcription factors. However, the molecular mechanism of the role of regulated Gli protein level in osteogenic specification of mesenchyme still remains elusive. Here, we show by conditionally inactivating Suppressor of Fused ( Sufu ), a critical repressor of Hedgehog signaling, in Wnt1-Cre -mediated cranial neural crest (CNC) or Dermo1-Cre -mediated mesodermal lineages that Sufu restraint of Hedgehog activity level is critical for differentiation of preosteogenic mesenchyme. Ablation of Sufu results in failure of calvarial bone formation, including CNC-derived bones and mesoderm-derived bones, depending on the Cre line being used. Although mesenchymal cells populate to frontonasal destinations, where they are then condensed, Sufu deletion significantly inhibits the proliferation of osteoprogenitor cells, and these cells no longer differentiate into osteoblasts. We show that there is suppression of Runx2 and Osterix , the osteogenic regulators, in calvarial mesenchyme in the Sufu mutant. We show that down-regulation of several genes upstream to Runx2 and Osterix is manifested within the calvarial primordia, including Bmp2 and its downstream genes Msx1/2 and Dlx5 By contrast, we find that Gli1 , the Hedgehog activity readout gene, is excessively activated in mesenchyme. Deletion of Sufu in CNC leads to a discernible decrease in the repressive Gli3 form and an increase in the full-length Gli2. Finally, we demonstrate that simultaneous deletion of Gli2 and Sufu in CNC completely restores calvarial bone formation, suggesting that a sustained level of Hedgehog activity is critical in specification of the osteogenic mesenchymal cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Hedgehog (Hh) signaling is thought to occur in primary cilia, but the molecular basis of Gli2 and Gli3 activation by Hh signaling in cilia is unknown. Similarly, how ciliary gene mutations result in reduced Gli3 processing that generates a repressor is also not clear. Here we show that Hh signaling inhibits Gli2 and Gli3 phosphorylation by protein kinase A (PKA) in cilia. The cilia related gene Talpid3 (Ta3) mutation results in the reduced processing and phosphorylation of Gli2 and Gli3. Interestingly, Ta3 interacts and colocalizes with PKA regulatory subunit PKARIIβ at centrioles in the cell. The centriolar localization and PKA binding regions are located in the N- and C-terminal regions of Ta3, respectively. PKARIIβ fails to localize at centrioles in some Ta3 mutant cells. Therefore, our study provides the direct evidence that Gli2 and Gli3 are dephosphorylated and activated in cilia and that impaired Gli2 and Gli3 processing in Ta3 mutant is at least in part due to a decrease in Gli2 and Gli3 phosphorylation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Sonic hedgehog (Shh) signaling plays a key role in regulation of normal development. The negative feedback mechanism mediated by the transcriptional factor, Gli3, acts to finely tune Shh signaling, providing tight control of normal developmental processes. Hyperactivation of Shh signaling often leads to many human malignancies, including basal cell carcinoma and medulloblastoma (MB). However, how tumor cells sustain the aberrant activation of Shh signaling is still not completely understood. We recently revealed that during MB formation, tumor cells express Nestin, a type VI intermediate filament protein, which maintains uncontrolled Shh signaling by abolishing negative feedback by Gli3. Therefore, Nestin expression is a necessary step for MB formation. These findings highlight the novel function of Nestin in regulating Shh signaling, as well as the important role of a disrupted negative feedback mechanism in MB tumorigenesis. Further, restoration of the intrinsic negative feedback by repressing Nestin expression represents a promising approach to treat MB as well as other Shh signaling associated malignancies., (Published by Elsevier Inc.)

Before birth, B cells develop in the fetal liver (FL). In this study, we show that Gli3 activity in the FL stroma is required for B cell development. In the Gli3-deficient FL, B cell development was reduced at multiple stages, whereas the Sonic hedgehog (Hh [Shh])-deficient FL showed increased B cell development, and Gli3 functioned to repress Shh transcription. Use of a transgenic Hh-reporter mouse showed that Shh signals directly to developing B cells and that Hh pathway activation was increased in developing B cells from Gli3-deficient FLs. RNA sequencing confirmed that Hh-mediated transcription is increased in B-lineage cells from Gli3-deficient FL and showed that these cells expressed reduced levels of B-lineage transcription factors and B cell receptor (BCR)/pre-BCR-signaling genes. Expression of the master regulators of B cell development Ebf1 and Pax5 was reduced in developing B cells from Gli3-deficient FL but increased in Shh-deficient FL, and in vitro Shh treatment or neutralization reduced or increased their expression, respectively., (© 2017 Solanki et al.)

During early fetal development, paracrine Hedgehog (HH) ligands secreted from the foregut epithelium activate Gli transcription factors in the surrounding mesenchyme to coordinate formation of the respiratory system, digestive track and the cardiovascular network. Although disruptions to this process can lead to devastating congenital defects, the underlying mechanisms and downstream targets, are poorly understood. We show that the zinc finger transcription factor Osr1 is a novel HH target as Osr1 expression in the foregut mesenchyme depends on HH signaling and the effector of HH pathway Gli3 binds to a conserved genomic loci near Osr1 promoter region. Molecular analysis of mouse germline Osr1 mutants reveals multiple functions of Osr1 during foregut development. Osr1 mutants exhibit fewer lung progenitors in the ventral foregut. Osr is then required for the proper branching of the primary lung buds, with mutants exhibiting miss-located lung lobes. Finally, Osr1 is essential for proper mesenchymal differentiation including pulmonary arteries, esophageal and tracheal smooth muscle as well as tracheal cartilage rings. Tissue specific conditional knockouts in combination with lineage tracing indicate that Osr1 is required cell autonomously in the foregut mesenchyme. We conclude that Osr1 is a novel downstream target of HH pathway, required for lung specification, branching morphogenesis and foregut mesenchymal differentiation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The Hedgehog (Hh) signaling pathway is activated in many cancers and is a promising target for therapeutic development. Deletions in the receptor Patched (PTCH) or activating mutations in Smoothened (SMO) have been reported in basal cell carcinoma and medulloblastoma, but are largely absent in most tumor types. Therefore, the mechanism of pathway activation in most cancers, including hematological malignancies, remains unknown. In normal tissues, Hh pathway activation via PTCH/SMO causes an increase in the downstream transcriptional activator GLI1 and a decrease in the GLI3 transcriptional repressor (GLI3R). In this article, we confirm that the Hh pathway is active in acute myeloid leukemia (AML), however, this activity is largely independent of SMO. Epigenetic and gene expression analysis of The Cancer Genome Atlas AML data set reveals that GLI3 expression is silenced in most AML patient samples, and the GLI3 locus is abnormally methylated. We show that GLI3R is required for the therapeutic effect of SMO antagonists in AML samples and restoration of GLI3R suppresses the growth of AML. We additionally demonstrate that GLI3R represses AML growth by downregulating AKT expression. In summary, this study provides the first evidence that GLI3R plays an essential role in SMO-independent Hh signaling in AML, and suggests that GLI3R could serve as a potential biomarker for patient selection in SMO antagonist clinical trials. Furthermore, these data support rational combinations of hypomethylating agents with SMO antagonists in clinical trials., (© 2017 by The American Society of Hematology.)

Background: Polydactyly is a group of congenital limb malformations that show high degree of phenotypic variability and genetic heterogeneity., Results: In the present study, four genomic regions (exons of GLI3, SHH, and noncoding sequences of preZRS and ZRS) involved in hedgehog (Hh) signaling pathway were sequenced for 102 unrelated Chinese children with nonsyndromic polydactyly. Two GLI3 variants (c.2844 G > G/A; c.1486C > C/T) and four preZRS variants (chr7:156585336 A>G; chr7:156585421 C>A; chr7: 156585247 G>C; chr7:156585420 A > C) were observed in 2(2.0%) and 6(5.9%) patients, respectively. These variants are not over-represented in the Chinese healthy population. All the 8 cases showed preaxial polydactyly in hands. Additionally, no specific patterns of malformation predicted mutations in other candidate genes or sequences., Conclusions: This is the first report of the assessment of the frequency of GLI3/SHH/preZRS/ZRS in Chinese patients to show any higher possibility of mutations or variants for the 4 genes or sequences in China. Developmental Dynamics 246:392-402, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here, we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation, resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies. Cancer Res; 76(18); 5573-83. ©2016 AACR., Competing Interests: No potential conflicts of interest were disclosed., (©2016 American Association for Cancer Research.)

The cortisol-inactivating enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) that catalyzes the intracellular inactivation of glucocorticoids plays a pivotal role in human pregnant maintenance and normal fetal development. Given the fact that the main components of Hedgehog (HH) signaling pathway are predominantly expressed in syncytial layer of human placental villi where 11β-HSD2 is robustly expressed, in the present study, we have investigated the potential roles and underlying mechanisms of HH signaling in 11β-HSD2 expression. Activation of HH signaling by a variety of approaches robustly induced 11β-HSD2 expression as well as the 11β-HSD2 activity, whereas suppression of HH signaling significantly attenuated 11β-HSD2 expression as well as the 11β-HSD2 activity in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in HH signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated HH-induced 11β-HSD2 expression and activity, and overexpression of GLI2 activator alone was sufficient to induce 11β-HSD2 expression and activity. Finally, GLI2 not only directly bound to the promoter region of gene hsd11b2 to transactivate hsd11b2 but also formed a heterodimer with RNA polymerase II, an enzyme that catalyzes the transcription of DNA to synthesize mRNAs, resulting in up-regulation of hsd11b2 gene transcription. Taken together, the present study has uncovered a hitherto uncharacterized role of HH/GLI2 signaling in 11β-HSD2 regulation, implicating that HH signaling through GLI2 could be required for the human pregnant maintenance and fetal development.

Hypothalamic hamartoma (HH) with gelastic epilepsy is a well-recognized drug-resistant epilepsy syndrome of early life.(1) Surgical resection allows limited access to the small deep-seated lesions that cause the disease. Here, we report the results of a search for somatic mutations in paired hamartoma- and leukocyte-derived DNA samples from 38 individuals which we conducted by using whole-exome sequencing (WES), chromosomal microarray (CMA), and targeted resequencing (TRS) of candidate genes. Somatic mutations were identified in genes involving regulation of the sonic hedgehog (Shh) pathway in 14/38 individuals (37%). Three individuals had somatic mutations in PRKACA, which encodes a cAMP-dependent protein kinase that acts as a repressor protein in the Shh pathway, and four subjects had somatic mutations in GLI3, an Shh pathway gene associated with HH. In seven other individuals, we identified two recurrent and three single brain-tissue-specific, large copy-number or loss-of-heterozygosity (LOH) variants involving multiple Shh genes, as well as other genes without an obvious biological link to the Shh pathway. The Shh pathway genes in these large somatic lesions include the ligand itself (SHH and IHH), the receptor SMO, and several other Shh downstream pathway members, including CREBBP and GLI2. Taken together, our data implicate perturbation of the Shh pathway in at least 37% of individuals with the HH epilepsy syndrome, consistent with the concept of a developmental pathway brain disease., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

We used Smo siRNA to inhibit hedgehog signaling pathway in embryonic day (E) 13 palatal shelves in organ culture. SiRNA 4 was chosen as the most efficient from four synthesized Smo siRNAs. Palatal shelf fusion rate of 4 μg/mL cyclopamine group was the lowest and significantly lower than that of blank control group (P<0.05), and that of siRNA 4 group was also lower than that of blank control group (P=0.183). At 48 h after transfection, Smo protein level of siRNA 4 group was 64.8% lower than that of blank control group (P<0.05), and Gli1 protein level of 4 μg/mL cyclopamine group was 68.9% lower than that of blank control group (P<0.05). Hedgehog signaling pathway inhibition decreased palatal fusion in organ culture, probably owing to downregulation of Smo and Gli1 proteins., (Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Hedgehog signaling plays very important roles in development and cancers. Vertebrates have three transcriptional factors, Gli1, Gli2 and Gli3. Among them, Gli3 is a very special transcriptional factor which closely resembles Cubitus interruptus (Ci, in Drosophila) structurally and functionally as a 'double agent' for Shh target gene expression. Here we show that Gli3 full-length, but not the truncated form, can be methylated at K436 and K595. This methylation is specifically catalyzed by Set7, a lysine methyltransferase (KMT). Methylation at K436 and K595 respectively increases the stability and DNA binding ability of Gli3, resulting in an enhancement of Shh signaling activation. Furthermore, functional experiments indicate that the Gli3 methylation contributes to the tumor growth and metastasis in non-small cell lung cancer in vitro and in vivo. Therefore, we propose that Set7 mediated methylation is a novel PTM of Gli3, which positively regulates the transactivity of Gli3 and the activation of Shh signaling.

Two major morphological changes occurred during the fin-to-limb transition: the appearance of the autopod, and the reduction of anterior skeletal elements. In the past decades, numerous approaches to the study of genetic developmental systems involved in patterning of fins/limbs among different taxa have provided clues to better understand the mechanism of the fin-to-limb transition. In this article, I discuss recent progress toward elucidating the evolutionary origin of the autopod and the mechanism through which the multiple-basal bones of ancestral fins were reduced into a single bone (humerus/femur). A particular focus of this article is the patterning mechanism of the tetrapod limb and chondrichthyan fin controlled by gene networks involving the 5'Hox genes, Gli3 and Shh. These recent data provide possible scenarios that could have led to the transformation of fins into limbs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Hedgehog (HH) signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the transcriptional effectors of the HH pathway in mouse embryonic stem (ES) cells. We found that both GLI3 and GLI1 bind to the pluripotency factor NANOG. The ectopic expression of NANOG inhibits GLI1-mediated transcriptional responses in a dose-dependent fashion. In differentiating ES cells, the presence of NANOG reduces the transcriptional response of cells to HH. Finally, we found thatGli1andNanogare co-expressed in ES cells at high levels. We propose that NANOG acts as a negative feedback component that provides stem cell-specific regulation of the HH pathway., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Midbrain dopaminergic (mDA) neurons modulate various motor and cognitive functions, and their dysfunction or degeneration has been implicated in several psychiatric diseases. Both Sonic Hedgehog (Shh) and Wnt signaling pathways have been shown to be essential for normal development of mDA neurons. Primary cilia are critical for the development of a number of structures in the brain by serving as a hub for essential developmental signaling cascades, but their role in the generation of mDA neurons has not been examined. We analyzed mutant mouse lines deficient in the intraflagellar transport protein IFT88, which is critical for primary cilia function. Conditional inactivation of Ift88 in the midbrain after E9.0 results in progressive loss of primary cilia, a decreased size of the mDA progenitor domain, and a reduction in mDA neurons. We identified Shh signaling as the primary cause of these defects, since conditional inactivation of the Shh signaling pathway after E9.0, through genetic ablation of Gli2 and Gli3 in the midbrain, results in a phenotype basically identical to the one seen in Ift88 conditional mutants. Moreover, the expansion of the mDA progenitor domain observed when Shh signaling is constitutively activated does not occur in absence of Ift88. In contrast, clusters of Shh-responding progenitors are maintained in the ventral midbrain of the hypomorphic Ift88 mouse mutant, cobblestone. Despite the residual Shh signaling, the integrity of the mDA progenitor domain is severely disturbed, and consequently very few mDA neurons are generated in cobblestone mutants. Our results identify for the first time a crucial role of primary cilia in the induction of mDA progenitors, define a narrow time window in which Shh-mediated signaling is dependent upon normal primary cilia function for this purpose, and suggest that later Wnt signaling-dependent events act independently of primary cilia., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

GLI proteins convert Sonic hedgehog (Shh) signaling into a transcriptional output in a tissue-specific fashion. The Shh pathway has been extensively studied in the limb bud, where it helps regulate growth through a SHH-FGF feedback loop. However, the transcriptional response is still poorly understood. We addressed this by determining the gene expression patterns of approximately 200 candidate GLI-target genes and identified three discrete SHH-responsive expression domains. GLI-target genes expressed in the three domains are predominately regulated by derepression of GLI3 but have different temporal requirements for SHH. The GLI binding regions associated with these genes harbor both distinct and common DNA motifs. Given the potential for interaction between the SHH and FGF pathways, we also measured the response of GLI-target genes to inhibition of FGF signaling and found the majority were either unaffected or upregulated. These results provide the first characterization of the spatiotemporal response of a large group of GLI-target genes and lay the foundation for a systems-level understanding of the gene regulatory networks underlying SHH-mediated limb patterning., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Proper lineage progression and diversification of neural progenitor cells (NPCs) ensures the generation of projection neuron (PN) subtypes in the mammalian neocortex. Here, we show that Suppressor of Fused (Sufu) controls PN specification by maintaining the identity of NPCs in the embryonic neocortex. Deletion of Sufu in NPCs of the E10.5 mouse neocortex led to improper specification of progenitors and a reduction in intermediate progenitors (IPs) during corticogenesis. We found that Sufu deletion resulted in unstable Gli2 and Gli3 activity, leading to the ectopic activation of Sonic hedgehog (Shh) signaling. The role of Sufu in maintaining progenitor identity is critical at early stages of corticogenesis, since deletion of Sufu at E13.5 did not cause similar abnormalities. Our studies revealed that Sufu critically modulates Shh signaling at early stages of neurogenesis for proper specification and maintenance of cortical NPCs to ensure the appropriate generation of cortical PN lineages., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Although chorionic plate-derived mesenchymal stem cells (CP-MSCs) were shown to promote liver regeneration, the mechanisms underlying the effect remain unclear. Hedgehog (Hh) signaling orchestrates tissue reconstruction in damaged liver. MSCs release microRNAs mediating various cellular responses. Hence, we hypothesized that microRNAs from CP-MSCs regulated Hh signaling, which influenced liver regeneration. Livers were obtained from carbon tetrachloride (CCl4)-treated rats transplanted with human CP-MSCs (Tx) or saline (non-Tx). Sonic Hh, one of Hh ligands, increased in CCl4-treated liver, whereas it decreased in CP-MSC-treated liver with CCl4. The expression of Hh-target genes was significantly downregulated in the Tx. Reduced expansion of progenitors and regressed fibrosis were observed in the liver of the Tx rats. CP-MSCs suppressed the expression of Hh and profibrotic genes in co-cultured LX2 (human hepatic stellate cell) with CP-MSCs. MicroRNA-125b targeting smo was retained in exosomes of CP-MSCs. CP-MSCs with microRNA-125b inhibitor failed to attenuate the expression of Hh signaling and profibrotic genes in the activated HSCs. Therefore, these results demonstrated that microRNA-125b from CP-MSCs suppressed the activation of Hh signaling, which promoted the reduced fibrosis, suggesting that microRNA-mediated regulation of Hh signaling contributed to liver regeneration by CP-MSCs.

The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells., Significance Statement: Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic acid modulates the chromatin state of cells to be permissive for motoneuron generation and directly suppresses the induction of GLI3, a negative regulator of SHH signaling. Therefore, our data point to a novel mechanism by which retinoic acid exposure can bypass the requirement for extrinsic SHH treatment during motoneuron induction., (Copyright © 2015 the authors 0270-6474/15/3511462-20$15.00/0.)

The GLI transcription factors, GLI1, GLI2, and GLI3, transduce Hedgehog and non-hedgehog signals and are involved in regulating development and tumorgenesis. Surprisingly, they were recently found to modulate important functions of mature liver. However, less is known about their mutual interactions and possible target genes in mature hepatocytes. To get a deeper insight into these interactions cultured mouse hepatocytes were transfected with siRNAs against each GLI factor. RNA was extracted at different times and the expression levels of the genes of interest were determined by quantitative real-time PCR. The time-dependent data were analysed by a fuzzy logic-based modelling approach. The results indicated that the GLI factors constitute an interconnected network. GLI2 inhibited GLI1 expression and was coupled with GLI3 by a positive feedback loop. The regulatory activity between GLI1 and GLI3 was more complex switching between a positive and a negative feedback loop depending on whether the level of GLI2 is low or high, respectively. Generally, this network structure enables a dynamic behaviour. When GLI2 is low, it may keep GLI1 and GLI3 activity balanced favouring the appropriate modulation of transcription factors like the Ppars and Srebp1. When GLI2 is high, it may prevent an uncontrolled amplification that may lead to cancer. In conclusion, the three GLI factors in mature hepatocytes form an interactive transcriptional network that is involved in the control of target genes associated with metabolic zonation as well as with lipid and drug metabolism. Its structure in mature cells seems different from embryonic cells.

Protein kinase A (PKA) accumulates at the base of the cilium where it negatively regulates the Hedgehog (Hh) pathway. Although PKA activity is essentially controlled by the cAMP produced by adenylyl cyclases, the influence of these enzymes on the Hh pathway remains unclear. Here, we show that adenylyl cyclase 5 and adenylyl cyclase 6 (AC5 and AC6, also known as ADCY5 and ADCY6, respectively) are the two isoforms most strongly expressed in cerebellar granular neuron precursors (CGNPs). We found that overexpression of AC5 and AC6 represses, whereas their knockdown activates, the Hh pathway in CGNPs and in the embryonic neural tube. Indeed, AC5 and AC6 concentrate in the primary cilium, and mutation of a previously undescribed cilium-targeting motif in AC5 suppresses its ciliary location, as well as its capacity to inhibit Hh signalling. Stimulatory and inhibitory Gα proteins, which are engaged by the G-protein-coupled receptors (GPCRs), control AC5 and AC6 activity and regulate the Hh pathway in CGNPs and in the neural tube. Therefore, we propose that the activity of different ciliary GPCRs converges on AC5 and AC6 to control PKA activity and, hence, the Hh pathway., (© 2015. Published by The Company of Biologists Ltd.)

Notochord-derived Sonic Hedgehog (Shh) is essential for dorsoventral patterning of the overlying neural tube. Increasing concentration and duration of Shh signal induces progenitors to acquire progressively more ventral fates. We show that Notch signalling augments the response of neuroepithelial cells to Shh, leading to the induction of higher expression levels of the Shh target gene Ptch1 and subsequently induction of more ventral cell fates. Furthermore, we demonstrate that activated Notch1 leads to pronounced accumulation of Smoothened (Smo) within primary cilia and elevated levels of full-length Gli3. Finally, we show that Notch activity promotes longer primary cilia both in vitro and in vivo. Strikingly, these Notch-regulated effects are Shh independent. These data identify Notch signalling as a novel modulator of Shh signalling that acts mechanistically via regulation of ciliary localisation of key components of its transduction machinery., (© 2015. Published by The Company of Biologists Ltd.)

Sonic hedgehog (Shh) is a morphogenic protein that operates through the Gli transcription factor-dependent canonical pathway to orchestrate normal development of many tissues. Because aberrant levels of Gli activity lead to a wide spectrum of diseases ranging from neurodevelopmental defects to cancer, understanding the regulatory mechanisms of Shh canonical pathway is paramount. During early stages of spinal cord development, Shh specifies neural progenitors through the canonical signaling. Despite persistence of Shh as spinal cord development progresses, Gli activity is switched off by unknown mechanisms. In this study we find that Shh inverts its action on Gli during development. Strikingly, Shh decreases Gli signaling in the embryonic spinal cord by an electrical activity- and cAMP-dependent protein kinase-mediated pathway. The inhibition of Gli activity by Shh operates at multiple levels. Shh promotes cytosolic over nuclear localization of Gli2, induces Gli2 and Gli3 processing into repressor forms, and activates cAMP-responsive element binding protein that in turn represses gli1 transcription. The regulatory mechanisms identified in this study likely operate with different spatiotemporal resolution and ensure effective down-regulation of the canonical Shh signaling as spinal cord development progresses. The developmentally regulated intercalation of electrical activity in the Shh pathway may represent a paradigm for switching from canonical to noncanonical roles of developmental cues during neuronal differentiation and maturation.

Uncontrolled Hedgehog (Hh) signaling is the cause of several malignancies, including the pediatric cancer medulloblastoma, a neuroectodermal tumor affecting the cerebellum. Despite the development of potent Hh pathway antagonists, medulloblastoma drug resistance is still an unresolved issue that requires the identification of novel drug targets. Following up on our observation that histone deacetylase 6 (HDAC6) expression was increased in Hh-driven medulloblastoma, we found that this enzyme is essential for full Hh pathway activation. Intriguingly, these stimulatory effects of HDAC6 are partly integrated downstream of primary cilia, a known HDAC6-regulated structure. In addition, HDAC6 is also required for the complete repression of basal Hh target gene expression. These contrasting effects are mediated by HDAC6's impact on Gli2 mRNA and GLI3 protein expression. As a result of this complex interaction with Hh signaling, global transcriptome analysis revealed that HDAC6 regulates only a subset of Smoothened- and Gli-driven genes, including all well-established Hh targets such as Ptch1 or Gli1. Importantly, medulloblastoma cell survival was severely compromised by HDAC6 inhibition in vitro and pharmacologic HDAC6 blockade strongly reduced tumor growth in an in vivo allograft model. In summary, our data describe an important role for HDAC6 in regulating the mammalian Hh pathway and encourage further studies focusing on HDAC6 as a novel drug target in medulloblastoma., (©2014 American Association for Cancer Research.)

Cholesterol modification of Hedgehog (Hh) ligands is fundamental for the activity of Hh signaling, and cholesterol biosynthesis is also required for intracellular Hh signaling transduction. Here, we investigated the roles and underlying mechanism of Hh signaling in metabolism of cholesterol. The main components of the Hh pathway are abundantly expressed in both human cytotrophoblasts and trophoblast-like cells. Activation of Hh signaling induces the conversion of cholesterol to progesterone (P4) and estradiol (E2) through up-regulating the expression of steroidogenic enzymes including P450 cholesterol side chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1), and aromatase. Moreover, inhibition of Hh signaling attenuates not only Hh-induced expression of steroidogenic enzymes but also the conversion of cholesterol to P4 and E2. Whereas Gli3 is required for Hh-induced P450scc expression, Gli2 mediates the induction of 3β-HSD1 and aromatase. Finally, in ovariectomized nude mice, systemic inhibition of Hh signaling by cyclopamine suppresses circulating P4 and E2 levels derived from a trophoblast-like choricarcinoma xenograft, and attenuates uterine response to P4 and E2. Together these results uncover a hitherto uncharacterized role of Hh signaling in metabolism of cholesterol., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Sonic hedgehog (Shh) functions as a conserved morphogen in the development of various organs in metazoans ranging from Drosophila to humans. Here, we have investigated the potential roles and underlying mechanisms of Shh signaling in murine placentation. Immunostaining revealed the abundant expression of the main components of Shh pathway in both the trophectoderm of blastocysts and developing placentas. Disruption of Shh led to impaired vascularogenesis of yolk sac, less branching and malformation of placental labyrinth, thereby leading to a robust decrease in capacity of transplacental passages. Moreover, placenta-specific gene incorporation by lentiviral transduction of mouse blastocysts and blastocyst transplantation robustly knocked down the expression of Gli3 and Gli2 in placenta but not in embryos. Finally, Gli3 knockdown in Shh(-/-) placentas partially rescued the defects of both yolk sac and placental labyrinth, and robustly restored the capacity of transplacental passages. Gli2 knockdown in Shh(+/)(-) placentas affected neither the capacity of tranplacental passages nor the vascularogenesis of yolk sac, however, it partially phenocopied the labyrinthine defects of Shh(-/-) placentas. Taken together, these results uncover that both Shh/Gli2 and Shh/Gli3 signals are required for proper development of murine placentas and are possibly essential for pregnant maintenance.

Binding of the Hedgehog (Hh) protein signal to its receptor, Patched, induces accumulation of the seven-pass transmembrane protein Smoothened (Smo) within the primary cilium and of the zinc finger transcription factor Gli2 at the ciliary tip, resulting ultimately in Gli-mediated changes in nuclear gene expression. However, the mechanism by which pathway activation is communicated from Smo to Gli2 is not known. In an effort to elucidate this mechanism, we identified Dlg5 (Discs large, homolog 5) in a biochemical screen for proteins that preferentially interact with activated Smo. We found that disruption of Smo-Dlg5 interactions or depletion of endogenous Dlg5 leads to diminished Hh pathway response without a significant impact on Smo ciliary accumulation. We also found that Dlg5 is localized at the basal body, where it associates with another pathway component, Kif7. We show that Dlg5 is required for Hh-induced enrichment of Kif7 and Gli2 at the tip of the cilium but is dispensable for Gpr161 exit from the cilium and the consequent suppression of Gli3 processing into its repressor form. Our findings suggest a bifurcation of Smo activity in Hh response, with a Dlg5-independent arm for suppression of Gli repressor formation and a second arm involving Smo interaction with Dlg5 for Gli activation., (© 2015 Chong et al.; Published by Cold Spring Harbor Laboratory Press.)

To feed or breathe, the oral opening must connect with the gut. The foregut and oral tissues converge at the primary mouth, forming the buccopharyngeal membrane (BPM), a bilayer epithelium. Failure to form the opening between gut and mouth has significant ramifications, and many craniofacial disorders have been associated with defects in this process. Oral perforation is characterized by dissolution of the BPM, but little is known about this process. In humans, failure to form a continuous mouth opening is associated with mutations in Hedgehog (Hh) pathway members; however, the role of Hh in primary mouth development is untested. Here, we show, using Xenopus, that Hh signaling is necessary and sufficient to initiate mouth formation, and that Hh activation is required in a dose-dependent fashion to determine the size of the mouth. This activity lies upstream of the previously demonstrated role for Wnt signal inhibition in oral perforation. We then turn to mouse mutants to establish that SHH and Gli3 are indeed necessary for mammalian mouth development. Our data suggest that Hh-mediated BPM persistence may underlie oral defects in human craniofacial syndromes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Dysregulation of the Hedgehog (Hh)-Gli signaling pathway is implicated in a variety of human cancers, including basal cell carcinoma (BCC), medulloblastoma (MB) and embryonal rhabdhomyosarcoma (eRMS), three principle tumors associated with human Gorlin syndrome. However, the cells of origin of these tumors, including eRMS, remain poorly understood. In this study, we explore the cell populations that give rise to Hh-related tumors by specifically activating Smoothened (Smo) in both Hh-producing and -responsive cell lineages in postnatal mice. Interestingly, we find that unlike BCC and MB, eRMS originates from the stem/progenitor populations that do not normally receive active Hh signaling. Furthermore, we find that the myogenic lineage in postnatal mice is largely Hh quiescent and that Pax7-expressing muscle satellite cells are not able to give rise to eRMS upon Smo or Gli1/2 overactivation in vivo, suggesting that Hh-induced skeletal muscle eRMS arises from Hh/Gli quiescent non-myogenic cells. In addition, using the Gli1 null allele and a Gli3 repressor allele, we reveal a specific genetic requirement for Gli proteins in Hh-induced eRMS formation and provide molecular evidence for the involvement of Sox4/11 in eRMS cell survival and differentiation.

Objective: Hedgehog signaling proteins were assessed in patients with cervical carcinoma receiving chemoradiation. Associations between five Hedgehog proteins and prognosis were studied., Methods: In all, 131 cases of cervical carcinomas (FIGO stages I-IV) were immunohistochemically (IHC) analyzed for Patched (PTCH), Smoothened (SMO), and GLI1, GLI2 and GLI3 protein expression. Associations between Hedgehog protein expressions, clinicopathological factors, and clinical outcome data were examined., Results: Positive IHC staining for the five Hedgehog proteins was recorded in 8% to 37% of the tumor cells. The highest frequency was noted for SMO and the lowest for GLI1. There was a significant association between low SMO- and GLI2-expression and KRAS-mutation. Tumors with overexpressed SMO had a higher frequency of residual tumor or local recurrences than tumors with low SMO expression. Patients with tumors expressing PTCH in more than 75% of the cells had significantly (P=0.023) better recurrence-free survival than patients with tumors with low expression. The opposite situation was true for SMO. For GLI2, there was a statistically significant difference with regard to overall (P=0.004) and distant (P=0.015) relapse rate for groups with expression of GLI2 in the range of 5-25% compared to higher rates., Conclusions: A predictive and prognostic value was found for PTCH, SMO, and GLI2 with regard to residual carcinoma, local recurrences, and for GLI2 distant relapses. The Hedgehog signaling pathway also seems to play an important role in cervical carcinogenesis together with HPV16-infection and KRAS-mutation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh), a morphogen that regulates the activity of Gli transcription factors. However, Shh expression is not observed during the first 12 hr of limb development. During this phase, the limb bud is prepatterned into anterior and posterior regions through the antagonistic actions of transcription factors Gli3 and Hand2. We demonstrate that precocious activation of Shh signaling during this early phase interferes with the Gli3-dependent specification of anterior progenitors, disturbing establishment of signaling centers and normal outgrowth of the limb. Our findings illustrate that limb development requires a sweet spot in the level and timing of pathway activation that allows for the Shh-dependent expansion of posterior progenitors without interfering with early prepatterning functions of Gli3/Gli3R or specification of anterior progenitors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Hedgehog signaling plays an important role in embryonic development, organogenesis and cancer. In the adult liver, Hedgehog signaling in non-parenchymal cells has been found to play a role in certain disease states such as fibrosis and cirrhosis. However, whether the Hedgehog pathway is active in mature healthy hepatocytes and is of significance to liver function are controversial., Findings: Two types of mice with distinct conditional hepatic deletion of the Smoothened gene, an essential co-receptor protein of the Hedgehog pathway, were generated for investigating the role of Hedgehog signaling in mature hepatocytes. The knockout animals (KO) were inconspicuous and healthy with no changes in serum transaminases, but showed a slower weight gain. The liver was smaller, but presented a normal architecture and cellular composition. By quantitative RT-PCR the downregulation of the expression of Indian hedgehog (Ihh) and the Gli3 transcription factor could be demonstrated in healthy mature hepatocytes from these mice, whereas Patched1 was upregulated. Strong alterations in gene expression were also observed for the IGF axis. While expression of Igf1 was downregulated, that of Igfbp1 was upregulated in the livers of both genders. Corresponding changes in the serum levels of both proteins could be detected by ELISA. By activating and inhibiting the transcriptional output of Hedgehog signaling in cultured hepatocytes through siRNAs against Ptch1 and Gli3, respectively, in combination with a ChIP assay evidence was collected indicating that Igf1 expression is directly dependent on the activator function of Gli3. In contrast, the mRNA level of Igfbp1 appears to be controlled through the repressor function of Gli3, while that of Igfbp2 and Igfbp3 did not change. Interestingly, body weight of the transgenic mice correlated well with IGF-I levels in both genders and also with IGFBP-1 levels in females, whereas it did not correlate with serum growth hormone levels., Conclusions: Our results demonstrate for the first time that Hedgehog signaling is active in healthy mature mouse hepatocytes and that it has considerable importance for IGF-I homeostasis in the circulation. These findings may have various implications for mouse physiology including the regulation of body weight and size, glucose homeostasis and reproductive capacity.

Gli proteins are transcriptional effectors of the Hedgehog (Hh) pathway in both normal development and cancer. We describe a program of multisite phosphorylation that regulates the conversion of Gli proteins into transcriptional activators. In the absence of Hh ligands, Gli activity is restrained by the direct phosphorylation of six conserved serine residues by protein kinase A (PKA), a master negative regulator of the Hh pathway. Activation of signaling leads to a global remodeling of the Gli phosphorylation landscape: the PKA target sites become dephosphorylated, while a second cluster of sites undergoes phosphorylation. The pattern of Gli phosphorylation can regulate Gli transcriptional activity in a graded fashion, suggesting a phosphorylation-based mechanism for how a gradient of Hh signaling in a morphogenetic field can be converted into a gradient of transcriptional activity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Imiquimod (IMQ), a nucleoside analogue of the imidazoquinoline family, is used in the topical treatment of basal cell carcinoma (BCC) and other skin diseases. It is reported to be a TLR7 and TLR8 agonist and, as such, initiates a Th1 immune response by activating sentinel cells in the vicinity of the tumour. BCC is a hedgehog (HH)-driven malignancy with oncogenic glioma-associated oncogene (GLI) signalling activated in a ligand-independent manner. Here we show that IMQ can also directly repress HH signalling by negatively modulating GLI activity in BCC and medulloblastoma cells. Further, we provide evidence that the repressive effect of IMQ on HH signalling is not dependent on TLR/MYD88 signalling. Our results suggest a mechanism for IMQ engaging adenosine receptors (ADORAs) to control GLI signalling. Pharmacological activation of ADORA with either an ADORA agonist or IMQ resulted in a protein kinase A (PKA)-mediated GLI phosphorylation and reduction in GLI activator levels. The activation of PKA and HH pathway target gene downregulation in response to IMQ were abrogated by ADORA inhibition. Furthermore, activated Smoothened signalling, which positively signals to GLI transcription factors, could be effectively counteracted by IMQ. These results reveal a previously unknown mode of action of IMQ in the treatment of BCC and also suggest a role for ADORAs in the regulation of oncogenic HH signalling.

Sonic hedgehog (SHH), a key regulator of embryonic neurogenesis, signals directly to neural stem cells (NSCs) in the subventricular zone (SVZ) and to astrocytes in the adult mouse forebrain. The specific mechanism by which the GLI2 and GLI3 transcriptional activators (GLI2(A) and GLI3(A)) and repressors (GLI2(R) and GLI3(R)) carry out SHH signaling has not been addressed. We found that the majority of slow-cycling NSCs express Gli2 and Gli3, whereas Gli1 is restricted ventrally and all three genes are downregulated when NSCs transition into proliferating progenitors. Surprisingly, whereas conditional ablation of Smo in postnatal glial fibrillary acidic protein-expressing cells results in cell-autonomous loss of NSCs and a progressive reduction in SVZ proliferation, without an increase in glial cell production, removal of Gli2 or Gli3 does not alter adult SVZ neurogenesis. Significantly, removing Gli3 in Smo conditional mutants largely rescues neurogenesis and, conversely, expression of a constitutive GLI3(R) in the absence of normal Gli2 and Gli3 abrogates neurogenesis. Thus unattenuated GLI3(R) is a primary inhibitor of adult SVZ NSC function. Ablation of Gli2 and Gli3 revealed a minor role for GLI2(R) and little requirement for GLI(A) function in stimulating SVZ neurogenesis. Moreover, we found that similar rules of GLI activity apply to SHH signaling in regulating SVZ-derived olfactory bulb interneurons and maintaining cortical astrocyte function. Namely, fewer superficial olfactory bulb interneurons are generated in the absence of Gli2 and Gli3, whereas astrocyte partial gliosis results from an increase in GLI3(R). Thus precise titration of GLI(R) levels by SHH is critical to multiple functions of adult NSCs and astrocytes.

The primary cilium is required for Hedgehog signaling. So far, all known ciliogenic proteins regulate Hedgehog signaling through their role in ciliogenesis. Here we show that the mouse DZIP1 regulates Hedgehog signaling through two mechanisms. First, DZIP1 interacts with GLI3, a transcriptional regulator for Hedgehog signaling, and prevents GLI3 from entering the nucleus. Second, DZIP1 is required for ciliogenesis. We show that DZIP1 colocalizes and interacts with CEP164, a protein localizing at appendages of the mother centrioles, and IFT88, a component of the intraflagellar transport (IFT) machinery. Functionally, both CEP164 and Ninein appendage proteins fail to localize to ciliary appendages in Dzip1 mutant cells; IFT components are not recruited to the basal body of cilia. Importantly, the accumulation of GLI3 in the nucleus is independent of loss of primary cilia in Dzip1 mutant cells. Therefore, DZIP1 is the first known ciliogenic protein that regulates Hedgehog signaling through a dual mechanism and that biochemically links IFT machinery with Hedgehog pathway components.

Anterioposterior vertebrate limb patterning is controlled by opposing action between Sonic Hedgehog (Shh) and the Gli3 transcriptional repressor. Unexpectedly, Gli3(Δ699) mutant mice, which are thought to express only a Gli3 repressor and not the full-length activator, exhibit limb phenotypes inconsistent with those of Shh mutant mice. Therefore, it remains debatable whether Shh patterns the anterioposterior limb primarily by inhibiting generation of the Gli3 repressor. However, one caveat is that Gli3(Δ699) may not be as potent as the natural form of Gli3 repressor because of the nature of the mutant allele. In the present study, we created a conditional Gli3 mutant allele that exclusively expresses Gli3 repressor in the presence of Cre recombinase. Using this mutant, we show that the phenotypes of mouse limbs expressing only the Gli3 repressor exhibit no or single digit, resembling those of Shh mutant limbs. Consistent with the limb phenotypes, the expression of genes dependent on Shh signaling is also inhibited in both mutants. This inhibition by the Gli3 repressor is independent of Shh. Thus, our study clarifies the current controversy and provides important genetic evidence to support the hypothesis that Shh patterns the anterioposterior limb primarily through the inhibition of Gli3 repressor formation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Preaxial polydactyly (PPD) is a common congenital abnormality and its classification varies among geneticists and hand surgeons. For example, the triphalangeal thumb, preaxial polysyndactyly, and the mirror hand deformity are considered as forms of PPD only in the genetics literature. Preaxial polydactyly is an error in the anteroposterior axis of the development of the upper limb. In this paper, the development of this axis is detailed and all molecular events that are known to lead to PPD are reviewed. Finally, based on the review, PPD is viewed as a spectrum of severity of embryonic events.

Limb development relies on an exquisite coordination between growth and patterning, but the underlying mechanisms remain elusive. Anterior-posterior and proximal-distal specification initiates in early limb bud concomitantly with the proliferative expansion of limb cells. Previous studies have shown that limb bud growth initially relies on fibroblast growth factors (FGFs) produced in the apical ectodermal ridge (AER-FGFs), the maintenance of which relies on a positive-feedback loop involving sonic hedgehog (Shh) and the BMP antagonist gremlin 1 (Grem1). The positive cross-regulation between Shh and the HoxA and HoxD clustered genes identified an indirect effect of Hox genes on the maintenance of AER-FGFs but the respective function of Shh and Hox genes in this process remains unknown. Here, by uncoupling Hox and Shh function, we show that HoxA and HoxD genes are required for proper AER-FGFs expression, independently of their function in controlling Shh expression. In addition, we provide evidence that the Hox-dependent control of AER-FGF expression is achieved through the regulation of key mesenchymal signals, namely Grem1 and Fgf10, ensuring proper epithelial-mesenchymal interactions. Notably, HoxA and HoxD genes contribute to both the initial activation of Grem1 and the subsequent anterior expansion of its expression domain. We propose that the intricate interactions between Hox genes and the FGF and Shh signaling pathways act as a molecular network that ensures proper limb bud growth and patterning, probably contributing to the coordination of these two processes.

Sonic hedgehog (Shh) signal, mediated by the Gli family of transcription factors, plays an essential role in the growth and patterning of the limb. Through analysis of the early limb bud transcriptome, we identified a posteriorly-enriched gene, Hyaluronic Acid Synthase 2 (Has2), which encodes a key enzyme for the synthesis of hyaluronan (HA), as a direct target of Gli transcriptional regulation during early mouse limb development. Has2 expression in the limb bud is lost in Shh null and expanded anteriorly in Gli3 mutants. We identified an ∼3kb Has2 promoter fragment that contains two strong Gli-binding consensus sequences, and mutation of either site abrogated the ability of Gli1 to activate Has2 promoter in a cell-based assay. Additionally, this promoter fragment is sufficient to direct expression of a reporter gene in the posterior limb mesenchyme. Chromatin immunoprecipitation of DNA-Gli3 protein complexes from limb buds indicated that Gli3 strongly binds to the Has2 promoter region, suggesting that Has2 is a direct transcriptional target of the Shh signaling pathway. We also showed that Has2 conditional mutant (Has2cko) hindlimbs display digit-specific patterning defects with longitudinally shifted phalangeal joints and impaired chondrogenesis. Has2cko limbs show less capacity for mesenchymal condensation with mislocalized distributions of chondroitin sulfate proteoglycans (CSPGs), aggrecan and link protein. Has2cko limb phenotype displays striking resemblance to mutants with defective chondroitin sulfation suggesting tight developmental control of HA on CSPG function. Together, our study identifies Has2 as a novel downstream target of Shh signaling required for joint patterning and chondrogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hedgehog signal is re-activated in several cancers. In this study, we examined the role of Gli3 on malignant phenotype of tumorigenicity for colorectal cancer and its relationship with p53, WNT and ERK/AKT signals. Gli3 expression was detected in HT29 and SW480 (p53-mutant) cells, but not in DLD-1 (p53-mutant) or HCT116 (p53-wild type) cells by reverse transcription-polymerase chain reaction and immunocytochemistry. Full-length Gli3 transfection increased anchor-independent growth for all cells regardless of p53 status, with upregulation of adhesion-related genes. Exogenous Sonic-Hedgehog increased activator-type of Gli3 and colony formation in Gli3-positive HT29 and SW480 cells. After implantation of Gli3-FL or mock-transfectant DLD-1 cells into SCID mice, tumor formation was highly observed in only Gli3-FL-transfectant group. In clinical specimens, Gli3 expression was detected in subsets of colorectal cancer and related with poorly-differentiated histological type, while Sonic-Hedgehog was present with high incidence. In conclusion, activator Gli3 signal augments tumorigenicity of colorectal cancer irrespective of p53 status., (© 2012 Japanese Cancer Association.)

Programs that control early lineage fate decisions and transitions from embryonic to adult human cell types during development are poorly understood. Using human pluripotent stem cells (hPSCs), in the present study, we reveal reduction of Hedgehog (Hh) signaling correlates to developmental progression of hematopoiesis throughout human ontogeny. Both chemical- and gene-targeting–mediated inactivation of Hh signaling augmented hematopoietic fate and initiated transitions from embryonic to adult hematopoiesis, as measured by globin regulation in hPSCs. Inhibition of the Hh pathway resulted in truncation of Gli3 to its repressor, Gli3R, and was shown to be necessary and sufficient for initiating this transition. Our results reveal an unprecedented role for Hh signaling in the regulation of adult hematopoietic specification, thereby demonstrating the ability to modulate the default embryonic programs of hPSCs.

Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2Δ43) caused mislocalization of Evc/Evc2Δ43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2(-/-) chondrocytes. Moreover, Evc silencing in Sufu(-/-) cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation.

In this study, we examined the expression of Sonic Hedgehog, Patched, Gli1, Gli2, Gli3 and Myocardin in the developing bladders of male and female normal and megabladder (mgb-/-) mutant mice at embryonic days 12 through 16 by in situ hybridization. This analysis indicated that each member of the Sonic Hedgehog signaling pathway as well as Myocardin displayed distinct temporal and spatial patterns of expression during normal bladder development. In contrast, mgb-/- bladders showed both temporal and spatial changes in the expression of Patched, Gli1 and Gli3 as well as a complete lack of Myocardin expression. These changes occurred primarily in the outer mesenchyme of developing mgb-/- bladders consistent with the development of an amuscular bladder phenotype in these animals. These results provide the first comprehensive analysis of the Sonic Hedgehog signaling pathway during normal bladder development and provide strong evidence that this key signaling cascade is critical in establishing radial patterning in the developing bladder. In addition, the lack of detrusor smooth muscle development observed in mgb-/- mice is associated with bladder-specific temporospatial changes in Sonic Hedgehog signaling coupled with a lack of Myocardin expression that appears to result in altered patterning of the outer mesenchyme and poor initiation and differentiation of smooth muscle cells within this region of the developing bladder.

Background & Aims: A fraction of gastrointestinal stromal tumor (GIST) cells overexpress the platelet-derived growth factor receptor (PDGFR)A, although most overexpress KIT. It is not known if this is because these receptor tyrosine kinases have complementary oncogenic potential, or because of heterogeneity in the cellular origin of GIST. Little also is known about why Hedgehog (HH) signaling is activated in some GIST. HH binds to and inactivates the receptor protein patched homolog (PTCH)., Methods: Ptch was conditionally inactivated in mice (to achieve constitutive HH signaling) using a Cre recombinase regulated by the lysozyme M promoter. Cre-expressing cells were traced using R26R-LacZ reporter mice. Tumors were characterized by in situ hybridization, immunohistochemistry, immunoblot, and quantitative reverse-transcriptase polymerase chain reaction analyses. Cell transformation was assessed by soft agar assay., Results: Loss of Ptch from lysozyme M-expressing cells resulted in the development of tumors of GIST-like localization and histology; these were reduced when mice were given imatinib, a drug that targets KIT and PDGFRA. The Hh signaling pathway was activated in the tumor cells, and Pdgfrα, but not Kit, was overexpressed and activated. Lineage tracing revealed that Cre-expressing intestinal cells were Kit-negative. These cells sometimes expressed Pdgfrα and were located near Kit-positive interstitial cells of Cajal. In contrast to KIT, activation of PDGFRA increased anchorage-independent proliferation and was required for tumor formation in mice by cells with activated HH signaling., Conclusions: Inactivation of Ptch in mice leads to formation of GIST-like tumors that express Pdgfrα, but not Kit. Activation of Pdgfrα signaling appears to facilitate tumorigenesis., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)