Proliferation of Murine Midbrain Neural Stem Cells Depends upon an Endogenous Sonic Hedgehog (Shh) Source

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.     

Kinetic and structural studies on interactions between heparin or heparan sulfate and proteins of the hedgehog signaling pathway

Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signaling proteins, thereby playing an essential role in the regulation of many physiological processes. These interactions are important for both normal signal transduction and regulation of the tissue distribution of signaling molecules. In this study, we use surface plasmon resonance (SPR) to study interactions of HS and structurally related heparin with proteins in the Hedgehog signaling pathway. SPR analysis shows that heparin binds with different affinities to active fragments of the proteins Hedgehog (Hh), Interference Hedgehog (Ihog), Cam-related/Down-regulated by Oncogenes (CDO), and Sonic Hedgehog (Shh). Solution competition studies show that the minimum size of a heparin oligosaccharide capable of interacting with Ihog is larger than a tetrasaccharide and for interacting with Shh is larger than an octasaccharide. In comparison with heparin, Ihog and Shh exhibited a lower affinity for HS than for heparin, and CDO and Hh exhibit negligible binding to HS. This study clearly demonstrates Shh and Ihog are heparin and HS binding proteins and that both molecules preferentially bind heparin or HS having a high level of sulfation.     

The decoupling of Smoothened from Galphai proteins has little effect on Gli3 protein processing and Hedgehog-regulated chick neural tube patterning

The Hedgehog (Hh) signal is transmitted by two receptor molecules, Patched (Ptc) and Smoothened (Smo). Ptc suppresses Smo activity, while Hh binds Ptc and alleviates the suppression, which results in activation of Hh targets. Smo is a seven-transmembrane protein with a long carboxyl terminal tail. Vertebrate Smo has been previously shown to be coupled to Gα_i proteins, but the biological significance of the coupling in Hh signal transduction is not clear. Here we show that although inhibition of Gα_i protein activity appears to significantly reduce Hh pathway activity in Ptc^−/− mouse embryonic fibroblasts and the NIH3T3-based Shh-light cells, it fails to derepress Shh- or a Smo-agonist-induced inhibition of Gli3 protein processing, a known in vivo indicator of Hh signaling activity. The inhibition of Gα_i protein activity also cannot block the Sonic Hedgehog (Shh)-dependent specification of neural progenitor cells in the neural tube. Consistent with these results, overexpression of a constitutively active Gα_i protein, Gα_i2QL, cannot ectopically specify the neural cell types in the spinal cord, whereas an active Smo, SmoM2, can. Thus, our results indicate that the Smo-induced Gα_i activity plays an insignificant role in the regulation of Gli3 processing and Shh-regulated neural tube patterning.     

The 3-beta-hydroxysteroid-Delta(8), Delta(7)-isomerase EBP inhibits cholesterylation of Smoothened

Hedgehog (Hh) pathway plays a central role in vertebrate embryonic development and carcinogenesis. The G-protein coupled receptor-like protein Smoothened (SMO) is one of the major members in Hh pathway. Covalent modification of cholesterol on the 95th asparagine (D95) of human SMO, which is regulated by Hh and PTCH1, is critical for SMO activation. However, it is not known whether SMO cholesterylation is regulated by other proteins. In this study, we identified Emopamil binding protein (EBP, also known as 3-beta-hydroxysteroid-Delta(8),Delta(7)-isomerase) as a SMO-interacting protein. Overexpression of EBP suppressed SMO cholesterylation and Hh pathway activity, whereas genetic disruption of EBP enhanced SMO cholesterylation and the downstream signaling. EBP-mediated inhibition of SMO cholesterylation was independent of its isomerase activity, but dependent on the C-terminus of EBP that was required for SMO binding. The X-linked dominant chondrodysplasia punctate 2 (CDPX2)-associated EBP mutants inhibited SMO cholesterylation too. Together, this study shows that EBP modulates SMO cholesterylation through direct binding and suggests a possible mechanism of CDPX2 pathogenesis.     

Negative regulation of Hedgehog signaling by the cholesterogenic enzyme 7-dehydrocholesterol reductase

Cholesterol regulates Hedgehog (Hh) signaling during early vertebrate development. Smith-Lemli-Opitz syndrome (SLOS) is caused by defects in 7-dehydrocholesterol reductase (DHCR7), an enzyme catalyzing the final step of cholesterol biosynthesis. Many developmental malformations attributed to SLOS occur in tissues and organs where Hh signaling is required for development, but the precise role of DHCR7 deficiency in this disease remains murky. We report that DHCR7 and Sonic Hedgehog (Shh) are co-expressed during midline development in Xenopus embryos. DHCR7 has previously been implicated to function as a positive regulator of Hh signaling that acts to regulate the cholesterol adduction of Hh ligand or to affect Hh signaling in the responding cell. We present gain- and loss-of-function analyses suggesting that DHCR7 functions as a negative regulator of Hh signaling at the level or downstream of Smoothened (Smo) and affects intracellular Hh signaling. Our analysis also raises the possibility that the human condition SLOS is caused not only by disruption of the enzymatic role of DHCR7 as a reductase in cholesterol biosynthesis, but may also involve defects in DHCR7 resulting in derepression of Shh signaling.     

Sonic Hedgehog基因及其在发育过程中的调控作用

Sonic Hedgehog(Shh)基因属于Hedgehog(Hh)基因家族,该家族最早在果蝇体内被发现,进化上呈高度保守状态。Sonic Hedgehog定位在7号染色体长臂远端(7q36),其通过细胞表面特殊受体Patched(Ptc)和Smoothened(Smo)被接收和传导,从而激活锌指蛋白C i/G li家族。Sonic Hedgehog基因作为重要的形态发生素,在胚胎发育、机体器官组织形成的过程中发挥了重要的作用,它的缺失或者失活会导致一系列严重的遗传疾病。其与体节、神经管、消化道、头面部、上下肢芽的发育以及肿瘤形成等有密切关系。本文主要就Sonic Hedgehog基因及其在发育中的调控作用作一综述。     

Roles for Hedgehog signaling in androgen production and prostate ductal morphogenesis

Previous studies have demonstrated that the Hedgehog (Hh) signaling pathway plays a critical role in the development and patterning of many endodermally derived tissues. We have investigated the role of Sonic hedgehog (Shh) in formation of the prostate gland by examining the urogenital phenotype of Shh mutant fetuses. Consistent with earlier work reporting an essential role for Shh in prostate induction, we have found that Shh mutant fetuses display abnormal urogenital development and fail to form prostate buds. Unexpectedly, however, we have discovered that this prostate defect could be rescued by three different methods: renal grafting, explant culture in the presence of androgens, and administration of dihydrotestosterone (DHT) to pregnant mice, indicating that the prostate defect in Shh mutants is due to insufficient levels of androgens. Furthermore, we find that the inhibition of Hh pathway signaling by treatment with cyclopamine does not block prostate formation in explant culture, but instead produces morphological defects consistent with a role for Hh signaling in ductal patterning. Taken together, our studies indicate that the initial organogenesis of the prostate proceeds independently of Shh, but that Shh or other Hh ligands may play a role in subsequent events that pattern the prostate.     

Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.     

Multiple roles for Hedgehog signaling in zebrafish pituitary development

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.     

Heparan sulfate proteoglycans exert positive and negative effects in Shh activity

Hedgehog (Hh) proteins are morphogens involved in short- and long-range effects during early embryonic development. Genetic analysis in fly and vertebrate embryos showed that heparan sulfate proteoglycans (HSPGs) are required for Hh transport and signaling. To further understand how HSPGs regulate Sonic hedgehog (Shh), we performed experiments using cell culture and biochemical assays. When the synthesis of HSPGs was reduced, a decrease in Shh activity was observed. Contrary to that, addition of a peptide that competes the binding of Shh to HSPGs resulted in augmentation of Shh activity. From these results, we concluded that HSPGs exert positive and negative effects in Shh activity. This dual effect correlates with the finding that Shh interacts preferentially with two HSPGs. The current model for the role of HSPGs in Shh diffusion is discussed in view of our findings.     

Sonic Hedgehog-induced proliferation requires specific Gα inhibitory proteins

Proliferation of cerebellar granular neuronal precursors (CGNPs) is mediated by Sonic Hedgehog (Shh), which activates the Patched and Smoothened (Smo) receptor complex. Although its protein sequence suggests that Smo is a G protein coupled receptor (GPCR), the evidence that this receptor utilizes heterotrimeric G proteins as downstream effectors is controversial. In Drosophila, Gα(i) is required for Hedgehog (Hh) activity, but the involvement of heterotrimeric G proteins in vertebrate Shh signaling has not yet been established. Here, we show that Shh-induced proliferation of rat CGNPs is enhanced strongly by the expression of the active forms of Gα(i/o) proteins (Gα(i1), Gα(i2), Gα(i3), and Gα(o)) but not by members of another class (Gα(12)) of heterotrimeric G proteins. Additionally, the mRNAs of these different Gα(i) members display specific expression patterns in the developing cerebellum; only Gα(i2) and Gα(i3) are substantially expressed in the outer external granular layer, where CGNPs proliferate. Consistent with this, Shh-induced proliferation of CGNPs is reduced significantly by knockdowns of Gα(i2) and Gα(i3) but not by silencing of other members of the Gα(i/o) class. Finally, our results demonstrate that Gα(i2) and Gα(i3) locate to the primary cilium when expressed in CGNP cultures. In summary, we conclude that the proliferative effects of Shh on CGNPs are mediated by the combined activity of Gα(i2) and Gα(i3) proteins.     

Sonic hedgehog is produced by follicular dendritic cells and protects germinal center B cells from apoptosis

The Hedgehog (Hh) signaling pathway is involved in the development of many tissues during embryogenesis, but has also been described to function in adult self-renewing tissues. In the immune system, Sonic Hedgehog (Shh) regulates intrathymic T cell development and modulates the effector functions of peripheral CD4(+) T cells. In this study we investigate whether Shh signaling is involved in peripheral B cell differentiation in mice. Shh is produced by follicular dendritic cells, mainly in germinal centers (GCs), and GC B cells express both components of the Hh receptor, Patched and Smoothened. Blockade of the Hh signaling pathway reduces the survival, and consequently the proliferation and Ab secretion, of GC B cells. Furthermore, Shh rescues GC B cells from apoptosis induced by Fas ligation. Taken together, our data suggest that Shh is one of the survival signals provided by follicular dendritic cells to prevent apoptosis in GC B cells.     

The whereabouts of a morphogen: direct evidence for short- and graded long-range activity of hedgehog signaling peptides

Sonic Hedgehog (Shh) and Indian Hedgehog (Ihh) are members of the Hedgehog (Hh) family of signaling molecules known to be involved in embryonic patterning and morphogenesis. The Hh proteins undergo an autocatalytic cleavage to yield an N-terminal and a C-terminal peptide, with the signaling capacities confined to the N peptide. Drosophila Hh-N has been shown to act via both short- and long-range signaling. In vertebrates, however, attempts to directly demonstrate Shh (SHH) or Ihh (IHH) proteins at a distance from producing cells have been largely unsuccessful. Furthermore, the fact that the Hh N peptides occur in a cholesterol-modified, membrane-tethered form is not easily reconciled with long-range signaling. This study used optimized immunohistochemistry combined with tissue separation and biochemical analyses in vivo and in vitro to determine the range of action of SHH and IHH in the mouse embryo. In all embryonic structures studied, we detect signaling peptides in producing cells, but we also find that ligands move over considerable distances depending on the tissue. These data provide direct evidence for the presence of Hedgehog signaling peptides in target compartments, suggesting a direct long-range action without a need for secondary mediators. Visualization of Hedgehog proteins in target tissues was achieved only under conditions that allowed proteoglycan/glycosaminoglycan (PG/GAG) preservation. Furthermore, we show that induced changes of the composition of PG/GAG in the tooth alter SHH signaling. These data suggest a crucial role for PG/GAGs in Hedgehog movement.     

Midline signaling and evolution of the forebrain in chordates: a focus on the lamprey Hedgehog case

Lampreys are agnathans (vertebrates without jaws). They occupy a key phylogenetic position in the emergence of novelties and in the diversification of morphology at the dawn of vertebrates. We have used lampreys to investigate the possibility that embryonic midline signaling systems have been a driving force for the evolution of the forebrain in vertebrates. We have focused on Sonic Hedgehog/Hedgehog (Shh/Hh) signaling. In this article, we first review and summarize our recent work on the comparative analysis of embryonic expression patterns for Shh/Hh, together with Fgf8 (fibroblast growth factor 8) and Wnt (wingless-Int) pathway components, in the embryonic lamprey forebrain. Comparison with nonvertebrate chordates on one hand, and jawed vertebrates on the other hand, shows that these morphogens/growth factors acquired new expression domains in the most rostral part of the neural tube in lampreys compared to nonvertebrate chordates, and in jawed vertebrates compared to lampreys. These data are consistent with the idea that changes in Shh, Fgf8 or Wnt signaling in the course of evolution have been instrumental for the emergence and diversification of the telencephalon, a part of the forebrain that is unique to vertebrates. We have then used comparative genomics on Shh/Hh loci to identify commonalities and differences in noncoding regulatory sequences across species and phyla. Conserved noncoding elements (CNEs) can be detected in lamprey Hh introns, even though they display unique structural features and need adjustments of parameters used for in silico alignments to be detected, because of lamprey-specific properties of the genome. The data also show conservation of a ventral midline enhancer located in Shh/Hh intron 2 of all chordates, the very species which possess a notochord and a floor plate, but not in earlier emerged deuterostomes or protostomes. These findings exemplify how the Shh/Hh locus is one of the best loci to study genome evolution with regards to developmental events.     

A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube.

Cell pattern in the ventral neural tube is organized by Sonic hedgehog (Shh) secreted by floor plate cells. To assay the range of direct Shh action, we developed a general method for blocking transduction of Hedgehog (Hh) signals through ectopic expression of a deleted form of the Hh receptor Patched (Ptc), termed Ptc(Deltaloop2). We validated this method in Drosophila and used mouse Ptc1(Deltaloop2) (mPtc1(Deltaloop2)) to block Shh transduction in the chick neural tube. mPtc1(Deltaloop2) expression caused cell-autonomous ventral-to-dorsal switches in progenitor identity and neuronal fate throughout the ventral neural tube, supporting a gradient mechanism whereby Shh acts directly and at long range. mPtc1(Deltaloop2) expression also caused the abnormal spread of Shh to more dorsal cells, indicating that Shh in the neural tube, like Hh in Drosophila, induces a feedback mechanism that limits its range of action.     

Inhibition of the hedgehog pathway targets the tumor-associated stroma in pancreatic cancer

Purpose: The Hedgehog (Hh) pathway has emerged as an important pathway in multiple tumor types and is thought to be dependent on a paracrine signaling mechanism. The purpose of this study was to determine the role of pancreatic cancer-associated fibroblasts (human pancreatic stellate cells, HPSCs) in Hh signaling. In addition, we evaluated the efficacy of a novel Hh antagonist, AZD8542, on tumor progression with an emphasis on the role of the stroma compartment. Experimental Design: Expression of Hh pathway members and activation of the Hh pathway were analyzed in both HPSCs and pancreatic cancer cells. We tested the effects of Smoothened (SMO) inhibition with AZD8542 on tumor growth in vivo using an orthotopic model of pancreatic cancer containing varying amounts of stroma. Results: HPSCs expressed high levels of SMO receptor and low levels of Hh ligands, whereas cancer cells showed the converse expression pattern. HPSC proliferation was stimulated by Sonic Hedgehog with upregulation of downstream GLI1 mRNA. These effects were abrogated by AZD8542 treatment. In an orthotopic model of pancreatic cancer, AZD8542 inhibited tumor growth only when HPSCs were present, implicating a paracrine signaling mechanism dependent on stroma. Further evidence of paracrine signaling of the Hh pathway in prostate and colon cancer models is provided, demonstrating the broader applicability of our findings. Conclusion: Based on the use of our novel human-derived pancreatic cancer stellate cells, our results suggest that Hh-targeted therapies primarily affect the tumor-associated stroma, rather than the epithelial compartment. Mol Cancer Res; 10(9); 1147-57. ?2012 AACR.