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The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis

Nadia Dahmane1,*, Pilar Sánchez1, Yorick Gitton1, Verónica Palma1, Tao Sun1, Mercedes Beyna1, Howard Weiner2 and Ariel Ruiz i Altaba1,{dagger}

1 Skirball Institute of Biomolecular Medicine, Developmental Genetics Program and Department of Cell Biology, NYU School of Medicine, 540 First Avenue, New York, NY 10016, USA
2 Department of Neurosurgery, NYU School of Medicine, 540 First Avenue, New York, NY 10016, USA
* Present address: Institute for Developmental Biology, CNRS, Marseille, France



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  		Fig. 1. Perinatal expression of Gli and Shh in the mouse dorsal brain. (A,D,G) Expression of Gli1 (A), Gli2 (D) and Gli3 (G) in the vz/svz of the cerebral neocortex (Ctx) and striatum (Str) at embryonic day (E) 15.5. The three Gli genes are also co-expressed in the vz of the striatum and olfactory bulb (D and not shown). (B,E,H) Expression of Gli1 (B), Gli2 (E) and Gli3 (H) in the cerebral cortex (Ctx) at E17.5. Gli gene expression is detected near the ventricle (v) with Gli1 also expressed in scattered cells within the cortical plate. (C,F,I) Expression of Gli1 (C), Gli2 (F) and Gli3 (I) in the midbrain at E17.5. Expression of the Gli genes is regionalized in the vz of the tectum (Tct) and tegmentum (Tgt). Expression is also detected in the EGL and Purkinje layer of the cerebellum (Cb). Shh is also detected in the tegmentum as well as in the amygdala (not shown). (J) RT-PCR analyses of Shh expression in the parietal neocortex of E14.5-postnatal day (P)3 mice (right) and of its expression at E17.5 in the neocortex (Nctx), striatum (Str), superior colliculus (SC), inferior colliculus (IC) and cerebellum (Cb). A no RT control is also added (Nctx-RT). Hprt is used to control for RNA levels. (K-M) Shh is expressed in a layer-specific manner in the P2 neocortex (Ctx; K,L) and cingulate cortex (Cc; K), as well as in the tectum (Tct) and cerebellum (Cb), where it is found in the Purkinje layer. Shh is also expressed in the hippocampal dentate gyrus (Dg, also detected by RT-PCR, not shown). Similar expression was detected at P1 and P5. (N,O) Gli1 expression is found in the vz/svz of the cortex and in scattering cells (O) in a P5 mouse brain. Gli1 expression is also prominent in the dentate gyrus (Dg) of the hippocampus (Hip). Similar expression was detected at P1 and P5. (P) Expression of the oligodendrocyte precursor marker Pdgfr{alpha} in scattered cells and in a subset of cells near the ventricle. (Q-S) High magnification of the similar expression patterns of Gli1 (Q), Ptch1 (R) and Pdgfr{alpha} (S) in scattering cells in the P5 cortex. Arrows point to sites of expression. v, ventricle. All panels show sagittal sections except N and the inset in K, which display coronal sections.

 


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  		Fig. 2. SHH upregulates Gli1 transcription and promotes proliferation of vz cells in perinatal mouse neocortical explants. (A) RT-PCR analyses of gene expression in untreated control (con) or SHH-treated (+SHH) mouse E17.5 cerebral cortical explants and meninges (m). One-fifth the concentration of SHH (1 nM) induced a smaller increase in Gli1 expression (not shown). The expression of the housekeeping gene Hprt is used as internal quantitative control. Dorsal brain-derived SHH could affect adjacent cells of the meninges, which express Gli1. (B) RT-PCR analyses of gene expression in untreated control or SHH-treated mouse P3 cerebral cortical explants. At this time, the choroid plexus expresses low levels of both Gli1 and Shh (not shown). (C) Sketch of a lateral view of a ~P3 mouse brain showing the position of explanted tissue within the parietal neocortex. (D,E) In situ hybridization analyses on cryostat sections of P3 mouse cerebral cortex explants left untreated (D) or grown in the presence of SHH (E). Each panel shows a representative section. vs, ventricular surface. (F-J) Localization of BrdU-positive cells in the vz of a P3 mouse cortical explants left untreated (F), or treated with SHH (G), anti-SHH blocking monoclonal antibody (H), dideoxyforskolin (I) or forskolin (J). ps, pial surface; vs, ventricular surface. (K) Quantification of cell proliferation by SHH treatment or inhibition of SHH signaling in P3 mouse cortical explants. Numbers of cells±s.e.m. are given. n>10 sections of six independent explants counted for each condition. Ten independent explants were used for RT-PCR in two separate experiments, and four or five independent explants for in situ analyses.

 


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  		Fig. 3. SHH induces gene expression and proliferation in tectal explants. (A) Sketch of a lateral view of a ~P2 mouse brain showing the position of explanted tissue within the tectum. (B) RT-PCR analyses of gene expression in untreated control (con) or SHH-treated (+SHH) mouse P2 superior (SC) or inferior (IC) colliculi explants. Expression of Gli1 and Ptch1 is clearly upregulated. The low expression of Gli1 is barely detectable in the IC but is clearly seen after additional cycles. (C,D) Increase in the number of BrdU-positive cells in IC explants treated with SHH (C) when compared with an untreated IC explant (D). ps, pial surface; vs, ventricular surface. Seven independent explants were used for RT-PCR from each the IC and SC. (E) Quantification of cell proliferation by SHH treatment or inhibition of SHH signaling on P2 mouse IC explants. Numbers of cells±s.e.m. are given. n>10 sections of five independent explants counted for each condition.

 


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  		Fig. 4. SHH induces proliferation of nestin-positive cells in the postnatal mouse cerebral neocortex. (A,B) Co-labeling of P3 mouse cortical dissociated cells with anti-nestin and anti-BrdU antibodies. In both control (A) and SHH-treated (B) samples, dividing cells express cytoplasmic nestin (arrows). Nuclei are counterstained with DAPI. Response to SHH induction in dissociated cells included upregulation of Ptch1 (not shown). (C,D) Labeling of dissociated cells with anti-GFAP and anti-BrdU antibodies. BrdU-labeled cells (arrows) do not express GFAP in control (C) or SHH-treated (D) samples. Nuclei are counterstained with DAPI. (E) Quantification of labeling results with dissociated cells. Numbers are percentages of single- or double-labeled cells±s.e.m. over the total number of cells as seen by DAPI staining. n>1000 cells counted for each sample.

 


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  		Fig. 5. Decreased precursor proliferation in the brain of Shh null embryos. (A) Morphological appearance of an E18.5 Shh null (right) and a wild-type littermate (left). Arrow points to the position of the forebrain, immediately posterior to the proboscis. (B,C) Details of the ventricular areas of the neocortex of wild-type (B) and in the forebrain of E18.5 Shh null embryos seen in sagittal sections (C) showing the marked decrease in proliferation in the vz/ svz in the mutant. (D,E) Expression of nestin in the vz/svz of both wild-type (D) and mutant (E) cortices. (F,G) Expression of the neuronal tubulin marker Tuj1 (red) in the neocortical plate of a wild-type embryo (F) and in the cortex region of a Shh-null sibling (G). (H,I) The characteristic proliferation of EGL cells in the E18.5 wild-type cerebellum (H) is missing in a sibling mutant brain (I), where no structure is morphologically recognizable as a cerebellum adjacent to the posterior choroid plexus (chp). (J,K) Proliferation of interfollicular cells appears normal in the wild-type (J) and mutant (K) epidermis (ep). Hair follicles (hf) are absent in the mutants. (L) Lateral views of dissected E15.5 wild-type (bottom) and Shh mutant (top) brains showing the existence of a morphologically defined cortex (Ctx), tectum (Tct) and medulla (Me) areas, as well as an elongated proboscis (Pb) anteriorly. The corresponding parts of the mutant brain and the wild-type littermate are indicated by white lines. Note the apparent presence of superior (SC) and inferior (IC) colliculi in both brains, but the apparent absence of the cerebellum (Cb, arrowhead) in the mutant. The olfactory bulb (OB) is also indicated in the normal brain. D, dorsal; Nctx, neocortex; V, ventral. (M) Camera lucida drawings of the outlines of sagittal sections of the brains of an E18 wild-type embryo (left) and a Shh-null sibling (right) at scale. The red lines and arrowheads indicate the source and deduced action of SHH protein in the neocortex, tectum and cerebellum.

 


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  		Fig. 6. GLI gene expression in human brain tumors, cell lines and effects of cyclopamine. (A,B) RT-PCR analyses of independent brain tumor samples. Note the varying levels of GLI1 and GLI2 expression and its general correlation with the levels of PTCH1. HFB, human fetal brain RNA, used here as control; PNETs, primitive neuroectodermal tumors. Varying levels of two GLI2 bands in B represent previously described differentially spliced forms (arrows) (Tanimura et al., 1998). Levels of expression are interpreted in relation to that of discoidin domain receptor 1 (DDR1) mRNA, used to measure the relative amount of tumor in a given sample (Weiner et al., 2000), assuming homogenous expression per tumor cell. Expression of the housekeeping gene GAPDH served as positive control to quantify the total amount of mRNA. A total of 22 DDR1-positive primary tumors were tested by RT-PCR. Of these, nine glioblastoma multiformes from the frontal, parietal or temporal lobes expressed GLI1 (9/9), GLI2 (7/9), GLI3 (9/9), PTCH1 (7/9) and SHH (7/9). Co-expression of GLI1 and SHH could suggest that the origin of these gliomas is the GLI1- and SHH-positive SVZ of the lateral ventricle (N. D., D. Lim, A. Álvarez-Buylla, A. R. A, unpublished); one gliosarcoma from the temporal lobe and one anaplastic oligodendroglioma from the parietal lobe expressed all these genes; one low grade glioma from the insula expressed GLI1, GLI3, PTCH1 but not GLI2 or SHH; four PNETs from the posterior fossa expressed GLI1, GLI3 and PTCH1; one PNET from the thalamus expressed all of these genes; and six PNETs from the cerebellum expressed all genes except SHH. Ages of the individuals with gliomas ranged from 20 to 74 years and those with PNETs from 2 to 38 years. There was no correlation of gene expression with gender. (C-H) In situ hybridization of cortical glioma (C-F) and cerebellar PNET (G,H) tumor sections showing the expression of GLI1 (C,E,G) coincident with that of PTCH1 (F,H). Sense GLI1 RNA probes did not show specific hybridization (D; six tumors tested). The localization of regions with tumor was determined by the high levels of DDR1 expression and the histopathological examination of Hematoxylin- and Eosin-stained sections (not shown). Matched slides of the same tumor are C,D, E,F and G,H. GBM, glioblastoma multiforme; LGA, low grade astrocytoma. A total of 22 DDR1-positive tumors were tested by in situ hybridization. Of these, seven GBMs from the temporal and parietal lobes expressed GLI1 (7/7) and PTCH1 (6/7); one oligodendroastrocytoma from the temporal lobe and one oligodendroglioma from the frontal lobe expressed both genes; two low grade gliomas from the cerebellum and centrum ovale expressed GLI1 (2/2) and PTCH1 (1/2); five juvenile pilocytic astrocytomas from the cerebellum, thalamus and hypothalamus expressed GLI1 (4/5) and PTCH1 (2/5); one anaplastic oligodendroglioma from the frontal lobe expressed both genes; two anaplastic astrocytomas from the frontal lobe expressed GLI1 (2/2) and PTCH1 (1/2); and three PNETs from the cerebellum, posterior fossa and occipital lobe expressed both genes. The age of individuals with glial tumors ranged from 6 to 60 years and those with PNETs from 3 to 17 years. There was no correlation of gene expression with gender. As controls, one ependymoma from the fourth ventricle and one hemangioma from the cerebellum did not express GLI1 or PTCH1. (I) RT-PCR analyses of human brain tumor cell lines testing for the expression of GLI1, PTCH1 and GAPDH. All cells tested express both GLI1 and PTCH1. (J) Percentage inhibition of the proliferation of four human glioma cell lines by cyclopamine at the concentrations indicated as measured by BrdU incorporation.

 


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  		Fig. 7. Gli1 induces hyperplasia in the CNS of frog embryos. (A) Histological appearance of a Gli1-induced hyperplasia at the level of the hindbrain after sectioning and staining with Hematoxylin and Eosin. The unilateral development of hyperplasia is a consequence of the injection of Gli1 RNA into one cell at the two-cell stage, using the uninjected half of the brain as internal control. (B) Section of a Gli1-injected embryo showing the ectopic differentiation of HNF-3ß-positive cells within the hyperplastic region at the level of the midbrain. (C) Lineage tracing of a GLI1-induced hyperplasia in the neural tube. The section shows X-gal staining, indicating the development of a tumor from cells inheriting the co-injected GLI1 and lacZ RNAs. (D) Analyses of BrdU incorporation in a GLI1-injected embryo showing a large increase in the number of BrdU-positive cells in the hyperpastic side (arrows) versus the uninjected, control side. (E,F) Endogenous expression of Pdgfr{alpha} mRNA in the midventricular zone of the diencephalon (E), and its ectopic expression within the CNS hyperplasia of a Gli1-injected embryo (F). (G,H) Constitutive expression of endogenous Gli1 mRNA in hyperplastic regions (G, spinal cord; H, hindbrain) induced by injected human GLI1. (I,J) Hyperplasia resulting from the of injection of human GLI1 RNA (I) or it complete absence after injection of GLI1 RNA plus morpholino oligonucleotide anti-frog Gli1. X-Gal-stained cross sections are shown. The distribution of X-gal-labeled cells in J is as expected for animal pole injection. n, notochord; ov, otic vesicle; s, somites. All panels show representative cross sections of embryos at stages ~34-38 except A, which show sections of embryos at stage ~40. Arrows indicate induced CNS hyperplasia and/or sites of gene expression within them. Broken lines show the axis of normal bilateral symmetry. The CNS and notochord are independently outlined. The size of the notochord in all sections serves as scale. fp, floor plate; n, notochord; s, somite; v, ventricle; v', secondary ventricle.

 

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