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First published online January 12, 2006
doi: 10.1242/10.1242/dev.02228

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The Sonic hedgehog pathway independently controls the patterning, proliferation and survival of neuroepithelial cells by regulating Gli activity

¹ Instituto de Biología Molecular de Barcelona, CSIC, Parc Científic de Barcelona, C/Josep Samitier 1-5, Barcelona, Spain.
² National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK.

View larger version (51K): [in a new window]   Fig. 1. Transfection of Ptc1loop2 inhibits neural cell proliferation and survival. HH11/12 stage chick embryos were electroporated in ovo with a bi-cistronic vector containing Ptc1loop2 and GFP, or with the empty vector (pCIG-GFP) as control, and assayed 12 and 24 hours post-electroporation (PE) for the indicated markers. Transfected side of the neural tube is to the right side of the figures. (A-D) 24 hours PE of Ptc1loop2 caused reduction of pH3-immunostaining on the transfected side, compared with the control contralateral side (A). BrdU incorporation is reduced in GFP-positive cells. Electroporated GFP-positive cells that have entered S phase of the cell cycle are yellow (B). TUNEL staining (brown) is increased in Ptc1loop2 (C) and activated caspase 3-immunostaining is also increased (D). (E-H) Ptc1loop2 neural tubes 12 hours PE show a reduction in pH3 immunostaining (E). BrdU incorporation is reduced in GFP-positive cells (F). TUNEL (G) and caspase 3 immunostaining (H) are not increased on the electroporated side 12 hours PE. (I-L) Control embryos transfected with the empty vector (pCIG) identified by GFP (green) and analyzed 12 hours PE, show an equal number of pH3-immunostaining cells on the electroporated side compared with non-electroporated side (I), GFP-positive cells can incorporate BrdU, as indicated by the elevated proportion of double-labeled yellow cells (J). TUNEL staining (K) and activated caspase 3 immunostaining (L) are the same on transfected side and control contralateral side. (M) Quantitative analysis of pH3-immunostained cells in electroporated and non-electroporated sides of the neural tube. 12 hours PE there was no significant changes in pH3 immunostaining; however, 24 hours PE, the number of pH3 cells in Ptc1loop2 EP embryos was significantly reduced on the transfected side of the neural tube. Error bars indicate s.d. (N) Quantitative analysis of Ptc1loop2-GFP/BrdU double labeled cells in ventral and dorsal neural tubes, after a 4-hour BrdU pulse indicated a decrease of 15% in ventral regions and 50% in dorsal regions of the number of cells incorporating BrdU by 12 hours PE, and a 60% decrease in ventral regions and 40% decrease in dorsal regions by 24 hours PE. Error bars indicate s.d. (O) Quantitative analysis of activated caspase 3-positive cells in electroporated compared with non-electroporated side. 12 hours PE there was no increase in caspase 3 immunostaining, while 24 hours PE there was a 12 fold increase in caspase 3 immunostaining in neural tubes transfected with Ptc1loop2. Error bars indicate s.d. (M-O) *P<0.05; **P<0.005; ***P<0.0001 control versus treated. (P) Summary of the effect of Ptc1loop2 on neuroepithelial cells. (Q) Schematic representation of the Shh pathway.

View larger version (82K): [in a new window]   Fig. 2. Gli transcriptional activation is required for neural cell survival. HH11/12 stage chick embryos were electroporated in ovo with a bi-cistronic vector containing the indicated plasmids and assayed, at the indicated times, for activated caspase 3 immunostaining as a marker for apoptotic cell death. (A-D) Repressor forms of Gli (Gli3R) cause high levels of apoptosis prior to inducing cell fate changes. (A,B) At 6 hours PE, Pax7 expression (red) is normal; however, caspase 3 immunostaining is highly activated on the electroporated side (B). (C,D) At 12 hours PE, Pax7 is ectopically expressed in ventral regions of the electroporated neural tube (C), together with an increased level of caspase 3 immunostaining (D). (E) Representation of Gli-variants transfected into the neural tube. Gli3AHIGH acts as a constitutive transcriptional activator. Gli3R acts as a constitutive transcriptional repressor form. Gli-ZnF inhibits both Gli repression and activation. (F-H) Expression of Gli-ZnF results in lack of activation of Nkx2.2 (F) and normal expression of Pax7 (G); however, Gli-ZnF causes increased caspase 3 immunostaining (H). (I-K) Blocking Gli-transcriptional activity does not rescue Ptc1loop2-induced apoptosis. Embryos were co-electroporated with Gli-ZnF/Myc and Ptc1loop2/GFP. (I) Immunostaining with anti-Myc (red) and GFP fluorescence (green) revealed a high proportion of cells co-transfected with both plasmids. (J,K) At 24 hours PE of Gli-ZnF+ Ptc1loop2, expression of Pax7 was normal (J); however, caspase 3 immunostaining was increased (K). (L) Gli-activator proteins induce expression of the survival gene Bcl2. At 12 hours PE of Gli3AHIGH, there is increased Bcl2 immunostaining (red) in electroporated cells (green). (M,N) Gli-activator proteins rescue Ptc1loop2-induced cell fate changes and apoptosis. (M) Embryos electroporated with Ptc1loop2+Gli3AHIGH demonstrate cell-autonomous downregulation of dorsal Pax7. Moreover, there is no increase in the level of activated caspase 3 (N). (O) Neural tubes electroporated with Gli3AHIGH were collected 12 and 24 hours PE for western blot analysis. Low levels of Bcl2 protein (29 kDa) were detected in control sides (WT). Expression of Bcl2 was moderately increased by 12 hours PE (GliA). A twofold increase in protein levels was detected 24 hours PE (GliA). Uniformity of protein loading was confirmed by uniform levels of a 35 kDa marker band. Chemiluminiscence was quantified using a Biorad Analyzer. (P,Q) Transfection of human BCL2 rescues Gli3R induced apoptosis but not cell fate changes. Embryos co-transfected with human BCL2+Gli3R had ectopic ventral expression of Pax7 (P); however, the levels of activated caspase 3 were normal in human BCL2+Gli3R co-electroporated neural tubes (Q). (R) Summary of Shh/Gli activities on neuroepithelial cell survival. (S) Quantitative analysis of caspase 3-positive cells in electroporated versus non-electroporated sides of the neural tube. 12 hours PE there was a significant increase in apoptosis induced by Gli3R this was prevented by co-electroporation of BCL2. Ptc1loop2 also significantly increase apoptosis at 12 hours PE which was prevented by the co-electroporation of either Gli-ZnF, Gli3AHIGH or human BCL2. Error bars indicate s.d. *P<0.05; **P<0.005; ***P<0.0001 control versus treated.

View larger version (105K): [in a new window]   Fig. 3. Loss of Shh/Ptc1-signaling does not inhibit or promote neuron differentiation. (A,B) HH stage 11/12 chick embryos electroporated with Ptc1loop2, were analyzed 24 hours later for GFP expression (green) and molecular markers for neural differentiation (red). (A) Immunostaining with the Tuj1 antibody (red) revealed the presence of Ptc1loop2-transfected cells expressing Tuj1 (arrows indicate co-expressing cells). (B) Immunostaining with NeuN revealed the presence of transfected cells expressing NeuN (yellow cells). (C,D) Loss of Shh/Ptc1 signaling causes cell fate changes 24 hours PE. (C) At 12 hours PE of Ptc1loop2, Pax7 expression is comparable on both sides of the neural tube. (D) At 24 hours PE, Pax7 expression is ventrally induced on the transfected side of the neural tube.

View larger version (49K): [in a new window]   Fig. 4. Repressor forms of Gli (Gli3R) cause cell cycle arrest. HH stage 11/12 embryos electroporated with the indicated plasmids, were analyzed for cell proliferation by immunostaining with the mitosis marker pH3 and by incorporation of BrdU for 1 hour, in ovo. (A-C) Repressor forms of Gli (Gli3R) cause cell cycle arrest. (A) At 12 hours PE, the number of mitotic cells is reduced in the electroporated side. (B) Few electroporated cells can enter the S phase of the cell cycle, as revealed by the number of GFP-expressing cells (green) that can incorporate BrdU (red) and thus are double labeled (yellow). (C) At 24 hours PE, the electroporated side of the neural tube is reduced and there is a clear reduction in the number of GFP/BrdU double-labeled cells. (D,E) Embryos co-transfected with Gli3R+human BCL2 and analyzed 12 hours PE display reduced pH3 staining (D) and reduced BrdU incorporation (E). (F) Embryos transfected with Bcl2 have normal rates of proliferation. (G-I) Embryos transfected with Gli-ZnF have a normal sized neural tube, and a normal rate of proliferation. (G) At 12 hours PE, pH3 immunostaining is comparable on both sides of the neural tube. A high proportion of Gli-ZnF electroporated cells can incorporate BrdU, as assessed by double labeling, either at 12 hours PE (H) or 24 hours PE (I). (J-L) Blocking Gli-transcriptional activity rescues Ptc1loop2-induced cell cycle arrest and the size reduction in the neural tube. At 12 hours PE of Ptc1loop2+Gli-ZnF, pH3-immunostained cells are comparable on both sides of the neural tube (J). A high proportion of Ptc1loop2+Gli-ZnF electroporated cells can incorporate BrdU, as assessed by double labeling, either at 12 hours PE (K) or at 24 hours PE (L). (M,N) Quantitative analysis of GFP-expressing cells (green) that have incorporated BrdU (red), after a 1 hour BrdU pulse. Analysis was carried out by separating dorsal and ventral halves of the neural tube. At 24 hours PE, the number of cells incorporating BrdU increased 15% in ventral regions and 50% in dorsal regions in Gli3AHIGH transfected embryos. (O) Summary of Shh/Gli activities on proliferation of neuroepithelial cells. *P<0.05; **P<0.005; ***P<0.0001 control versus treated.

View larger version (38K): [in a new window]   Fig. 5. Flow cytometry analysis of cell cycle phase distribution of transfected cells. HH stage 11/12 embryos were electroporated with pCIG, pCIG+human BCL2, Gli3R and Gli3R+human BCL2-containing vectors. At 12 hours PE, neural tubes were dissected out, GFP-expressing cells separated by flow cytometry and cell cycle phases analyzed by Hoescht staining. (A-C) Representative examples of cell cycle profile of cells expressing pCIG (A), Gli3R (B) and Gli3R+human BCL2 (C). The sub-G1 cell population observed in Gli3R-electroporated cells corresponds to dead and dying cells (B). (D) Cell cycle phase distribution of transfected cells. At 12 hours PE, in Gli3R transfected embryos there was a 12% increase in the number of cells accumulating at G0/G1 phase of the cell cycle. (E,F) Gli3-R repressed transcription of genes involved on G1 transition. (E) In situ hybridization with a chick cyclin D1 probe, 12 hours PE of Gli3R+human BCL2. The ventral domain of cyclin D1 expression is lacking on the electroporated side of the neural tube. (F) In situ hybridization with a chick N-myc probe, 12 hours PE of Gli3R+human BCL2. N-myc expression is repressed on the electroporated side. (G) Summary of Shh/Gli activities on cell cycle progression of neuroepithelial cells.

View larger version (49K): [in a new window]   Fig. 6. Gli3AHIGH transfection induces cell fate changes and overproliferation. HH11/12 stage embryos were electroporated with Gli3AHIGH and assayed 24 hours PE for the indicated markers. (A,B) Electroporation of Gli3AHIGH causes cell-autonomous cell-fate changes, including repression of the dorsal marker Pax7 (A) and activation of the motoneuron marker MNR2 (B). (C,D) Electroporation of Gli3AHIGH causes overgrowth of the electroporated side, together with increased pH3 staining (C) and increased BrdU incorporation (D). (E) Quantitative analysis of Gli3AHIGH/BrdU double-labeled cells in ventral and dorsal neural tubes, after a 1 hour BrdU pulse. At 24 hours PE, the number of cells incorporating BrdU increased 58% in ventral regions and 75% in dorsal regions in Gli3AHIGH transfected embryos. Error bars indicate s.d. *P<0.05; **P<0.005; ***P<0.0001 control versus treated. (F-H) Flow cytometry analysis of cell cycle phase distribution of transfected cells. pCIG and Gli3AHIGH vectors were electroporated in ovo. At 24 hours PE, neural tubes were dissected out, GFP-expressing cells separated by flow cytometry and cell cycle phases analyzed by Hoescht staining. (F,G) Representative examples of the cell cycle profile of cells expressing pCIG (F) or Gli3AHIGH (G). (H) At 24 hours PE, in Gli3AHIGH transfected embryos there was a 9% decrease in the number of cells accumulating at G0/G1 phase of the cell cycle. (I) In situ hybridization with a chick cyclin D1 probe in an embryo electroporated with Gli3AHIGH, analyzed 24 hours PE. Electroporated side is to the right. cyclin D1 expression is upregulated. (J) Summary of Shh/Gli activities on cell cycle progression of neuroepithelial cells.