Cadherin-7 enhances Sonic Hedgehog signalling by preventing Gli3 repressor formation during neural tube patterning

Abstract

Sonic Hedgehog (Shh) is a ventrally enriched morphogen controlling dorsoventral patterning of the neural tube. In the dorsal spinal cord, Gli3 protein bound to suppressor-of-fused (Sufu) is converted into Gli3 repressor (Gli3R), which inhibits Shh-target genes. Activation of Shh signalling prevents Gli3R formation, promoting neural tube ventralization. We show that cadherin-7 (Cdh7) expression in the intermediate spinal cord region is required to delimit the boundary between the ventral and the dorsal spinal cord. We demonstrate that Cdh7 functions as a receptor for Shh and enhances Shh signalling. Binding of Shh to Cdh7 promotes its aggregation on the cell membrane and association of Cdh7 with Gli3 and Sufu. These interactions prevent Gli3R formation and cause Gli3 protein degradation. We propose that Shh can act through Cdh7 to limit intracellular movement of Gli3 protein and production of Gli3R, thus eliciting more efficient activation of Gli-dependent signalling.

Keywords: Cadherin-7 (Cdh7); Gli3; Gli3 repressor; Sonic Hedgehog; neural tube patterning; suppressor-of-fused.

Figure 1.

The concentration-dependent regulation of Cdh7 expression by Shh controls specification of the Pax7⁺/Pax7⁻ neural tube boundary. (a–j) Immunostaining (a,c–e,g–j) or in situ hybridization (b,f) analyses using anti-Shh mAb (a,e), Cdh7 RNA probe (b,f), anti-Cdh7 mAb (c,g), anti-Pax7 mAb (d,h), anti-Isl1/2 mAb (i) or anti-Lim1/2 mAb (j) on transversal sections of HH st. 17 (a–d) or st. 23 (e–j) chick embryonic spinal cord. Cdh7 mRNA and Cdh7 protein are detectable in the intermediate region of the developing neural tube (black and white brackets in (b,f) and (c,g), respectively), as well as in the floor plate (f) in (f,g). At HH st. 23, Cdh7 transcription in the intermediate region is restricted to neural progenitors in the periventricular region (black brackets in (f), whereas Cdh7 protein is expressed throughout the neural tube wall (white brackets in (g)). The dorsal border of Cdh7 expression in the intermediate spinal cord abuts the ventral border of Pax7 expression in the dorsal spinal cord (cf. (c,g) with (d,h)) and coincides with the boundary between the dorsal and the ventral neural tube. At these stages, Shh is detectable in the neural tube floor plate (f) and the underlying notochord (n) (a,e). Isl1/2 is detectable in the dI3 and MN regions (i), and Lim1/2 is detectable in the dI2, dI4, dI6, V0, V1 and V2 regions (j) in HH st. 23 embryonic spinal cord; (k) shows a schematic diagram of the progenitor and neuronal domains and of the expression patterns of Shh, Cdh7, Pax7, Lim1/2 and Isl1/2 in the chick embryonic spinal cord. The ventral-to-dorsal gradient of Shh protein and the different Shh concentrations at which expression of Pax7, Cdh7, Lim1/2 and Isl1/2 is induced in spinal cord explants are shown on the right. See text for details: f, floor plate; n, notochord; Scale bars, 50 µm. (l–w) Adjacent transversal sections of HH st. 23 chick neural tube following unilateral electroporation of a chick Shh-expressing plasmid at HH st. 10 and immunostaining using anti-Shh (l,r), anti-Cdh7 (m,s), anti-Pax7 (n,t) anti-Isl1/2 (o,u) or anti-Lim1/2 (p,v) antibodies. The electroporated side is shown on the left; (q,w) shows merging of Shh (red), Cdh7 (green) and Pax7 (blue) staining as in (l,r), (m,s) and (n,t). Shh overexpression in the ventral neural tube causes coordinated dorsal shift of both Cdh7⁺ and Pax7⁺ domains. Shh overexpression in dorsal regions represses Cdh7 expression in the intermediate spinal cord and ectopically activates it in the roof of the neural tube. Green brackets in (l,q,r,w): ectopic Shh-expressing region. Red squares in (w); endogenous Shh-expressing region. White brackets in (m,q,s,w) and green squares in (w): endogenous and ectopic Cdh7-expressing regions. White arrows in (n,q,t,w) point to the boundaries between the Pax7⁺ and Pax7⁻ region. f, floor plate. Scale bar, 100 µm. (x–ac) Immunofluorescence analyses performed with anti-Cdh7 mAb (red signal, (x,z)), anti-GFP mAb (green signal, (y,z)) or anti-Shh mAb (red signal, (ab)) on transversal sections of HH st. 20 chick spinal cord tissue, following unilateral co-electroporation of plasmids expressing SmoM2 and EGFP at HH st. 10. The electroporated side is shown on the right. Quantification of the fraction of Cdh7⁺/EGFP⁺ cells in the dorsal and the intermediate spinal cord is shown in (aa). At least 10 sections from five different embryos were used for these analyses. Error bars represent s.e.m.; *p < 0.005 according to Student's t-test. SmoM2 expression causes Cdh7 upregulation in the dorsal spinal cord, but represses Cdh7 expression in the intermediate spinal cord. SmoM2 expression results in Shh upregulation within the ventral, but not the dorsal spinal cord (ab). Hoechst 33342 staining is shown in blue in (ac). Scale bars, 25 μm. (ad) Immunostaining using antibodies against Isl1/2, Lim1/2, Pax7 and Cdh7 on HH st. 10 explants of presumptive intermediate spinal cord, which were cultured for 20 h with 0, 2 or 4 nM rN-Shh. Scale bar, 100 µm. Quantification of the percentage of cells positive for the indicated markers is shown in (ae). At least each 500 cells were counted for these analyses. Error bars represent s.e.m. rN-Shh treatments cause Pax7 repression and dose-dependent upregulation of Cdh7-Lim1/2 (at 2 nM rN-Shh) or Isl1/2 (at 4 nM rN-Shh). (af) Immunostaining using antibodies against Isl1/2, Lim1/2, Pax7 and Cdh7 on HH st. 10 explants of presumptive intermediate spinal cord, which were cultured for 20 h with 0, 0.5 or 0.75 µM SAG. SAG treatments cause Pax7 repression and dose-dependent upregulation of Cdh7-Lim1/2 (0.5 µM SAG) or Isl1/2 (0.75 µM SAG). Scale bar, 100 µm.

Figure 2.

Shh-dependent Cdh7 expression controls specification of the Pax7⁺/Pax7⁻ neural tube boundary. (a–c) Immunostaining analysis with anti-Pax7 (red staining) and anti-GFP (green staining) mAbs on HH st. 15 chick neural tube, following unilateral electroporation of a control plasmid driving expression of a chimeric GFP protein fused to a nuclear localization signal; (c) shows a higher magnification of boxed regions in (a) and (b). Yellow and blue circles indicate Pax7-negative and Pax7-positive electroporated cells, respectively, within the intermediate spinal cord region (bracket). Within this region, most of the GFP-expressing cells are Pax7-positive. f, floor plate. Scale bars, 50 µm. (d–g) Immunostaining with anti-Pax7 (red staining) and anti-GFP (green staining) antibodies on HH st. 15 neural tube, following unilateral electroporation at HH st. 10 of a plasmid driving expression of both Cdh7 and a chimeric GFP protein fused to a nuclear localization signal; (f) shows a higher magnification of (d,e) at the level of the dorsal spinal cord, while (g) shows further enlargement of the electroporated intermediate spinal cord region. Cells overexpressing Cdh7 (green cells in (e), (f) and (g)) within the intermediate or the dorsal spinal cord are mostly negative or positive for Pax7, respectively. Brackets indicate the dorsal half of the spinal cord in (d), and the intermediate (bottom bracket) or the dorsal (top bracket) spinal cord regions in (f). In (f,g), yellow arrows point to electroporated cells that are Pax7⁻, whereas blue arrows point to electroporated Pax7⁺ cells. f, floor plate; so, somite. Scale bar, 100 µm for (e); 50 µm for f; 25 µm for (g). (h) Quantification of the percentage of the electroporated Pax7⁻ or Pax7⁺ cells within the intermediate spinal cord region, following electroporation of expression plasmids (EP) encoding for both Cdh7 and GFP (blue bars) or GFP only (orange bars). At least 13 sections from three different embryos were used for these analyses. Error bars represent s.e.m.; *p < 0.005 according to Student's t-test. (i–l) Immunostaining with anti-Dbx1 (red staining) and anti-GFP (green staining) antibodies on HH st. 15 neural tube, following unilateral electroporation at HH st. 10 of a plasmid driving expression of both Cdh7 and a chimeric GFP protein fused to a nuclear localization signal; (l) shows a higher magnification of the boxed area (i,j,k). Blue arrows point to electroporated Dbx1⁺ cells. Scale bar, 40 µm for (k); 20 µm for (l); (m) shows the percentage of the electroporated cells ectopically expressing Dbx1 following electroporation of EP encoding for both Cdh7 and GFP (blue bars) or GFP only (orange bars). At least 15 sections from five different embryos were used for these analyses. Error bars represent s.e.m.; *p < 0.005 according to Student's t-test. (n,o) siRNA-mediated knock-down of Cdh7 protein in L-Cdh7 cells with stable expression of Cdh7, as shown by immunoblotting (n) and immunostaining (o) analyses. In (o), cells electroporated with plasmid expressing siRNA-Cdh7 and GFP are labelled in green, while red staining shows Cdh7 expression. Scale bar, 50 µm. (p–r) Immunostaining with anti-Pax7 (red staining, p,r) or anti-GFP (green staining, q,r) mAbs in transversal sections of HH st. 15 neural tube following unilateral co-electroporation of a plasmid encoding for siRNA-control and GFP at HH st. 10. The expression domain of Pax7 is not affected in the electroporated side. Scale bar, 50 µm. (s–x) Immunostaining with anti-Pax7 (red staining) or anti-GFP (green staining) antibodies on HH st. 15 neural tube following unilateral co-electroporation of a plasmid encoding for siRNA-Cdh7 and GFP at HH st. 10. Images in (v), (w) and (x) show high magnifications of the boxed areas in (s), (t) and (u), respectively. Hoechst 33342 (blue) staining is shown in (u,x). Cdh7 knock-down causes ventral ectopic expression of Pax7 in siRNA-Cdh7/GFP-expressing cells within the intermediate spinal cord region. f, floor plate; so, somite. Scale bars, 50 µm for (u); 25 µm for (x). (y–ac) Immunostaining with anti-Cdh7 (red staining) or anti-GFP (green staining) mAbs on HH st. 15 chick neural tube following unilateral co-electroporation of a plasmid encoding for siRNA-Cdh7 and for GFP at HH st. 10. Images in (ab) and (ac) show high magnifications of the boxed areas in (y) and (aa), respectively. In the electroporated side (shown on the right), Cdh7 expression is abrogated in GFP-positive cells. f, floor plate. Scale bars, 50 µm.

Figure 3.

Cdh7 enhances Shh signalling by acting at the level of Gli3FL. (a) RLA quantification in HH st. 10 explants of presumptive intermediate spinal cord, which were dissected from embryos electroporated with GFP (control) or Cdh7 expression plasmids along with GBS-luc reporter and internal control plasmids, and treated with the indicated doses of rN-Shh as described in the electronic supplementary material, Material and methods. Cdh7 overexpression significantly enhances RLA in the presence of 2 nM rN-Shh, but not in untreated explants or in the presence of 4 nM rN-Shh. Results are shown as the mean of more than 12 independent experiments. Error bars represent s.e.m.; *p < 0.005 according to Student's t-test. (b) RLA quantification in explants that were treated as in (a) but also electroporated with siRNA-Cdh7. Cdh7 knock-down abolishes the effects of Cdh7 overexpression. (c) Schematic diagram of Myc-tagged Cdh7 full-length and two deletion constructs (delC and delN) as used for the assays shown in (d). (d) RLA quantification in explants that were treated as in (a) but electroporated with Cdh7 deletion constructs in place of full-length Cdh7. Cdh7 requires both its extracellular and intracellular regions to enhance Shh signalling. (e) RLA quantification in explants that were treated as in (a) but electroporated with constructs encoding for Ncdh or Cdh20 instead of Cdh7. Cdh7 ability to enhance Shh signalling is not shared by other cadherins. (f) RLA quantification in explants that were treated as in (a) but also exposed to DMSO or 25 nM cyclopamine-KAAD. Smo inhibition abrogates the ability of Cdh7 to enhance Shh signalling. (g) RLA quantification in explants that were treated as in (a) but electroporated with either SmoM2 or both SmoM2 and Cdh7 expression constructs. Cdh7 can further enhance Shh signalling in the presence of both SmoM2 and 2 nM rN-Shh. (h) RLA quantification in explants that were treated as in (a) but electroporated with either Gli3FL or both Gli3FL and Cdh7 expression constructs. Cdh7 can enhance Shh signalling in combination with both Gli3FL and 2 nM rN-Shh.

Figure 4.

Cdh7 binds Shh and associates with Gli3FL and Sufu. (a) Immunoblotting with anti-Cdh7 mAb (left blot) or anti-Shh pAb (right blot) showing immunoprecipitation assays with lysates of NIH3T3 cells transiently expressing chick Cdh7 that were incubated with rN-Shh, followed by pull-down with anti-Shh or anti-Cdh7 mAbs. (b) Immunoblotting with anti-Cdh7 mAb (left blot) or anti-Shh pAb (right blot) showing the results of immunoprecipitation assays with HH st. 17 chick embryo lysates, following pull-down with anti-Shh or anti-Cdh7 mAbs. Both anti-Shh and anti-Cdh7 mAbs can co-immunoprecipitate both N-Shh and Cdh7. (c) Immunoblotting with anti-Ncdh pAb (left blot) or anti-Shh pAb (right blot) showing immunoprecipitation assays with lysates of NIH3T3 cells transiently expressing chick Ncdh that were incubated with rN-Shh, followed by pull-down with anti-Shh or anti-Ncdh mAbs. No co-immunoprecipitation of N-Shh and Ncdh is detectable. (d) Immunoblotting with anti-Myc pAb (left blot) or anti-Shh pAb (right blot) showing immunoprecipitation assays with lysates of NIH3T3 cells transiently expressing Myc-tagged chick Cdh20 that were incubated with rN-Shh, followed by pull-down with anti-Shh or anti-Myc mAbs. No co-immunoprecipitation of N-Shh and Cdh20 is detectable. (e) AP staining of control (left) or Cdh7-expressing (right) COS-7 cells that were incubated with conditioned medium from 293 cells expressing N-Shh-AP. N-Shh-AP signal is detectable only in Cdh7-expressing cells. Scale bar, 200 µm. (f) Saturation binding curve (inset) and Scatchard analysis of N-Shh-AP binding to Cdh7, showing a dissociation constant (Kd) of 4.8 nM. (g) Schematic diagram of Myc-tagged Cdh7 full-length and deletion constructs as used for the assays shown in (h). (h) Immunoblotting with anti-Myc pAb showing immunoprecipitation assays with lysates from COS-7 cells transiently expressing Cdh7 constructs shown in (f). Lysates were incubated with rN-Shh, followed by pull-down with anti-Shh mAb. Only constructs containing CR1 and CR2 domains are pulled down by anti-Shh mAb. The blot at the bottom shows the expression levels of each Cdh7 construct in lysates used for immunoprecipitation (input). (i–m) Immunoblotting with anti-Cdh7 mAb (i), anti-Gli3FL pAb (j), anti-Sufu pAb (k), anti-Gli3 pAb (l) or anti-GSK3β pAb (m) showing immunoprecipitation assays with NIH3T3 cells transiently expressing Cdh7 that were incubated with the indicated doses of rN-Shh, followed by pull-down with anti-Cdh7 mAb. Following cell treatment with rN-Shh, anti-Cdh7 mAb can co-immunoprecipitate Gli3FL and Sufu along with Cdh7, but not Gli3R and GSK3β. Note that anti-Gli3FL pAb used for (j) does not react with Gli3R, while anti-Gli3 pAb used for (l) reacts with both Gli3FL and Gli3R, and only the Gli3R 80 kDa band is shown. (n–p) Immunoblotting with anti-Gli3FL pAb (n), anti-Cdh7 mAb (o) or anti-Ncdh pAb (p) showing immunoprecipitation assays with HH st. 17 spinal cord lysates using anti-Cdh7 or anti-Ncdh mAbs. Anti-Cdh7 mAb, but not anti-Ncdh mAb, can co-immunoprecipitate both Gli3FL (n) and Cdh7 (o).

Figure 5.

Shh promotes aggregation of Cdh7. (a–d) Immunofluorescence analysis performed with anti-Cdh7 mAb (red signal) using NIH3T3 cells transiently expressing Cdh7 that were cultured for 24 h in the indicated concentrations of rN-Shh. rN-Shh promotes Cdh7 aggregation at the cell membrane. Scale bar, 10 µm. (b) High magnifications of the yellow boxed areas in (a). (c) Distribution of fluorescence intensities in the representative images shown in (b) of Cdh7-expressing cells cultured without or with 2 nM or 4 nM rN-Shh. Charts report the number of pixels falling within a given range of fluorescence intensity. Cdh7 signal varies much more broadly in rN-Shh-treated samples in comparison with the untreated sample. (d) Box-and-whisker plots of the standard deviation of fluorescence intensities in Cdh7-expressing cells treated with the indicated concentrations of rN-Shh. Standard deviation of Cdh7 signal is significantly higher in rN-Shh-treated cells in comparison with control samples. Whiskers represent the distribution of the standard values for the fluorescence intensity of each image. The lower and higher whiskers indicate the minimum and maximum values, respectively. The bottom and top of the box represent the first and third quartiles, respectively, and the band inside the box indicates the second quartile (the median). At least 15 representative cells for each experimental condition were used for this analysis; *p < 0.005 according to Student's t-test. (e–h) Immunofluorescence analysis performed with anti-Ncdh mAb (red signal) using NIH3T3 cells transiently expressing Ncdh that were cultured for 24 h in the indicated concentrations of rN-Shh. rN-Shh treatments do not cause Ncdh aggregation at the cell membrane. (f) High magnifications of the yellow boxed areas in (e). (g) Distribution of fluorescence intensities in the representative images shown in (e) of Ncdh-expressing cells cultured without or with 2 nM or 4 nM rN-Shh. Charts report the number of pixels falling within a given range of fluorescence intensity. Ncdh signal spans a similar range of intensities in rN-Shh-treated samples in comparison with the untreated sample. (h) Box-and-whisker plots of the standard deviation of fluorescence intensities in Ncdh-expressing cells treated with the indicated concentrations of rN-Shh. Standard deviation of Ncdh signal is not significantly different in rN-Shh-treated cells in comparison with control samples. (i–v) Immunofluorescence analyses performed with anti-Cdh7 Ab ((i–l), red signal), anti-β-catenin Ab ((m–p), red signal) or anti-Ncdh Ab ((q–t), red signal) on transversal sections of HH st. 20 chick spinal cord tissue, showing aggregation of Cdh7 (blue arrows in (l)), but not β-catenin (p) or Ncdh (t), in cells within the intermediate spinal cord region. (k,o,s) show high magnification images of (i,m,q), respectively; (l,p,t) show high magnification images of the yellow boxed regions in (k,o,s), respectively. Hoechst 33342 staining is shown in blue in (j,n,r). Scale bar, 100 µm in (i), 50 µm in (k) and 10 µm in (l,p,t). (u,v) Distribution of fluorescence intensities in the representative images shown in (l,p,t). (v) Box-and-whisker plots of the standard deviation of fluorescence intensities in (l,p,t). Standard deviation of Cdh7 signal is significantly higher than that of β-catenin or Ncdh signal; *p < 0.005 according to Student's t-test. (w–ac) Immunofluorescence analysis performed with anti-Cdh7 mAb (red signal) on transversal sections of HH st. 23 chick spinal cord, which shows ectopic expression of Cdh7 in the dorsal neural tube following unilateral electroporation of a Cdh7-expressing construct at HH st. 10. The electroporated side is shown on the left. Lower levels of Cdh7 aggregates are detectable in dorsal spinal cord cells within ectopic Cdh7 expression (w,x,y) in comparison with Cdh7 aggregates present in the intermediate spinal cord (z,aa); (x,y,aa) show high magnification images of the boxed regions in (w,z) as indicated. Scale bar, 50 µm. (ab) Distribution of fluorescence intensities in the representative images shown (x,y,aa). (ac) Box-and-whisker plots of the standard deviation of fluorescence intensities in (x,y,aa). Standard deviation of Cdh7 signal is significantly higher in the intermediate than in the dorsal spinal cord region; *p < 0.005 according to Student's t-test.

Figure 5.

Shh promotes aggregation of Cdh7. (a–d) Immunofluorescence analysis performed with anti-Cdh7 mAb (red signal) using NIH3T3 cells transiently expressing Cdh7 that were cultured for 24 h in the indicated concentrations of rN-Shh. rN-Shh promotes Cdh7 aggregation at the cell membrane. Scale bar, 10 µm. (b) High magnifications of the yellow boxed areas in (a). (c) Distribution of fluorescence intensities in the representative images shown in (b) of Cdh7-expressing cells cultured without or with 2 nM or 4 nM rN-Shh. Charts report the number of pixels falling within a given range of fluorescence intensity. Cdh7 signal varies much more broadly in rN-Shh-treated samples in comparison with the untreated sample. (d) Box-and-whisker plots of the standard deviation of fluorescence intensities in Cdh7-expressing cells treated with the indicated concentrations of rN-Shh. Standard deviation of Cdh7 signal is significantly higher in rN-Shh-treated cells in comparison with control samples. Whiskers represent the distribution of the standard values for the fluorescence intensity of each image. The lower and higher whiskers indicate the minimum and maximum values, respectively. The bottom and top of the box represent the first and third quartiles, respectively, and the band inside the box indicates the second quartile (the median). At least 15 representative cells for each experimental condition were used for this analysis; *p < 0.005 according to Student's t-test. (e–h) Immunofluorescence analysis performed with anti-Ncdh mAb (red signal) using NIH3T3 cells transiently expressing Ncdh that were cultured for 24 h in the indicated concentrations of rN-Shh. rN-Shh treatments do not cause Ncdh aggregation at the cell membrane. (f) High magnifications of the yellow boxed areas in (e). (g) Distribution of fluorescence intensities in the representative images shown in (e) of Ncdh-expressing cells cultured without or with 2 nM or 4 nM rN-Shh. Charts report the number of pixels falling within a given range of fluorescence intensity. Ncdh signal spans a similar range of intensities in rN-Shh-treated samples in comparison with the untreated sample. (h) Box-and-whisker plots of the standard deviation of fluorescence intensities in Ncdh-expressing cells treated with the indicated concentrations of rN-Shh. Standard deviation of Ncdh signal is not significantly different in rN-Shh-treated cells in comparison with control samples. (i–v) Immunofluorescence analyses performed with anti-Cdh7 Ab ((i–l), red signal), anti-β-catenin Ab ((m–p), red signal) or anti-Ncdh Ab ((q–t), red signal) on transversal sections of HH st. 20 chick spinal cord tissue, showing aggregation of Cdh7 (blue arrows in (l)), but not β-catenin (p) or Ncdh (t), in cells within the intermediate spinal cord region. (k,o,s) show high magnification images of (i,m,q), respectively; (l,p,t) show high magnification images of the yellow boxed regions in (k,o,s), respectively. Hoechst 33342 staining is shown in blue in (j,n,r). Scale bar, 100 µm in (i), 50 µm in (k) and 10 µm in (l,p,t). (u,v) Distribution of fluorescence intensities in the representative images shown in (l,p,t). (v) Box-and-whisker plots of the standard deviation of fluorescence intensities in (l,p,t). Standard deviation of Cdh7 signal is significantly higher than that of β-catenin or Ncdh signal; *p < 0.005 according to Student's t-test. (w–ac) Immunofluorescence analysis performed with anti-Cdh7 mAb (red signal) on transversal sections of HH st. 23 chick spinal cord, which shows ectopic expression of Cdh7 in the dorsal neural tube following unilateral electroporation of a Cdh7-expressing construct at HH st. 10. The electroporated side is shown on the left. Lower levels of Cdh7 aggregates are detectable in dorsal spinal cord cells within ectopic Cdh7 expression (w,x,y) in comparison with Cdh7 aggregates present in the intermediate spinal cord (z,aa); (x,y,aa) show high magnification images of the boxed regions in (w,z) as indicated. Scale bar, 50 µm. (ab) Distribution of fluorescence intensities in the representative images shown (x,y,aa). (ac) Box-and-whisker plots of the standard deviation of fluorescence intensities in (x,y,aa). Standard deviation of Cdh7 signal is significantly higher in the intermediate than in the dorsal spinal cord region; *p < 0.005 according to Student's t-test.

Figure 6.

Cdh7 interacts with Sufu and collaborates with Shh to prevent Gli3R formation. (a–s) Immunostaining analysis with anti-Cdh7 (green signal) or anti-Sufu (red signal) antibodies using NIH3T3 cells transiently expressing Cdh7 that were cultured for 24 h in the indicated concentrations of rN-Shh. rN-Shh promotes association of Cdh7 aggregates with Sufu (d,e,f,j,k,l); (m,n) show high magnification images of the boxed areas in (l); (o,p) show high magnification images of the boxed areas in (j,k), respectively. Scale bar, 50 µm in (i,j), 10 µm in (n). (q,r) Surface plots showing quantification of fluorescence intensities in (o,p); (s) shows the merging of the plots in (q,r) (t–as). Immunostaining analysis with anti-GFP (green signal) or anti-V5 (red signal) antibodies using NIH3T3 cells transiently expressing a Gli3 chimeric construct tagged with GFP at the N-terminus and V5 at the C-terminus. Both control (t–af) or cells transiently expressing Cdh7 (ag–as) were treated for 18 h with the indicated concentrations of rN-Shh. Nuclear GFP staining is detectable in both control and Cdh7-expressing cells in the absence of rN-Shh (t–v,ag–ah), but not in cells treated with 4 nM rN-Shh (z–ab,am–ao). At 2 nM rN-Shh, nuclear GFP localization is present in control cells (w–y) but not in Cdh7-expressing cells (aj–al), suggesting that Cdh7 can effectively prevent Gli3R formation at these lower rN-Shh levels. Scale bar, 50 µm. (ac–ae) Surface plots of fluorescence intensities in (w–y); (af) shows the merging of the plots in (ac–ae). (ap–as) Surface plots of fluorescence intensities in (aj, ak, al); (as) shows the merging of the plots in (aj–al). (at–ay) Immunoblotting of nuclear extracts of NIH3T3 cells transiently expressing a Gli3 construct tagged with V5 at the C-terminus, using antibodies against the Gli3 N-terminal region (at,aw) or V5 (au,ax). Blots in (at,aw) show the 80 kDa Gli3R band, while the 190 kDa Gli3FL band is shown in (au,ax). Control (at–av) or Cdh7-expressing cells (au–ax) were treated with the indicated concentrations of rN-Shh. In the absence of rN-Shh, nuclear extracts from both control and Cdh7-expressing cells clearly show the 80 kDa Gli3 N-terminus-positive band, indicating that Gli3R formation takes place in these conditions. The 190 kDa V5-positive band is weakly detectable in the nuclear extract from control cells. Treatments of control cells with increasing rN-Shh levels cause progressive decrease of the ratio between the 80 kDa Gli3 N-terminus-positive and 190 kDa V5-positive bands, indicating dose-dependent inhibition of Gli3FL processing to Gli3R. In Cdh7-expressing cells, the 80 kDa band is clearly decreased and the 190 kDa band is undetectable in rN-Shh-treated samples, indicating that Cdh7 effectively prevents Gli3R production and leads to Gli3 degradation in the presence of rN-Shh. Transfection efficiency in each condition is shown by immunoblotting using anti-GFP mAb (av,ay), following co-transfection of a GFP control plasmid.

Figure 7.

Models of Cdh7 roles in the regulation of Shh signalling and neural tube patterning. (a) Model of Cdh7-dependent specification of the Pax7⁺/Pax7⁻ neural tube boundary via regulation of Gli3R production and Pax7 expression. Shh promotes Cdh7 expression in the intermediate spinal cord region and collaborates with Cdh7 to prevent processing of Gli3 to Gli3R and Pax7 expression. Dorsal to the Cdh7 expression domain, Gli3 is converted into Gli3R, indirectly leading to Pax7 expression (dashed arrow). Repression of Cdh7 expression by Pax7 helps to maintain a sharp boundary between the Cdh7⁺/Pax7⁻ ventral spinal cord and the Cdh7⁻/Pax7⁺ dorsal spinal cord. BMPs and Wnts promote both Gli3 and Pax7 expression in the dorsal spinal cord. (b,c) Models of the regulation of Gli-dependent signalling in cells exposed to low/moderate levels of Shh in the absence (dorsal spinal cord cell, (b)) or in the presence (ventral spinal cord cell, (c)) of Cdh7. In the dorsal spinal cord (Cdh7 non-expressing cell), low/moderate Shh levels do not effectively prevent the Gli3FL/Sufu complex from transiting through the base of the primary cilium, where Gli3FL becomes partially phosphorylated and converted into Gli3R, leading to expression of dorsal transcription factors (D-TFs) and repression of intermediate and ventral neural tube TFs (I-TFs, V-TFs) (b). In ventral spinal cord cells (Cdh expressing cell), low/moderate Shh levels collaborate with Cdh7 to retain the Gli3FL/Sufu complex in the cytoplasm, thus preventing Gli3R formation and leading to Gli3 degradation and expression of I-TFs (c). Higher Shh levels are needed for expression of V-TFs (not shown). See text for further details.