spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 9 July 2008
doi: 10.1242/dev.015891

Development 135, 2717-2727 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Komada, M.
Right arrow Articles by Ishibashi, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Komada, M.
Right arrow Articles by Ishibashi, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Hedgehog signaling is involved in development of the neocortex

Munekazu Komada1, Hirotomo Saitsu1,*, Masato Kinboshi1, Takashi Miura1, Kohei Shiota1,2 and Makoto Ishibashi1,{dagger}

1 Departmant of Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
2 Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.


Figure 1
View larger version (100K):
[in this window]
[in a new window]

  		Fig. 1. The size of the dorsal telencephalon is reduced in Shh-CKO and Smo-CKO embryos. Dorsal views of wild-type (A), Shh-CKO mutant (A') and Smo-CKO (A'') mutant brains at E18.5. The appearance of the mutant brains was grossly normal. Coronal sections of wild-type (B), Shh-CKO (B') and Smo-CKO (B'') telencephalons at E18.5 were stained with HE. These panels illustrated that the size of the dorsal telencephalon was slightly, but significantly reduced in the mutants (see Table 1). Note that the thickness of the mutant cortices is slightly thinner than that of the wild-type cortex (asterisk). The angle of the lower extension of the lateral ventricle (arrow in B) is almost perpendicular in wild type, while that of the mutants is medially inclined (arrow in B',B''). By contrast, the ventral parts were not apparently affected. In situ hybridization of Smo on E18.5 wild-type (C) and Smo-CKO mutant (C') embryo brains. The expression of Smo was undetected specifically in the dorsal telencephalon in the mutant brain. Scale bar: 1 mm in A-A''; 200 µm in B-B''; 100 µm in C,C'.

 

Figure 2
View larger version (53K):
[in this window]
[in a new window]

  		Fig. 2. Proliferation of progenitor/stem cells in the dorsal pallium is reduced and apoptosis is increased in the absence of Shh signaling. BrdU-incorporation analysis at E13.5 (A-C) and E15.5 (A'-C'). Cells were counted in 100 µm-wide sampling boxes (white boxes). Examples of anti-BrdU labeling (red) of parasagittal sections of the dorsal pallium of wild-type (A,A'), Shh-CKO mutant (B,B') and Smo-CKO mutant (C,C') embryos. (D) Both Shh-CKO and Smo-CKO embryos showed significantly decreased BrdU incorporation index at E13.5 (13.2±1.26% and 14.4±0.29%, respectively; n=3) compared with wild type (16.8±0.47%, n=3). At E15.5, Smo-CKO showed significantly decreased index (7.78±0.10%; n=3), while Shh-CKO showed comparable index (12.04±1.15%, n=3) to wild-type embryos (11.75±1.60%, n=3). Parasagittal sections of E13.5 dorsal pallium were immunostained with anti-cleaved caspase 3 antibody (E-G) and cells were counted on the entire sections (H). There were a few stained cells (red) in wild-type brain (E,H, 0.35±0.118%; n=3). Apoptotic cells were increased in the dorsal pallium of Shh-CKO (F,H, 1.33±0.075%; n=3) and Smo-CKO embryos (G,H, 1.07±0.210%; n=3). *P<0.01. Scale bar: 50 µm.

 

Figure 3
View larger version (81K):
[in this window]
[in a new window]

  		Fig. 3. Loss of Shh signaling leads to prolonged cell cycle length and reduced cell cycle exit of the neural progenitor/stem cells. (A-C') After 1-hour pulse labeling of BrdU, immunostaining of parasagittal sections was performed with anti-Ki67 (green) and anti-BrdU (red) antibodies at E13.5 (A-C) and E15.5 (A'-C'). (G) Cell cycle length was estimated as percentage of Ki67 and BrdU double-positive cells in all Ki67-positive cells. Smaller percentage represents longer cell cycle. At E13.5, both Shh-CKO and Smo-CKO embryos showed significantly prolonged cycle length (26.5±0.005% and 23.0±0.004%, respectively, n=3) compared with wild-type embryos (33.0±0.034%, n=3) (A-C,G). At E15.5, cell cycle length of Shh-CKO was not significantly prolonged (19.6±0.005%, n=3) whereas that of Smo-CKO was significantly longer (15.4±0.003%, n=3) than that of wild type (21.5±0.027%, n=3) (A'-C',G). (D-F') To estimate cell cycle exit, sections were stained with anti-Ki67 and anti-BrdU antibodies 24 hours after BrdU pulse (E13.5, D-F; E15.5, D'-F'). (H) Cell cycle exit was determined as a ratio of the cells that exited the cell cycle (red, BrdU+/Ki67-, no longer dividing) to all cells labeled with BrdU (red+yellow) after 24 hours labeling. In Shh-CKO embryos, the ratio was significantly decreased at E13.5 (0.131±0.008; wild type, 0.198±0.005; n=3), but not different at E15.5 (0.230±0.021, n=3; wild type, 0.205±0.016, n=3). In Smo-CKO embryos, the ratio was significantly decreased compared with wild type at both E13.5 and E15.5 (0.168±0.002 and 0.069±0.013, respectively; n=3). *P<0.01. Scale bar: 50 µm.

 

Figure 4
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 4. Positioning of postmitotic neurons was affected in CKO mutants. (A-C) Birth-date analysis was carried out by CldU and IdU double staining of E18.5 the dorsal pallium sections after a single pulse of CldU at E13.5 and IdU at E15.5. (D-I) Quantification of CldU- and IdU-positive cell index in each Bin. In wild-type embryos, E13.5-born neurons (CldU+, red) mostly settled down in the deep cortical layers with a peak in Bin4 (**29.92±2.45%; n=3, in D) and E15.5-born neurons (IdU+, green) distributed mainly to the superficial layer, Bin5 (#14.12±2.51%; n=3, in G). In Shh-CKO embryos, E13.5-neurons positioned similarly to wild type though neurons in Bin4 were significantly decreased (**11.24±1.89% in E compared with D; n=3, P<0.01). E15.5 neurons in Shh-CKO were positioned in the almost same pattern as in wild type (compare G with H). In Smo-CKO embryos, E13.5 neurons were differently scattered throughout the dorsal pallium (Bin1****, 4.06±0.669%; Bin2***, 18.38±3.69%; Bin5*, 28.07±0.76%; n=3, P<0.01 in F) compared with wild type (Bin1****, 0.09±0.09%; Bin2***, 2.56±1.86%; Bin5*, 14.00±3.29%; n=3, in D). E15.5 cells mostly remained in SVZ/VZ (Bin1 and 2 in I) and significantly decreased in CP (Bin3##, 3.63±1.97%; n=3, P<0.05 and Bin5#, 1.51±0.38%; n=3, P<0.01 in I) compared with wild type (Bin3##, 7.37±1.02% and Bin5#, 14.12±2.51%; n=3, in G). Scale bar: 50 µm.

 

Figure 5
View larger version (114K):
[in this window]
[in a new window]

  		Fig. 5. Shh signaling was required for formation of the deep cortical layer and subplate. Cresyl violet staining of the E18.5 dorsal pallium (A-C). In situ hybridization on parasagittal sections of the E18.5 dorsal pallium with Wnt7b (D-F) and Tbr1 probes (G-I). In wild type, Wnt7b and Tbr1 were intensely expressed in the deep cortical layer and SP, and weakly expressed in SVZ/VZ, but not in IZ (D,G). In Shh-CKO, the unstained gap of Wnt7b expression was not observed (E). Tbr1 expression was comparable with wild type (H). In Smo-CKO, Wnt7b expression in SP and SVZ/VZ was undetectable (F). Tbr1 expression in SP and SVZ/VZ was not detected either (I). MAP2 and chondroitin sulfate were detected by immunohistochemistry. In wild type and Shh-CKO, MAP2 (J-L) and chondroitin sulfate (CS56) (M-O) expression in SP neurons was detectable at E18.5 and E15.5, respectively (J,K; arrow in M, N). In Smo-CKO, MAP2 and Chondroitin sulfate were weakly expressed in SP neurons at E18.5 (L) and E15.5 (arrow in O), respectively. Scale bar: 100 µm.

 

Figure 6
View larger version (93K):
[in this window]
[in a new window]

  		Fig. 6. Overexpression of Shh in utero leads to expansion of CP and SVZ/VZ. pCIG-Shh expression vector was electroporated into the dorsal pallium in utero at E12.5. At E18.5, EGFP was detected mostly in CP on the electroporated side (A). Note that the dorsal pallium was expanded in a planar direction on the electroporated side (brackets in B). Ki67-positive proliferating cells in SVZ/VZ were greatly increased so that SVZ/VZ was almost twice the thickness as on the control side (bracket in C,C'). In situ hybridization on coronal sections of E18.5 dorsal pallium with Wnt7b (D,D') and Tbr1 probes (E,E'). On the electroporated side, the non-expression regions in SVZ/VZ were expanded compared with the control side (bracket in D-E'). TBR2-positive progenitors were greatly increased in the SVZ on the electroporated side (F,F') and their distribution was disturbed (arrows in F'). Scale bar: 100 µm in B; 50 µm in C-F'.

 

Figure 7
View larger version (139K):
[in this window]
[in a new window]

  		Fig. 7. Localization of Shh protein in the developing dorsal pallium. Shh protein was localized in MZ and SVZ/VZ of the wild-type dorsal pallium at E13.5 (A); there are few Shh-positive cells in Shh-CKO mutants (A'). At E15.5, Shh immunoreactivity was also found additionally in IZ and CP (B). Note that MZ and IZ were more intensely stained than other zones at E15.5. In Shh-CKO mutants, the Shh-immunoreactivity was sustained in IZ and MZ (B'). N-cadherin was intensely expressed in IZ (D), and Shh protein (C) was colocalized with N-cadherin in IZ (E). Reelin and calretinin, CR cell markers, were detected in some of Shh-positive cells in MZ at E13.5 (F,G, arrows). Shh protein was co-expressed in GABAergic interneurons in MZ at E15.5 (H, arrows). Sections were counterstained with DAPI (dark blue nuclei). The antibodies and colors used in each panel are as indicated. Scale bar: 100 µm in A-E; 50 µm in F,G.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008