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 29 March 2006
doi: 10.1242/dev.02339

Development 133, 1799-1809 (2006)
Published by The Company of Biologists 2006
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 Related articles in Development
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 Blaess, S.
Right arrow Articles by Joyner, A. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blaess, S.
Right arrow Articles by Joyner, A. L.
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?

Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region

Sandra Blaess1,2, JoMichelle D. Corrales1,2 and Alexandra L. Joyner1,2,3,*

1 Howard Hughes Medical Institute and Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, 540 First Avenue, New York, NY 10016, USA.
2 Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.
3 Department of Physiology and Neuroscience, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA.


Figure 1
View larger version (79K):

[in a new window]
  		Fig. 1. Mid/hindbrain phenotype in the absence of total or Gli2A-mediated Shh signaling. (A-L) Dorsal view of whole-mount brains (A-F), and Hematoxylin and Eosin staining of midline sagittal sections (G-L) of P0 wild-type (control) and cko embryos or E18.5 null embryos. (B,H) In Shh-null mutants, cerebral cortex (Ctx), dorsal midbrain (tectum, Tec), ventral Mb (tegmentum, Teg) and ventral anterior hindbrain (vHb) are not discernible, and the cerebellum (Cb) is abnormal. (H, inset) Calbindin-positive Cb Purkinje cells (red, arrow) and Hoechst staining (blue) of the area indicated by the square. A cell dense layer probably corresponds to the Cb external granule cells (arrowhead). (C,I) In Smo-En1 cko mutants, the size of the Mb/Cb is reduced, the ventricle is absent (arrow) and the Tec is not divided into superior (SC) and inferior colliculi (IC). (D,J) In Smo-Nes cko mutants, the IC is truncated (arrow) and the Cb is reduced in size. The Ctx is also reduced in size. (E,F,K,L) In Gli2-null and Gli2-En1 cko mutants, the Teg, vHb and Cb are reduced in size. The Tec thins in Gli2-null mutants because of hydrocephaly. (M-R) Hematoxylin and Eosin staining of midline sagittal sections of E12.5 wild-type (control) and mutant embryos. (N) In Shh-null mutants, the mes/r1 is severely reduced in size. Dorsal and ventral neural tube is joined at the isthmus (Is) (arrow). (O) A close apposition of ventral and dorsal isthmus (vIs, dIs) is visible in Smo-En1 cko mutants (arrow), and dorsal mes (d-mes) and r1 (d-r1) are truncated. (P) In Smo-Nes cko embryos, d-r1 and the posterior d-mes are reduced in size (arrow), but no obvious defect is observed in ventral mes (v-mes) or r1 (v-r1). (Q,R) There is no obvious dorsal phenotype in Gli2-null or Gli2-En1 cko mutants, but ventral mes/r1 is altered (arrows). Scale bars: 700 µm in A-F; 300 µm in G-L; 75 µm in M-R.

 

Figure 2
View larger version (131K):

[in a new window]
  		Fig. 2. Distinct temporal dependence of medial-to-lateral-derived ventral cell types on Shh signaling. (A-H) Immunohistochemistry for dopaminergic (Th, green) and serotonergic (5-HT, red) neurons on E18.5 sagittal sections. Sections are counterstained with Hoechst (blue), the mid/hindbrain is outlined. (A-H) Both cell types are greatly reduced in Smo-En1 cko and Gli2-En1 cko but not in Smo-Nes cko mutants (arrows). The number of serotonergic neurons in the posterior hindbrain is normal (arrowheads). Arrowhead in C indicates Th-positive neurons in the locus coeruleus. (I-P) In situ hybridization for Nkx2.2 and Isl1 on horizontal sections of E10.5 and E12.5 mes. (I-L) The ventral (V) populations of Isl1- and Nkx2.2-positive cells are not induced in Smo-En1 cko mes (K,L, red arrowheads). (M-P) In Smo-Nes cko mes, Isl1- and Nkx2.2-positive cells are generated but slightly reduced (O,P, red arrowheads). (J,L,N,P) Dorsal (D) Isl1-expressing cells in control and mutants (black arrowheads). (Q) The plane of section (I-P) is indicated in the schematic. Scale bars: 50 µm.

 

Figure 3
View larger version (62K):

[in a new window]
  		Fig. 3. Increased apoptosis following inactivation of Shh signaling at E9.0. TUNEL assay on control, Smo-En1 cko and Smo-Nes cko mutants. (A-D) Apoptotic cells (green) are increased in E9.5 Smo-En1 cko mutants, particularly in the anterior mes and around the isthmus (B, arrowheads) but not in the ventral midline (D, red arrowhead). (E,F) A few apoptotic cells are found in control and E12.5 Smo-Nes cko mutant embryos (arrowheads), but cell death is not increased in the mutants. Hoechst counterstaining is in blue. (G) Shown areas are indicated in the schematic. V, ventral; D, dorsal. Scale bars: 25 µm.

 

Figure 4
View larger version (62K):

[in a new window]
  		Fig. 4. Regulation of Gli3 repressor (Gli3R) levels is required for dorsal mes/r1 development and growth. (A-D) Hematoxylin and Eosin staining of E18.5 and E12.5 sagittal sections. (A,B) Dorsal mes/r1 development is severely affected in the Gli3Xt/Xt-null mutants. (A) At E18.5, IC and SC are not discernible and the Cb is reduced in size and abnormal. (B) The dorsal isthmus (d-Is) and r1 region are enlarged at E12.5. (C,D) The Shh-/- phenotype (compare Fig. 1H,N) is partially rescued by removing one copy of Gli3. Ventral (Teg/vHb and v-mes/r1) and dorsal structures are present and dorsal Mb appears to be organized into IC and SC. Insets in A and C show calbindin-positive Purkinje cells in the Cb. (E) The N-terminal Gli3R form is increased whereas full-length Gli3 (Gli3F) is decreased in Shh-/- brain compared with Shh+/+ or Shh+/- mes/r1 at E12.5. Gli3F is slightly reduced in Shh+/- mes/r1. Whole brain extracts were used for Shh-/- mutants, as the size of the mes/r1 is severely reduced by E12.5. Scale bars: 300 µm in A,C; 75 µm in B,D.

 

Figure 5
View larger version (134K):

[in a new window]
  		Fig. 5. Changing requirement of Gli2A- and Gli3R-mediated Shh signaling in maintaining gene expression domains in ventral and dorsal mes/r1. RNA in situ hybridization with Shh, Gli1, Gli3 and Pax7 probes. The analysis was performed on representative sections along the AP axis of the mes/r1; sections shown are at the level of the posterior mes (see Fig. 3G). V, ventral; D, dorsal. The mes is outlined where necessary. (A-E) Shh is induced and maintained in the mes ventral midline (VM) of Smo cko and Gli2-En1 cko mutants (B,D,E, arrowheads) and comparable with controls (A, inset in E), but is absent from the mes VM in Gli2-null mutants (C, red arrowhead). Inset in C shows that Shh is induced in the midline of the ventral diencephalon (di) (arrow) similar to control embryos (A). (F-J) The ventrolateral expression of Gli1 observed in control sections (F, inset in J) is lost in Smo cko mutants (G,J, red arrowheads), but is induced weakly in Gli2-null and Gli2-En1 cko mutants (H,I, arrowheads). Gli1 is expressed in mesenchyme (m) in E9.5 control and mutants. (K-T) Gli3 and Pax7 are expressed in dorsolateral mes in control (K,P) but are extended ventrally to the VM in Smo-En1 cko mes (L,Q, arrows). Expression of both markers remains dorsally restricted in Smo-Nes cko mutants (O,T, arrows). In Gli2-null and Gli2-En1 cko mutant mes, Pax7 expression remains restricted (R,S, arrows), but Gli3 expression is expanded ventrally, albeit in a dorsal-to-ventral gradient (M,N). This results in overlapping Gli3 and Gli1 expression domains (H,I,M,N, arrowheads). Scale bars: 25 µm.

 

Figure 6
View larger version (80K):

[in a new window]
  		Fig. 6. Reduction in Fgf8 expression and signaling in the absence of Shh. RNA in situ hybridization for Fgf8, Spry1 and Wnt1. Analysis was performed on midline sections, as ventral and dorsal isthmus are fused just off the midline in Smo-En1 cko mutants (B, inset). (A-F) Dorsal and most posterior mes, isthmus (Is) and r1 are shown (see Fig. 3G) and are outlined where necessary. The thickness of this region is reduced in E10.5 mutants. (A-D) The Fgf8 and Spry1 domains are severely reduced in Smo-En1 cko mutants (B,D arrows). (E,F) Wnt1 is expanded posteriorly in Smo-En1 cko mutants (arrows). (G-J) Posterior mes, Is and r1 of E12.5 control and Smo-Nes cko mutants. The dorsal Fgf8 and Spry1 domains are slightly reduced in the Smo-Nes cko mutants and Fgf8 is shifted posteriorly (arrows). Scale bars: 25 µm.

 

Figure 7
View larger version (41K):

[in a new window]
  		Fig. 7. Changing temporal requirement for total and Gli2A- and Gli3R-mediated Shh signaling in mes/r1 development. Schematic representation of wild-type, Shh-null, Smo-En1 cko, Smo-Nes cko, Gli2-null, Gli2-En1 cko and Gli2-Nes cko mutant mes/r1 phenotypes. The neural tube at E9.5 or E12.5 at the level of the posterior mes is depicted on the left-hand side; a schematic drawing of the midbrain, cerebellum and anterior ventral hindbrain at E18.5/P0 is indicated on the right side of each panel. The range of Gli2A-mediated Shh signaling (green arrow) and Gli3R-mediated Shh signaling (red line) is indicated. Light green dots indicate weak Gli1 expression/GliA; the purple gradient shows Gli3R levels (dark to light indicating high to low).

 

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 2006