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 September 2004
doi: 10.1242/dev.01424

Development 131, 5327-5339 (2004)
Published by The Company of Biologists 2004
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
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 Burstyn-Cohen, T.
Right arrow Articles by Kalcheim, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Burstyn-Cohen, T.
Right arrow Articles by Kalcheim, C.
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?

Canonical Wnt activity regulates trunk neural crest delamination linking BMP/noggin signaling with G1/S transition

Tal Burstyn-Cohen, Jonathan Stanleigh, Dalit Sela-Donenfeld* and Chaya Kalcheim{dagger}

Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel



View larger version (63K):

[in a new window]
  		Fig. 1. BMP signaling controls G1/S transition in the neural tube in an axial-specific manner. Neural tube sections opposite dissociating somites (A-C) or segmental plate mesoderm (D,E). Hoechst+ nuclei are blue and BrdU+ nuclei are pink. CHO pellets are delineated by white dots and asterisks (*). In A, there is an intact area showing the neural tube with emigrating NC cells (arrowheads); B represents a nearby section from the same embryo at a level where CHO-noggin cells were implanted (*). No NC cells emigrate and BrdU incorporation is partially inhibited. (C) Implanted CHO-control cells (*) have no effect on NC delamination (arrowheads) and BrdU incorporation. (D,E) Noggin overexpression (E) has no effect on neuroepithelial cell proliferation at segmental plate levels of the axis when compared with control CHO (D). (F) Percentage of BrdU+/total nuclei (±s.d) at dissociating and segmental plate levels of the axis in embryos grafted with CHO-noggin cells or electroporated (EP) with a noggin-expressing vector. Numbers in parenthesis represent the embryos quantified. Scale bar: 25 µm in A-C; 40 µm in D,E.

 


View larger version (100K):

[in a new window]
  		Fig. 2. BMP-dependent NC delamination from explanted neural primordia is inhibited by the G1/S transition blocker mimosine. (A) Control, and (B) 600 µm mimosine, (C) 100 ng/ml BMP4 and (D) mimosine+BMP4 treatment. Left panels are phase-contrast images and right panels depict BrdU immunoreactivity. (E) Data quantification. NC delamination is significantly stimulated by BMP4 (P<0.005) and inhibited by mimosine (P<0.005) relative to controls. Mimosine prevented BMP4-induced NC delamination (P<0.002 relative to BMP4 alone) and BrdU incorporation. Scale bar: 75 µm.

 


View larger version (149K):

[in a new window]
  		Fig. 3. Wnt1, but not Wnt3a, is an immediate downstream target of BMP signaling in the dorsal neural tube. (A-F) Expression of Wnt1 mRNA. (A) An embryo aged 20 somites with decreasing to no signal opposite the segmental plate mesoderm. Rostral is towards the top. (B) Graft of CHO-noggin cells inhibits Wnt1 transcription 10 hours later; (C,D) cross-sections of marked regions in B at grafted and intact levels, respectively. Asterisk (*) marks grafted cells. (E) Unilateral noggin electroporation (EP, *electroporated hemi tube) inhibits Wnt1 transcription 10 hours later. (F) A BMP4-coated bead implanted on the neural tube at the segmental plate level induces ectopic and premature Wnt1 expression (arrowhead). (G-I) Expression of Wnt3a mRNA. (G,H) Whole-mount in situ hybridization of 15 and 21 somite stage (SS) embryos, respectively. Rostral is towards the left. There is a rostrocaudal gradient of expression along the neural tube in the younger embryo with no signal adjacent to the segmental plate mesoderm, but uniform rostrocaudal signal at 21 somites and onwards (not shown). (I) Noggin overexpression (*) has no effect on Wnt3a mRNA following 10 hours incubation. Scale bar: 40 µm in C,D,I.

 


View larger version (50K):

[in a new window]
  		Fig. 4. Wnt1 transcription in the neural tube is modulated by the developing somites. Dissociating somites were unilaterally grafted in the place of the unsegmented mesoderm. (A) There is premature expression of Wnt1 mRNA (arrowheads) in the caudal hemi-tube adjacent to the graft (*) at the level of the caudal segmental plate (cSP). (B) Opposite the rostral segmental plate (rSP), Wnt1 transcription is apparent in the intact hemi-tube of this embryo at the age of 23 somites. At this level, the graft (*) resulted in expansion of the Wnt1 expression domain with associated emigration of NC cells that retained Wnt1 expression (arrow). (C) Grafting of a fragment of sclerotomal tissue (Scl) had no effect on Wnt1 transcription or on NC delamination. Scale bar: 25 µm.

 


View larger version (111K):

[in a new window]
  		Fig. 5. The effects of loss and gain of function of Wnt signaling on BrdU incorporation and NC delamination. (A-I) Transverse sections of embryos that received control GFP (A-C), Xdd1 (D-F) or Dep+ (G-I). Upper panels represent GFP immunostaining, central panels are BrdU immunoreactivity, and lower panels depict Hoechst-stained nuclei. (A-C) Electroporation with a control GFP-encoding vector (green) into the hemi neural tube reveals no change in BrdU incorporation (red) when compared with contralateral side. Emigration of GFP+/BrdU+ NC cells is shown (arrowheads). (D-F) Transfection with Xdd1-GFP (green) caused a reduction in number of BrdU+ nuclei (red) and no GFP+ crest cells exiting the treated side of the tube. In D,F, GFP-negative NC cells emigrate from the transfected side. Emigration is normal from the contralateral hemi-tube. (G-I) Dep+ had no effect on either BrdU incorporation or NC emigration. Dep+/GFP+/BrdU+ delaminating cells (arrowheads). NC delamination is also normal from the control side, albeit not seen in this particular section. (J-M) Neural tube explants that received control GFP (J,K) or Wnt1-GFP at 3 µg/µl (L,M). (J,L) An overlay of GFP immunofluorescence (green) onto the phase-contrast images. The white lines depict the border of the tube explants. A larger number of Wnt1/GFP+ NC cells delaminate from the tube and migrate on the substrate when compared with GFP controls. (K,M) GFP in green, BrdU immunolabeling in red and Hoechst nuclear staining in blue. Yellow cells co-express GFP and BrdU. DM, dermomyotome; NT, neural tube; No, notochord; Scl, sclerotome. Scale bar: 35 µm in A-I; 200 µm in J-M.

 


View larger version (157K):

[in a new window]
  		Fig. 7. Expression of cyclin D1 under normal conditions and upon inhibition of Wnt signaling. (A,B) Normal expression patterns of cyclin D1 in the neural tube at (A) a segmental plate (SP) level and (B) opposite dissociating somites (DS). In A, cyclin D1 mRNA is expressed throughout the neural tube, except for the floor plate and dorsal midline. (B) At more rostral regions, cyclin D1 signal becomes apparent in premigratory and early emigrating NC cells (arrowheads). (C,D) Electroporation of ß-catenin fused to the engrailed repressor domain downregulates unilaterally expression of cyclin D1 as shown in whole-mount (C, electroporation between arrows) and (D) transverse section (*treated hemi-tube). Scale bar: 35 µm in A,B,D.

 


View larger version (157K):

[in a new window]
  		Fig. 6. The effects of inhibiting Wnt signaling on expression of dorsal tube-specific genes. (A,B,D-F) Whole-mount in situ hybridization following electroporation with (A) control GFP or (B,D-F) dNLEF1 to the left hemi-tubes and 10 hours reincubation. While the control vector had no effect on bilateral transcription of Cad6B (A), Pax3, Msx1 or RhoB (not shown), there was unilateral downregulation of Cad6B, Pax3 and Msx1, but not of RhoB in dNLEF1-treated hemi-tubes (transfected areas depicted between arrows). Results were confirmed by transverse section analysis. (C) Transverse section of an embryo that received Xdd1-GFP and was analyzed for expression of Cad6B in combination with GFP immunostaining. Downregulation of Cad6B (blue) in dorsal hemi-tube expressing Xdd1-GFP (brown). NC cells emigrating from the treated side are devoid of Xdd1-GFP. (G,H) Transverse sections following electroporation with dNLEF1 to show bilaterally symmetrical expression of Sox9 and Foxd3. The hemi-tubes in the transfected sides are slightly narrower (*). Similar results were obtained for all tested genes (RhoB, Sox9, Cad6B, Msx1, Pax3 and Foxd3) upon transfection with ß-catenin/engrailed or Xdd1 (not shown). (I) High magnification of the dorsal hemi-tube of an embryo electroporated with Xdd1-GFP to show that individual cells expressing Sox9mRNA (upper panel) co-express GFP (middle panel). The lower panel is an overlay of the upper two. EC, ectoderm; NT, neural tube. Scale bar: 40 µm in C,G,H; 20 µm in I.

 


View larger version (131K):

[in a new window]
  		Fig. 8. ß-Catenin overexpression rescues NC delamination in noggin-treated neural tubes. (A,B) Neural tubes that received (A) GFP only or (B) ß-catenin/GFP were placed onto monolayers of control CHO cells (control). (C,D) Neural tubes that received (C) GFP only or (D) ß-catenin/GFP were placed onto monolayers of noggin-producing CHO cells. GFP+NC cells (green) that incorporated BrdU (red) emigrated from the neural primordia onto the substrates in A,B,D, but not in C. CHO cells on the substrate have background fluorescence that is clearly distinct from the vibrant green fluorescence of the electroporated progenitors. Scale bar: 20 µm.

 


View larger version (29K):

[in a new window]
  		Fig. 9. A model of NC delamination that integrates data from the present and previous studies (see Discussion). Opposite the segmental plate mesoderm, high levels of noggin result in low BMP activity, no Wnt1 transcription, low Cyclin D1 and no NC cells emigrating from the caudal tube. G1/S transition and cell proliferation at this level are independent of BMP/Wnt1. With ongoing development, opposite mature epithelial and dissociating somites, a factor emitted by the dorsomedial region of the paraxial mesoderm inhibits noggin transcription in the dorsal tube, thereby relieving BMP activity from inhibition. BMP4 in turn positively regulates Wnt1 transcription. Wnt signaling, via the canonical pathway, positively modulates transcription of cyclin D1, G1/S transition and NC cell delamination. Maintenance of Pax3, Cad6B and Msx1 transcription in the dorsal tube is also regulated by the BMP/Wnt signaling pathway but their possible involvement in NC delamination awaits further testing. RhoB, at variance, is downstream of BMP but not of Wnt activities. The possible role of RhoB in NC delamination in vivo is still unknown. If it promotes delamination, it might act either via a parallel pathway (pink arrow), be upstream of Wnt1 or of Cyclin D1, or interact at a post-transcriptional level with molecules along the Wnt pathway.

 

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 2004