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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/dev.00434

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow A corrigendum has been published
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 Yokota, C.
Right arrow Articles by Heasman, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yokota, C.
Right arrow Articles by Heasman, J.
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?

A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements via the FGF receptor

Chika Yokota1, Matt Kofron1, Mike Zuck1, Douglas W. Houston1, Harry Isaacs2, Makoto Asashima3, Chris C. Wylie1 and Janet Heasman1,*

1 Division of Developmental Biology, Cincinnati Children's Research Foundation, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA
2 Department of Biology, University of York, York YO10 5YW, UK
3 Department of Life Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan



View larger version (72K):

[in a new window]
  		Fig. 1. Xnr3 MO-injected embryos have gastrulation and convergent extension defects. (A) Uninjected animal caps and caps injected with Xnr3 morpholino (MO) were dissected at the late blastula stage and photographed at the late neurula stage (stage 20). The MO blocks elongation movements caused by ectopic expression of full-length Xnr3 mRNA in the range 100-300 pg (second panel), but not those caused by Xnr3 ORF (fourth panel). Xnr3 ORF mRNA consists of the open reading frame only, and lacks the binding site. (B) Sibling, control (top row) and Xnr3 MO-injected (bottom row) embryos at late gastrula (stage 12), neurula (stage 17), and tailbud (stage 30). A total of 20 ng of MO was injected into two dorsal marginal cells at the 4-cell stage. (C) MO injection (0-20 ng) caused a range of defects, scored as five classes of phenotype at stage 38. Numbers on right side of embryos indicate five classes. Class 1: normal embryo. Class 2: embryo has slightly shortened axis. Class 3: closed blastopore and neural fold, normal head and a slightly dorsally curved trunk and shortened axis. Class 4: slightly opened neural folds, dorsally curved trunk and shortened axis. Class 5: open neural folds, dorsally curved trunk and shortened axis. (D) The morphology of Keller explants of uninjected, and 20 ng of Xnr3 MO-injected embryos dissected at sibling stage 10.5 and cultured until sibling stage 24. (E) Xnr3 ORF mRNA rescues elongation of MO-injected Keller explants. Left column is uninjected explant, middle is MO-injected, and right column is MO + ORF mRNA-injected explant. 20 ng of Xnr3 MO and/or 200 pg of Xnr3ORF mRNA were injected into the dorsal marginal two cells of the 4-cell-stage embryo and explants were dissected at stage 10.5.

 


View larger version (48K):

[in a new window]
  		Fig. 2. Organizer genes are expressed but some neural markers are reduced in Xnr3– embryos. (A) Organizer gene expression was analyzed at gastrula and early neurula stages (stage 10, 10.5, 11, 12, 14). Xnr3 MO does not affect the expression level of organizer genes. (B) Neural markers were analyzed at stage 28 NCAM and en2 expression was repressed in MO-injected embryos. 10 ng (for late markers), or 20 ng (for both early and late markers) was injected into the dorsal marginal two cells at the 4-cell stage. In each case, ornithine decarboxylase (ODC) was used as a loading control (data not shown), and expression of each gene was normalized to the level of ODC expression. (C) chd and gsc are not expressed in the correct region in MO-injected embryos. Expression pattern of chd (top two rows), and gsc (bottom two rows) in uninjected (upper row) and MO-injected (lower row) embryos. chd was expressed normally in MO-injected embryos at stage 11, and continued to be expressed around the dorsal rim of the blastopore even at stage 14. Gsc expression in MO-injected embryos also remained adjacent to the blastopore at stage 14. 20 ng of MO was injected into the two dorsal animal cells of stage 8 albino embryos, and the expression pattern was analyzed by whole-mount in situ hybridization.

 


View larger version (69K):

[in a new window]
  		Fig. 3. The expression of Xbra is absent in the dorsal marginal region of MO-injected embryos at the gastrula stage and from the notochord region at the neurula stage. (A) The expression of Xbra in uninjected control (A), Xnr3 MO-injected (B), and Xdd1h mRNA-injected (C) embryos. Dorsal expression of Xbra was reduced at stage 11.5 (arrow) and stage 15 in the MO-injected region in Xnr3– embryos (middle column), whereas it occurred normally at stage 11.5 in Xdd1 mRNA-injected embryos (third column). Xbra expression in Xdd1 mRNA-injected embryos was lost in the neurula stage (stage 15). (B) Xnr3-induced animal cap elongation is blocked by the expression of dominant negative dishevelled mRNA (Xdd1). Xdd1 expression alone does not cause elongation of caps. These samples were frozen and subjected to real-time PCR analysis for the expression of Xbra, MyoD, NCAM and nrp1 (Fig. 3B right hand side). All these markers continue to be induced by Xnr3 in animal caps in the presence of Xdd1. (C) The dorsal reduction of Xbra expression is specific in Xnr3 MO-injected embryos. The expression of Xbra was analyzed in dorsally and ventrally injected embryos. Xbra expression in ventrally injected embryos was slightly delayed at the injected region, but was otherwise normal. Arrowheads indicate areas where Xbra expression is missing from the blastopore ring. 20 ng of MO was injected together with NLS-lacZ RNA into two dorsal or ventral marginal two cells in 8-cell-stage embryos.

 


View larger version (47K):

[in a new window]
  		Fig. 4. Over-expression of Xnr3 induces convergent extension movement and the expression of Xbra, eFGF, MyoD and NCAM in animal caps. (A) Morphology of animal cap explants injected at the 2-cell stage with a dose response of Xnr3 mRNA (125 pg-1 ng), dissected at late blastula (stage 9) and photographed at the late neurula stage (stage 20). Elongation is dose responsive. Convergent extension occurs during the neurula stages. (B) Xbra, eFGF, eomesodermin, MyoD and NCAM expression is induced in animal caps injected with Xnr3 mRNA. 250 or 500 pg Xnr3 RNA was injected at the animal pole into two cells of the 2-cell-stage embryo. Animal caps were dissected at stage 9. Gene expressions were analyzed by real-time RT-PCR at stage 11 (for eomesodermin, Xbra and eFGF), and stage 28 (for NCAM and MyoD). In each case, ornithine decarboxylase (ODC) was used as a loading control (data not shown), and each bar was normalized to the level of ODC expression. ND, not done.

 


View larger version (49K):

[in a new window]
  		Fig. 5. A dominant negative FGFR, XFD, suppresses phenotypes caused by Xnr3 over-expression. (A) Morphology of animal caps. Elongation movements caused by Xnr3 over-expression (Xnr3) was suppressed by co-injection of XFD (Xnr3+XFD). (B) MyoD and NCAM expression in animal caps. XFD repressed the induction of MyoD in Xnr3 over-expressing animal caps. Xnr3 (500 pg), XFD (500 pg) or Xnr3+XFD (500 pg each) were injected animally into two cells of 2-cell-stage embryos, and animal caps were dissected from stage 9 embryos. Gene expressions were analyzed by real-time RT-PCR at stage 20. In each case, ornithine decarboxylase (ODC) was used as a loading control (data not shown), and expression was normalized to the level of ODC expression. MyoD expression, but not NCAM expression was inhibited by coinjection of XFD mRNA with Xnr3 mRNA. (C) Phenotypes of Xnr3, XFD or Xnr3+XFD mRNA-injected embryos. XFD rescues both the head abnormalities and finger-like protrusions of Xnr3-injected embryos. Embryos were injected with 500 pg of each mRNA at the animal pole (2 cells at the 2-cell stage). (D) XFD blocks animal cap responses to FGF, activin and Xnr3. Animal caps from wild-type and XFD mRNA over-expressing embryos were treated with FGF (top row) or activin (middle row) as described in Materials and Methods or co-injected with Xnr3 mRNA. Caps were dissected at the late blastula stage and cultured until the late neurula stage. XFD blocked responses to all three treatments.

 


View larger version (64K):

[in a new window]
  		Fig. 6. Xnr3 function in convergent extension requires maternal FGFR1 receptor. (A) Animal caps dissected from FGFR1- late blastulae were unable to elongate in the presence of basic FGF, and this defect was specifically rescued by the injection of 75 pg of synthetic FGFR1 mRNA at the 2-cell stage. (B) Convergent extension movement in Xnr3-overexpressing animal caps was inhibited by the depletion of maternal FGF receptor, FGFR1. FGFR1– caps were also inhibited from responding to basic FGF but not activin. The experiment was repeated with the same result. (C) FGFR1 depletion repressed the induction of MyoD (upper histogram) but not of NCAM (lower histogram) in Xnr3 over-expressing animal caps. In contrast, FGFR1 depletion did not prevent the induction of MyoD by activin. Gene expressions were analyzed by real-time RT-PCR system at stage 20. In each case, ornithine decarboxylase (ODC) was used as a loading control (data not shown), and each bar was normalized to the level of ODC expression. (D) The phenotype of FGFR1– embryos. Oocytes were injected with 3 or 4 ng of antisense FGFR1 oligo, fertilized by the host transfer technique and photographed at the tailbud stage. There was a dose response of gastrulation and convergent extension abnormalities, with dorsally curved axes and open neural folds. (E) Histogram of RT-PCR analyses for Xbra and chordin in sibling embryos of the embryos shown in D, frozen at the early (left histogram) and mid-gastrula (right histogram) stages. FGFR1 depletion (FGFR1– high=4 ng dose of oligo) prevents the expression of Xbra and this was rescued by the re-introduction of FGFR1 mRNA. In contrast, dorsal mesodermal markers such as chordin were little affected by FGFR1 depletion (upper histogram). (F) Xnr3 induces activation of ERK2. Animal caps were isolated from stage 9.5 embryos injected with eFGF (5 pg), FRL1 (2 ng), Xnr3 (500 pg) or Xnr1 (500 pg), cultured until stage 10 and subjected to immunoblotting for phosphorylated (activated) ERK2. {alpha}-tubulin was used as a loading control. Oocytes, untreated or incubated in progesterone (+Prog.), were included as negative and positive controls, respectively.

 


View larger version (55K):

[in a new window]
  		Fig. 7. eFGF depletion, FRL1 and cmXnr2 overexpression increase Xnr3 induced convergent extension activity and gene expression. (A) Animal caps depleted of eFGF with 20 ng of eFGF morpholino (eFGF MO) were able to elongate in the presence of Xnr3 mRNA (right) and to express Xbra at the early gastrula stage (histogram). The experiment was repeated with the same result. (B) FRL1 acts synergistically with Xnr3. 500 pg of Xnr3 mRNA synergized with 500 pg FRL1 mRNA to cause excessive elongation of animal caps (left). RHS shows embryo injection experiments. 500 pg of FRL1 or 50 pg of Xnr3 mRNA injected into 2 ventral cells at the 8-cell stage did not cause finger-like protrusions in whole embryos. Injection of 50 pg Xnr3 mRNA together with 500 pg FRL1 mRNA caused extensive protrusion formation (arrowheads). (C) Animal caps taken from embryos exposed to secreted cmXnr2 as well as injected with Xnr3 mRNA, elongated significantly and showed a synergistic increase in the expression of Xbra and MyoD but not of goosecoid compared with Xnr3-overexpressing caps. Inset diagram shows that Xnr3 mRNA was injected animally at the two-cell stage, and cmXnr2 mRNA was injected into 4 vegetal cells, at the 8-cell stage. Caps were cut at the late blastula stage.

 

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 2003