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Def6 is required for convergent extension movements during zebrafish gastrulation downstream of Wnt5b signaling.

Goudevenou K, Martin P, Yeh YJ, Jones P, Sablitzky F - PLoS ONE (2011)

Bottom Line: Wnt signaling results in downstream activation of Rho GTPases that in turn regulate actin cytoskeleton rearrangements essential for co-ordinated CE cell movement.Here we show that def6, a novel GEF, regulates CE cell movement during zebrafish gastrulation.In addition, by knocking down both def6 and Wnt11, we show that def6 synergises with the Wnt11 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Queen's Medical Centre, The University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
During gastrulation, convergent extension (CE) cell movements are regulated through the non-canonical Wnt signaling pathway. Wnt signaling results in downstream activation of Rho GTPases that in turn regulate actin cytoskeleton rearrangements essential for co-ordinated CE cell movement. Rho GTPases are bi-molecular switches that are inactive in their GDP-bound stage but can be activated to bind GTP through guanine nucleotide exchange factors (GEFs). Here we show that def6, a novel GEF, regulates CE cell movement during zebrafish gastrulation. Def6 morphants exhibit broadened and shortened body axis with normal cell fate specification, reminiscent of the zebrafish mutants silberblick and pipetail that lack Wnt11 or Wnt5b, respectively. Indeed, def6 morphants phenocopy Wnt5b mutants and ectopic overexpression of def6 essentially rescues Wnt5b morphants, indicating a novel role for def6 as a central GEF downstream of Wnt5b signaling. In addition, by knocking down both def6 and Wnt11, we show that def6 synergises with the Wnt11 signaling pathway.

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Def6 RNA rescues the CE movement defects observed in wnt5b morphants.Embryos injected with def6 RNA (150 pg), wnt5b MO (5 ng) alone and together at the 1–2 cell stage were fixed at tail-bud stage (10 hpf), and stained for dlx3 (marks the anterior borders of the neural plate) and ntl (marks the presumptive notochord) expression. (A–C) Expression of dlx3 shows restoration of the wider neural plate in the embryos co-injected with def6 RNA and wnt5b MO. Def6 RNA was also sufficient to rescue the reduced anterior extension of the presumptive notochord as revealed by ntl expression. (D) Representative embryo at tail-bud stage viewed from the dorsal side with anterior to top. The measures taken are shown. The black line indicates the width of the embryo whereas the red double-headed arrow is the width of the neural plate. (E) The ratio of the width of the neural plate divided by the width of the embryo was quantified for each category of embryos. ANOVA single factor indicated a significant (p<0.001) difference between the three groups of embryos. Two-tailed Student's t-tests showed a significant (p<0.001, three asterisks) increase in this ratio in wnt5b morphants versus def6 RNA-injected embryos and statistical significance (p<0.001; three asterisks) in wnt5b MO-injected embryos versus embryos co-injected with def6 RNA and wnt5b MO. There was no statistical difference (N.S) between def6 RNA only versus rescued embryos. (F) Embryos at 24 hpf were morphologically analysed and separated into four categories according to their phenotype: normal (blue box), mild (purple box), moderate (green box), and severe (red box). (G) The phenotypes of the embryos from three independent experiments were scored and the percentages of normal (blue bar), mild (green bar), moderate (yellow bar) and severe (red bar) phenotypes were plotted on the graph.
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pone-0026548-g008: Def6 RNA rescues the CE movement defects observed in wnt5b morphants.Embryos injected with def6 RNA (150 pg), wnt5b MO (5 ng) alone and together at the 1–2 cell stage were fixed at tail-bud stage (10 hpf), and stained for dlx3 (marks the anterior borders of the neural plate) and ntl (marks the presumptive notochord) expression. (A–C) Expression of dlx3 shows restoration of the wider neural plate in the embryos co-injected with def6 RNA and wnt5b MO. Def6 RNA was also sufficient to rescue the reduced anterior extension of the presumptive notochord as revealed by ntl expression. (D) Representative embryo at tail-bud stage viewed from the dorsal side with anterior to top. The measures taken are shown. The black line indicates the width of the embryo whereas the red double-headed arrow is the width of the neural plate. (E) The ratio of the width of the neural plate divided by the width of the embryo was quantified for each category of embryos. ANOVA single factor indicated a significant (p<0.001) difference between the three groups of embryos. Two-tailed Student's t-tests showed a significant (p<0.001, three asterisks) increase in this ratio in wnt5b morphants versus def6 RNA-injected embryos and statistical significance (p<0.001; three asterisks) in wnt5b MO-injected embryos versus embryos co-injected with def6 RNA and wnt5b MO. There was no statistical difference (N.S) between def6 RNA only versus rescued embryos. (F) Embryos at 24 hpf were morphologically analysed and separated into four categories according to their phenotype: normal (blue box), mild (purple box), moderate (green box), and severe (red box). (G) The phenotypes of the embryos from three independent experiments were scored and the percentages of normal (blue bar), mild (green bar), moderate (yellow bar) and severe (red bar) phenotypes were plotted on the graph.

Mentions: Given the similarities observed in terms of phenotype in the wnt5b and def6 morphants, rescue experiments were performed in order to determine whether it was possible to rescue wnt5b MO-induced defects with def6 RNA. Embryos were injected at the 1–2 cell stage with 150 pg GFP-tagged def6 RNA and 5 ng wnt5b MO alone or together, fixed at 10 hpf and stained for dlx3 and ntl expression. GFP-tagged def6 RNA was sufficient to rescue the perturbed convergence of the anterior neural plate as revealed by dlx3 expression. Also, GFP-tagged def6 RNA rescued the wnt5b MO-induced extension defect of presumptive notochord cells to the anterior of the embryo, as revealed by ntl expression (Figure 8A–C). Morphometric analysis of the distance between the borders of the anterior neural plate indicated that the significant increase in wnt5b morphants compared to controls was rescued by GFP-tagged def6 RNA (Figure 8E). Morphological scoring of embryos at 24 hpf indicated that whereas greater than 50% of the embryos were severely affected by the wnt5b MO, less than 10% were severely affected after rescue (Figure 8G). These results demonstrate that GFP-tagged def6 RNA rescued the wnt5b MO-induced defects, placing def6 downstream of Wnt5b in the non-canonical Wnt signaling pathway.


Def6 is required for convergent extension movements during zebrafish gastrulation downstream of Wnt5b signaling.

Goudevenou K, Martin P, Yeh YJ, Jones P, Sablitzky F - PLoS ONE (2011)

Def6 RNA rescues the CE movement defects observed in wnt5b morphants.Embryos injected with def6 RNA (150 pg), wnt5b MO (5 ng) alone and together at the 1–2 cell stage were fixed at tail-bud stage (10 hpf), and stained for dlx3 (marks the anterior borders of the neural plate) and ntl (marks the presumptive notochord) expression. (A–C) Expression of dlx3 shows restoration of the wider neural plate in the embryos co-injected with def6 RNA and wnt5b MO. Def6 RNA was also sufficient to rescue the reduced anterior extension of the presumptive notochord as revealed by ntl expression. (D) Representative embryo at tail-bud stage viewed from the dorsal side with anterior to top. The measures taken are shown. The black line indicates the width of the embryo whereas the red double-headed arrow is the width of the neural plate. (E) The ratio of the width of the neural plate divided by the width of the embryo was quantified for each category of embryos. ANOVA single factor indicated a significant (p<0.001) difference between the three groups of embryos. Two-tailed Student's t-tests showed a significant (p<0.001, three asterisks) increase in this ratio in wnt5b morphants versus def6 RNA-injected embryos and statistical significance (p<0.001; three asterisks) in wnt5b MO-injected embryos versus embryos co-injected with def6 RNA and wnt5b MO. There was no statistical difference (N.S) between def6 RNA only versus rescued embryos. (F) Embryos at 24 hpf were morphologically analysed and separated into four categories according to their phenotype: normal (blue box), mild (purple box), moderate (green box), and severe (red box). (G) The phenotypes of the embryos from three independent experiments were scored and the percentages of normal (blue bar), mild (green bar), moderate (yellow bar) and severe (red bar) phenotypes were plotted on the graph.
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Related In: Results  -  Collection

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pone-0026548-g008: Def6 RNA rescues the CE movement defects observed in wnt5b morphants.Embryos injected with def6 RNA (150 pg), wnt5b MO (5 ng) alone and together at the 1–2 cell stage were fixed at tail-bud stage (10 hpf), and stained for dlx3 (marks the anterior borders of the neural plate) and ntl (marks the presumptive notochord) expression. (A–C) Expression of dlx3 shows restoration of the wider neural plate in the embryos co-injected with def6 RNA and wnt5b MO. Def6 RNA was also sufficient to rescue the reduced anterior extension of the presumptive notochord as revealed by ntl expression. (D) Representative embryo at tail-bud stage viewed from the dorsal side with anterior to top. The measures taken are shown. The black line indicates the width of the embryo whereas the red double-headed arrow is the width of the neural plate. (E) The ratio of the width of the neural plate divided by the width of the embryo was quantified for each category of embryos. ANOVA single factor indicated a significant (p<0.001) difference between the three groups of embryos. Two-tailed Student's t-tests showed a significant (p<0.001, three asterisks) increase in this ratio in wnt5b morphants versus def6 RNA-injected embryos and statistical significance (p<0.001; three asterisks) in wnt5b MO-injected embryos versus embryos co-injected with def6 RNA and wnt5b MO. There was no statistical difference (N.S) between def6 RNA only versus rescued embryos. (F) Embryos at 24 hpf were morphologically analysed and separated into four categories according to their phenotype: normal (blue box), mild (purple box), moderate (green box), and severe (red box). (G) The phenotypes of the embryos from three independent experiments were scored and the percentages of normal (blue bar), mild (green bar), moderate (yellow bar) and severe (red bar) phenotypes were plotted on the graph.
Mentions: Given the similarities observed in terms of phenotype in the wnt5b and def6 morphants, rescue experiments were performed in order to determine whether it was possible to rescue wnt5b MO-induced defects with def6 RNA. Embryos were injected at the 1–2 cell stage with 150 pg GFP-tagged def6 RNA and 5 ng wnt5b MO alone or together, fixed at 10 hpf and stained for dlx3 and ntl expression. GFP-tagged def6 RNA was sufficient to rescue the perturbed convergence of the anterior neural plate as revealed by dlx3 expression. Also, GFP-tagged def6 RNA rescued the wnt5b MO-induced extension defect of presumptive notochord cells to the anterior of the embryo, as revealed by ntl expression (Figure 8A–C). Morphometric analysis of the distance between the borders of the anterior neural plate indicated that the significant increase in wnt5b morphants compared to controls was rescued by GFP-tagged def6 RNA (Figure 8E). Morphological scoring of embryos at 24 hpf indicated that whereas greater than 50% of the embryos were severely affected by the wnt5b MO, less than 10% were severely affected after rescue (Figure 8G). These results demonstrate that GFP-tagged def6 RNA rescued the wnt5b MO-induced defects, placing def6 downstream of Wnt5b in the non-canonical Wnt signaling pathway.

Bottom Line: Wnt signaling results in downstream activation of Rho GTPases that in turn regulate actin cytoskeleton rearrangements essential for co-ordinated CE cell movement.Here we show that def6, a novel GEF, regulates CE cell movement during zebrafish gastrulation.In addition, by knocking down both def6 and Wnt11, we show that def6 synergises with the Wnt11 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Queen's Medical Centre, The University of Nottingham, Nottingham, United Kingdom.

ABSTRACT
During gastrulation, convergent extension (CE) cell movements are regulated through the non-canonical Wnt signaling pathway. Wnt signaling results in downstream activation of Rho GTPases that in turn regulate actin cytoskeleton rearrangements essential for co-ordinated CE cell movement. Rho GTPases are bi-molecular switches that are inactive in their GDP-bound stage but can be activated to bind GTP through guanine nucleotide exchange factors (GEFs). Here we show that def6, a novel GEF, regulates CE cell movement during zebrafish gastrulation. Def6 morphants exhibit broadened and shortened body axis with normal cell fate specification, reminiscent of the zebrafish mutants silberblick and pipetail that lack Wnt11 or Wnt5b, respectively. Indeed, def6 morphants phenocopy Wnt5b mutants and ectopic overexpression of def6 essentially rescues Wnt5b morphants, indicating a novel role for def6 as a central GEF downstream of Wnt5b signaling. In addition, by knocking down both def6 and Wnt11, we show that def6 synergises with the Wnt11 signaling pathway.

Show MeSH
Related in: MedlinePlus