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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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Synergy between def6 and wnt11 MO-mediated knockdown results in severe phenotype.Zebrafish embryos were injected with wnt11 MO (1.5 ng) and def6 MO (1.5 ng) separately and together. Development was assessed at different stages. (A–C) Tail-bud stage (10 hpf), the anterior-most structure is indicated with an arrowhead. (D–F) 28 hpf, co-injection with def6 and wnt11 MOs results in no forebrain structures anterior to the eyes (D'–F') the same embryos are shown from the front; note complete fusion of the eyes in the double-knockdown embryos. (G–I) 3 dpf, double knockdown embryos have their eyes completely fused in comparison to def6 or wnt11 MO-injected embryos. (J) The phenotypes of 28 hpf embryos were scored morphologically into 4 categories. Representative embryos of normal (blue box), mild (purple box), moderate (green box) and severely affected (red box) morphants are shown. (K) The phenotypes of the embryos from three independent experiments were scored and the percentages of normal (blue bar), mild (purple bar), moderate (green bar) and severe (red bar) are indicated.
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pone-0026548-g009: Synergy between def6 and wnt11 MO-mediated knockdown results in severe phenotype.Zebrafish embryos were injected with wnt11 MO (1.5 ng) and def6 MO (1.5 ng) separately and together. Development was assessed at different stages. (A–C) Tail-bud stage (10 hpf), the anterior-most structure is indicated with an arrowhead. (D–F) 28 hpf, co-injection with def6 and wnt11 MOs results in no forebrain structures anterior to the eyes (D'–F') the same embryos are shown from the front; note complete fusion of the eyes in the double-knockdown embryos. (G–I) 3 dpf, double knockdown embryos have their eyes completely fused in comparison to def6 or wnt11 MO-injected embryos. (J) The phenotypes of 28 hpf embryos were scored morphologically into 4 categories. Representative embryos of normal (blue box), mild (purple box), moderate (green box) and severely affected (red box) morphants are shown. (K) The phenotypes of the embryos from three independent experiments were scored and the percentages of normal (blue bar), mild (purple bar), moderate (green bar) and severe (red bar) are indicated.

Mentions: Although ectopic def6 expression was unable to rescue the wnt11 MO-induced phenotype, def6 and Wnt11 could still function together in parallel or overlapping pathways. To test this hypothesis, decreasing concentrations of wnt11 and def6 MOs were tested; suboptimal amounts, that do not induce obvious phenotypes by themselves, were co-injected into 1–2 cell stage zebrafish embryos. 1.5 ng of wnt11 MO or 1.5 ng of def6 MO alone induced, at most, a very mild phenotype, whereas co-injection of both MOs at these concentrations induced severe CE movement defects (Figure 9). These results suggest that def6 functions in a parallel or overlapping pathway with Wnt11, or, alternatively, that they both have a common target downstream of Wnt11.


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)

Synergy between def6 and wnt11 MO-mediated knockdown results in severe phenotype.Zebrafish embryos were injected with wnt11 MO (1.5 ng) and def6 MO (1.5 ng) separately and together. Development was assessed at different stages. (A–C) Tail-bud stage (10 hpf), the anterior-most structure is indicated with an arrowhead. (D–F) 28 hpf, co-injection with def6 and wnt11 MOs results in no forebrain structures anterior to the eyes (D'–F') the same embryos are shown from the front; note complete fusion of the eyes in the double-knockdown embryos. (G–I) 3 dpf, double knockdown embryos have their eyes completely fused in comparison to def6 or wnt11 MO-injected embryos. (J) The phenotypes of 28 hpf embryos were scored morphologically into 4 categories. Representative embryos of normal (blue box), mild (purple box), moderate (green box) and severely affected (red box) morphants are shown. (K) The phenotypes of the embryos from three independent experiments were scored and the percentages of normal (blue bar), mild (purple bar), moderate (green bar) and severe (red bar) are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198796&req=5

pone-0026548-g009: Synergy between def6 and wnt11 MO-mediated knockdown results in severe phenotype.Zebrafish embryos were injected with wnt11 MO (1.5 ng) and def6 MO (1.5 ng) separately and together. Development was assessed at different stages. (A–C) Tail-bud stage (10 hpf), the anterior-most structure is indicated with an arrowhead. (D–F) 28 hpf, co-injection with def6 and wnt11 MOs results in no forebrain structures anterior to the eyes (D'–F') the same embryos are shown from the front; note complete fusion of the eyes in the double-knockdown embryos. (G–I) 3 dpf, double knockdown embryos have their eyes completely fused in comparison to def6 or wnt11 MO-injected embryos. (J) The phenotypes of 28 hpf embryos were scored morphologically into 4 categories. Representative embryos of normal (blue box), mild (purple box), moderate (green box) and severely affected (red box) morphants are shown. (K) The phenotypes of the embryos from three independent experiments were scored and the percentages of normal (blue bar), mild (purple bar), moderate (green bar) and severe (red bar) are indicated.
Mentions: Although ectopic def6 expression was unable to rescue the wnt11 MO-induced phenotype, def6 and Wnt11 could still function together in parallel or overlapping pathways. To test this hypothesis, decreasing concentrations of wnt11 and def6 MOs were tested; suboptimal amounts, that do not induce obvious phenotypes by themselves, were co-injected into 1–2 cell stage zebrafish embryos. 1.5 ng of wnt11 MO or 1.5 ng of def6 MO alone induced, at most, a very mild phenotype, whereas co-injection of both MOs at these concentrations induced severe CE movement defects (Figure 9). These results suggest that def6 functions in a parallel or overlapping pathway with Wnt11, or, alternatively, that they both have a common target downstream of Wnt11.

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