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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 def6 MO-induced phenotype.(A–C) Def6 splice MO was injected alone (2.5 ng) or together with def6 RNA (150 pg). As a control, def6 RNA was also injected alone (150 pg). Embryos are shown at the 1-somite stage. (D) The angle between the anterior- and posterior-most embryonic structures was measured in at least 20 embryos and the average angle is shown on the graph in degrees. ANOVA single factor and two-tailed Student's t-tests showed a significant (p<0.001; three asterisks) increase in the angle after injection of 2.5 ng def6 MO and a significant (p<0.001; three asterisks) decrease in the angle after addition of def6 RNA. The angle measured between injected controls and rescued embryos was also statistically significant (p<0.05; one asterisk), suggestive of a partial rescue. (E) The phenotypes of the embryos from three independent experiments were scored at 3 dpf and the percentages of normal/mild (blue bar), moderate (green bar) and severe (red bar) morphology are shown. Representative images of embryos are shown in Figure 2 panel E.
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pone-0026548-g003: Def6 RNA rescues the def6 MO-induced phenotype.(A–C) Def6 splice MO was injected alone (2.5 ng) or together with def6 RNA (150 pg). As a control, def6 RNA was also injected alone (150 pg). Embryos are shown at the 1-somite stage. (D) The angle between the anterior- and posterior-most embryonic structures was measured in at least 20 embryos and the average angle is shown on the graph in degrees. ANOVA single factor and two-tailed Student's t-tests showed a significant (p<0.001; three asterisks) increase in the angle after injection of 2.5 ng def6 MO and a significant (p<0.001; three asterisks) decrease in the angle after addition of def6 RNA. The angle measured between injected controls and rescued embryos was also statistically significant (p<0.05; one asterisk), suggestive of a partial rescue. (E) The phenotypes of the embryos from three independent experiments were scored at 3 dpf and the percentages of normal/mild (blue bar), moderate (green bar) and severe (red bar) morphology are shown. Representative images of embryos are shown in Figure 2 panel E.

Mentions: To further verify the specificity of the def6 MO-induced phenotype, a rescue experiment was carried out using in vitro transcribed GFP-tagged def6 RNA. When injected alone, GFP-tagged def6 RNA (150 pg) did not affect embryonic development (Figure 3A). 150 pg of GFP-tagged def6 RNA, when co-injected with 2.5 ng of def6 MO, restored the body length of embryos at tail-bud stage (Figure 3C). The increase in the angle between the anterior- and posterior-most embryonic structures observed in def6 morphants was significantly decreased (p<0.001) upon co-injection with GFP-tagged def6 RNA (Figure 3D). The MO-injected and rescued embryos were further scored at 3 dpf for morphological abnormalities, with an increase from 16.1% to 50.5% of embryos with a normal to mild phenotype after rescue (Figure 3E). These results showed that the def6 MO induced defects were specific to def6 MO-mediated knockdown.


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 def6 MO-induced phenotype.(A–C) Def6 splice MO was injected alone (2.5 ng) or together with def6 RNA (150 pg). As a control, def6 RNA was also injected alone (150 pg). Embryos are shown at the 1-somite stage. (D) The angle between the anterior- and posterior-most embryonic structures was measured in at least 20 embryos and the average angle is shown on the graph in degrees. ANOVA single factor and two-tailed Student's t-tests showed a significant (p<0.001; three asterisks) increase in the angle after injection of 2.5 ng def6 MO and a significant (p<0.001; three asterisks) decrease in the angle after addition of def6 RNA. The angle measured between injected controls and rescued embryos was also statistically significant (p<0.05; one asterisk), suggestive of a partial rescue. (E) The phenotypes of the embryos from three independent experiments were scored at 3 dpf and the percentages of normal/mild (blue bar), moderate (green bar) and severe (red bar) morphology are shown. Representative images of embryos are shown in Figure 2 panel E.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198796&req=5

pone-0026548-g003: Def6 RNA rescues the def6 MO-induced phenotype.(A–C) Def6 splice MO was injected alone (2.5 ng) or together with def6 RNA (150 pg). As a control, def6 RNA was also injected alone (150 pg). Embryos are shown at the 1-somite stage. (D) The angle between the anterior- and posterior-most embryonic structures was measured in at least 20 embryos and the average angle is shown on the graph in degrees. ANOVA single factor and two-tailed Student's t-tests showed a significant (p<0.001; three asterisks) increase in the angle after injection of 2.5 ng def6 MO and a significant (p<0.001; three asterisks) decrease in the angle after addition of def6 RNA. The angle measured between injected controls and rescued embryos was also statistically significant (p<0.05; one asterisk), suggestive of a partial rescue. (E) The phenotypes of the embryos from three independent experiments were scored at 3 dpf and the percentages of normal/mild (blue bar), moderate (green bar) and severe (red bar) morphology are shown. Representative images of embryos are shown in Figure 2 panel E.
Mentions: To further verify the specificity of the def6 MO-induced phenotype, a rescue experiment was carried out using in vitro transcribed GFP-tagged def6 RNA. When injected alone, GFP-tagged def6 RNA (150 pg) did not affect embryonic development (Figure 3A). 150 pg of GFP-tagged def6 RNA, when co-injected with 2.5 ng of def6 MO, restored the body length of embryos at tail-bud stage (Figure 3C). The increase in the angle between the anterior- and posterior-most embryonic structures observed in def6 morphants was significantly decreased (p<0.001) upon co-injection with GFP-tagged def6 RNA (Figure 3D). The MO-injected and rescued embryos were further scored at 3 dpf for morphological abnormalities, with an increase from 16.1% to 50.5% of embryos with a normal to mild phenotype after rescue (Figure 3E). These results showed that the def6 MO induced defects were specific to def6 MO-mediated knockdown.

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