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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 morphants display defects associated with abnormal gastrulation movements.(A–C) Zebrafish embryos uninjected (wt), or injected with 2.5 or 5 ng def6 splice MO, respectively, are shown at the 1-somite stage. (D) The angle between the anterior- and posterior- most end was measured at the 1-somite stage and the average angle is depicted in degrees. 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 versus wt and an additional significant (p<0.001; three asterisks) increase in 5 ng def6 MO-injected embryos when compared to wt. (E) Def6 MO-injected embryos show a reduction in overall length at 3 dpf (wt at the top; increasing severity of phenotype in def6 MO-injected embryos towards the bottom, defined as mild, moderate and severe, respectively). (F) The length of 20 morphants injected with 2.5 ng def6 MO was measured and normalised to the length of the wt control embryos. Two-tailed Student's t-tests showed a significant (p<0.001; three asterisks) decrease in length after injection of 2.5 ng def6 MO versus wt embryos. (G) The phenotypes of the embryos were scored at 3 dpf and the percentages of normal/mild (blue bar), moderate (green bar) and severe (red bar) morphology are shown.
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pone-0026548-g002: Def6 morphants display defects associated with abnormal gastrulation movements.(A–C) Zebrafish embryos uninjected (wt), or injected with 2.5 or 5 ng def6 splice MO, respectively, are shown at the 1-somite stage. (D) The angle between the anterior- and posterior- most end was measured at the 1-somite stage and the average angle is depicted in degrees. 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 versus wt and an additional significant (p<0.001; three asterisks) increase in 5 ng def6 MO-injected embryos when compared to wt. (E) Def6 MO-injected embryos show a reduction in overall length at 3 dpf (wt at the top; increasing severity of phenotype in def6 MO-injected embryos towards the bottom, defined as mild, moderate and severe, respectively). (F) The length of 20 morphants injected with 2.5 ng def6 MO was measured and normalised to the length of the wt control embryos. Two-tailed Student's t-tests showed a significant (p<0.001; three asterisks) decrease in length after injection of 2.5 ng def6 MO versus wt embryos. (G) The phenotypes of the embryos were scored at 3 dpf and the percentages of normal/mild (blue bar), moderate (green bar) and severe (red bar) morphology are shown.

Mentions: Zebrafish embryos were injected at the 1–2 cell stage with 2.5 and 5ng def6 MO and development was monitored at specific intervals during development. The first defects could be morphologically identified at the end of gastrulation, with no observable phenotype occurring during the epiboly stages. At the 1-somite stage, def6 morphants failed to extend normally around the yolk, resulting in a shorter anterior-posterior axis when compared to uninjected control siblings (Figure 2A–C). The angle was measured between the anterior- and posterior-most parts of the uninjected, 2.5 ng and 5 ng def6 MO-injected embryos, with a significant increase in the angle between uninjected and 2.5 ng or 5 ng def6 MO-injected embryos (Figure 2D). The severity of the knockdown phenotype was greater after injection of 5 ng of def6 MO, indicating that the def6 MO acted in a dose-dependent manner. The smaller dose of the def6 MO was used for subsequent experiments. In addition to the reduced embryonic axis of injected embryos at the end of gastrulation, their body length was also shorter at 3 dpf in comparison to controls (Figure 2E). To validate this observation, the overall length of the def6 morphants was measured from anterior to posterior at 3 dpf. A significant (p<0.001) decrease was present in the body length of MO-injected embryos compared to control siblings (Figure 2F), together with an increased severity of phenotype (Figure 2G). These results are consistent with def6 MO-mediated knockdown leading to cell movement defects during gastrulation that result in a decrease in the body length of injected embryos.


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 morphants display defects associated with abnormal gastrulation movements.(A–C) Zebrafish embryos uninjected (wt), or injected with 2.5 or 5 ng def6 splice MO, respectively, are shown at the 1-somite stage. (D) The angle between the anterior- and posterior- most end was measured at the 1-somite stage and the average angle is depicted in degrees. 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 versus wt and an additional significant (p<0.001; three asterisks) increase in 5 ng def6 MO-injected embryos when compared to wt. (E) Def6 MO-injected embryos show a reduction in overall length at 3 dpf (wt at the top; increasing severity of phenotype in def6 MO-injected embryos towards the bottom, defined as mild, moderate and severe, respectively). (F) The length of 20 morphants injected with 2.5 ng def6 MO was measured and normalised to the length of the wt control embryos. Two-tailed Student's t-tests showed a significant (p<0.001; three asterisks) decrease in length after injection of 2.5 ng def6 MO versus wt embryos. (G) The phenotypes of the embryos were scored at 3 dpf and the percentages of normal/mild (blue bar), moderate (green bar) and severe (red bar) morphology are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198796&req=5

pone-0026548-g002: Def6 morphants display defects associated with abnormal gastrulation movements.(A–C) Zebrafish embryos uninjected (wt), or injected with 2.5 or 5 ng def6 splice MO, respectively, are shown at the 1-somite stage. (D) The angle between the anterior- and posterior- most end was measured at the 1-somite stage and the average angle is depicted in degrees. 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 versus wt and an additional significant (p<0.001; three asterisks) increase in 5 ng def6 MO-injected embryos when compared to wt. (E) Def6 MO-injected embryos show a reduction in overall length at 3 dpf (wt at the top; increasing severity of phenotype in def6 MO-injected embryos towards the bottom, defined as mild, moderate and severe, respectively). (F) The length of 20 morphants injected with 2.5 ng def6 MO was measured and normalised to the length of the wt control embryos. Two-tailed Student's t-tests showed a significant (p<0.001; three asterisks) decrease in length after injection of 2.5 ng def6 MO versus wt embryos. (G) The phenotypes of the embryos were scored at 3 dpf and the percentages of normal/mild (blue bar), moderate (green bar) and severe (red bar) morphology are shown.
Mentions: Zebrafish embryos were injected at the 1–2 cell stage with 2.5 and 5ng def6 MO and development was monitored at specific intervals during development. The first defects could be morphologically identified at the end of gastrulation, with no observable phenotype occurring during the epiboly stages. At the 1-somite stage, def6 morphants failed to extend normally around the yolk, resulting in a shorter anterior-posterior axis when compared to uninjected control siblings (Figure 2A–C). The angle was measured between the anterior- and posterior-most parts of the uninjected, 2.5 ng and 5 ng def6 MO-injected embryos, with a significant increase in the angle between uninjected and 2.5 ng or 5 ng def6 MO-injected embryos (Figure 2D). The severity of the knockdown phenotype was greater after injection of 5 ng of def6 MO, indicating that the def6 MO acted in a dose-dependent manner. The smaller dose of the def6 MO was used for subsequent experiments. In addition to the reduced embryonic axis of injected embryos at the end of gastrulation, their body length was also shorter at 3 dpf in comparison to controls (Figure 2E). To validate this observation, the overall length of the def6 morphants was measured from anterior to posterior at 3 dpf. A significant (p<0.001) decrease was present in the body length of MO-injected embryos compared to control siblings (Figure 2F), together with an increased severity of phenotype (Figure 2G). These results are consistent with def6 MO-mediated knockdown leading to cell movement defects during gastrulation that result in a decrease in the body length of injected embryos.

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