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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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The def6 MO-mediated knockdown phenotype induces CE movement defects.Uninjected (wt) or embryos injected with def6 MO were fixed at tail-bud stage and in situ hybridisations were carried out with probes to hgg1 and dlx3 (A,B). ImageJ software was utilised to analyse the staining patterns, measuring the posterior shift of the hgg1 staining (red double-headed arrow) in relation to the arc formed by dlx3 expression (yellow dotted arc) (C), and measuring the width of the dlx3 staining (blue double-headed arrow) at a constant distance (1/4 of the embryo width) from the dlx3 arc when the embryo was positioned dorsally (D). (E and F) The measured distances were plotted as the average posterior shift (E) or width (F) as a percentage of the total width of the embryo. Two-tailed Student's t-tests were carried out between groups indicated, and were of statistical significance (p<0.001; three asterisks). This experiment has been repeated at least three times; a representative experiment is depicted here. Zebrafish embryos uninjected (wt) or injected with def6 MO (2.5 ng) were also stained for hgg1/ntl (G, H; statistical analysis shown in Panel I), ntl (J, K) and myoD (L-O-) expression. Images G, H, L, M are embryos at tail-bud stage. Images J. K show embryos at 24 hpf. Images N, O show embryos at the 10-somite stage. G, H, L-O are dorsal views with anterior to the top; J, K are lateral views with anterior to the left.
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pone-0026548-g005: The def6 MO-mediated knockdown phenotype induces CE movement defects.Uninjected (wt) or embryos injected with def6 MO were fixed at tail-bud stage and in situ hybridisations were carried out with probes to hgg1 and dlx3 (A,B). ImageJ software was utilised to analyse the staining patterns, measuring the posterior shift of the hgg1 staining (red double-headed arrow) in relation to the arc formed by dlx3 expression (yellow dotted arc) (C), and measuring the width of the dlx3 staining (blue double-headed arrow) at a constant distance (1/4 of the embryo width) from the dlx3 arc when the embryo was positioned dorsally (D). (E and F) The measured distances were plotted as the average posterior shift (E) or width (F) as a percentage of the total width of the embryo. Two-tailed Student's t-tests were carried out between groups indicated, and were of statistical significance (p<0.001; three asterisks). This experiment has been repeated at least three times; a representative experiment is depicted here. Zebrafish embryos uninjected (wt) or injected with def6 MO (2.5 ng) were also stained for hgg1/ntl (G, H; statistical analysis shown in Panel I), ntl (J, K) and myoD (L-O-) expression. Images G, H, L, M are embryos at tail-bud stage. Images J. K show embryos at 24 hpf. Images N, O show embryos at the 10-somite stage. G, H, L-O are dorsal views with anterior to the top; J, K are lateral views with anterior to the left.

Mentions: As the def6 MO-induced phenotype did not affect dorso-ventral patterning, it was necessary to determine whether the shortened body axis observed could be a result of impaired CE movements during gastrulation. Double in situ hybridisation experiments were performed with a series of well-characterised markers widely used to study CE movements. These markers include: dlx3 (distal-less homeobox gene 3), which labels the borders of neural and non-neural ectoderm, hgg1 (hatching gland 1) which marks the polster, the anterior-most end of the prechordal plate, and ntl, which marks the presumptive notochord. At the end of gastrulation, expression of dlx3 showed an enlarged neural plate in def6 morphants (Figure 5A and B), suggesting impaired CE in the neural ectoderm. In def6 MO-injected embryos, the prechordal plate, marked by hgg1 expression, was positioned posteriorly with respect to dlx3 expression in the anterior edges of the neural plate, suggesting that the most anterior axial mesendodermal tissues were affected (Figure 5A and B). The posterior shift of hgg1 expression was highly significant, as assessed by measurement relative to the arc formed by dlx3 expression (Figure 5C and E). In addition, the neural plate width, measured at a constant distance (1/4 of embryo width) from the dlx3 arc, was significantly increased in def6 morphants (Figure 5D and F).


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)

The def6 MO-mediated knockdown phenotype induces CE movement defects.Uninjected (wt) or embryos injected with def6 MO were fixed at tail-bud stage and in situ hybridisations were carried out with probes to hgg1 and dlx3 (A,B). ImageJ software was utilised to analyse the staining patterns, measuring the posterior shift of the hgg1 staining (red double-headed arrow) in relation to the arc formed by dlx3 expression (yellow dotted arc) (C), and measuring the width of the dlx3 staining (blue double-headed arrow) at a constant distance (1/4 of the embryo width) from the dlx3 arc when the embryo was positioned dorsally (D). (E and F) The measured distances were plotted as the average posterior shift (E) or width (F) as a percentage of the total width of the embryo. Two-tailed Student's t-tests were carried out between groups indicated, and were of statistical significance (p<0.001; three asterisks). This experiment has been repeated at least three times; a representative experiment is depicted here. Zebrafish embryos uninjected (wt) or injected with def6 MO (2.5 ng) were also stained for hgg1/ntl (G, H; statistical analysis shown in Panel I), ntl (J, K) and myoD (L-O-) expression. Images G, H, L, M are embryos at tail-bud stage. Images J. K show embryos at 24 hpf. Images N, O show embryos at the 10-somite stage. G, H, L-O are dorsal views with anterior to the top; J, K are lateral views with anterior to the left.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198796&req=5

pone-0026548-g005: The def6 MO-mediated knockdown phenotype induces CE movement defects.Uninjected (wt) or embryos injected with def6 MO were fixed at tail-bud stage and in situ hybridisations were carried out with probes to hgg1 and dlx3 (A,B). ImageJ software was utilised to analyse the staining patterns, measuring the posterior shift of the hgg1 staining (red double-headed arrow) in relation to the arc formed by dlx3 expression (yellow dotted arc) (C), and measuring the width of the dlx3 staining (blue double-headed arrow) at a constant distance (1/4 of the embryo width) from the dlx3 arc when the embryo was positioned dorsally (D). (E and F) The measured distances were plotted as the average posterior shift (E) or width (F) as a percentage of the total width of the embryo. Two-tailed Student's t-tests were carried out between groups indicated, and were of statistical significance (p<0.001; three asterisks). This experiment has been repeated at least three times; a representative experiment is depicted here. Zebrafish embryos uninjected (wt) or injected with def6 MO (2.5 ng) were also stained for hgg1/ntl (G, H; statistical analysis shown in Panel I), ntl (J, K) and myoD (L-O-) expression. Images G, H, L, M are embryos at tail-bud stage. Images J. K show embryos at 24 hpf. Images N, O show embryos at the 10-somite stage. G, H, L-O are dorsal views with anterior to the top; J, K are lateral views with anterior to the left.
Mentions: As the def6 MO-induced phenotype did not affect dorso-ventral patterning, it was necessary to determine whether the shortened body axis observed could be a result of impaired CE movements during gastrulation. Double in situ hybridisation experiments were performed with a series of well-characterised markers widely used to study CE movements. These markers include: dlx3 (distal-less homeobox gene 3), which labels the borders of neural and non-neural ectoderm, hgg1 (hatching gland 1) which marks the polster, the anterior-most end of the prechordal plate, and ntl, which marks the presumptive notochord. At the end of gastrulation, expression of dlx3 showed an enlarged neural plate in def6 morphants (Figure 5A and B), suggesting impaired CE in the neural ectoderm. In def6 MO-injected embryos, the prechordal plate, marked by hgg1 expression, was positioned posteriorly with respect to dlx3 expression in the anterior edges of the neural plate, suggesting that the most anterior axial mesendodermal tissues were affected (Figure 5A and B). The posterior shift of hgg1 expression was highly significant, as assessed by measurement relative to the arc formed by dlx3 expression (Figure 5C and E). In addition, the neural plate width, measured at a constant distance (1/4 of embryo width) from the dlx3 arc, was significantly increased in def6 morphants (Figure 5D and F).

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