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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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Craniofacial defects in def6 and wnt5b MO-injected embryos.(A–C) 4 dpf, the distance between the eyes is indicated with a double-headed arrow. Def6 and wnt5b MO-injected embryos exhibit a ‘hammerhead’-like phenotype. (D–F) Alcian Blue staining of the cartilage in the head region of 4 dpf embryos. Meckel's cartilage is indicated with a black arrowhead and does not extend anteriorly beyond the eyes in def6 and wnt5b MO-injected embryos. The ceratohyal is indicated with a red arrowhead and is more posteriorly shifted in the two groups of morphants. Image E; representative image of 105/109 embryos, Image F; representative image of 89/92 embryos. (G) Representative wt embryo and measures taken are shown on the left. Line A was drawn as a baseline for further measurements and also served to normalise distance B. Line B is the distance from line A to the anterior end of the ceratohyal. The ratio of distance B divided by distance A is indicated on the graph. Two- tailed Student's t-tests indicated a significant (p<0.001; three asterisks) decrease in this ratio in the def6 MO-injected embryos versus wt siblings and between wnt5b MO-injected embryos versus wt siblings. (H) Representative wt embryo and the measures taken are shown on the image on the left. Line A, as before, was used to normalise distance C. Line C is the distance from line A to the anterior end of Meckel's cartilage. The ratio of this distance is shown on the graph. Injecting 2.5 ng of def6 MO resulted in a significant (p<0.001; three asterisks) decrease in this ratio as determined by two-tailed Student's t-tests; similarly injection of 5 ng wnt5b MO resulted in a significant (p<0.001; three asterisks) decrease in this ratio.
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pone-0026548-g007: Craniofacial defects in def6 and wnt5b MO-injected embryos.(A–C) 4 dpf, the distance between the eyes is indicated with a double-headed arrow. Def6 and wnt5b MO-injected embryos exhibit a ‘hammerhead’-like phenotype. (D–F) Alcian Blue staining of the cartilage in the head region of 4 dpf embryos. Meckel's cartilage is indicated with a black arrowhead and does not extend anteriorly beyond the eyes in def6 and wnt5b MO-injected embryos. The ceratohyal is indicated with a red arrowhead and is more posteriorly shifted in the two groups of morphants. Image E; representative image of 105/109 embryos, Image F; representative image of 89/92 embryos. (G) Representative wt embryo and measures taken are shown on the left. Line A was drawn as a baseline for further measurements and also served to normalise distance B. Line B is the distance from line A to the anterior end of the ceratohyal. The ratio of distance B divided by distance A is indicated on the graph. Two- tailed Student's t-tests indicated a significant (p<0.001; three asterisks) decrease in this ratio in the def6 MO-injected embryos versus wt siblings and between wnt5b MO-injected embryos versus wt siblings. (H) Representative wt embryo and the measures taken are shown on the image on the left. Line A, as before, was used to normalise distance C. Line C is the distance from line A to the anterior end of Meckel's cartilage. The ratio of this distance is shown on the graph. Injecting 2.5 ng of def6 MO resulted in a significant (p<0.001; three asterisks) decrease in this ratio as determined by two-tailed Student's t-tests; similarly injection of 5 ng wnt5b MO resulted in a significant (p<0.001; three asterisks) decrease in this ratio.

Mentions: The CE movement defects observed in def6 MO-injected embryos strongly resembled previously published ppt/wnt5b mutants and/or morphants [11], [29]. At tail-bud stage, the embryonic axis failed to move around the yolk (Figure 6A–C, arrowheads). This effect was statistically significant (Figure 6D), with the def6 MO having a stronger effect than the wnt5b MO at the concentrations of MO tested. At later stages, embryos were shorter with truncated tails (Figure 6E–G). At 4dpf, def6 morphants developed the ‘hammerhead’-like phenotype, a hallmark of ppt/wnt5b mutants (Figure 7A–C). A direct side-by-side comparison of def6 and wnt5b morphants was therefore undertaken. Alcian blue staining of the cartilaginous structures in the region of the head indicated impaired growth of the head skeleton in both def6 and wnt5b morphants (Figure 7D–F). In detail, Meckel's cartilage (Figure 7D–F, black arrowhead) did not extend as far anteriorly beyond the eyes as in wt embryos. Also, the ceratohyal was posteriorly shifted and thicker in def6 and wnt5b MO-injected embryos in comparison to wt embryos (Figure 7 D–F, red arrowhead). Morphometric analysis indicated that both of these changes were significantly different between def6 morphants and wild-type controls (Figure 7G and H), similar to wnt5b morphants.


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)

Craniofacial defects in def6 and wnt5b MO-injected embryos.(A–C) 4 dpf, the distance between the eyes is indicated with a double-headed arrow. Def6 and wnt5b MO-injected embryos exhibit a ‘hammerhead’-like phenotype. (D–F) Alcian Blue staining of the cartilage in the head region of 4 dpf embryos. Meckel's cartilage is indicated with a black arrowhead and does not extend anteriorly beyond the eyes in def6 and wnt5b MO-injected embryos. The ceratohyal is indicated with a red arrowhead and is more posteriorly shifted in the two groups of morphants. Image E; representative image of 105/109 embryos, Image F; representative image of 89/92 embryos. (G) Representative wt embryo and measures taken are shown on the left. Line A was drawn as a baseline for further measurements and also served to normalise distance B. Line B is the distance from line A to the anterior end of the ceratohyal. The ratio of distance B divided by distance A is indicated on the graph. Two- tailed Student's t-tests indicated a significant (p<0.001; three asterisks) decrease in this ratio in the def6 MO-injected embryos versus wt siblings and between wnt5b MO-injected embryos versus wt siblings. (H) Representative wt embryo and the measures taken are shown on the image on the left. Line A, as before, was used to normalise distance C. Line C is the distance from line A to the anterior end of Meckel's cartilage. The ratio of this distance is shown on the graph. Injecting 2.5 ng of def6 MO resulted in a significant (p<0.001; three asterisks) decrease in this ratio as determined by two-tailed Student's t-tests; similarly injection of 5 ng wnt5b MO resulted in a significant (p<0.001; three asterisks) decrease in this ratio.
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Related In: Results  -  Collection

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pone-0026548-g007: Craniofacial defects in def6 and wnt5b MO-injected embryos.(A–C) 4 dpf, the distance between the eyes is indicated with a double-headed arrow. Def6 and wnt5b MO-injected embryos exhibit a ‘hammerhead’-like phenotype. (D–F) Alcian Blue staining of the cartilage in the head region of 4 dpf embryos. Meckel's cartilage is indicated with a black arrowhead and does not extend anteriorly beyond the eyes in def6 and wnt5b MO-injected embryos. The ceratohyal is indicated with a red arrowhead and is more posteriorly shifted in the two groups of morphants. Image E; representative image of 105/109 embryos, Image F; representative image of 89/92 embryos. (G) Representative wt embryo and measures taken are shown on the left. Line A was drawn as a baseline for further measurements and also served to normalise distance B. Line B is the distance from line A to the anterior end of the ceratohyal. The ratio of distance B divided by distance A is indicated on the graph. Two- tailed Student's t-tests indicated a significant (p<0.001; three asterisks) decrease in this ratio in the def6 MO-injected embryos versus wt siblings and between wnt5b MO-injected embryos versus wt siblings. (H) Representative wt embryo and the measures taken are shown on the image on the left. Line A, as before, was used to normalise distance C. Line C is the distance from line A to the anterior end of Meckel's cartilage. The ratio of this distance is shown on the graph. Injecting 2.5 ng of def6 MO resulted in a significant (p<0.001; three asterisks) decrease in this ratio as determined by two-tailed Student's t-tests; similarly injection of 5 ng wnt5b MO resulted in a significant (p<0.001; three asterisks) decrease in this ratio.
Mentions: The CE movement defects observed in def6 MO-injected embryos strongly resembled previously published ppt/wnt5b mutants and/or morphants [11], [29]. At tail-bud stage, the embryonic axis failed to move around the yolk (Figure 6A–C, arrowheads). This effect was statistically significant (Figure 6D), with the def6 MO having a stronger effect than the wnt5b MO at the concentrations of MO tested. At later stages, embryos were shorter with truncated tails (Figure 6E–G). At 4dpf, def6 morphants developed the ‘hammerhead’-like phenotype, a hallmark of ppt/wnt5b mutants (Figure 7A–C). A direct side-by-side comparison of def6 and wnt5b morphants was therefore undertaken. Alcian blue staining of the cartilaginous structures in the region of the head indicated impaired growth of the head skeleton in both def6 and wnt5b morphants (Figure 7D–F). In detail, Meckel's cartilage (Figure 7D–F, black arrowhead) did not extend as far anteriorly beyond the eyes as in wt embryos. Also, the ceratohyal was posteriorly shifted and thicker in def6 and wnt5b MO-injected embryos in comparison to wt embryos (Figure 7 D–F, red arrowhead). Morphometric analysis indicated that both of these changes were significantly different between def6 morphants and wild-type controls (Figure 7G and H), similar to wnt5b morphants.

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