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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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Related in: MedlinePlus

Knockdown of def6 does not alter mesodermal cell fate specification and anterior-posterior patterning.Uninjected and def6 MO-injected embryos were fixed at 6 hpf or 10 hpf and in situ hybridisation was carried out with the indicated probes. Chordin (chd; A and B; 21/21 embryos) and goosecoid (gsc; E and F; 15/15 embryos) are expressed in the dorsal mesoderm and specify dorsal cell fates. Bone morphogenetic proteins (bmp2b; C and D; 18/18 embryos; bmp4; I and J; 15/15 embryos) are involved in ventral cell fate specification. The non-axial marker caudal homeobox transcription factor 4 (cdx4; I and J; 19/19 embryos) and the mesendodermal marker no-tail (ntl; K and L; 31/31 embryos) are also shown. The expression pattern of all these genes in wt and def6 morphants was indistinguishable at 6 hpf, indicating normal cell fate specification in def6 MO-injected embryos. At 10 hpf, expression of the anterior specific genes krox20 (M and N; 37/41 embryos) and pax2 (O and P; 16/19 embryos,) persisted in def6 MO-injected embryos indicating that no anterior structures were deleted. The expression domain of these markers was posteriorly shifted and expanded in def6 MO-injected embryos in comparison to wt siblings (M'–P') when viewed from the dorsal side. Lateral views (A–F, I, J, M–P), animal pole views (G, H, K, L) and dorsal views (M'–P') with anterior to the top are shown.
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pone-0026548-g004: Knockdown of def6 does not alter mesodermal cell fate specification and anterior-posterior patterning.Uninjected and def6 MO-injected embryos were fixed at 6 hpf or 10 hpf and in situ hybridisation was carried out with the indicated probes. Chordin (chd; A and B; 21/21 embryos) and goosecoid (gsc; E and F; 15/15 embryos) are expressed in the dorsal mesoderm and specify dorsal cell fates. Bone morphogenetic proteins (bmp2b; C and D; 18/18 embryos; bmp4; I and J; 15/15 embryos) are involved in ventral cell fate specification. The non-axial marker caudal homeobox transcription factor 4 (cdx4; I and J; 19/19 embryos) and the mesendodermal marker no-tail (ntl; K and L; 31/31 embryos) are also shown. The expression pattern of all these genes in wt and def6 morphants was indistinguishable at 6 hpf, indicating normal cell fate specification in def6 MO-injected embryos. At 10 hpf, expression of the anterior specific genes krox20 (M and N; 37/41 embryos) and pax2 (O and P; 16/19 embryos,) persisted in def6 MO-injected embryos indicating that no anterior structures were deleted. The expression domain of these markers was posteriorly shifted and expanded in def6 MO-injected embryos in comparison to wt siblings (M'–P') when viewed from the dorsal side. Lateral views (A–F, I, J, M–P), animal pole views (G, H, K, L) and dorsal views (M'–P') with anterior to the top are shown.

Mentions: The reduced extension of the embryonic axis observed in def6 MO-injected embryos suggested impairment of CE movements during gastrulation. However, it could also imply incorrect mesoderm cell specification at the onset of gastrulation. These two processes, although very different, occur at the same time and produce similar phenotypes. To test whether cell fate specification was affected by the def6 MO, whole mount in situ hybridisation was carried out using a panel of dorsal, ventral and mesendodermal markers, all known to be involved in cell fate specification. The expression pattern of the dorsalising factors chordin (chd; Figure 4A and B) and goosecoid (gsc; Figure 4E and F) remained unchanged in def6 MO-injected embryos when compared to wild-type siblings at shield stage. The expression of bone morphogenetic proteins (BMPs) bmp2b (Figure 4C and D) and bmp4 (Figure 4G and H) were similar in both def6 morphants and wild-type siblings, indicating that knockdown of def6 did not affect ventral cell fate specification. In addition, the expression pattern of the non-axial mesodermal marker, cdx4, remained unaffected in def6-MO injected embryos versus uninjected controls (Figure 4I and J). Finally, the expression pattern of the mesendodermal marker no-tail (ntl) was similar in def6 morphants and wild-type embryos (Figure 4K and L), further confirming that mesoderm induction occurs normally in def6 MO-injected embryos. Taken together, these results support the notion that altered cell fate does not account for the CE movement defect observed in def6 MO-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)

Knockdown of def6 does not alter mesodermal cell fate specification and anterior-posterior patterning.Uninjected and def6 MO-injected embryos were fixed at 6 hpf or 10 hpf and in situ hybridisation was carried out with the indicated probes. Chordin (chd; A and B; 21/21 embryos) and goosecoid (gsc; E and F; 15/15 embryos) are expressed in the dorsal mesoderm and specify dorsal cell fates. Bone morphogenetic proteins (bmp2b; C and D; 18/18 embryos; bmp4; I and J; 15/15 embryos) are involved in ventral cell fate specification. The non-axial marker caudal homeobox transcription factor 4 (cdx4; I and J; 19/19 embryos) and the mesendodermal marker no-tail (ntl; K and L; 31/31 embryos) are also shown. The expression pattern of all these genes in wt and def6 morphants was indistinguishable at 6 hpf, indicating normal cell fate specification in def6 MO-injected embryos. At 10 hpf, expression of the anterior specific genes krox20 (M and N; 37/41 embryos) and pax2 (O and P; 16/19 embryos,) persisted in def6 MO-injected embryos indicating that no anterior structures were deleted. The expression domain of these markers was posteriorly shifted and expanded in def6 MO-injected embryos in comparison to wt siblings (M'–P') when viewed from the dorsal side. Lateral views (A–F, I, J, M–P), animal pole views (G, H, K, L) and dorsal views (M'–P') with anterior to the top are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198796&req=5

pone-0026548-g004: Knockdown of def6 does not alter mesodermal cell fate specification and anterior-posterior patterning.Uninjected and def6 MO-injected embryos were fixed at 6 hpf or 10 hpf and in situ hybridisation was carried out with the indicated probes. Chordin (chd; A and B; 21/21 embryos) and goosecoid (gsc; E and F; 15/15 embryos) are expressed in the dorsal mesoderm and specify dorsal cell fates. Bone morphogenetic proteins (bmp2b; C and D; 18/18 embryos; bmp4; I and J; 15/15 embryos) are involved in ventral cell fate specification. The non-axial marker caudal homeobox transcription factor 4 (cdx4; I and J; 19/19 embryos) and the mesendodermal marker no-tail (ntl; K and L; 31/31 embryos) are also shown. The expression pattern of all these genes in wt and def6 morphants was indistinguishable at 6 hpf, indicating normal cell fate specification in def6 MO-injected embryos. At 10 hpf, expression of the anterior specific genes krox20 (M and N; 37/41 embryos) and pax2 (O and P; 16/19 embryos,) persisted in def6 MO-injected embryos indicating that no anterior structures were deleted. The expression domain of these markers was posteriorly shifted and expanded in def6 MO-injected embryos in comparison to wt siblings (M'–P') when viewed from the dorsal side. Lateral views (A–F, I, J, M–P), animal pole views (G, H, K, L) and dorsal views (M'–P') with anterior to the top are shown.
Mentions: The reduced extension of the embryonic axis observed in def6 MO-injected embryos suggested impairment of CE movements during gastrulation. However, it could also imply incorrect mesoderm cell specification at the onset of gastrulation. These two processes, although very different, occur at the same time and produce similar phenotypes. To test whether cell fate specification was affected by the def6 MO, whole mount in situ hybridisation was carried out using a panel of dorsal, ventral and mesendodermal markers, all known to be involved in cell fate specification. The expression pattern of the dorsalising factors chordin (chd; Figure 4A and B) and goosecoid (gsc; Figure 4E and F) remained unchanged in def6 MO-injected embryos when compared to wild-type siblings at shield stage. The expression of bone morphogenetic proteins (BMPs) bmp2b (Figure 4C and D) and bmp4 (Figure 4G and H) were similar in both def6 morphants and wild-type siblings, indicating that knockdown of def6 did not affect ventral cell fate specification. In addition, the expression pattern of the non-axial mesodermal marker, cdx4, remained unaffected in def6-MO injected embryos versus uninjected controls (Figure 4I and J). Finally, the expression pattern of the mesendodermal marker no-tail (ntl) was similar in def6 morphants and wild-type embryos (Figure 4K and L), further confirming that mesoderm induction occurs normally in def6 MO-injected embryos. Taken together, these results support the notion that altered cell fate does not account for the CE movement defect observed in def6 MO-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