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Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish.

Lin CW, Yen ST, Chang HT, Chen SJ, Lai SL, Liu YC, Chan TH, Liao WL, Lee SJ - PLoS ONE (2010)

Bottom Line: During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish.Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO).The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, National Taiwan University, Taipei, Taiwan, Republic of China.

ABSTRACT
During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish. The association of different layers relies on E-cadherin based cellular junctions, whose stability can be affected by actin turnover. Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO). Knockdown of cfl1 interfered with epibolic movement of deep cell layer (DEL) but not in the enveloping layer (EVL) and the defect could be specifically rescued by overexpression of cfl1. It appeared that the uncoordinated movements of DEL and EVL were regulated by the differential expression of cfl1 in the DEL, but not EVL as shown by in situ hybridization. The dissociation of DEL and EVL was further evident by the loss of adhesion between layers by using transmission electronic and confocal microscopy analyses. cfl1 morphants also exhibited abnormal convergent extension, cellular migration and actin filaments, but not involution of hypoblast. The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays. These results suggest that proper actin turnover mediated by Cfl1 is essential for adhesion between DEL and EVL and cell movements during gastrulation in zebrafish.

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Related in: MedlinePlus

Cfl1 MOs efficiently block the translation of GFP fusion constructs containing respective MO binding site.Embryos at 1-cell stage were injected with 330 pg pCS2+ XLT fused with a fragment of cfl1 gene sequence from −22 to +195 or −52 to +51 in the absence or presence of 7.5 ng cfl1 tMO1 or tMO2, respectively. The expression of GFP was examined at 10 hpf and photographed under epifluorescent microscopy. The representative photos for embryos treated without cfl1 MO (A) and with tMO1 (B) are shown and the percentages of injected embryos expressing GFP are shown in average ± standard deviation (C).
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pone-0015331-g003: Cfl1 MOs efficiently block the translation of GFP fusion constructs containing respective MO binding site.Embryos at 1-cell stage were injected with 330 pg pCS2+ XLT fused with a fragment of cfl1 gene sequence from −22 to +195 or −52 to +51 in the absence or presence of 7.5 ng cfl1 tMO1 or tMO2, respectively. The expression of GFP was examined at 10 hpf and photographed under epifluorescent microscopy. The representative photos for embryos treated without cfl1 MO (A) and with tMO1 (B) are shown and the percentages of injected embryos expressing GFP are shown in average ± standard deviation (C).

Mentions: To examine whether the zygotic Cfl1 expression can be inhibited by cfl1 MOs, we inserted a stretch of cfl1 nucleotides ∼200-bp, which contains their respective MO target site, into a pCS2+ XLT vector with a green fluorescent protein (GFP) gene and co-injected these constructs with their respective MO. Embryos (81.0±13.8%) injected with the pCS2+ XLT with tMO1 binding site (tMO1 plasmid) expressed GFP fluorescence (Fig. 3A). By contrast, none of embryos co-injected with tMO1 showed GFP fluorescence (Fig. 3B). Similarly, embryos (79.4±11.9%) injected with the pCS2+ XLT with tMO2 binding site (tMO2 plasmid) expressed GFP fluorescence, but not in those embryos co-injected with tMO2 (Fig. 3C). Both MOs appeared to be equally effectively, we thus used tMO1 for the rest of experiments unless otherwise stated.


Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish.

Lin CW, Yen ST, Chang HT, Chen SJ, Lai SL, Liu YC, Chan TH, Liao WL, Lee SJ - PLoS ONE (2010)

Cfl1 MOs efficiently block the translation of GFP fusion constructs containing respective MO binding site.Embryos at 1-cell stage were injected with 330 pg pCS2+ XLT fused with a fragment of cfl1 gene sequence from −22 to +195 or −52 to +51 in the absence or presence of 7.5 ng cfl1 tMO1 or tMO2, respectively. The expression of GFP was examined at 10 hpf and photographed under epifluorescent microscopy. The representative photos for embryos treated without cfl1 MO (A) and with tMO1 (B) are shown and the percentages of injected embryos expressing GFP are shown in average ± standard deviation (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008747&req=5

pone-0015331-g003: Cfl1 MOs efficiently block the translation of GFP fusion constructs containing respective MO binding site.Embryos at 1-cell stage were injected with 330 pg pCS2+ XLT fused with a fragment of cfl1 gene sequence from −22 to +195 or −52 to +51 in the absence or presence of 7.5 ng cfl1 tMO1 or tMO2, respectively. The expression of GFP was examined at 10 hpf and photographed under epifluorescent microscopy. The representative photos for embryos treated without cfl1 MO (A) and with tMO1 (B) are shown and the percentages of injected embryos expressing GFP are shown in average ± standard deviation (C).
Mentions: To examine whether the zygotic Cfl1 expression can be inhibited by cfl1 MOs, we inserted a stretch of cfl1 nucleotides ∼200-bp, which contains their respective MO target site, into a pCS2+ XLT vector with a green fluorescent protein (GFP) gene and co-injected these constructs with their respective MO. Embryos (81.0±13.8%) injected with the pCS2+ XLT with tMO1 binding site (tMO1 plasmid) expressed GFP fluorescence (Fig. 3A). By contrast, none of embryos co-injected with tMO1 showed GFP fluorescence (Fig. 3B). Similarly, embryos (79.4±11.9%) injected with the pCS2+ XLT with tMO2 binding site (tMO2 plasmid) expressed GFP fluorescence, but not in those embryos co-injected with tMO2 (Fig. 3C). Both MOs appeared to be equally effectively, we thus used tMO1 for the rest of experiments unless otherwise stated.

Bottom Line: During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish.Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO).The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, National Taiwan University, Taipei, Taiwan, Republic of China.

ABSTRACT
During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish. The association of different layers relies on E-cadherin based cellular junctions, whose stability can be affected by actin turnover. Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO). Knockdown of cfl1 interfered with epibolic movement of deep cell layer (DEL) but not in the enveloping layer (EVL) and the defect could be specifically rescued by overexpression of cfl1. It appeared that the uncoordinated movements of DEL and EVL were regulated by the differential expression of cfl1 in the DEL, but not EVL as shown by in situ hybridization. The dissociation of DEL and EVL was further evident by the loss of adhesion between layers by using transmission electronic and confocal microscopy analyses. cfl1 morphants also exhibited abnormal convergent extension, cellular migration and actin filaments, but not involution of hypoblast. The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays. These results suggest that proper actin turnover mediated by Cfl1 is essential for adhesion between DEL and EVL and cell movements during gastrulation in zebrafish.

Show MeSH
Related in: MedlinePlus