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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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Cfl1 is required cell-autonomously for proper epiboly cell migration.Labeled cells from donor embryos injected with rhodamine-dextran in combined with StdMO (A, C) or cfl1 tMO1 (B, D) were transplanted into control embryos (A, B) and cfl1 morphants (C, D) respectively, and recorded under a confocal microscope. Pseudopods (arrowheads) and polygonal cell shape were observed in StdMO-injected cells transplanted in both control hosts (A) and cfl1 morphants (C), while blebbing-like structure (arrows) and rounded cell shape were observed in cfl1 tMO1-injected cells transplanted in both control hosts (B) and cfl1 morphants (D). The photographs shown are representative of at least 10 embryos in each experiment. Migration curvilinear velocity (Vcl) and straight line velocity (Vsl) of these StdMO- and cfl1 tMO1-injected cells in control hosts or cfl1 morphants were recorded by time-lapse epifluorescent microscopy and analyzed by SimplePCI software, respectively (E). Values between groups were compared using unpaired Student's t-test, and those showing a significant (* p<0.05) difference are denoted by different letters.
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pone-0015331-g011: Cfl1 is required cell-autonomously for proper epiboly cell migration.Labeled cells from donor embryos injected with rhodamine-dextran in combined with StdMO (A, C) or cfl1 tMO1 (B, D) were transplanted into control embryos (A, B) and cfl1 morphants (C, D) respectively, and recorded under a confocal microscope. Pseudopods (arrowheads) and polygonal cell shape were observed in StdMO-injected cells transplanted in both control hosts (A) and cfl1 morphants (C), while blebbing-like structure (arrows) and rounded cell shape were observed in cfl1 tMO1-injected cells transplanted in both control hosts (B) and cfl1 morphants (D). The photographs shown are representative of at least 10 embryos in each experiment. Migration curvilinear velocity (Vcl) and straight line velocity (Vsl) of these StdMO- and cfl1 tMO1-injected cells in control hosts or cfl1 morphants were recorded by time-lapse epifluorescent microscopy and analyzed by SimplePCI software, respectively (E). Values between groups were compared using unpaired Student's t-test, and those showing a significant (* p<0.05) difference are denoted by different letters.

Mentions: To further evaluate the cell autonomy of Cfl1 functions, cells from embryos injected with MOs and rhodamine-dextran, were transplanted into untreated host (control) embryos and cfl1 morphants respectively. Only those transplanted cells undergoing epiboly were selected for further analysis. The protrusive activities of these transplanted cells were examined by confocal microscopy, and representative snapshots were taken to show the polygonal shape and pseudopod formation by StdMO-injected cells in control hosts (arrow heads in Fig. 11A; Supplementary Movie S7) and in cfl1 morphants (Fig. 11C; Supplementary Movie S8), while cfl1 tMO1-injected cells remained rounded with blebbing-like structures in control hosts (arrow in Fig. 11B; Supplementary Movie S9) and in cfl1 morphants (Fig. 11D; Supplementary Movie S10). Furthermore, to reveal the migration velocity and direction of movement, we took time-lapse recordings under an epifluorescence microscope to trace cells from both StdMO- and cfl1 MO-injected embryos transplanted into untreated or cfl1 morphants. After analysis, we found that both the exact velocity (Vcl: curvilinear velocity; curvilinear distance/time) and directed velocity (Vsl: straight line velocity; straight line distance/time) were significantly reduced in cfl1 tMO1-injected cells compared to StdMO-injected cells when transplanted into an untreated host embryo (Fig. 11E). This indicates that cofilin functions cell autonomously in transplanted cell migration. In the same logic, both Vcl and Vsl of cfl1 tMO1-injected cells significantly reduced when transplanted into cfl1 morphants. However, the Vcl and Vsl of StdMO-injected cells is also significantly reduced when transplanted into cfl1 morphants, although was not as severe as cfl1 tMO1-injected cells in the same background (Vsl, Fig. 11E).


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 is required cell-autonomously for proper epiboly cell migration.Labeled cells from donor embryos injected with rhodamine-dextran in combined with StdMO (A, C) or cfl1 tMO1 (B, D) were transplanted into control embryos (A, B) and cfl1 morphants (C, D) respectively, and recorded under a confocal microscope. Pseudopods (arrowheads) and polygonal cell shape were observed in StdMO-injected cells transplanted in both control hosts (A) and cfl1 morphants (C), while blebbing-like structure (arrows) and rounded cell shape were observed in cfl1 tMO1-injected cells transplanted in both control hosts (B) and cfl1 morphants (D). The photographs shown are representative of at least 10 embryos in each experiment. Migration curvilinear velocity (Vcl) and straight line velocity (Vsl) of these StdMO- and cfl1 tMO1-injected cells in control hosts or cfl1 morphants were recorded by time-lapse epifluorescent microscopy and analyzed by SimplePCI software, respectively (E). Values between groups were compared using unpaired Student's t-test, and those showing a significant (* p<0.05) difference are denoted by different letters.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015331-g011: Cfl1 is required cell-autonomously for proper epiboly cell migration.Labeled cells from donor embryos injected with rhodamine-dextran in combined with StdMO (A, C) or cfl1 tMO1 (B, D) were transplanted into control embryos (A, B) and cfl1 morphants (C, D) respectively, and recorded under a confocal microscope. Pseudopods (arrowheads) and polygonal cell shape were observed in StdMO-injected cells transplanted in both control hosts (A) and cfl1 morphants (C), while blebbing-like structure (arrows) and rounded cell shape were observed in cfl1 tMO1-injected cells transplanted in both control hosts (B) and cfl1 morphants (D). The photographs shown are representative of at least 10 embryos in each experiment. Migration curvilinear velocity (Vcl) and straight line velocity (Vsl) of these StdMO- and cfl1 tMO1-injected cells in control hosts or cfl1 morphants were recorded by time-lapse epifluorescent microscopy and analyzed by SimplePCI software, respectively (E). Values between groups were compared using unpaired Student's t-test, and those showing a significant (* p<0.05) difference are denoted by different letters.
Mentions: To further evaluate the cell autonomy of Cfl1 functions, cells from embryos injected with MOs and rhodamine-dextran, were transplanted into untreated host (control) embryos and cfl1 morphants respectively. Only those transplanted cells undergoing epiboly were selected for further analysis. The protrusive activities of these transplanted cells were examined by confocal microscopy, and representative snapshots were taken to show the polygonal shape and pseudopod formation by StdMO-injected cells in control hosts (arrow heads in Fig. 11A; Supplementary Movie S7) and in cfl1 morphants (Fig. 11C; Supplementary Movie S8), while cfl1 tMO1-injected cells remained rounded with blebbing-like structures in control hosts (arrow in Fig. 11B; Supplementary Movie S9) and in cfl1 morphants (Fig. 11D; Supplementary Movie S10). Furthermore, to reveal the migration velocity and direction of movement, we took time-lapse recordings under an epifluorescence microscope to trace cells from both StdMO- and cfl1 MO-injected embryos transplanted into untreated or cfl1 morphants. After analysis, we found that both the exact velocity (Vcl: curvilinear velocity; curvilinear distance/time) and directed velocity (Vsl: straight line velocity; straight line distance/time) were significantly reduced in cfl1 tMO1-injected cells compared to StdMO-injected cells when transplanted into an untreated host embryo (Fig. 11E). This indicates that cofilin functions cell autonomously in transplanted cell migration. In the same logic, both Vcl and Vsl of cfl1 tMO1-injected cells significantly reduced when transplanted into cfl1 morphants. However, the Vcl and Vsl of StdMO-injected cells is also significantly reduced when transplanted into cfl1 morphants, although was not as severe as cfl1 tMO1-injected cells in the same background (Vsl, Fig. 11E).

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