Limits...
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.

Show MeSH

Related in: MedlinePlus

Knockdown of cfl1 causes convergent and extension defects.(A, B) Embryos were injected with Q-rhodamine with or without cfl1 tMO1 and incubated in the dark. Cells of the lateral (A) and dorsal blastomere margins (B) are marked as shown in red fluorescence at the shield stage and observed until 10.5 hpf. Representative photographs taken at 6.5, 8.5 and 10.5 hpf for both the untreated control and cfl1 MO-injected embryos are shown. (C) As shown in the panel A with an 8.5-hpf control embryo, the radius of the embryo (R) and the migration distance of the labeled cells (r) were measured. The degree of convergence (θ) was calculated by applying the following equation: θ =  sin−1(r/R), and then plotted against the stage of the embryo in hpf. (D) As shown in panel B with an 8.5-hpf control embryo, the angle (φ) between the two arrows of the anterior and posterior ends of the marked axial mesoendoderm connecting the center of the embryo was measured in each embryo to estimate the degree of extension. The degree of extension (φ) was then plotted against the stage of the embryo. (E, F) WISH against cstl1b (expressed in the prechordal plate as indicated by asterisks) and dlx3b (expressed in paraxial mesoderm as indicated by arrows) of bud-stage zebrafish embryos was performed. Representative photographs in frontal view are shown for an untreated (E) and a cfl1 tMO1-injected embryo (F).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3008747&req=5

pone-0015331-g008: Knockdown of cfl1 causes convergent and extension defects.(A, B) Embryos were injected with Q-rhodamine with or without cfl1 tMO1 and incubated in the dark. Cells of the lateral (A) and dorsal blastomere margins (B) are marked as shown in red fluorescence at the shield stage and observed until 10.5 hpf. Representative photographs taken at 6.5, 8.5 and 10.5 hpf for both the untreated control and cfl1 MO-injected embryos are shown. (C) As shown in the panel A with an 8.5-hpf control embryo, the radius of the embryo (R) and the migration distance of the labeled cells (r) were measured. The degree of convergence (θ) was calculated by applying the following equation: θ =  sin−1(r/R), and then plotted against the stage of the embryo in hpf. (D) As shown in panel B with an 8.5-hpf control embryo, the angle (φ) between the two arrows of the anterior and posterior ends of the marked axial mesoendoderm connecting the center of the embryo was measured in each embryo to estimate the degree of extension. The degree of extension (φ) was then plotted against the stage of the embryo. (E, F) WISH against cstl1b (expressed in the prechordal plate as indicated by asterisks) and dlx3b (expressed in paraxial mesoderm as indicated by arrows) of bud-stage zebrafish embryos was performed. Representative photographs in frontal view are shown for an untreated (E) and a cfl1 tMO1-injected embryo (F).

Mentions: To further investigate how Cfl1 affects cell movements during gastrulation, we conducted cell tracing assays using caged Q-rhodamine dextran [32]. Caged Q-rhodamine was injected with or without the cfl1 tMO1 into one-cell stage embryos and later uncaged by brief exposure to ultraviolet light to mark a selected group of cells. To monitor dorsal convergence movements, we marked cells in the lateral blastoderm margin at 90° from the dorsal embryonic shield, and fluorescent cells were traced during gastrulation. The marked cells in the control StdMO-injected embryos moved dorso-anteriorly, and had extended along the anterior-posterior (AP) axis at 10.5 hpf (Fig. 8A, upper panels), as previously reported [33], [34]. By contrast, the anterior migration and convergence of lateral mesendodermal cells toward the dorsal side were notably impaired in cfl1 morphants (Fig. 8A, lower panels). Measuring the degree of convergence (θ =  sin−1(r/R)) from the origin of the marked cells in control and treated embryos at different times after recording and plotting the results against the time (hpf) showed that the fitted slope of cfl1 tMO1-injected embryos (y = 10.7x - 69.7) clearly deviated from that of StdMO-injected ones (y = 15.6x - 96.2) (Fig. 8C). To examine the effect of cfl1 tMO1 on AP extension movements, cells of dorsal embryonic shields were marked and monitored as previously described in StdMO- and cfl1 tMO1-injected embryos (Fig. 8B). In control StdMO-injected embryos, marked cells were found in the dorsal axial hypoblast along the entire AP axis at 10.5 hpf (Fig. 8B, upper panels) which is consistent with a previous report [34]. By contrast, the anterior movement of marked cells was perturbed, exhibiting a shortened axial mesoendoderm in cfl1 tMO1-injected embryos (Fig. 8B, lower panels). Measuring the angle (ϕ) between two arrows of the anterior and posterior ends of the marked axial mesoendoderm connecting to the center of the embryos (Fig. 8B) in control and treated embryos at different times after recording and plotting the results against times of measurement demonstrated that the fitted slope (33.2x - 215.8) of cfl1 tMO1-injected embryos was also lower than that of StdMO-injected ones (y = 41.7x - 261.9) (Fig. 8D). Furthermore, we also conducted WISH to reveal the anterior edge of the neural plate and prechordal plate by probing against cathepsin L 1 b (cstl1b) and distal-less homeobox gene 3b (dlx3b), respectively. The body axis was severely shortened and broadened in tMO1-treated embryos (Fig. 8E) compared to that of control embryos (Fig. 8F).


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)

Knockdown of cfl1 causes convergent and extension defects.(A, B) Embryos were injected with Q-rhodamine with or without cfl1 tMO1 and incubated in the dark. Cells of the lateral (A) and dorsal blastomere margins (B) are marked as shown in red fluorescence at the shield stage and observed until 10.5 hpf. Representative photographs taken at 6.5, 8.5 and 10.5 hpf for both the untreated control and cfl1 MO-injected embryos are shown. (C) As shown in the panel A with an 8.5-hpf control embryo, the radius of the embryo (R) and the migration distance of the labeled cells (r) were measured. The degree of convergence (θ) was calculated by applying the following equation: θ =  sin−1(r/R), and then plotted against the stage of the embryo in hpf. (D) As shown in panel B with an 8.5-hpf control embryo, the angle (φ) between the two arrows of the anterior and posterior ends of the marked axial mesoendoderm connecting the center of the embryo was measured in each embryo to estimate the degree of extension. The degree of extension (φ) was then plotted against the stage of the embryo. (E, F) WISH against cstl1b (expressed in the prechordal plate as indicated by asterisks) and dlx3b (expressed in paraxial mesoderm as indicated by arrows) of bud-stage zebrafish embryos was performed. Representative photographs in frontal view are shown for an untreated (E) and a cfl1 tMO1-injected embryo (F).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015331-g008: Knockdown of cfl1 causes convergent and extension defects.(A, B) Embryos were injected with Q-rhodamine with or without cfl1 tMO1 and incubated in the dark. Cells of the lateral (A) and dorsal blastomere margins (B) are marked as shown in red fluorescence at the shield stage and observed until 10.5 hpf. Representative photographs taken at 6.5, 8.5 and 10.5 hpf for both the untreated control and cfl1 MO-injected embryos are shown. (C) As shown in the panel A with an 8.5-hpf control embryo, the radius of the embryo (R) and the migration distance of the labeled cells (r) were measured. The degree of convergence (θ) was calculated by applying the following equation: θ =  sin−1(r/R), and then plotted against the stage of the embryo in hpf. (D) As shown in panel B with an 8.5-hpf control embryo, the angle (φ) between the two arrows of the anterior and posterior ends of the marked axial mesoendoderm connecting the center of the embryo was measured in each embryo to estimate the degree of extension. The degree of extension (φ) was then plotted against the stage of the embryo. (E, F) WISH against cstl1b (expressed in the prechordal plate as indicated by asterisks) and dlx3b (expressed in paraxial mesoderm as indicated by arrows) of bud-stage zebrafish embryos was performed. Representative photographs in frontal view are shown for an untreated (E) and a cfl1 tMO1-injected embryo (F).
Mentions: To further investigate how Cfl1 affects cell movements during gastrulation, we conducted cell tracing assays using caged Q-rhodamine dextran [32]. Caged Q-rhodamine was injected with or without the cfl1 tMO1 into one-cell stage embryos and later uncaged by brief exposure to ultraviolet light to mark a selected group of cells. To monitor dorsal convergence movements, we marked cells in the lateral blastoderm margin at 90° from the dorsal embryonic shield, and fluorescent cells were traced during gastrulation. The marked cells in the control StdMO-injected embryos moved dorso-anteriorly, and had extended along the anterior-posterior (AP) axis at 10.5 hpf (Fig. 8A, upper panels), as previously reported [33], [34]. By contrast, the anterior migration and convergence of lateral mesendodermal cells toward the dorsal side were notably impaired in cfl1 morphants (Fig. 8A, lower panels). Measuring the degree of convergence (θ =  sin−1(r/R)) from the origin of the marked cells in control and treated embryos at different times after recording and plotting the results against the time (hpf) showed that the fitted slope of cfl1 tMO1-injected embryos (y = 10.7x - 69.7) clearly deviated from that of StdMO-injected ones (y = 15.6x - 96.2) (Fig. 8C). To examine the effect of cfl1 tMO1 on AP extension movements, cells of dorsal embryonic shields were marked and monitored as previously described in StdMO- and cfl1 tMO1-injected embryos (Fig. 8B). In control StdMO-injected embryos, marked cells were found in the dorsal axial hypoblast along the entire AP axis at 10.5 hpf (Fig. 8B, upper panels) which is consistent with a previous report [34]. By contrast, the anterior movement of marked cells was perturbed, exhibiting a shortened axial mesoendoderm in cfl1 tMO1-injected embryos (Fig. 8B, lower panels). Measuring the angle (ϕ) between two arrows of the anterior and posterior ends of the marked axial mesoendoderm connecting to the center of the embryos (Fig. 8B) in control and treated embryos at different times after recording and plotting the results against times of measurement demonstrated that the fitted slope (33.2x - 215.8) of cfl1 tMO1-injected embryos was also lower than that of StdMO-injected ones (y = 41.7x - 261.9) (Fig. 8D). Furthermore, we also conducted WISH to reveal the anterior edge of the neural plate and prechordal plate by probing against cathepsin L 1 b (cstl1b) and distal-less homeobox gene 3b (dlx3b), respectively. The body axis was severely shortened and broadened in tMO1-treated embryos (Fig. 8E) compared to that of control embryos (Fig. 8F).

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