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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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Knockdown of cfl1 causes epiboly defects.Embryos were injected with or without cfl1 MO and photographed at 10 h post-fertilization (hpf). The cfl1 MO caused epiboly defects of different severities. (A) A sham-injected embryo reached 100% epiboly. (B) A cfl1 morphant with a malformed tail bud. (C) A cfl1 morphant that reached 90% epiboly. (D) A cfl1 morphant that reached 50% epiboly. Scale bar, 200 µm.
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pone-0015331-g002: Knockdown of cfl1 causes epiboly defects.Embryos were injected with or without cfl1 MO and photographed at 10 h post-fertilization (hpf). The cfl1 MO caused epiboly defects of different severities. (A) A sham-injected embryo reached 100% epiboly. (B) A cfl1 morphant with a malformed tail bud. (C) A cfl1 morphant that reached 90% epiboly. (D) A cfl1 morphant that reached 50% epiboly. Scale bar, 200 µm.

Mentions: To further investigate the role of Cfl1 during embryogenesis in zebrafish, we separately injected embryos with two non-overlapping antisense MOs, tMO1 and tMO2 targeting the start codon and neighboring 5′-untranslated region (UTR) sequence of cfl1 mRNA at −22 to +3 and −50 to −26, respectively, to block translation of cfl1. At 10 hours post fertilization (hpf), untreated embryos had completed epiboly and had entirely enclosed the yolk sphere (Fig. 2A). By contrast, knockdown of cfl1 by tMO1 delayed or even arrested epiboly progression (Fig. 2B–C) in a dose-dependent manner and similar inhibition of epiboly was observed using the non-overlapping tMO2 (Table 1). Embryos injected with 10 ng tMO1, showed severe retardation in epiboly progression, and some of them could not complete gastrulation and ceased at about the 50% epiboly stage at 10 hpf (Fig. 2D). Embryos injected with lower tMO1 dosages had milder epiboly defects, most of them achieved 80%∼90% epiboly (Fig. 2C), and some of them only exhibited a malformed tail-bud (Fig. 2B). To further confirm that the severely retarded embryos are developmentally arrested, we monitored those 10 ng tMO1-injected embryos, which did not reach 100% epiboly at 10 hpf, until 12 hpf when the un-injected control embryos developed to 6-somite stage, and found that 235 out of 247 of those embryos (95.6±5.4%) still arrested at 60∼80% epiboly in three trials.


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 epiboly defects.Embryos were injected with or without cfl1 MO and photographed at 10 h post-fertilization (hpf). The cfl1 MO caused epiboly defects of different severities. (A) A sham-injected embryo reached 100% epiboly. (B) A cfl1 morphant with a malformed tail bud. (C) A cfl1 morphant that reached 90% epiboly. (D) A cfl1 morphant that reached 50% epiboly. Scale bar, 200 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3008747&req=5

pone-0015331-g002: Knockdown of cfl1 causes epiboly defects.Embryos were injected with or without cfl1 MO and photographed at 10 h post-fertilization (hpf). The cfl1 MO caused epiboly defects of different severities. (A) A sham-injected embryo reached 100% epiboly. (B) A cfl1 morphant with a malformed tail bud. (C) A cfl1 morphant that reached 90% epiboly. (D) A cfl1 morphant that reached 50% epiboly. Scale bar, 200 µm.
Mentions: To further investigate the role of Cfl1 during embryogenesis in zebrafish, we separately injected embryos with two non-overlapping antisense MOs, tMO1 and tMO2 targeting the start codon and neighboring 5′-untranslated region (UTR) sequence of cfl1 mRNA at −22 to +3 and −50 to −26, respectively, to block translation of cfl1. At 10 hours post fertilization (hpf), untreated embryos had completed epiboly and had entirely enclosed the yolk sphere (Fig. 2A). By contrast, knockdown of cfl1 by tMO1 delayed or even arrested epiboly progression (Fig. 2B–C) in a dose-dependent manner and similar inhibition of epiboly was observed using the non-overlapping tMO2 (Table 1). Embryos injected with 10 ng tMO1, showed severe retardation in epiboly progression, and some of them could not complete gastrulation and ceased at about the 50% epiboly stage at 10 hpf (Fig. 2D). Embryos injected with lower tMO1 dosages had milder epiboly defects, most of them achieved 80%∼90% epiboly (Fig. 2C), and some of them only exhibited a malformed tail-bud (Fig. 2B). To further confirm that the severely retarded embryos are developmentally arrested, we monitored those 10 ng tMO1-injected embryos, which did not reach 100% epiboly at 10 hpf, until 12 hpf when the un-injected control embryos developed to 6-somite stage, and found that 235 out of 247 of those embryos (95.6±5.4%) still arrested at 60∼80% epiboly in three trials.

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