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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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Spatial and temporal expression of cfl1 during embryogenesis.(A–N) Representative whole-mount in situ hybridization photographs are shown to reveal the expression patterns of cfl1 at the designated stages from 1-cell to 5 day post-fertilization (dpf) as denoted at the lower left corner of each panel. (O) A representative cryo-section photograph of an embryo at the 70% epiboly stage underwent WISH against cfl1. The cryo-section was taken from the box region as depicted in the embryo carton shown on the right. b, brain; ba, bronchial arches; ll, lateral line system; pd, pronephric duct; pa, pharyngeal arches; EVL, enveloping layer; DEL, deep cell layer. Scale bars: 400 µm for the 4- and 5-dpf embryos, 200 µm for the others and 50 µm for the cryo-section photograph.
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pone-0015331-g005: Spatial and temporal expression of cfl1 during embryogenesis.(A–N) Representative whole-mount in situ hybridization photographs are shown to reveal the expression patterns of cfl1 at the designated stages from 1-cell to 5 day post-fertilization (dpf) as denoted at the lower left corner of each panel. (O) A representative cryo-section photograph of an embryo at the 70% epiboly stage underwent WISH against cfl1. The cryo-section was taken from the box region as depicted in the embryo carton shown on the right. b, brain; ba, bronchial arches; ll, lateral line system; pd, pronephric duct; pa, pharyngeal arches; EVL, enveloping layer; DEL, deep cell layer. Scale bars: 400 µm for the 4- and 5-dpf embryos, 200 µm for the others and 50 µm for the cryo-section photograph.

Mentions: The unsynchronized movements of EVL and DEL in cfl1 morphants led us to suspect that cfl may be expressed differentially and result in different responses upon knockdown of cfl1. The RT-PCR analysis showed that cfl1 was expressed throughout development and was present in all adult tissues examined (data not shown). To gain the temporal and spatial profiles of cfl1 during embryogenesis, we thus performed whole-mount in situ hybridization (WISH) in different stages of embryos (Fig. 5). The WISH analysis revealed that the expression domains of cfl1 during early development were ubiquitous until the sphere stage (Fig. 5A–D). The cfl1's expression was notably reduced at 30% epiboly (Fig. 5E) and the shield stages (Fig. 5F), and it was absent from the future ventral side of the embryo in the bud stage (Fig. 5G). cfl1 was strongly expressed throughout the embryo body during early segmentation period (Fig. 5H); it was later restricted to the central nervous system, lateral line, and pronephric duct at 26∼31 hpf (Fig. 5I and J); and expression domains were evident in the brain, pharyngeal arches, lateral line, and pronephric duct in the larval stages (3∼5 days post-fertilization, Fig. 5K–N). We were in particular interest in the expression of cfl1 at different cell layers of a gastrulating embryo, thus we performed cryo-section for those 70% epiboly stage embryos underwent WISH against cfl1. Interestingly, we found that the cfl1 expression domain was restricted to blastomere boundaries of the DEL, but not those of the EVL (Fig. 5O).


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)

Spatial and temporal expression of cfl1 during embryogenesis.(A–N) Representative whole-mount in situ hybridization photographs are shown to reveal the expression patterns of cfl1 at the designated stages from 1-cell to 5 day post-fertilization (dpf) as denoted at the lower left corner of each panel. (O) A representative cryo-section photograph of an embryo at the 70% epiboly stage underwent WISH against cfl1. The cryo-section was taken from the box region as depicted in the embryo carton shown on the right. b, brain; ba, bronchial arches; ll, lateral line system; pd, pronephric duct; pa, pharyngeal arches; EVL, enveloping layer; DEL, deep cell layer. Scale bars: 400 µm for the 4- and 5-dpf embryos, 200 µm for the others and 50 µm for the cryo-section photograph.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015331-g005: Spatial and temporal expression of cfl1 during embryogenesis.(A–N) Representative whole-mount in situ hybridization photographs are shown to reveal the expression patterns of cfl1 at the designated stages from 1-cell to 5 day post-fertilization (dpf) as denoted at the lower left corner of each panel. (O) A representative cryo-section photograph of an embryo at the 70% epiboly stage underwent WISH against cfl1. The cryo-section was taken from the box region as depicted in the embryo carton shown on the right. b, brain; ba, bronchial arches; ll, lateral line system; pd, pronephric duct; pa, pharyngeal arches; EVL, enveloping layer; DEL, deep cell layer. Scale bars: 400 µm for the 4- and 5-dpf embryos, 200 µm for the others and 50 µm for the cryo-section photograph.
Mentions: The unsynchronized movements of EVL and DEL in cfl1 morphants led us to suspect that cfl may be expressed differentially and result in different responses upon knockdown of cfl1. The RT-PCR analysis showed that cfl1 was expressed throughout development and was present in all adult tissues examined (data not shown). To gain the temporal and spatial profiles of cfl1 during embryogenesis, we thus performed whole-mount in situ hybridization (WISH) in different stages of embryos (Fig. 5). The WISH analysis revealed that the expression domains of cfl1 during early development were ubiquitous until the sphere stage (Fig. 5A–D). The cfl1's expression was notably reduced at 30% epiboly (Fig. 5E) and the shield stages (Fig. 5F), and it was absent from the future ventral side of the embryo in the bud stage (Fig. 5G). cfl1 was strongly expressed throughout the embryo body during early segmentation period (Fig. 5H); it was later restricted to the central nervous system, lateral line, and pronephric duct at 26∼31 hpf (Fig. 5I and J); and expression domains were evident in the brain, pharyngeal arches, lateral line, and pronephric duct in the larval stages (3∼5 days post-fertilization, Fig. 5K–N). We were in particular interest in the expression of cfl1 at different cell layers of a gastrulating embryo, thus we performed cryo-section for those 70% epiboly stage embryos underwent WISH against cfl1. Interestingly, we found that the cfl1 expression domain was restricted to blastomere boundaries of the DEL, but not those of the EVL (Fig. 5O).

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