In zebrafish, there are three homologous genes of ADF/cofilins: cofilin 1 (cfl1, non-muscle form, AY398324), cofilin 1-like (cfl1l, AY398323) and cofilin 2 (cfl2, muscle form, NM_205700). We were interested in cofilins, which are expressed during early embryogenesis and have the potential to regulate gastrulation cell migration. According to the available expression patterns in ZFIN, only cfl1 and cfl1l fit into this category, as cfl2 is mainly expressed in somites and later in myotomes and lenses. By a preliminary screening using an antisense morpholino oligo (MO), we found that knockdown of cfl1l had a minimum effect on gastrulation (our unpublished data). By contrast, the cfl1 MOs showed severe interference with the progression of gastrulation (data will be described later). Thus, we had isolated the zebrafish cfl1 gene by PCR cloning from zebrafish embryonic complementary DNAs. To compare its sequence identities and similarities of zebrafish Cfl1 to cofilins of other animal species, we aligned its amino acid sequence of with that of the non-muscle form of cofilins in mammals (including bovine, human, mouse, and rat), chick, western clawed frog, fish (including fugu and medaka), and the ADFs in the fruit fly (TSR) and nematode (UNC-60) (Fig. 1A). Zebrafish Cfl1 was highly similar to those cofilins examined (82%∼90%) except for the fruit fly (50%) and nematode (56%) (Fig. 1B). It was most similar to Cfl1 of medaka with 90% similarity. As shown in the phylogenetic tree (Fig. 1C), zebrafish Cfl1 was grouped together with the chordates, while the invertebrate ADFs (fly and nematode) were placed in an out-group cluster.

To determine the chromosomal position of cfl1, we conducted a syntenic analysis of three cofilin genes (Fig. 1D). The cfl1 in zebrafish, human, and mouse are located on chromosomes 14, 11, and 9, respectively. The same genes are found in the vicinity, which are efemp2 (EGF-containing fibulin-like extracellular matrix protein 2) and fibp (fibroblast growth factor (acidic) intracellular binding protein), but zebrafish cfl1 is arranged in a reversed orientation compared with those of human and mouse. These analyses suggest that zebrafish cfl1 is evolutionarily related to mammalian cofilin 1 genes.

The primary amino acid sequence of Cfl1 contains 165 amino acids with a molecular weight of ∼18 kDa. The characteristic secondary structures are depicted in Fig. 1E. Cfl1 has six α-helices and seven β-sheets with a phosphorylation site at Ser 3 and a nuclear localization sequence (NLS, at residues 21∼38). A three-dimensional ribbon structure of Cfl1 created by SWISS-MODEL (http://www.swissmodel.expasy.org) is shown in Fig. 1F. Taken together, the zebrafish cfl1 we isolated has high sequence and functional domain similarities compared to its orthologs from other species.

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.

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.

To confirm that the defects we observed in cfl1 morphants were specifically due to the loss of Cfl1 activity, we examined whether overexpression of cfl1 could rescue the cfl1 morphant defects. cfl1 was successfully overexpressed as revealed by the presence of GFP fluorescence in zebrafish embryos injected with mRNAs prepared from a PCS2+ XLT vector containing the full cfl1 coding sequence (data not shown). More importantly, the inhibition of epiboly could be significantly (P<0.05) reduced in embryos co-injected with 100 pg cfl1-egfp mRNAs and 7.5 ng tMO₁ (Table 2). Furthermore, depending on the sequence specificity of MOs, MOs may have caused secondary effects like p53-dependent apoptosis [31]. To examine whether the effect of the cfl1 MO was mediated by p53, we co-injected the cfl1 tMO₁ (7.5 ng) with a p53 MO (7.5 ng) into zebrafish embryos and found that both epiboly progression and mortality of co-injected embryos did not significantly (P>0.05) differ from those embryos injected with only the cfl1 tMO₁ (Table 2).

To examine the effects of cfl1 knockdown on the actin cytoskeleton structure, the filamentous actin (F-actin) was stained by rhodamine phalloidin, and to better differentiate individual cells nuclei were visualized by a nuclear dye DAPI (4′,6-diamidino-2-phenylindole). The double-stained embryos were examined under confocal microscopy (Fig. 4). At the lower magnification, the margins of EVL and DEL did not appear to migrate together during epiboly in cfl1 morphants (Fig. 4D) compared to the synchronized movements in control embryos (Fig. 4A). At a higher magnification, it clearly showed that the EVL was 2 or 3 rows of cells ahead of the DEL (Fig. 4E, F). This uncoordinated migration between the EVL and DEL was even more obvious in embryos injected with a higher dosage (10 ng) of cfl1-tMO₁ (data not shown). At the higher magnification, the YSL actin cable (arrow) and cell boundary actin were notably intense and condensed in cfl1 morphants (Fig. 4E) compared to control embryos (Fig. 4B).

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).

It is known that a close contact association exists between EVL and DEL [6]. We therefore examined whether the unsynchronized movements of EVL and DEL is due to the loss of their association by transmission electron microscopy (TEM). Clear disruptions of EVL-DEL attachments were observed in cfl1 morphants as evidenced by spaces in part of the contact zones between the DEL and EVL (Fig. 6B) compared to that in control embryos (Fig. 6A).

To further compare dynamic interactions between the DEL and EVL, we performed live imaging analysis of a gastrulating embryo from 7.5 to 8.5 hpf under confocal microscopy. We focused on the dorsolateral sites of embryos injected with membrane-bound GFP-GAP43 mRNAs and the StdMO or cfl1 tMO₁. While both the DEL and EVL showed highly coordinated migration toward posterior sites in control embryos (Fig. 7, left column; Supplementary Movie S1), cells of the DEL in cfl1-morphants did not form tight attachments and showed a clear gap with EVL cells (Fig. 7, right column; Supplementary Movie S2). In addition, a very dynamic change in cell shape was observed in DEL cells of control embryos, but not in those of cfl1 morphants (Supplementary Movie S2).

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).

To examine whether the internalization of epiblast (i.e. involution) was affected by the reduction of cfl1 translation, we have treated embryos with 5.0 or 7.5 ng of tMO₁ or stdMO, fix and subjected them to WISH against cstl1b. The appearance of cstl1b is an indication of formation of prechordal plate and thus the occurrence of involution. In two independent trials, involution occurred in all embryos treated with both dosages of tMO₁ tested (Fig. 9A–F for 7.5 ng, n = 46; Fig. 7G–L for 5 ng, n = 49) as those embryos injected with stdMO (Fig. 9M–R, n = 55).

Cellular migration requires actin-based pseudopod extensions in which Cfl1 might participate. To examine the role of Cfl1 in the formation of pseudopods during gastrulation, we applied time-lapse recording to monitor dynamic changes in migrating cells during the 75%∼90%-epiboly stages by targeting the apparent margins of involuting cells. After the germ-ring stage, both lateral (mesendodermal cells) and prechordal plate cells (progenitor cells) began involution from a lateral or dorsal site, respectively. Concomitantly, lateral cells converged toward the dorsal site while prechordal plate cells extended to the anterior. During a 15-min time-lapse recording, StdMO-injected embryos formed a normal number of pseudopods per cell in both lateral cells (6.7±1.1, Supplementary Movie S3) and prechordal plate cells (6.6±1.1, Supplementary Movie S5). By contrast, the number of pseudopods per cell in cfl1 tMO₁-injected embryos was significantly (p<0.05) lower in both lateral cells (2.8±0.8, Supplementary Movie S4) and prechordal plate cells (3.5±0.5, Supplementary Movie S6). Representative images are shown in Fig. 10A–D, and the average number of protrusions formed in each treatment is presented in Fig. 10E.

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).

All animal handling procedures were approved by the use of laboratory animal committee at National Taiwan University, Taipei, Taiwan (IACUC Approval ID: 97 Animal Use document No. 55).

Wild-type zebrafish (AB/TU) were raised under a 14-h light/10-h dark cycle at 28.5°C. Embryos were collected at 15∼20-min intervals after spawning, washed, and incubated in 0.3x Danieau's buffer (by diluting 1x Danieau's buffer consisting of 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO₄, 0.6 mM Ca(NO₃)₂, and 5.0 mM HEPES (pH 7.6) with double-distilled water) supplemented with 50 µg/mL streptomycin and 50 µg/mL penicillin G at 28.5°C until observation or fixation.

Antisense MOs were purchased from Gene Tools (Philomath, OR) or Open Biosystems (Huntsville, AL). The standard MO (StdMO; sequence: 5′-CCTCTTACCTCAGTTACAATTTATA-3′) without sequence homology to any known zebrafish DNA sequences was used as a control treatment. To knock down zebrafish cfl1 expression, two translational-blocking MOs (tMO), tMO₁ (targeting the start codon and neighboring 5′-untranslated region (UTR) sequence of cfl1 mRNA, −22 to +3; sequence: 5′-CATGGCTGTGTCTCTGTGCTAGTCG-3′) and tMO₂ (targeting 5′-UTR sequences of cfl1 mRNA, −50 to −26; sequence: 5′-TCTGGGTTGGAATTGAATGAGCTGT-3′) were used. To counter p53 activity, a p53 MO (sequence: 5′-GCGCCATTGCTTTGCAAGAATTG-3′) was used. To examine the translation blocking efficiency of cfl1 tMO₁ and tMO₂, two pCS2+ XLT constructs were generated with an insertion of cfl1 gene fragmen from −43 to +189 and −52 to +51, respectively. The cfl1 tMO₁ and tMO₂ were co-injected with respective pCS2+ XLT plasmid and the % of embryos expressing GFP fluorescence was determined. To prepare cfl1 expression vector, the full coding region of cfl1 was inserted into pCS2+ XLT vector, which contains the green fluorescent protein (GFP) sequence following the insertion site. The cfl1-gfp construct was linearized by NotI, and the capped RNA was transcribed according to the manufacturer's instruction using a mMESSAGE mMACHINE® SP6 Kit (Applied Biosystems, Foster City, CA 94404).

Glass capillaries (1.14×0.50 mm, O.D. × I.D., World Precision Instrument, Sarasota, FL) were pulled using a horizontal puller (P-97, Sutter Instrument, Navato, CA). Embryos were collected after spawning, and then immobilized by an injection trough on a 100-mm 1% agar plate. An injection pipette was forced through the chorion, into the yolk cell and reached the area adjacent to the blastomeres where 2.3–4.6 nL of the desired solution was ejected using a Nanoliter injector (World Precision Instrument). Before injection, all solutions were diluted by 1x Danieau's buffer with phenol red (0.25% (w/v), pH 7.4) to the desired concentrations. Microinjection was completed before the embryos had reached the 4-cell stage. After injection, embryos were recovered from the trough and cultured at 28.5°C for further experiments.

Embryos injected with MOs were grown in 0.3x Danieau's buffer supplemented with 0.2 mM 1-phenyl-2-thiourea (Sigma) to inhibit pigment formation [53], fixed at the desired developmental stages with 4% fresh paraformaldehyde in phosphate-buffer saline (PBS) overnight. After being manually dechorionated with fine forceps, embryos then transferred and stored into 100% methanol at −20°C. WISH was performed according to Thisse et al. [54], [55] using a DIG-labeled, SP6-RNA polymerase-made riboprobes hybridized to cfl1 or cfl1l. For cryosections, stained samples were immersed in PBS containing 30% sucrose overnight, and embedded in Optimal Cutting Temperature (OCT) compound embedding medium (Sakura, Tokyo, Japan) at −20°C, and 10-µm frozen cross-sections were cut with a CM 1900 rapid sectioning cryostat (Leica, Heidelberg, Germany) and attached to poly-L-lysine-coated slides (Electron Microscopy Sciences, Ft. Washington, PA). Stained embryos and sections were observed under a stereomicroscope (Mz75, Leica Microsystems) and a DM5000B DIC system, respectively. Photographs were taken using a digital camera (Coolpix 995, Nikon, Melville, NY).

Embryos injected with MOs were dechorionated at 8 hpf, fixed with 2% fresh paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.4) at 8.5 hpf for 2 h at room temperature, washed by the same buffer, and then post-fixed with 1% OsO₄ in the same buffer for 2 h on ice. They were then rinsed with distilled water, stained with 0.5% aqueous uranyl acetate overnight at room temperature, dehydrated with ethanol series then 100% acetone, and embedded in Spurr's low-viscosity embedding mixture (Electron Microscopy Sciences). Ultrathin sections were cut with diamond knives, double-stained with uranyl acetate and lead citrate, and examined with a Hitachi H-7650 transmission electron microscope (Toronto, Ontario, Canada).

Embryos injected with designated MOs and membrane-bound GFP-GAP43 messenger RNA (mRNA) were dechorionated and mounted in 1% low-melting-point (LMP) agarose (AMRESCO, Solon, OH) at 6.5 hpf. Embryos then were monitored in regions of the ventral leading edges under confocal microscope (TCS SP5, Leica Microsystems). Time-lapse images of suitable embryos were taken at 10-s intervals for 10 min.

Embryos were injected with 1% (w/v) caged Q-rhodamine dextran (Invitrogen, Carlsbad, CA) and 7.5 ng cfl1 tMO₁ dissolved in injection buffer mixed with 1% Q-rhodamine were injected into embryos at one cell stage. The embryos were grown in dark until 50% epiboly and immobilized at an injection through on a 50 mm 1% agar plate. To uncage Q-rhodamine, a beam of ultraviolet light through a DAPI filter set under an automatic upright microscope system (DM5000B, Leica Microsystems, Wetzlar, Germany) with a 40X water objective, was directed for 1∼3 s at the lateral blastoderm margin [33]. Both dark and bright field images were taken using a CoolSNAP fx charge-coupled device (CCD) camera (Roper Scientific, Tucson, AZ). Images were collected and analyzed using Simple PCI Imagine System software (Compix, Sewickley, PA).

Embryos injected with MOs were dechorionated and mounted in 0.8% LMP agarose at the sphere stage for lateral cell observations, or at the shield stage for prechordal plate cell observations. Differential interference contrast (DIC) optical time-lapse images were acquired and analyzed by the Simple PCI Imagine System software for 15 min at 15-s intervals under the DM5000B system with a 40X water immersion objective.

Donor embryos were injected with dextran, tetramethylrhodamine (10,000 MW, anionic, fixable, Invitrogen) and designated MOs. After the 1k-cell stage, host embryos were dechorionated by 10 mg/mL protease (Sigma, St. Louis, MO) and mounted in 0.8% LMP agarose. Cells from donor embryos were then transplanted into host embryos at approximately the sphere stage using CellTram vario (Eppendorf, Hamburg, Germany). The single focal plane time-lapse recordings of transplanted cells were collected at 30-s intervals for 1 h during late gastrulation (at 7∼9 hpf) using the 40x water-immersion objective under the DM5000B system and analyzed by the SimplePCI Imagine System software. The transplanted cells were also observed under confocal microscopy (TCS SP5, Leica Microsystems) and 10-min movies were recorded at 15-s intervals.