Disruption of blastomeric F-actin: a potential early biomarker of developmental toxicity in zebrafish

Abstract

The expression of at least some biomarkers of toxicity is generally thought to precede the appearance of frank pathology. In the context of developmental toxicity, certain early indicators may be predictive of later drastic outcome. The search for predictive biomarkers of toxicity in the cells (blastomeres) of an early embryo can benefit from the fact that for normal development to proceed, the maintenance of blastomere cellular integrity during the process of transition from an embryo to a fully functional organism is paramount. Actin microfilaments are integral parts of blastomeres in the developing zebrafish embryo and contribute toward the proper progression of early development (cleavage and epiboly). In early embryos, the filamentous actin (F-actin) is present and helps to define the boundary of each blastomere as they remain adhered to each other. In our studies, we observed that when blastomeric F-actin is depolymerized by agents like gelsolin, the blastomeres lose cellular integrity, which results in abnormal larvae later in development. There are a variety of toxicants that depolymerize F-actin in early mammalian embryos, the later consequences of which are, at present, not known. We propose that very early zebrafish embryos (~5-h old) exposed to such toxicants will also respond in a like manner. In this review, we discuss the potential use of F-actin disruption as a predictive biomarker of developmental toxicity in zebrafish.

Fig. 1

Correlation between blastomeric F-actin integrity and embryonic development in zebrafish. Zebrafish gelsolin (scinla or C/L-gelsolin) cDNA was constructed in pCS2 vectors (BamHI/EcoRI sites) [29]. Capped synthetic mRNAs were prepared using the SP6 mMESSAGE mMACHINE kit (Ambion, Austin, TX) after linearizing the plasmid with Not I that was used as the template to synthesize the mRNA for C/L-gelsolin in vitro. C/L-gelsolin mRNA that was transcribed in vitro was purified using RNA purification columns (Ambion, TX) (Fig. 1) and diluted in Danieau buffer immediately before microinjection. Synthesized C/L-gelsolin mRNA was injected into the yolk mass of one- to two-cell embryos. Post injection (4 h), damaged embryos were discarded and the rest were allowed to grow at 28°C for up to 28 h. Embryos were fixed at different stages in 4% paraformaldehyde for further processing. Zebrafish embryos (5-h old, late blastulae/gastrulae) were fixed overnight in 4% paraformaldehyde in phosphate buffer saline (PBS) at 4°C. For actin immunostaining, embryos were washed three times for 5 min each in 0.5% Triton in PBS; followed by 30 min incubation in blocking solution (10% normal goat serum, 1% DMSO, and 0.1% Triton in PBS). Embryos were then incubated for 30 min in blocking solution containing Rhodamine-Phalloidin (Molecular Probes, Eugene, OR). They were then washed three times for 5 min with PBS containing 0.1% Triton (PBT). Zebrafish embryos were mounted on agarose-coated dishes in PBT medium. Images acquired using a Leica stereomicroscope with UV illumination show a control embryo, b embryo injected with 25 pg C/L-gelsolin mRNA, and c embryo injected with 50 pg C/L-gelsolin mRNA. The dorsal side (upper hemisphere) shows the blastomeres and the ventral side (lower hemisphere) contains the yolk. Arrows indicate the actin ring (purse string). Enlarged views of a region of the dorsal side of the embryos in a, b, and c are presented in d, e, and f, respectively, showing the F-actin architecture. Asterisks indicate some blastomeres. Bright-field images of representative embryos at 28 hpf are shown in g control embryo, h embryo injected with 25 pg C/L-gelsolin mRNA, and i embryo injected with 50 pg C/L-gelsolin mRNA

Fig. 2

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces blastomeric F-actin depolymerization in zebrafish embryos. Embryos at 2 hpf (with intact chorions) were treated with 0.1% dimethylsulfoxide (DMSO) (n = 50) or 5 ng/ml TCDD (n = 50) for 4 h. Half of the embryos (n = 25) from each group were then washed three times with phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde. The rest were allowed to develop further. Embryos (6 hpf) fixed in 4% paraformaldehyde were processed for F-actin staining with rhodamine-phalloidin as described in Fig. 1 legend. Images show a control embryo and b embryo treated with TCDD. The dorsal side (upper Hemisphere) of the embryos shows the blastomeres and the ventral side (lower hemisphere) contains the yolk. Arrows indicate the actin ring (purse string). Enlarged views of a region of the dorsal side of the embryos in a and b are presented in c and d, respectively, showing the F-actin architecture. Asterisks indicate some blastomeres. Bright-field images of representative embryos at 78 hpf are shown in e control embryo and f TCDD-treated embryo. Magnified views of the images in e and f are presented in g and h, respectively, showing the pericardium (large arrow) and the heart (small arrow)

Fig. 3

Actin cytoskeleton is modulated by a variety of signaling pathways. Both polymerization (formation of filamentous actin or F-actin) and depolymerization (formation of monomeric globular actin or G-actin) of actin are modulated by Rho and Rac GTPases downstream of known signaling pathways, as well as by the expression of actin-binding proteins. A number of drugs can also directly bind to the actin cytoskeleton and modulate its architecture. Modulation of actin dynamics in turn regulates a number of cellular physiological processes, e.g., cell shape, cell division (cytokinesis), cell–cell adhesion/interaction, cell survival, and cell motility or migration. Any alteration in these processes in the early embryos can contribute to developmental toxicity