NIR-fs Laser Photoporation of MOs or dextran into single cells of zebrafish embryos.

(A) Experimental setup for targeted introduction of biomolecules with femtosecond laser irradiation. Specifications are described in Materials and Methods. (B) Schematic representation of targeted delivery to single cells of zebrafish embryos. FITC-MO, FITC-dextran, or mini-ruby dextran was injected into the chorion cavity of anesthetized embryos mounted in methylcellulose solution. The femtosecond laser pulse train was focused on the surface of single cells. (C) FITC-MO was detected in three targeted single cells (arrowheads). Inset: FITC-MO was localized to the nucleus (N) of the targeted cell. (D) Success rate of delivery of FITC-MO into 28-hpf zebrafish epithelial cells. (E, F) Success rate of delivery of mini-ruby dextran into 25- to 26-hpf (E) and 30-hpf (F) zebrafish epithelial cells. (G) Mini-ruby dextran was detected in a single targeted cell (arrowhead) of a 25- to 26-hpf zebrafish embryo. Inset: Magnification of the targeted cell. (H) At 24 h after introduction of FITC-dextran, FITC fluorescence was detected in newly divided cells (arrowheads). n, number of individual experiments. Scale bars: 250 μm (C, G); 50 μm (insets in C, G); 100 μm (H).

Manipulation of the fate of targeted cells in zebrafish embryos.

Targeted introduction of dnPKA mRNA induced floor plate marker gene expression in single dorsal neurons of zebrafish embryos. (A) Schematic illustration of dnPKA mRNA delivery to single dorsal neurons of zebrafish embryos. dnPKA mRNA was injected into the cavity between the chorion and the surface of embryo, and dorsal neurons in the neural keel (center) were irradiated at 13–14 hpf with a femtosecond laser pulse (red triangle). Irradiated embryos were fixed at 24 hpf (right panel) and processed for in situ hybridization. (B–E) Expression of the floor plate marker spon1b in embryos injected with control EGFP RNA (B) and dnPKA RNA (C–E) at 24 hpf. (B–D) Lateral view; (E) transverse section. In the control embryo (B), spon1b transcripts were detected only in the floor plate. In the dnPKA RNA-injected embryo, ectopic spon1b signals were observed in individual cells (arrowheads in C, E). (D) Expression of the floor plate marker spon1b (purple) and localization of biotin-dextran (brown) in embryos injected with dnPKA RNA at 24 hpf. Ectopic expression of spon1b was only detected in cells photoporated with biotin-dextran (black arrowhead). Although dextran was also observed in the adjacent dividing cell (open arrowhead), ectopic spon1b expression was barely detected in this cell. CNS, central nervous system; D, dorsal; FP, floor plate; V, ventral. Scale bars: 100 μm (B, C); 50 μm (D, E).