Generation of Naïve Blastoderm Explants from Zebrafish Embryos

Abstract

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively naïve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise naïve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Figure 1:. Procedure for zebrafish blastoderm explantation (step 4.3).

(A) Hold the forceps in the non-dominant hand (orange) closed against the yolk to stabilize the embryo while pinching the blastoderm at approximately ½ of its height using the forceps in the dominant hand (blue). (B) Run the orange forceps along the edge of the blue forceps gripping the embryo to slice through the blastoderm so that the first cut reaches approximately halfway across the blastoderm. (C) Rotate the embryo 90°, then place the blue forceps inside (but orthogonal to) the original cut and pinch to sever the remaining blastoderm. (D) Allow explanted blastoderm cells to heal in 3x Danieau’s solution for approximately 5 min before transferring into explant media.

Figure 2 (modified from):. Nodal ligands promote C&E morphogenesis and germ layer formation in zebrafish blastoderm explants.

(A) Diagram of injection and explantation of zebrafish embryos. (B-E) Representative bright-field images of live blastoderm explants of the indicated conditions/genotypes at the of equivalent the 2–4 somite stage. N = number of explants from two to four independent trials. (F) Time-lapse DIC series of a representative explant from a WT embryo injected with 10 pg ndr2 RNA. (G)Representative images of the whole-mount in situ hybridization for the transcripts indicated in explants from WT embryos injected with 10 pg ndr2 RNA. Scale bars are 200 μm.

Figure 3 (Modified from³¹):. Chimeric explants reveal that neuroectoderm specification does not require tissue-autonomous Nodal signaling ex vivo.

(A) Diagram of the procedure to generate chimeric zebrafish explants. (B-F) Whole-mount in situ hybridization for the mesoderm marker tbxta (top) and neuroectoderm marker sox2 (bottom) in explants from WT embryos injected with 100 pg ndr2 RNA (B), uninjected WT controls (C), MZoep−/− injected with 10 pg ndr2 (D), and chimeric explants containing neuroectoderm portions from WT (E) or MZoep−/− (F) embryos at the equivalent of the 2–4 somite stage. Fractions indicate the number of explants with the phenotype shown over the total number of explants examined. (G-J) Representative images of live Tg[lhx1a:gfp] explants from a single embryo (G-H) or combined with H2B-expressing blastoderms (I-J, magenta) of the conditions indicated at the equivalent of the 2–4 somite stage. N = number of explants from three independent trials. Scale bars are 200 μm.