In vivo assay for the developmental competence of embryo-derived zebrafish cell lines

We have produced chimeric zebrafish embryos by transplanting permanent embryo-derived cell lines into blastula-stage embryos. Furthermore, we have established a fluorescent in vivo assay to monitor the developmental effects and fate of such transplanted cells using confocal laser scanning microscopy. All zebrafish embryonic cell lines tested (ZEM2, ZF13, ZF29, ZF29-C1, and ZF29-C2), when introduced as small groups of less than 20 cells, survived at least several days after transplantation, whereas a control cell line isolated from adult goldfish fin (CAR) soon displayed necrosis and cell death. The ZEM2 cells, isolated from blastula-stage embryos, appeared to contribute to different germ layers, whereas the ZF29 cells derived from 22- to 28-somite embryos differentiated mainly into mesenchymal-like cells in vivo. Interestingly, when transplanted as groups of 50 to 150 cells, the ZF29 cells were capable of profound developmental effects, the most prominent being the induction of ectopic notochord and neural tissue, sometimes resulting in a partial secondary body axis. This effect was observed more frequently when cells were transplantated into young blastula-stage embryos, and occurred mostly in the prechordal head region of the embryo. In contrast, ZEM2 cells never induced ectopic tissues after transplantation. The methods and results described here will be useful in future efforts to select and characterize putative embryonic stem cell lines from zebrafish.