Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation

Abstract

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.

FIG. 1.

Chimera formation between medaka blastomeres and zebrafish embryos. (A and A') Micrograph of a 3-day-old embryo of medaka strain HB32C, showing black-pigmented melanophores (A) and autofluorescent guanophores (asterisks; A'). (B and B') Micrograph of a 3-day-old zebrafish embryo, showing black pigmentation in the eye (B) and the absence of autofluorescent cells (B'). (C) Micrograph of 2-day-old chimeric embryos, showing medaka blastomere-derived autofluorescing guanophores (asterisks) in zebrafish hosts. (E–I) Micrographs of a chimeric fry at 5 days postfertilization (dpf), showing the presence of 2 clusters of guanophores in the trunk (asterisk) and dorsal head surface (frame) of the chimera (E). The medaka guanophores (arrows) are yellow and brown in color under bright field optics (F), and are positive for yellow (G), green (H), and red fluorescence (I) under fluorescent optics. (J–L) Merged micrographs of a posthatching chimeric fry at 5 dpf, showing guanophores in the eye (circle). Color images available online at www.liebertonline.com/scd

FIG. 2.

Chimera formation between medaka embryonic stem (ES) cells and zebrafish embryos. (A and B) Micrograph of a zebrafish embryo host, showing transplanted medaka haploid ES cell line HX1 (green; A) and diploid ES cell line MES1 (red; B). (C and D) Micrograph of 1-day-old chimera, showing similar distribution of HX1 (C) and MES1 (D). (E and F) Micrograph of 2-day-old chimeras, showing wide distribution of HX1 (E) and MES1 (F). (G and H) Merged micrograph of a 3-day-old chimera, showing a similarly wide distribution of HX1 and MES1 donor cells. Scale bars, 100 μm. Color images available online at www.liebertonline.com/scd

FIG. 3.

Medaka ES cell donors adopt the zebrafish developmental program. Red fluorescent protein (RFP)-labeled MES1 cells were transplanted into zebrafish blastulae and monitored for differentiation into cardiomyocytes in the heart. (A–D) Micrographs of chimeras at the lateral view at 1 dpf (A and B) and 2 dpf (C, D). (D) Larger magnification of the area framed in (C), highlighting MES1-derived cardiomyocytes in the heart. (E) Micrographs of chimeras at the dorsal view, highlighting a reduction in the relative MES1 signal in 3 areas (a, b and c) and the heart (circled) from 5 dpf (E) to 10 dpf (F). One and the same chimera at different days of development is shown in (B–F). The anterior is to the left. Asterisks depict MES1 derivatives outside the heart. ey, eye; ht, heart; so, somites; tl, tail; ys, yolk sac. Micrographs are merges between red fluorescent optics and bright-field optics. Scale bars, 100 μm. Color images available online at www.liebertonline.com/scd

FIG. 4.

Molecular analyses of medaka ES cell differentiation in zebrafish host. (A) After transplantation at the blastula stage with medaka haploid ES cell line HX1, zebrafish, and medaka embryos were collected at day 1–7 (D1–D7) postfertilization and subjected to reverse transcription–polymerase chain reaction (RT-PCR) analysis of gene expression profiles by using primers specific to medaka cDNAs, except for β-actin primers that amplify the β-actin cDNA of both medaka and zebrafish. The haploid ES cell line HX1 clone HX1a and nontransplanted embryos of medaka and zebrafish were used for comparisons. For β-actin, PCR was run for 28 cycles with 25 ng of cDNA. For other genes, PCR was run for 38 cycles with 25 ng of cDNA (HX1 cells, zebrafish, and medaka embryos) and for 40 cycles with 100 ng of cDNA (chimeras). (B) Medaka embryos. Time course of gene expression was examined in developing embryos at indicated intervals in hours postfertilization. neg, negative control PCR without cDNA. For β-actin, PCR was run for 28 cycles with 25 ng of cDNA. For other genes, PCR was run for 35 cycles with 25 ng of cDNA except for mitf1, which was run for 38 cycles with 50 ng of cDNA.