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Fig. 2. Whole-mount in situ hybridization for ets-1 and ets-2 in early embryogenesis. (A) Localization of ets-1 transcript in the animal pole of a 4-cell embryo. The cleavage furrows are clearly indicated by residual dark-brown pigmentation. (B). Same experiment conducted for ets-2 transcripts. (C) Control experiment in which a 2-cell stage embryo was cut into halves prior to hybridization to exclude possible artifacts resulting from a poor penetration of the anti-digoxigenin antibody (ets-1 antisense probe). An, animal pole; Veg, vegetal pole. (D) ets-1 mRNA localization in a 32-cell stage blastula, showing the presence of transcripts In the animal blastomeres and in the upper part of vegetal blastomeres (blue arrowheads). (E) Same experiment carried out on a 32- cell stage embryo cut into halves prior to hybridization. Note the presence of the transcripts (dark-blue staining) in the roof of the blastocoel and in the marginal zone. An, animal; Veg, vegetal; BI, blastocoel. (F,G,H) Whole-mount inSitu localization of ets-1 (F) and ets-2 (G,H) in stage 8 (F and H) and stage 6 (G) embryos. In addition to the animal pole cytoplasmic localization of the transcripts, notice the nuclear signal which can be observed in stage 8 embryos, at the level of the vegetal part, in the almost colorless cytoplasmic background. The specificity of this signal is assessed by the fact that no nuclear staining is observed in embryos prior to MBT (compare F and H with G) and by the fact that a sense probe does not give any signal (not shown). The proliferative activity of these cells is demonstrated by the presence of duplicated nuclei (white arrowheads in F) and a mitotlc figure (white arrowhead in H), in cells which have not yet undergone cytokinesis. (I,J) 10um thick sections of whole-mount hybridizations on a 32-cell stage (ets-1) and 16-cell stage (ets-2) embryos respectively. The transcripts are essentially located below the cortex of the animal blastomeres, but a significant level of both ets-1 and ets-2 mRNAs is also observed at the level of the germ plasms (red arrows). AB, animal blastomere; BI, blastocoel; VB, vegetal blastomere. Bar, 100 um. |
