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A key feature of early vertebrate development is the formation of superficial, epithelial cells that overlie non-epithelial deep cells. In Xenopus, deep and superficial cells show a range of differences, including a different competence for primary neurogenesis. We show that the two cell populations are generated during the blastula stages by perpendicularly oriented divisions. These take place during several cell divisions, in a variable pattern, but at a percentage that varies little between embryos and from one division to the next. The orientation of division correlates with cell shape suggesting that simple geometric rules may control the orientation of division in this system. We show that dividing cells are molecularly polarised such that aPKC is localised to the external, apical, membrane. Membrane localised aPKC can be seen as early as the one-cell stage and during the blastula divisions, it is preferentially inherited by superficial cells. Finally, we show that when 64-cell stage isolated blastomeres divide perpendicularly and the daughters are cultured separately, only the progeny of the cells that inherit the apical membrane turn on the bHLH gene, ESR6e. We conclude that oriented cell divisions generate the superficial and deep cells and establish cell fate diversity between them.
Fig. 7. aPKC is apically localised and asymmetrically inherited during the perpendicular divisions. (A,B,E) Stage 8 aPKC localisation. (C,F) Stage 8 occludin localisation. (D) No primary antibody negative control. (G,H) Stage 8 aPKC and a tubulin double staining. (I) Transmitted light image of fertilised egg. (J,K) aPKC localisation in fertilised egg. In this case, a pigmented embryo was used to allow identification of animal hemisphere. (L,M) aPKC staining at the four-cell stage. (N) No primary antibody negative control at the four-cell stage. (A-H) No vitelline membrane. In I-M, embryos with vitelline membrane are shown (as they were less damaged), but the staining was the same in those without vitelline membrane. All images are from antibody stained sections, except G,H, which are stained as whole-mount preparations.
Fig. 8. Perpendicular divisions generate molecularly distinct daughter cells. (A-C) Perpendicular divisions in isolated blastomeres generate one daughter cell with localised aPKC and one daughter cell without. Blastomeres were stained with aPKC (red) and β1 integrin (green). A transmitted light image is shown below the fluorescent image. (A) An isolated blastomere. (B) An isolated blastomere during a perpendicular division. (C) Superficial (top) and deep (bottom) daughters generated after an isolated blastomere completes a perpendicular division. (D,E) The divisions generate differences that lead to later differences in gene expression. (D) An isolated 64-cell stage blastomere was left to divide, separated into a deep and a superficial cell, and cultured until control embryos reach stage 10. (E) Gene expression of the cultured blastomeres was analysed by quantitative real time RT-PCR. The y-axis shows expression in arbitrary units, with 100 being the same level as a stage 10 animal cap. Odc and XSox3 are equally expressed, while ESR6e is preferentially expressed in outer cell derived clones. The mean of three independent experiments is presented.
