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Fig. 2. Xath5 (X5) overexpression expands the expression of olfac- tory neural markers. RNA encoding -galactosidase ( -gal) alone (80 pg) or together with RNA for Xath5 (200 pg) or XNeuroD (50 pg) was injected into one cell of two-cell stage Xenopus embryos. Embryos were collected at neural plate (stage 20) or tail bud (stage 36) stages and assayed by in situ hybridization for neural- and olfactory-specific gene expression. The blue stain is the lineage tracer -gal, which identifies the injected side of the embryo. Embryos overexpressing Xath5 exhibited a striking expan- sion of Xomp2 expression on the injected side (A,C) compared with the uninjected side (B,C). D: Conversely, control embryos injected with -gal alone had symmetrical expression of Xomp2 within the olfactory epithe- lium on the injected and uninjected side. XNeuroD overexpression did not cause an expansion of Xomp2 expression but instead caused either a loss (E) or reduction (F, arrowhead) in Xomp2 expression. Embryos injected with RNA for -gal and Xath5 and assayed for N-tubulin expres- sion showed ectopic neurogenesis on the injected side of the embryo (arrowheads in G), whereas N-tubulin staining was predominantly re- stricted to the developing nervous system on the uninjected side (H). Embryos injected with -gal and Xath5 (I) or -gal alone (J) were ana- lyzed for Xebf2 expression at stage 20. Xebf2 expression was expanded on the injected side in embryos expressing -gal and Xath5 (bracket, I) but unchanged between the injected and uninjected sides in embryos overexpressing -gal alone (arrow, J). Embryos in A, B, G, and H are shown in a lateral view with the anterior to the left. In C, I, and J, embryos are shown in an anterior view, injected side to the right. |
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Fig. 3. Xath5-induced expansion of Xebf2 expression within the developing olfactory placode occurs independent of cellular proliferation. A: Anterior view of stage 24-26 embryos analyzed by whole-mount in situ hybridization for expression of Xebf2, which labels the developing olfactory placode. Embryos overexpressing Xath5 show an expansion of Xebf2 expression on the injected side when incubated in the absence (A) or presence (C) of the DNA synthesis inhibitors hydroxyurea and aphi-docolin (HUA). Control embryos show no change in Xebf2 expression when incubated in the absence (B) or presence (D) of HUA. E: Stage 246 embryos stained with anti-phosphohistone H3 (HP3) antibody (bright red fluorescent spots), showing that extensive proliferation occurs in untreated embryos injected with Xath5 and beta-gal (E, lateral; F, anterior) or beta-gal alone (I, lateral; J, anterior). There is a dramatic decrease in HP3-positive cells (note very few bright red fluorescent spots) in the HUA-treated embryos injected with Xath5 beta-gal (G, lateral; H, anterior) or beta-gal alone (K, lateral; L, anterior). In A, the injected side is on the left. E, G, I, and K show lateral views of the trunk on the injected side of a whole embryo, with anterior to the right. F, H, J, and L show anterior views of the head. |
