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Fig. 4. Developmental expression of the Xenopus laevis Dact-4 Spemann organizer gene. (A) RT-PCR showing expression of Dact-4 mRNA at selected developmental stages. Expression at egg (E) and 16-cell (16c) stages indicates the presence of maternal transcripts; Histone 4 (H4) was used as loading control. (B) RT-PCR performed on dorsal (D) and ventral (V) marginal zones dissected from stage 10 Xenopus gastrula embryos. Dact-4 transcripts are strongly expressed on the dorsal side. Stage 10 whole embryos were used as positive controls and histone H4 as a loading control. -RT was used as a negative control. (C) Xenopus - embryos were injected at the 2-cell stage with β-Catenin morpholino (MO) and assessed for Dact-4 and Chordin mRNA expression through RT-qPCR, at stage 10. β-catenin MO reduces Dact-4 expression by 40% in injected embryos. Chordin, whose expression relies on early Wnt/β-catenin signals and lacks maternal expression, was used as a positive control. Expression levels were normalized to ornithine decarboxylase (ODC) transcripts. The experiment was performed in biological triplicate, and average values are shown. Error bars represent standard deviation. (D–G) whole-mount in situ hybridizations at stages 9 (blastula), 10 (gastrula), 16 (neurula), and 24 (tailbud), showing Dact-4 expression pattern at different developmental stages. The embryo shown in (E) was bisected along the Animal-Vegetal axis using a surgical scalpel, splitting the Organizer region in two halves. The half-gastrula embryos were then subjected to in situ hybridization. BCNE, blastula Chordin-Noggin expressing center; nt, neural tube; ov, otic vesicle; pn, pronephros; ppe, pre-placodal ectoderm; psm, pre-somitic mesoderm; SO, Spemann organizer; som, somite. (H) Schematic showing the procedure for Xenopus animal cap immunofluorescence (IF). Embryos were injected at 4-cell stage with Dact-4-Flag mRNA, cultured until stage 8, and animal caps were dissected and processed for IF. (I–K) Dissected animal caps were stained with anti-flag antibody, to determine the subcellular localization of injected Dact-4-Flag. Nuclei were counterstained with Dapi. Note that Dact-4, like other paralogues of the family, localizes both to the nucleus and the cytoplasm. Scale bar represents 10 μm. |
