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dact4 (dishevelled binding antagonist of beta catenin 4) gene expression in Xenopus laevis embyo, NF stage 22, assayed via in situ hybridization, ventral view, anterior left, |
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Figure S3. In situ hybridization for Dact-4 in Xenopus embryos. (A-C) Xenopus embryos were fixed at the indicated developmental stages, and processed for in situ hybridization using a DIG- labeled probe specific for Dact4. At stage 16 (panel A, embryo shown in frontal view), Dact 4 is detected in the floor plate (fp), pre-placodal ectoderm (ppe) and somites (som). At stage 22, Dact4 is expressed in the eye, somites and presomitic mesoderm (psm), otic vesicle (ov), the pronephros (pn) (panel B, lateral view). At this stage, Dact4 is also present around the proctodeum (panel C, bottom view). |
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| Gene |
Clone |
Species |
Stages |
Anatomy |
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dact4
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laevis
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NF stage 9
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neuroectoderm
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dorsal
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dact4
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laevis
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NF stage 10
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ectoderm
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mesoderm
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involuting marginal zone mesenchymal layer
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upper blastopore lip
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dorsal
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dact4
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laevis
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NF stage 16
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presomitic mesoderm
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somite
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head somite
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neural tube
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preplacodal ectoderm
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[+]
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dact4
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laevis
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NF stage 8
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nucleus
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cytoplasm
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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. |
