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Experiment details for pax8

Buisson I et al. (2014) Assay

Pax8 and Pax2 are specifically required at different steps of Xenopus pronephros development.

Gene Clone Species Stages Anatomy
pax8.L laevis NF stage 18 dorsal lateral plate mesoderm

  Fig. 8. MoPax8 knockdown results in cell proliferation defect in the KF that is rescued by activation of the canonical wnt pathway. (A) Embryos were injected at the 4-cell stage in the equatorial region of both left blastomeres and cultured until stage 18. They were then injected with BrdU into the archenteron, cultured for an additional one hour and fixed for in situ hybridization and immunostaining. On transverse sections, we combined in situ hybridization for pax8 (a, a) with immunolocalization for BrdU (b, b) and Pax8 (d, d), and DAPI staining (c, c). MoPax8 injected side (ad), uninjected side (ad). The outlined area in a and a corresponds to the KF determined by the expression of pax8 mRNA. The number of BrdU stained nuclei was counted in this region Scale bar: 80 m. (B) Histogram showing the BrdU labeling index (LI) in MoC and MoPax8 injected embryos. For each embryo, uninjected side is compared with the injected side. The LI represents the proportion of BrdU-labeled cells over the total number of cells in the KF area. BrdU LI is significantly decreased by Pax8 depletion. (C) Histogram showing the BrdU LI in embryos co-injected with MoPax8 and TCF-VP16-GR. Dexamethasone was added (+DEX) or not at stage 15. Activation of TCF-VP16-GR rescues the effect of MoPax8 on the BrdU LI. n=number of analyzed embryos in total. For each embryo, 4 sections were analyzed. Average values from three independent experiments. **P<0.005, ***P<0.001.

Gene Clone Species Stages Anatomy
pax8.L laevis NF stage 18 somatic layer of lateral plate mesoderm , splanchnic layer of lateral plate mesoderm , lateral plate mesoderm

  Supplementary Material. Fig. S1 MoPax8 and MoPax2 block endogenous embryonic Pax8 and Pax2 expression, respectively. (A) Immunofluorescence with anti-Pax8 (a-b) and anti-Pax2 (c, d) antibodies performed on transverse sections of embryos injected with MoPax8.2 (a, a), MoC (b, b) or MoPax2 (c, d) in both left blastomeres at the 4-cell. The KF region of injected (a, b) and uninjected side (b, b) is shown at the late neurula stage 18. The dorsal part of a stage 25 embryo containing the neural tube (NT) and the medial part containing the pronephros (arrow) are shown in c and d, respectively. The dotted line delineates injected (left) and the uninjected (right) halves. Pax8 is detected both in the splanchnic and somatic layers of the KF on the uninjected side, while it is absent on the MoPax8 injected side (a, a). MoC has no effect on the endogenous Pax8 expression (b). Pax2 expression in the neural tube and in the pronephros is inhibited on the MoPax2 injected side (c, d). Scale bar in a and b: 100 m, scale bar in c and d: 400 m (B) location of the Mo target sequences used in this study on pax2 and pax8 pseudoalleles. Nucleotides that differ between the two pseudoalleles are in blue. The start codon is in red.

Gene Clone Species Stages Anatomy
pax8.L laevis NF stage 25 pronephric mesenchyme

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  Fig. 7. Expression of somitic, lateral plate, blood and endothelial marker genes in Pax8 morphants. (A) Expression of somitic (myoD and mlc) and lateral plate markers (foxf1). Embryos were injected with MoPax8 at the 4-cell stage, in the equatorial region of both left blastomeres. They were cultured until tailbud stage 25 (a, a, b, b) or neurula stage 18 (c, c), fixed and analyzed by whole-mount in situ hybridization with the indicated probes. For each embryo, injected (a, b, c) and uninjected (a, b, c) sides are shown. Interference with Pax8 function does not modify the expression pattern of neither myoD, mlc and foxf1. (B) Expression of blood (scl and lmo2) and endothelial markers (msr and flk1). Embryos were injected with MoPax8 (dg) or MoC (hk) at the 4-cell stage, in the equatorial region of both left blastomeres. They were cultured until tailbud stage 27 and processed for whole-mount in situ hybridization with the indicated probes. For each embryo, injected (dj, hk) and uninjected (dg, hk) sides are shown. Double in situs with pax8 and lmo2 probes are shown in whole mount (d, d) as well as in section performed at the level of the anterior (l) and posterior (m) part of the pronephros anlage and in the posterior region of the embryo (n). The dotted line delineates injected (left) and uninjected (right) halves. In Pax8 morphants, the expression domain of scl, lmo2, msr and flk1 is enlarged in the anterior region where the tubule anlage normally forms. Double in situ hybridization with pax8 and lmo2 probes shows a much stronger lmo2 signal dorsal to pax8 expression domain on the injected side at the level of the pronephros anlage (l, m). In A and B, pax8 is revealed in light blue while the other genes are revealed in dark blue. Scale bar: 200 m, (C) Expression of lmo2 in Pax8 morphants in the absence of cell division. Embryos were injected with MoPax8 at the 4-cell stage, in the equatorial region of both left blastomeres. Half of them were cultivated in the presence of HUA from neurula stage 18 onwards. They were processed for whole-mount in situ hybridization at stage 27. The enlargement of the lmo2 expression domain in the anterior region in response to the loss of Pax8 (o) is maintained in HUA treated embryos (p).