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Fig. 6. Pronephros development is altered after overexpression of Dg-δCyto protein. Dg-δCyto mRNA was delivered unilaterally into both blastomeres at eight cell-stage. (A, B) Whole-mount immunochemistry of embryos injected with Dg-δCyto mRNAs performed with the 3G8 and 4A6 antibodies. In the injected side, note the defects in the pronephric tubules (red arrowhead) and the anterior segment of the duct (blue arrowhead). (C–D′) Expression of pronephros marker genes in Dg-δcyto mRNA injected embryos. The expression of Pax-8 gene is unchanged at stage 35 in Dg-δcyto mRNA injected embryos (C′) compared to the control (C). The Pax-2 gene expression is also unmodified at stage 30 (D, D′). (E–O) Cryostat sections of pronephros areas were immunolabelled with the anti-laminin-1 antibodies (panels E, F, I, J). Detection of the GFP, which was used as tracer of Dg-δCyto proteins is shown in panels G and K. Panels H, L represent a merge between laminin-1 and Dg-δCyto proteins. The control and injected sides are shown at stage 29/30 and 33/34. Stages are indicated in the top right of each panel. At stage 29/30 and 33/34 in the injected side laminin-1 is detected around a small pronephros anlage (see yellow arrowheads in merge, panels H, L). Cells expressing Dg-δCyto (red fluorescence in panels G and K and white arrowhead in panel L) are excluded from this pronephros structure. (M–O) Two cells overexpressing the Dg-δCyto protein that are excluded from the pronephric area. Laminin-1 (M) and GFP-tagged Dg-δCyto (N) are detected at the surface of these cells (O). Both proteins are co-localized at the level of the cell membrane (O). |
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Fig. 3. Depletion of dystroglycan leads to a drastic diminution of circonvoluted tubules. (A–D) Microinjections were performed unilaterally at the 4-cell stage with Dg-Mo1 + 2 (16 ng) in both left blastomeres. Embryos were fixed at stage 40 and then development of pronephros was analysed by immunochemistry staining with monoclonal antibodies 3G8 for tubules and 4A6 for ducts. (A, B) Whole-mount immunochemistry. The pronephric tubules (red arrowhead) and the anterior duct segment (blue arrowhead) in panel B show severe developmental defects in the injected side compared to control side in panel A. (C, D) Cryostat sections of these embryos at level of the pronephros area. A single and large 3G8-positive tubule is present in the injected side (D) whereas in the control several tubules are detected (C). (E–J) Expression of pronephros marker genes in Dg-Mo injected embryos. Embryonic stage is indicated in the top right of each panel. The expressions of the Pax-8 (E–H) and Pax-2 (I, J) genes were detected by whole-mount in situ hybridization on embryos injected unilaterally with Dg-Mo1 + 2 (16 ng). The expression of the Pax-8 gene is similar in the control (right side of the embryo in panels E–G) and injected sides at all developmental stages. In spite of unchanged Pax-8 expression, some defects in the pronephros area are observed in the injected side: the duct is shorter and enlarged in stage 24 embryo (panel F, green arrowhead). In the control side of stage 35 embryo three nephrostomes (panel G, red arrowheads) are detected, in contrast in the injected side only a single structure is observed (H). In stage 35 embryo, the expression of the Pax-2 gene is not modified in the injected side (J) compared to the control (I). Here again a single structure is observed in the injected side (J) compared to the three nephrostomes observed in the control side (I). |
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Fig. 5. Depletion of Dg affects laminin-1 assembly and tubule formation in vitro. Two-cell stage embryos were injected with Dg-Mo1 + 2 (32 ng/embryo). Animal caps were treated with activin/retinoic acid for 3 h. (A, D) Immunodetection with the 3G8 antibodies and the anti-laminin antibodies. (A, B) Section of a control animal cap. 3G8 (blue arrowhead) and anti-laminin-1 (red arrowhead) antibodies staining are detected. Laminin-1 is present at the basal pole of cells of the tubules. Laminin-1 is also observed elsewhere in the explant where the tubules will be formed (purple arrowhead) (A). (C, D) Section of a Mo1 + 2 injected explant (C). Large cavities and mesenchyme like tissues are observed. (D) A diffuse fluorescence is observed (white arrowhead) suggesting that laminin-1 is accumulated but does not assemble basal lamina. (E–H) Expression of pronephros marker genes in animal caps. (E, G) The expression of the Pax-8 gene is similar in the control and Mo1 + 2 injected explant. (F, H) Pax-2 gene is detected in control and Mo1 + 2 injected explant. |
