May 1, 2008;
A function for dystroglycan in pronephros development in Xenopus laevis.
(Dg) is a laminin receptor that is expressed at the interface between the basement membrane and the cell membrane
. Dg has been reported to play a role in skeletal muscle
cell stability, morphogenesis of neuroepithelial tissues, and in regulating cytoskeletal organization, cell polarization, and cell signalling. In this study, we have focused our analysis on the expression of Dg-mRNA and protein at different developmental stages in the pronephros
of Xenopus laevis. In order to study its role, we performed loss-of-function experiments mediated by Dg antisense morpholinos and dominant negative mutant. We show that Dg expression is first detectable when epithelialization begins in the pronephric anlage and persists later during tubulogenesis. Loss-of-function experiments induced a disorganization of the basement membrane, a drastic reduction of pronephric tubules and duct
that can lead to a renal agenesis. A diminished proliferation of pronephric cell progenitors was also observed in Dg depleted embryos. Together, these data indicate that Dg plays a key role for laminin-1 assembly and pronephric cell anchoring to the basement membrane during early development of the pronephros
. They also indicate that Dg may induce a signal transduction pathway controlling cell proliferation needed for the formation of tubules and their growth.
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Fig. 1. Dystroglycan expression during Xenopus laevis pronephros development. Panels A–D show spatial expression of dystroglycan transcripts from stage 23 to 35, and schematic representation of the developing pronephros adapted from Vize et al. (1995). Whole-mount in situ hybridizations were performed using digoxigenin-labelled antisense αDg RNA probes. (A) Stage 23. Expression is confined to the developing pronephric tubule anlage. (B–D) Stage 28, 31 and 35. Dg transcripts are present in the nephrostomes, the pronephric tubules (red arrowhead) and in the pronephric duct (green arrowhead). Panels E–H show immunolocalization of Dg protein. Embryonic stages are indicated in the top right of each panel. (E) Stage 35. Dg is present in the pronephric tubules (red arrowhead), in the anterior and posterior segments of the pronephric duct (blue and green arrowheads). (F–H) Cryostat sections. (F) Stage 29/30. Dg protein is detected on the surface of cells into the pronephric bud (yellow arrowhead) and at the basal pole of cells surrounding the pronephric bud (white arrowhead). (G) Stage 36. The Dg is detected around the nephrostomes (yellow arrowhead) and the pronephric tubules (red arrowhead). (H) Stage 40. The Dg is present in the elongating tubules (white arrowhead), and duct (yellow arrowhead).
Fig. 2. Depletion of dystroglycan by antisense morpholino oligomers. (A) The positions of the antisense morpholino oligomers and ATG initiation codon are indicated. (B) Dg-Mo specificity in vivo; Western Blot analysis using anti-Dg antibody showing that the Dg-Mo interferes with in vivo translation in Xenopus embryos of Dg mRNA. Lane 1, uninjected embryos; Lanes 2–4, embryos were injected in both blastomeres at the two cell stage with 8 ng (lane 2), 16 ng (lane 3) and 18 ng (lane 4) of Dg-Mo1 + 2. At stage 28 dorsal tissues containing pronephric anlage were isolated and proteins were analysed by SDS-PAGE. α-Tubulin was used as a loading control. Significant reduction of Dg protein synthesis is observed between 8 ng and 18 ng of morpholino. (C) Frequency of the phenotypes obtained with the different concentrations of Mo analysed with the 3G8 and 4A6 antibodies. The number n indicates the total number of injected embryos. (D–G) Whole-mount immunodetection of Dg protein at stage 41 on embryos injected unilaterally at the 4-cell stage with Dg-Mo1 + 2 (16 ng, panel E and 18 ng, panel G in both left blastomeres). At the dose of 16 ng Dg-Mo1 + 2 a strong reduction of Dg immunoreactivity is observed in the injected side (E) compared to the control side (D). At 18 ng Dg-Mo1 + 2, Dg immunoreactivity is absent in the injected side (G) compared to the control (F). (H–K) The phenotype of pronephric tubules was rescued by coinjection with Dg full-length mRNA. (H, I) Pronephric tubule formation is inhibited in the Dg-Mo injected side of embryos (arrowhead in panel I) compared to the non-injected-side (arrowhead in panel H). (J–K) Pronephric tubule formation was significantly rescued in embryos injected with Dg-Mo1 + 2 (16 ng) plus Dg full-length (FL) mRNA (400 pg) (arrowhead in panel K) compared to the Dg-Mo1 + 2 injected side (arrow in panel I).
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).
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.
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).