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Dev Dyn
2009 Jun 01;2386:1332-45. doi: 10.1002/dvdy.21814.
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In vivo analyzes of dystroglycan function during somitogenesis in Xenopus laevis.
Hidalgo M
,
Sirour C
,
Bello V
,
Moreau N
,
Beaudry M
,
Darribère T
.
Abstract Dystroglycan (Dg) is a cell adhesion receptor for laminin that has been reported to play a role in skeletal muscle cell stability, cytoskeletal organization, cell polarity, and signaling. Here we show that Dg is expressed at both the notochord/somite and the intersomitic boundaries, where laminin and fibronectin are accumulated during somitogenesis. Inhibition of Dg function with morpholino antisense oligonucleotides or a dominant negative mutant results in the normal segmentation of the presomitic mesoderm but affects the number, the size, and the integrity of somites. Depletion of Dg disrupts proliferation and alignment of myoblasts without affecting XMyoD and XMRF4 expression. It also leads to defects in laminin deposition at the intersomitic junctions, whereas expression of integrin beta1 subunits and fibronectin assembly occur normally. Our results show that Dg is critical for both proliferation and elongation of somitic cells and that the Dg-cytoplasmic domain is required for the laminin assembly at the intersomitic boundaries. Developmental Dynamics 238:1332-1345, 2009. (c) 2008 Wiley-Liss, Inc.
Figure 1. Dystroglycan transcript and protein expressions. A-C: Lateral view, whole-mount in situ hybridization at stage 21 (A), 23 (B), and 27 (C). Dg mRNAs are detected in the dorsal region, posteriorly in the presomitic mesoderm and in the mesoderm in a series of stripes. D: Stage 40, lateral view, in toto immunodetection. The staining of the Dg protein is associated with the intersomitic junctions and the notochord (white arrow). mu m; b, brain; ov, otic vesicle; psm, presomitic mesoderm; s, somites; va, visceral arches; p, pronephros and duct. Scale bar = 400 mu m.
Figure 6. Dystroglycan depletion does not disrupt the myogenic signaling pathway. A-C: Sections of control explant at stage 24 equivalent. A: Immunostaining with the 12/101 antibodies. B: Hoechst. Some nuclei are aligned. C: Merge. D-F: Sections of morpholino (MO) -injected explant at stage 24 equivalent. D: Immunodetection with the 12/101 antibodies. No staining is observed. E: Hoechst. F: Merge. G-H prime : Whole-mount in situ hybridization of XMyoD. G: Stage 24, dorsal view of unilaterally injected embryo (injected side on the right). H: Stage 28, lateral view of the control side. H prime : Stage 28, lateral view of the injected side. The intersomitic junctions are disorganized (white arrows). I: Stage 24, frontal section, immunodetection of the XMyoD protein (injected side at the right). J,K: Stage 24 equivalent, whole-mount in situ hybridization of XMyoD. J: Control explant. K: MO-injected explant. L,M: Stage 24 equivalent, immunodetection of the MyoD protein. L: Control explant. M: MO-injected explant. N-N prime : Stage 28, whole-mount in situ hybridization of XMRF4. N: Lateral view of the control side. N prime : Lateral view of the injected side. O-P prime : Stage 28, immunodetection of the MRF4 protein. O: Frontal section (injected side on the right). P: Control explant. P prime : MO-injected explant. WT, wild-type; Mo, injected side. Scale bar = 70 mu m.
Fig. 3. Somites are disorganized and reduced when dystroglycan is depleted. A–E: Embryos were injected with 28 ng of Dg morpholino (MO) into the left side at the two-cell stage and stained with the 12/101 antibodies. A–D: Dorsal views of stage 24 (A), 28 (B), 32 (C), and 45 (D). E: Statistical analysis of the somite number at different developmental stages. Bars indicate SE (t-test: P stage24 = 0.0004; P stage28 = 0.0014; P stage32 = 0.005; P stage41 = 0.047). F–I: An embryo at stage 24. F,G: Lateral view of the control side. The segmentation of the paraxial mesoderm is clearly visible. H,I: Lateral view of the injected side. The repetitive units often lack their characteristic chevron pattern. J–L: Stage 24, transversal section (injected side on the right). J: Hoechst (blue). K: Immunodetection with the 12/101 antibodies (red). L: Merge. The injected sides show a reduction of the somite size. M: Stage 24, frontal section, immunode- tection with the 12/101 antibodies (injected side at the right). The segmentation is not affected; the somites are less compact and less cohe- sive. WT, wild-type; Mo, injected side. Scale bar = 200 um.
Fig. 5. Dystroglycan depletion disrupts myotome cells alignment and intersomitic cohesion. A,B–C: Stage 24. Control embryo sectioned and stained with Hoechst (blue, A), and immunostained with the 12/101 antibodies (red, B); merged image (C). Arrows show cells undergoing their rotation. D–L: Embryos were unilaterally injected at the two-cell stage with 28 ng of Dg morpholino (MO). At stage 24, embryos were fixed, sectioned and double stained with the 12/101 antibodies (red) and Hoechst (blue). D–F: Frontal section. The nuclei are randomly distributed on the injected side (lower part of the panel). G–I: Para-sagittal section, control side of the embryo. G: The nuclei exhibit a typical oval/elongated shape and are aligned in each somite. H: The somites are strongly cohesive the ones with the others. The 12/101 staining is concentrated at the intersomitic junctions (white arrows). I: Merge. J–L: Parasagittal section of the injected side. J: The nuclei are randomly distributed. K: The somites are less compact and less cohesive; the 12/101 staining is absent at the intersomitic junctions (white ar- rows); within somites, the cell alignment ap- pears affected. L: Merge. M–P: Embryos were unilaterally injected at the two-cell stage with Dg MO and mRNAs encoding a membrane-tagged green fluorescent protein (GFP). M: Stage 24, frontal section, control side. Cells are aligned parallel to the notochord and somite boundaries are well delimited (white arrows). N: Stage 24, frontal section, injected side. The somites are disorganized. Cells fail to extend and align within each somite. The intersomitic junctions fail to form correctly (white arrows). O: Stage 24, para-sagittal section, control side. Cells are aligned. Somite boundaries exhibit their characteristic chevron pattern (white arrows). P: Stage 24, para-sagittal section, injected side. Cells fail to align and boundaries are not well delimited (white arrows). Q: Frontal section of a rescued embryo stained with Hoechst (blue) and the 12/101 antibodies (red). R: Frontal section of an embryo injected with a five-nucleotide mismatch Dg-MO and stained with Hoechst (blue) and the 12/101 antibodies (red). The somite morphology and their number are not affected. Cells are aligned parallel to the notochord. WT, wild-type; Mo, injected side. Scale bar = 70 um.
Fig. 7. Dg depletion and Dg without the cytoplasmic domain prevents laminin deposition at the somite boundaries. A–E: Immunodetection of the laminin. A: Frontal section of control embryo, stage 24. The laminin is present around the notochord, the neural tube, and at the intersomitic junctions. B: Frontal section of the ventral region of a unilaterally morpholino (MO) -injected embryo, stage 24. Laminin is detected at the intersomitic boundaries in the uninjected side (left) and absent in the injected side (right). C: Frontal section of the dorsal region of a unilaterally Mo-injected embryo, stage 28. The laminin is absent in the injected side (right) but present at the intersomitic junctions of the control side. D,E: Immunodetection of the laminin in explants. D: Laminin is deposited in control explant. E: MO-treated explant. Laminin was not detected. F: Frontal section, stage 28. Immunodetection of the integrins (injected side on the right). The integrins are localized at the intersomitic junctions in both the control and injected sides. G: Frontal section, stage 28. Immunodetection of the fibronectin (injected side on the right). The fibronectin is detected at the somite boundaries and around the notochord. H,I: MO-injected explants. H: Immunodetection of the integrins. I: Immunodetection of the fibronectin. J–N: Dg-deltaCyto mRNA was delivered unilater- ally and effects of the overexpression of the Dg-cytoplasmic mutant was analyzed at stage 24 on frontal section. J–L: Immunochemistry with Hoechst (blue) and the anti-laminin antibodies (red). J: Laminin is detected around the notochord and at the intersomitic boundaries in the control side (left). Laminin is absent at the intersomitic junctions in the injected side (right). K: Magnification of the control side. The nuclei exhibit a typical oval/elongated shape and are aligned in each somite. The laminin is present at both the somite/notochord and intersomitic boundaries. Somites are cohesives. L: Magnification of the Dg-deltaCyto mRNA-injected side. The nuclei are randomly distributed. The laminin is present at the somite/notochord boundaries, but its deposition at the inter- somitic junctions is severely disrupted. The fluorescence appears sparse within the putative somites (white arrows). M,N: Immunodetection of the green fluorescent protein (GFP) -tagged Dg-deltaCyto (green) and the laminin (red). M: The Dg-deltaCyto proteins are detected at the surface of somitic cells (white arrows). N: Magnification of one cell expressing the Dg-deltaCyto protein. Somitic cells expressing the Dg-deltaCyto proteins (white arrows) were also stained by the anti-laminin antibodies (blue arrows) and both proteins are co-localized at the level of the plasma membrane. Ch, notochord; TN, neural tube; WT, wild-type; Mo, injected side. Scale bar = 100 um.
