Lunardi A et al. (2006), Dystroglycan is required for proper retinal lay...

XB-ART-861

Dev Biol 2006 Feb 15;2902:411-20. doi: 10.1016/j.ydbio.2005.11.044.

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Dystroglycan is required for proper retinal layering.

Lunardi A , Cremisi F , Dente L .

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Dystroglycan (DG) is a transmembrane receptor linking the extracellular matrix to the internal cytoskeleton. Its structural function has been mainly characterized in muscle fibers, but DG plays signaling and developmental roles also in different tissues and cell types. We have investigated the effects of dystroglycan depletion during eye development of Xenopus laevis. We have injected a specific morpholino (Mo) antisense oligonucleotide in the animal pole of one dorsal blastomere of embryos at four cells stage. Mo-mediated loss of DG function caused disruption of the basal lamina layers, increased apoptosis and reduction of the expression domains of specific retinal markers, at early stages. Later in development, morphants displayed unilateral ocular malformations, such as microphtalmia and retinal delayering with photoreceptors and ganglion cells scattered throughout the retina or aggregated in rosette-like structures. These results recall the phenotypes observed in specific human diseases and suggest that DG presence is crucial at early stages for the organization of retinal architecture.

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Species referenced: Xenopus laevis
Genes referenced: crx crybb1 dag1 gal.2 itgb1 lama1 nppa nrp1 otx2 prkci prkcz rax rbpms2 tubb2b zic1
???displayArticle.antibodies??? BrdU Ab2 Dag1 Ab2 Itgb1 Ab1 Lama1 Ab1 Prkcz Ab1 Tuba4b Ab5
???displayArticle.morpholinos??? dag1 MO3

???displayArticle.disOnts??? congenital muscular dystrophy [+]

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	Fig. 1. Dystroglycan gene expression during early Xenopus laevis retinogenesis. Panels A�D are sections of in situ whole-mount hybridized embryos from stage 19 to stage 28. (A) Tangential section showing DG mRNA expression localized in the anterior neural plate at stage 19. (B�D) Transversal sections showing X-DG mRNA expression at stage 24 (B), at stage 26 (C) and at stage 28 (D) in the optic stalk, in the presumptive pigmented epithelium and in the lens vesicle (C, D). Panels E� IV are cryostat sections showing. X-DG immunolocalization. (E) X-DG detection at stage 28 in the neural tube and in the retina at the level of the presumptive pigmented epithelium, the optic stalk and all around the sensorial layer of the ectoderm. (F, G) High magnification of retina sections at stage 32 showing the presence of X-DG in the endfeet of retinal precursors in the ciliary marginal zone and in the inner limiting membrane at the vitreal border of the retina (purple arrowheads in panel G). Green arrowheads indicate X-DG detection in the lens vesicle. (H� IV) Transverse sections of Xenopus retina at stage 45 showing X-DG immunoreactivity at the level of retinal pigmented epithelium (white arrowhead), inner limiting membrane (purple arrowhead) and lens epithelium (green arrowhead). Punctuated distribution of X-DG in the OPL is evidenced at high magnification in panels I, IV (same section, red detection) between the two nuclear layers, highlighted in panel IV by Hoechst staining. Abbreviations: anp, anterior neural plate; OS, optic stalk; ppe, presumptive pigmented epithelium; nr, neural retina; nrp, neural progenitors; lv, lens vesicle; d, dorsal; v, ventral; nt, neural tube; sle, sensorial layer of ectoderm; cmz, ciliary marginal zone; gcl, ganglion cell layer; inl, inner nuclear layer; onl, outer nuclear layer; opl, outer plexiform layer; ipl, inner plexiform layer; ilm, inner limiting membrane.
	Fig. 2. Results of DG loss-of-function experiments. (A) Stage 45 Xenopus embryo injected at four cells stage with 8 ng of Mo-X-DG plus 250 pg of GFP cRNA. The injected side is indicated by the green staining; white brackets highlight reduction of the eye size in the injected side (left) compared to the uninjected, control side (right). (B, C) Transversal section of a stage 32 injected embryo in which specific DG immunostaining (in red) is lost in neural tube (nt) and eye (E) of the Mo-X-DG-injected side and in some cells of the other side, where diffusion of the injected material sporadically happens. Fluorescent green labeling shows the injected side of the embryo in panel C.
	Fig. 3. Expression of eye markers in X-DG morphants. Xotx2 (A, C, E, EV), Xrx1 (B, D, F, FV) and b-crystallin (G, GV) mRNA expression was detected by BMP purple (blue staining) in Mo-X-DG-injected embryos at stage 14 (A, B), 25 (C, D) and 28 (E�GV). Nuclear beta-gal staining (red) was used to trace the side of injection. Panels A�D show both injected and uninjected sides in frontal views. Panels E�GV show lateral view of uninjected (E, F, G) and injected (EV, FV, GV) sides, respectively. No changes in the expression domains of Xotx2 (A) and Xrx1 (B) are detectable between the two sides at stage 14. White brackets highlight the decrease of Xotx2 (C) and Xrx1 (D) expression in the eye of the injected sides at stage 25. Decrease is evident also at stage 28 (compare panel E to EV and F to FV); beta-crystallin expression is also dramatically reduced in the injected side (compare panel G to GV). Mo-X-DG injection completely abolishes also Xotx2 rostral expression domain in panels E to EV.
	Fig. 4. Cryostat sections of Xenopus retina immunolabeled with polyclonal antibody against Lam1 (A�D and F: green labeling); monoclonal antibody against h1- integrin (A�G: red labeling) and polyclonal antibody against aPKC (H�K, red labeling). Control side (A, D, E, H, I) and injected side (B, C, F, G, J, K) of retinas at stage 32. The injected side in panels J and K was traced by GFP. Laminin and integrin were co-detected (yellow signal) in the Bruch�s membrane (BrM) at the bases of pigmented epithelium (PE), at the border of vitreal cavity in the inner limiting membrane (ILM) and in the lamina surrounding the lens. Severe disorganization of these laminae was detected in the injected sides of morphants (arrows in panels B, C, F). Integrin staining, which showed a radial-like pattern in the control sides (A, E) was affected in the injected sides of morphants (B, C, G). High magnifications (D�G) highlight the histology of the retina at its most marginal side near the lens. Localization of aPKC at the apical side of the retinal neural precursors (nrp), underneath the pigmented epithelium (PE) was visible either in control (H, I) or in Moinjected eye (J, K). At higher magnification, the aPKC staining of retinas in the control (I) and injected (K) sides was comparable.
	Fig. 5. Retinal delayering in DG morphants. (A�E) control sides; (AV�EV) Mo-X-DG-injected sides. Expression of markers for specific cell types in retinal sections from stage 45 embryos: (A, AV) Xotx5 mRNA; (B, BV) Xotx2 mRNA; (C, CV) hermes mRNA; (D, DV) N-tubulin. (E, EV) Hoechst nuclear staining of sections shown in panels D and DV. In panel A, blue and white arrowheads indicate photoreceptors and bipolar cells. Green arrowheads show rosette-like structures. Red arrowhead indicates ectopic hermes expression in panel CV. Yellow arrowheads indicate ectopic nervous fibers in panel DV and the corresponding position in panel EV.
	Fig. 6. Effects of the unilateral injection of Mo-X-DG (8 ng) at stage 4 cells on the apoptosis and cell proliferation of the eye. (A�F) Eye sections of stage 32 embryos. Pe: pigmented epithelium. Detection of GFP, which was used as tracer, is shown in panels B, D, F. Panels A�D show BrdU immunodetection (red nuclei) in the control side (A, C) and Mo-injected side (Mo, B, D) of the embryo. Hoechst counterstaining labels all nuclei in panels C, D. Panels E and F show TUNEL detection of apoptotic nuclei (brown staining) in control (E) and Mo-X-DG-injected side (F), respectively. Sections of Mo-X-DG injected eyes always displayed a significantly higher number of TUNEL-positive cells than sections of uninjected, contralateral eyes. Eyes injected with control-Mo had the same number of TUNEL-positive cells than uninjected eyes (not shown). The numbers of BrdU-positive nuclei in control and Mo-X-DGinjected (Mo) retinas, expressed as percent over the total number of cells analyzed (control: n = 550, Mo: n = 514), are compared in panel G. The average number of TUNEL-positive cells per eye section (n = 120 sections) in control and Mo-X-DG-injected retinas is compared in panel H. Bars indicate SE in panel G and SEM in panel H. Differences between control and Mo are significant in panel G (t test: P = 0.008904109) and in panel H (t test: P = 8.997E�06).