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Adhesive interactions between integrin receptors and the extracellular matrix (ECM) are intimately involved in regulating development of a variety of tissues within the organism. In the present study, we have investigated the relationships between beta(1) integrin receptors and focal adhesion associated proteins during eye development. We used specific antibodies to examine the distribution of beta(1) integrin ECM receptors and the cytoplasmic focal adhesion associated proteins, talin, vinculin, and paxillin in the developing Xenopus retina. Immunoblot analysis confirmed antibody specificity and indicated that beta(1) integrins, talin, vinculin, and paxillin were expressed in developing retina and in the retinal-derived Xenopus XR1 glial cell line. Triple-labeling immunocytochemistry revealed that talin, vinculin, paxillin, and phosphotyrosine proteins colocalized with beta(1) integrins at focal adhesions located at the termini of F-actin filaments in XR1 cells. In the retina, these focal adhesion proteins exhibited developmentally regulated expression patterns during eye morphogenesis. In the embryonic retina, immunoreactivities for focal adhesion proteins were expressed in neuroepithelial cells, and immunoreactivity was especially strong at the interface between the optic vesicle and overlying ectoderm. At later stages, these proteins were expressed throughout all retinal layers with higher levels of expression observed in the plexiform layers, optic fiber layer, and in the region of the inner and outer limiting membrane. Strong immunoreactivities for beta(1) integrin, paxillin, and phosphotyrosine were expressed in the radially oriented Müller glial cells at later stages of development. These results suggest that focal adhesion-associated proteins are involved in integrin-mediated adhesion and signaling and are likely to be essential in regulating retinal morphogenesis.
Fig. 1. Immunoblot analysis using monoclonal antibodies against focal adhesion proteins 1 integrin, talin, vinculin, and paxillin. Protein homogenates of stage (St) 40 Xenopus eye and XR1 cells were analyzed. A band of approximately 115 kDa was observed in homogenates from St 40 eyes (lane 1) and XR1 cells (lane 2) with 1 integrin monoclonal antibody under nonreducing conditions. Bands of 235 and 225 kDa were observed with anti-talin antibody in both samples under reducing conditions (lane 3, 4). Anti-vinculin antibody identified a band of approximately 116 kDa in both samples (lane 5, 6), and anti-paxillin antibody identified a band of approximately 68 kDa (lane 7, 8) in both samples under reducing conditions.
Fig. 2. Triple-labeling fluorescence analysis revealing focal adhesion
proteins in Xenopus XR1 retinal glial cells. Focal adhesions were characterized
as discrete streak-like patterns of immunoreactivity (IR).
A,E,I,M: The pattern of 1 integrin-IR. B,F,J,N: The pattern of talin-,
vinculin-, paxillin-, and phosphotyrosine (P-Tyr) -IRs, respectively, for the
same cell pictured to the left. C,G,K,O: The pattern of F-actin filaments
labeled with rhodamine-phalloidin in the same cell as in A, E, I, M,
respectively. D,H,L,P: The merged images, indicating talin, vinculin, paxillin,
and phosphotyrosine are each colocalized with 1 integrins and
colocalize at the termini of F-actin filaments. Scale bar 20 m in P
(applies to A).
Fig. 3. The changing patterns of expression of beta1 integrin-, talin-, and
vinculin-immunoreactivities (IRs) during retinal development. Fluorescence
images of 1 integrin- (A), talin- (G), and vinculin- (M) IRs.
Each series of images represent retinal tissue sections from Xenopus
laevis at stage (St) 25, 30, 37, 40, 47, and 65, respectively. RPE, retinal
pigment epithelium; OS, outer segments of photoreceptors; ONL, outer
nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL,
inner plexiform layer; GCL, ganglion cell layer; OFL, optic fiber layer.
Scale bars 20 m in O,R (applies to A).
Fig. 4. The changing patterns of expression of paxillin- and phosphotyrosine-
(P-Tyr) immunoreactivities (IRs) during retinal development.
Fluorescence images of paxillin- (A) and phosphotyrosine-IRs (G).
Each row of images represent retinal tissue sections from Xenopus laevis
at stage (St) 25, 30, 37, 40, 47, and 65, respectively. RPE, retinal pigment
epithelium; OS, outer segments of photoreceptors; ONL, outer nuclear
layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner
plexiform layer; GCL, ganglion cell layer; OFL, optic fiber layer. Scale
bars 20 m in I,L (applies to A).
Fig. 5. Confocal images illustrating colocalization of 1 integrin- and
glial fibrillary acidic protein- (GFAP) immunoreactivities (IRs) in Muller
glial cells. Cryostat section of St 47 Xenopus laevis were double-labeled
with anti- 1 integrin (A), and GFAP (B) antibodies. A precise colocalization
of the immunoreactivity was observed in the radially oriented Muller
cells (arrows) and in the astrocytes in the OFL (arrowheads). OPL, outer
plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; OFL,
optic fiber layer. Scale bar 20 m in B (applies to A,B).
tln1 (talin 1) gene expression in sectioned Xenopus laevis eyes, NF stage 65, as assayed by in situ hybridization. Superficial up.
vcl (vinculin) gene expression in sectioned Xenopus laevis eyes, NF stage 65, as assayed by in situ hybridization. Superficial up.
pxn (paxillin) gene expression in sectioned Xenopus laevis eyes, NF stage 47, as assayed by in situ hybridization. Superficial up.
