Integrin α2β1 mediates interactions between developing embryonic retinal cells and collagen

In the developing nervous system, the extracellular matrix provides a source of extrinsic cues to guide determination of cell fate, neuroblast migration, axon outgrowth and synapse formation. In the neural retina, undifferentiated neuroepithelial precursor cells contact extracellular matrix that contains multiple collagen types. Collagens have been shown to support retinal cell adhesion and neurite outgrowth, but the integrin receptors mediating neuronal responses are not understood. Here we provide evidence that integrin α2β1 acts as a collagen receptor in the developing avian retina and examine its expression pattern. Using a recently described monoclonal antibody, MEP-17, α2 protein was detected in the developing retina by immunofluorescence in tissue sections and dissociated cells, and by immunoprecipitation. At embryonic day 4 (E4), when the majority of retinal cells are undifferentiated neuroepithelial cells, α2 immunoreactivity in sections was widespread and about half of cells dissociated in culture were α2 positive. At E6, after the retinal ganglion cell layer had differentiated, immunoreactivity in sections decreased in the central, more developed portion of the retina and 25% of dissociated cells were α2 positive. E6 retinal ganglion cells, identified by neurofilament immunoreactivity, did not express detectable α2 immunoreactivity. Immunoprecipitation experiments using E6 extracts demonstrated that the α2 subunit was paired with the β1 integrin subunit. By E12, α2 immunoreactivity in sections was confined to the extreme peripheral retina, although the antigen may be masked since expression levels comparable to or slightly higher than E6 could be detected in dissociated cells and extracts. By employing function blocking antibodies, it was shown that α2β1 integrin is necessary for cell adhesion and process outgrowth by embryonic retinal cells on collagens I and IV. Although α2 expression continued through E12, α2 activity was down regulated with increasing embryonic age, since α2-dependent adhesion and outgrowth declined. These data suggest a role for α2β1 in neuroepithelial cell interactions with collagen rather than for axon extension by retinal ganglion cells.