Immunoprecipitation and mass spectrometry identify non-cell autonomous Otx2 homeoprotein in the granular and supragranular layers of mouse visual cortex

Plasticity in the visual cerebral cortex is regulated by the internalization of Otx2 homeoprotein into parvalbumin neurons in cortical layers II/III and IV. However the Otx2 locus is not active in these neurons and the protein is imported from external sources, including the choroid plexus. Because Otx1 and Otx2 may have redundant functions, we wanted to verify if part of the staining in parvalbumin neurons corresponds to Otx1 transported from cortical layer V neurons. It is demonstrated here that Otx staining in layer IV cells is maintained in Otx1-null mice. The immunoprecipitation of extracts from finely dissected granular and supragranular cortex (layers I-IV) gave immunoblots with a band corresponding to Otx2 and not Otx1. Moreover, high-resolution mass spectrometry analysis after immunoprecipitation identifies two peptides within the Otx2 homeodomain. One of these peptides is specific for Otx2 and is not found in Otx1. These results unambiguously establish that the staining in parvalbumin neurons revealed with the anti-Otx2 antibodies used in our previous studies identifies non-cell autonomous Otx2.