Spatiotemporal expression patterns of 6B4 proteoglycan/phosphacan in the developing rat retina

PURPOSE. TO investigate expression of 6B4 proteoglycan/phosphacan, the major constituent of chondroitin sulfate proteoglycan and a possible modulator of neural network formation in the developing central nervous system, in developing rat retina. METHODS. Changes in expression and localization of 6B4 proteoglycan in developing rat retina were investigated by reverse transcription-initiated polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot analysis. RESULTS. Semiquantitative RT-PCR revealed that mRNA expression of 6B4 proteoglycan in retinas peaked at postnatal day 14 (P14) and then decreased at P42. Immunohistochemical analyses using MAb 6B4, a monoclonal antibody against 6B4 proteoglycan, revealed faint immunoreactivity in the inner aspects of the retina at embryonal day 16 (E16). At birth, weak immunoreactivity was present in the nerve fiber layer (NFL) and inner plexiform layer (IPL). At P7 and P14, the NFL, IPL, and outer plexiform layer (OPL) stained intensely, but the ganglion cell layer (GCL) remained unstained. Between P21 and P42, immunoreactivity in the NFL and IPL weakened slightly. Immunoblot analyses showed a MAb 6B4 immunopositive band in the retinal soluble fraction treated with chondroitinase ABC. The amount of the immunopositive band increased rapidly as retinal development proceeded. Surprisingly, a significant amount of the immunopositive band was present in the retina even before digestion with chondroitinase ABC, indicating that at least part of 6B4 proteoglycan in rat retina exists in a non-proteoglycan form. CONCLUSIONS. The existence of 6B4 proteoglycan/phosphacan was thus demonstrated in Mt retina, although some biochemical parameters were different from those of the 6B4 proteoglycan seen in brain.