pRB Loss Induces Human Cone Precursor Proliferation and Inward Migration in Intact Cultured Retina

Abstract

Purpose : RB1 inactivation collaborates with human cone precursor circuitry to initiate cone precursor proliferation and development of retinoblastoma-like tumors in experimental settings (Xu et al., 2014). However, human cone precursors are centered at the outer boundary of the outer nuclear layer (ONL), while early tumors are frequently centered near the outer plexiform layer (OPL) or inner nuclear layer (INL) in retinoblastoma patients (Berry et al, 2015). This discrepancy has brought into question whether retinoblastomas originate in cone precursors or in an inner retinal cell type (Rootman et al., 2013). This study aimed to define the basis for the discrepant locations of early tumors and the putative cone precursor cell of origin.

Methods : Human fetal week 18-19 retinas were co-transduced with fluorescently labeled lentiviruses to knock down (KD) RB1 and express a cone specific fluorescent reporter. Retinas were live-imaged 9 or 13 days post-RB KD on a 2-photon microscope at 30-minute intervals for 27-65 hours. Cell movement was tracked using Imaris Image Analysis Software. Cell cycle entry was evaluated by immunofluorescence.

Results : At 12 days post RB KD, RXRγ+, ARR3+, and L/M Opsin+ cone precursors co-stained for proliferation markers Ki67 and EdU. Among many proliferating cone precursors several were displaced toward the OPL. By 30 days post-KD more proliferating RXRγ+ cells were observed in the INL suggesting that cone precursors might migrate towards the OPL after pRB loss. We tested this hypothesis using live imaging on intact developing retinas. A human cone-specific fluorescent reporter was constructed and its specificity validated by co-staining for cone specific markers. Retinas were co-transduced with lentivirus expressing the cone-specific fluorescent protein and RB1 shRNA. Live imaging identified co-transduced cone precursors that underwent cell division and moved toward the OPL. shRB-infected cone precursors that migrated towards the OPL were observed in multiple retinas.

Conclusions : pRB-depleted cone precursors divide and/or migrate towards the OPL. The cone precursors’ inward migration provides a possible explanation for the localization of early retinoblastoma tumors near the OPL even though cone precursors are normally centered in the outer ONL. This study provides further support for a cone precursor origin of retinoblastoma.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.