A Human Cone Precursor Program Underlying a Proliferative Response to RB Loss

Abstract

Purpose: Inactivating RB1 mutations are thought to collaborate with retinal cell type-specific circuitry to initiate retinoblastoma tumorigenesis. We have earlier shown that human cone precursors proliferate in response to RB loss, dependent upon intrinsically high levels of MDM2 and MYCN and low levels of p27 as compared to other retinal cell types, and can develop into retinoblastoma tumors. In contrast, mouse cone precursors fail to proliferate in response to RB loss, and lack prominent MDM2, yet have not been evaluated for other components implicated in retinoblastoma development. The goals of this study are to 1) define the spatio-temporal sequence of appearance of human cone precursor features implicated in retinoblastoma development, 2) determine whether these features appear in mouse cone precursors, and 3) test whether human-specific features such as high-level MDM2 expression can sensitize mouse cones to the loss of RB function.

Methods: Human fetal week (Fwk) 15 and Fwk 21 retinas and mouse post-natal day (P) 6, P10, and P20 retinas were used for immunostaining. Transgenic Red-Green-opsin Promoter (RGP)-MDM2 mice were mated with RGP-Cre and Rb1^l/l mice to generate mice predicted to have cone-directed RB loss and MDM2 expression.

Results: In developing human retinas, RB, MDM2, and MYCN were not detected in RXRγ+ cone precursors in the periphery but were prominent in central maturing cone precursors with expression initiating peripheral to the boundary of cone arrestin (ARR3) expression. In developing mouse retinas Rb and Mdm2 were barely detectable at any ages, while Mycn was detected at P10 but not at P20. RGP-MDM2 mice expressed high levels of MDM2 in cone cells, beginning at P8. No abnormal proliferation was detected at P8, P10, P15, or P20 in RGP-MDM2, RGP-Cre, Rb1^l/l mice. No tumors were observed in more than 40 RGP-MDM2, RGP-Cre, Rb1^l/l mice.

Conclusions: In human cone precursors, expression of RB, MDM2, and MYCN are first detected after the onset of RXRγ and before ARR3, and further increase during cone precursor maturation. Maturing mouse cone precursors fail to induce high-level Rb, Mdm2, or Mycn. Cone-directed MDM2 overexpression is not sufficient to sensitize RB deficient mouse cones to retinoblastoma. Additional factors specific to human cones such as high MYCN may be needed for cone precursor proliferation and retinoblastoma development in response to Rb loss.