In the mouse, the seven main classes of retinal cell types (rod, cone, bipolar, horizontal, amacrine, ganglion and Müller glial cells) are produced from multipotent progenitor cells over a 17-d interval starting at embryonic day (E) 11.5 and continuing through postnatal day (P) 9. The overall size of the retina and the proportion of each cell type contained therein is essential for proper visual processing; therefore, during retinal development, cell cycle exit and cell fate specification are coordinated to ensure that the adult retina forms appropriately 1,2 . When cell proliferation and cell fate specification become uncoupled, as in retinoblastoma 3 , microphthalmia 4,5 and some forms of retinal dysplasia 6,7 and degeneration 8 , vision is severely compromised. By studying individual proteins that integrate the decision to exit the cell cycle and to specify cell fate, we may begin to gain insights into the synchronization of these two important processes during neural development.Rb lies at the heart of the regulatory network that executes cell cycle exit during the G1 phase through interactions with the E2F transcription factor family. There is also evidence that Rb has a role in cell fate specification [9][10][11] . In support of this notion, Rb can bind more than 110 different proteins, several of which are tissue-restricted transcription factors 12 . It is conceivable that Rb binds to E2F and regulates cell cycle exit through its canonical pathway and then contributes to cell fate specification, differentiation or both through interactions with tissuerestricted transcription factors.To overcome the embryonic lethality of Rb1 -/-embryos, which die in utero around E13.5, we used an explant culture procedure to study the development of isolated whole retinas beyond E13.5. To complement and extend the explant culture studies in vivo, we inactivated the gene Rb1 in the retina by using a new tissue-specific Cre transgenic line (Chx10-cre) crossed to Rb1 lox mice. Finally, we injected a Cre-expressing replication-incompetent retrovirus into the eyes of newborn Rb1 lox mice to generate clones of cells lacking Rb. These experiments showed that Rb is required in a cell-autonomous manner for appropriate cell cycle exit and rod development in the mouse retina. This is the first example of a cell-autonomous role for an Rb family member in these two interrelated processes in the developing retina. RESULTS Rb expression during developmentTo identify the cells expressing Rb in the developing mouse retina, we immunolabeled retinal cryosections at six postnatal stages of development (P0, P3, P6, P9, P12 and P21). At P0, when approximately 35% of cells are still dividing and 65% are postmitotic 1,13 (R. Martins and M.A.D., unpublished results), Rb was expressed in the nuclei of dividing retinal progenitor cells in the outer neuroblastic layer and postmitotic differentiating neurons in the developing inner nuclear layer (Fig. 1a-d). To verify that the cells expressing Rb in the outer neuroblastic layer were actively dividin...
Chemotherapy combined with laser therapy and cryotherapy has improved the ocular salvage rate for children with bilateral retinoblastoma. However, children with late-stage disease often experience recurrence shortly after treatment. To improve the vision salvage rate in advanced bilateral retinoblastoma, we have developed and characterized two new rodent models of retinoblastoma for screening chemotherapeutic drug combinations. The first model is an orthotopic xenograft model in which green fluorescent protein^or luciferase-labeled human retinoblastoma cells are injected into the eyes of newborn rats.The second model uses a replication-incompetent retrovirus (LIA-E E1A ) encoding the E1A oncogene. Clonal, focal tumors arise from mouse retinal progenitor cells when LIA-E E1A is injected into the eyes of newborn p53 À/À mice. Using these two models combined with pharmacokinetic studies and cell culture experiments, we have tested the efficacy of topotecan combined with carboplatin and of topotecan combined with vincristine for the treatment of retinoblastoma. The combination of topotecan and carboplatin most effectively halted retinoblastoma progression in our rodent models and was superior to the current triple drug therapy using vincristine, carboplatin, and etoposide.Vincristine had the lowest LC 50 in culture but did not reduce tumor growth in our preclinical retinoblastoma models. Taken together, these data suggest that topotecan may be a suitable replacement for etoposide in combination chemotherapy for the treatment of retinoblastoma.
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