Extra-embryonic function of Rb is essential for embryonic development and viability

Abstract

The retinoblastoma (Rb) gene was the first tumour suppressor identified. Inactivation of Rb in mice results in unscheduled cell proliferation, apoptosis and widespread developmental defects, leading to embryonic death by day 14.5 (refs 2-4). However, the actual cause of the embryonic lethality has not been fully investigated. Here we show that loss of Rb leads to excessive proliferation of trophoblast cells and a severe disruption of the normal labyrinth architecture in the placenta. This is accompanied by a decrease in vascularization and a reduction in placental transport function. We used two complementary techniques-tetraploid aggregation and conditional knockout strategies-to demonstrate that Rb-deficient embryos supplied with a wild-type placenta can be carried to term, but die soon after birth. Most of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-null animals were virtually absent in rescued Rb-null pups. These findings identify and define a key function of Rb in extra-embryonic cell lineages that is required for embryonic development and viability, and provide a mechanism for the cell autonomous versus non-cell autonomous roles of Rb in development.

Rb regulates proliferation and rod photoreceptor development in the mouse retina

“…Restoration of Rb to the placenta of developing embryos partially rescued the embryonic lethality of Rb-deficient embryos 23 . Cre expression from the Mox2 promoter leads to recombination in the E6.5 embryo with no recombination in extraembryonic endoderm or trophoblast.…”

“…Cre expression from the Mox2 promoter leads to recombination in the E6.5 embryo with no recombination in extraembryonic endoderm or trophoblast. We isolated retinas from Rb1 lox/-Mox2-cre embryos 23 and their littermates at E18.5 and cultured them for 12 d. We then sectioned or dissociated the retinal explants and immunostained them with cell type-specific antibodies (Supplementary Table 1 online). As with the retinal explants of E13.5 Rb1 -/-embryos, all rod photoreceptor markers were markedly reduced in the retinas of Rb1 lox/-Mox2-cre embryos but not in those of Rb1 lox/+ Mox2-cre or Rb1 lox/-embryos ( Supplementary Fig.…”

“…For example, in Rb1-deficient embryos with wild-type placentas 23 , several of the hematopoietic and CNS defects initially identified in the Rb1-knockout mice were rescued by restoration of the Rb1 gene in the placenta. To test whether the proliferation and rod development defects in our Rb1-null explant cultures were cell autonomous or non-cell autonomous, we generated a series of replication-incompetent retroviruses carrying the oncogene E1A (Fig.…”

“…The generation of conditional knockout mice has enabled researchers to identify non-cell-autonomous effects of gene inactivation, but even this approach can be difficult to interpret, depending on the proportion of cells undergoing recombination. Many of the phenotypes initially identified in the Rb1-knockout embryos were non-cell autonomous 18,23 . To complement our studies on knockout and conditional knockout mice, we carried out clonal analysis using a Cre retrovirus to test the cell-autonomous role of Rb.…”

Retinoblastoma family genes

“…In particular, although ectopic proliferation continued in the CNS, apoptosis was suppressed, and erythrocyte developmental delays were almost completely suppressed. In contrast, both ectopic proliferation and apoptosis in the lens were unaffected, and the skeletal muscle defect was not restored (de Bruin et al, 2003b;Wu et al, 2003). In light of these findings, the suppression effects of the RbÀ/À phenotype by loss of the activating E2Fs will need to be revisited: does the removal of E2F 1 or 3 reduce CNS apoptosis through an indirect effect of restoring placental development?…”