The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.

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.

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...

Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions.

Retinoblastoma (Rb)-deficient embryos show severe defects in neurogenesis, erythropoiesis, and lens development and die at embryonic day 14.5. Our recent results demonstrated a drastic disorganization of the labyrinth layer in the placenta of Rbdeficient embryos, accompanied by reduced placental transport function. When these Rb ؊/؊ embryos were supplied with a wildtype placenta by using either tetraploid aggregation or genetic approaches, animals survived until birth. Here we analyze the role of extraembryonic Rb in regulating proliferation, apoptosis, and differentiation in the rescued animals at different developmental stages. Many of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb ؊/؊ animals, including the ectopic apoptosis in the CNS, were virtually absent in rescued Rb ؊/؊ pups. However, rescued animals died at birth with severe skeletal muscle defects. Like in Rb knockout embryos, rescued animals showed a marked increase in DNA replication and cell division in the CNS. In sharp contrast, the typical widespread neuronal apoptosis was absent in Rb-deficient embryos reconstituted with a normal placenta. In lens fiber cells, however, the inappropriate proliferation and apoptosis that is normally observed in Rb ؊/؊ embryos continued unabated in rescued animals. These results demonstrate that Rb function in extraembryonic lineages plays an important role in the survival of neuronal cells and in the differentiation of the erythroid lineage, providing mechanistic insight into the cell autonomous and nonautonomous functions of Rb during development.

The E2F family is conserved from C. elegans to mammals with some family members having transcription activation functions and others having repressor functions 1, 2 . Whereas C. elegans 3 and Drosophila melanogaster 4, 5 have a single E2F activator and repressor proteins, mammals evolved to have at least three activator and five repressor proteins 1,2,6 . Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a 3bki ; E2f3a 1ki ) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development. KeywordsE2F3a; E2F3b; Rb; development; proliferation; transcription and apoptosis Since the identification of the founding E2F family member, E2f1 7 , two distinct genes in lower eukaryotes and eight genes in higher eukaryotes have been identified to encode the signature DNA binding domain that endow these transcription factors with E2F 1,2,6 . Among the mammalian E2F activator subset, the E2f3 gene has emerged as the critical family member involved in the control of cell proliferation and development 8,9 . The E2f3 locus was originally thought to encode a single DNA binding activity, but was later shown to drive the expression of two related isoforms, E2f3a and E2f3b, from two distinct promoters 10 . Given the critical link between the E2f3 locus and the control of cell proliferation, we used homologous recombination to individually disrupt its two isoforms in mice and rigorously evaluate how their functions are integrated with that of other E2F activators. The inactivation of E2f3a or E2f3b was achieved by targeting exon 1a or 1b sequences, respectively, using LoxP-cre technology (Fig. 1a). Mice deleted for either exon 1a or exon 1b were identified by Southern blot and genomic PCR analysis (Fig. 1b). Specific ablation of E2f3a or E2f3b was confirmed by Western blot assays using total E2F3-specific antibodies (Fig. 1c).It was previously shown that inactivation of both E2f3a and E2f3b (E2f3 −/− ) in mice with a mixed strain background yielded offspring that developed rather normally 8, 9 , but we show here that breeding these mice into a pure strain background (∼98% pure) resulted in embryonic lethality ( Fig. 1e and Supplementary Fig. 1). Intercrossing E2f3 +/− mice of different pure backgrounds restored viability of E2f3 −/− mice, albeit with some observed strain-specific biase...

The inactivation of the retinoblastoma (Rb) tumor suppressor gene in mice results in ectopic proliferation, apoptosis, and impaired differentiation in extraembryonic, neural, and erythroid lineages, culminating in fetal death by embryonic day 15.5 (E15.5). Here we show that the specific loss of Rb in trophoblast stem (TS) cells, but not in trophoblast derivatives, leads to an overexpansion of trophoblasts, a disruption of placental architecture, and fetal death by E15.5. Despite profound placental abnormalities, fetal tissues appeared remarkably normal, suggesting that the full manifestation of fetal phenotypes requires the loss of Rb in both extraembryonic and fetal tissues. Loss of Rb resulted in an increase of E2f3 expression, and the combined ablation of Rb and E2f3 significantly suppressed Rb mutant phenotypes. This rescue appears to be cell autonomous since the inactivation of Rb and E2f3 in TS cells restored placental development and extended the life of embryos to E17.5. Taken together, these results demonstrate that loss of Rb in TS cells is the defining event causing lethality of Rb −/− embryos and reveal the convergence of extraembryonic and fetal functions of Rb in neural and erythroid development. We conclude that the Rb pathway plays a critical role in the maintenance of a mammalian stem cell population.[Keywords: Rb; development; placenta; stem cells] Supplemental material is available at http://www.genesdev.org. The retinoblastoma (Rb) tumor suppressor gene was identified more than two decades ago as the gene responsible for retinoblastoma, but has since been implicated in most human cancers. In contrast to retinoblastoma patients, inheritance of one deleted copy of Rb in mice did not induce retinoblastoma but did increase risk of pituitary and thyroid cancers (Jacks et al. 1992;Hu et al. 1994;Maandag et al. 1994;Williams et al. 1994;Robanus-Maandag et al. 1998;Yamasaki et al. 1998). Deletion of both copies of Rb in mice resulted in a broad range of severe abnormalities that lead to lethality by embryonic day 15.5 (E15.5) (Clarke et al. 1992;Jacks et al. 1992;Lee et al. 1992;Wu et al. 2003). Because Rb is normally expressed in all tissues of the mouse embryo, it was assumed that these developmental abnormalities were due to the absence of Rb protein in the tissues affected. Subsequent analysis of chimeric embryos suggested that Rb function is likely to be much more complex than initially suspected (Maandag et al. 1994;Lipinski et al. 2001). Indeed, recent findings showed that Rb-deficient embryos supplied with a wild-type placenta could develop to term and suggested a critical function of Rb in the placenta that might underlie many of the fetal developmental abnormalities observed in Rb −/− embryos Wu et al. 2003).Because Rb is involved in so many important pro-

We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll PTD/WT mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll PTD/WT mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.