Rb is critical in a mammalian tissue stem cell population

Abstract

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-

Hypoxic stress underlies defects in erythroblast islands in the Rb-null mouse

“…14 However, this concept was difficult to reconcile with the amelioration of Rb-null erythroid defects in mice provided with a wild-type placenta but harboring Rb-null macrophages. 9 Our current work provides an explanation for these seemingly contradictory results by showing that Rb-null macrophages harvested from healthy fetal liver or bone marrow are not defective for erythroblast island formation in the absence of exogenous stress, that hypoxia can disrupt erythroblast islands in vitro, that macrophage coculture does not affect the level of red cell enucleation, and that the Rb-null fetal liver is hypoxic at times when erythroid islands are increasing in size and number in wild-type fetal liver. 4 Thus, erythroblastic island defects in Rb-null mice and in ex vivo islands derived from these mice likely stem from the deleterious effects on macrophage-erythroblast interactions by hypoxia and the rapidly deteriorating microenvironment.…”

“…The induction of known HIF target genes in the E12.5 Rb-null fetal liver is consistent with ischemia (due to placental defects and anemia) being the cause, as opposed to the consequence, of the cell death that is first detected in E13.5 Rb-null fetal liver. 8,9,12 We also used the microarray data and quantitative real-time PCR to examine how loss of Rb affected expression of macrophagespecific genes that had been previously implicated in the erythroblast island defect in Rb-null fetal liver, 14 including c-Fms, myeloperoxidase (MPO), cathepsin S, complement components, We noted that Rb-null erythroblasts (D) failed to enucleate irrespective of macrophage contacts (arrow) and demonstrated increased size and abnormal chromatin structure as reported previously. 11,12 (E,F) Native erythroblast islands cultured in vitro from mice with the indicated Rb genotypes and at early or later stages of embryonic development were enumerated (F).…”

“…7 Mice carrying a targeted deletion of the Rb tumor suppressor gene in their germ line die at E13.5 to E14.5 of gestation, exhibiting extensive cell death in multiple tissues, including the fetal liver, and this has been attributed to systemic ischemia due to defective placental function and anemia. 8,9 The production of enucleated mature red blood cells appeared normal in mice derived from germ-line Rb-null chimeras 10 and in conditionally targeted E15.5 embryos in which Rb was not deleted in the placenta. 8 However, analysis of erythroblast maturation in vitro 11,12 or in Rb-null chimeric mice that had been challenged in vivo with phenylhydrazine to induce stress erythropoiesis recapitulated the erythroid maturation defects seen previously in the developing Rb-null fetal liver, including the failure to enucleate and up-regulate TER119.…”

The RB family is required for the self-renewal and survival of human embryonic stem cells

“…Thus, the RB pathway controls cellular processes that are critically linked to stem cell properties. Accordingly, RB and its two family members p107 and p130 are important regulators of adult stem cell and progenitor populations [10][11][12][13][14][15] .…”

Non-rigid Registration for Large Sets of Microscopic Images on Graphics Processors

“…In order to evaluate our optimization techniques and implementation methods, we applied our registration algorithm to a series of microscopic images of consecutive sections of (1) mouse placenta for a morphometric study on the role of the retinoblastoma gene and (2) mammary gland for studying the breast cancer tumor microenvironment [16]. For details about these sets of images, see Table 5.…”