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Rb is critical in a mammalian tissue stem cell population - PubMed

  • ️Mon Jan 01 2007

Rb is critical in a mammalian tissue stem cell population

Pamela L Wenzel et al. Genes Dev. 2007.

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.

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Figures

Figure 1.
Figure 1.

Three-dimensional image analysis reveals global disruption of Rb mutant placentas. (A, panels in first row) Representative 3D images derived from H&E-stained sections of whole placentas that were scanned, registered, and stitched together. A representative internal slice reveals the major cell layers of the placenta. (de) Decidua; (sp) spongiotrophoblast; (lb) labyrinth trophoblast. (Panels in second row) Higher-magnification (2× and 5×) cubic images of the labyrinth–spongiotrophoblast interface. Green lines represent the boundary between the labyrinth–spongiotrophoblast interface; dotted yellow lines encircle clusters of Rb−/− trophoblasts that are embedded within the labyrinth; arrows point to typical blood spaces visible in the Rb+/+ placenta. (Panels in third row) Volumetric rendering of the labyrinth layer (blue). Morphometric analysis of the labyrinth (blue) and spongiotrophoblast (yellow) layers are depicted by cubic images at high resolution. The magenta area represents the region of contact between the labyrinth and spongiotrophoblast layers; note the expanded interface zone between these two layers in the Rb−/− placenta. (Panels in fourth row) Morphometric analyses of spongiotrophoblast intrusions and extrusions into the labyrinth layer (blue) are visually rendered in orange and green, respectively. The genotypes of each placenta are indicated at the top (Rb+/+, n = 3; Rb−/−, n = 3). For a rotational view of whole placentas and magnified cubic images, see also

http://tinyurl.com/bc6ka

. (B) Morphometric analysis of Rb mutant placentas.

Figure 2.
Figure 2.

Embryos reconstituted with Rb+/+ and Rb−/Δ19 placentas. (A) Southern blot analysis of aggregated chimeric fetuses hybridized with an Rb-specific probe that differentiates between the RbΔ19 and Rb+/Rb− alleles (indicated on the right). (M) Lane loaded with DNA ladder marker; (H) RbΔ19/+ control DNA sample. Note the low contribution of YC5 ES cells (Rb+/+) to chimeric fetus number 1; all others fetuses contained >95% contribution from YC5 ES cells. (B) Placental sections from representative aggregated embryos with the indicated genotypes were stained with H&E. Note the clusters of small cuboidal trophoblast cells in Rb−/Δ19 placentas. Original magnification, 20×. (C) Genotypic analysis of aggregated diploid morula–ES cell embryos.

Figure 3.
Figure 3.

The CYP19-cre transgene drives expression of cre in TS cell lineages. (A) CYP19-cre transgenic mice were crossed to RosaLoxP/LoxP reporter mice and analyzed for β-galactosidase activity to establish the pattern and timing of cre expression in the E6.5 and E8.5 embryo (counterstained with eosin) and E13.5 (counterstained with nuclear fast red) fetus, yolk sac, and placenta. The dotted yellow line shows the incision site made for removal of the fetus for genotyping; note that the fetus is not shown for the E8.5 panels. (ec) Ectoplacental cone; (ee) extraembryonic ectoderm; (em) embryonic endoderm; (me) extraembryonic mesoderm; (ve) visceral endoderm; (bi) blood island; (de) decidua; (sp) spongiotrophoblast; (lb) labyrinth trophoblast; (cp) chorionic plate; (ys) yolk sac. (B, right) Placenta sections from E13.5 embryos were hybridized with a spongiotrophoblast-specific (Tpbp) RNA probe and were counterstained with hematoxylin. Micrographs of sections hybridized with a sense RNA Tpbp probe are not shown. (Left) An adjacent section was stained by X-gal and counterstained with nuclear fast red. Giant trophoblasts that did not express cre are indicated by arrows.

Figure 4.
Figure 4.

Rb ablation in the placenta causes lethality and disruption of labyrinth architecture. (A) Sections of placentas were stained with H&E (two left panels) or processed for immunofluorescence using BrdU-specific antibodies and counterstained with DAPI (two right panels). Yellow lines indicate the boundary between the labyrinth and spongiotrophoblast cell layers. The original magnification is indicated within the bottom panels for each column. (B) Genotypic analysis of embryos. (C) Pocket proteins were detected by Western blot in TS cells and mouse embryonic fibroblasts (MEFs). TKO is a cell line triply deleted for Rb, p107, and p130. (D) Expression of p107 and p130 in Rb+/+ and Rb−/− TS cells was measured by real-time RT–PCR using primers listed in Supplementary Figure S3. (E) Immunofluorescence of BrdU incorporation and phosphorylation of histone H3 was used as an indicator of DNA synthesis and mitosis, respectively, in cultured TS cells.

Figure 5.
Figure 5.

Deletion of Rb in labyrinth trophoblasts or spongiotrophoblasts does not affect embryonic development. (A,B) Whole mount of the fetus and sections of the yolk sac and placenta from CYP19lab-cre;Rosa+/LoxP (A) and Tpbp-cre;Rb−/LoxP;Rosa+/LoxP (B) embryos were stained with X-gal and counterstained with nuclear fast red. Inset represents a higher-magnification micrograph of a representative TS cell cluster, which does not stain positively with X-gal. (me) Extraembryonic mesoderm; (ve) visceral endoderm; (bi) blood island; (TS) cluster of cells with TS cell morphology. (C,D) In situ hybridization of placenta sections from E13.5 CYP19lab-cre; Rb−/LoxP;Rosa+/LoxP (C) or E13.5 Tpbp-cre;Rb−/LoxP;Rosa+/LoxP (D) embryos using an antisense RNA Tpbp probe. Micrographs of sections hybridized with a sense RNA Tpbp probe are not shown. Adjacent sections were stained with X-gal as indicated. Arrows point to a giant trophoblast. (sp) Spongiotrophoblast; (lb) labyrinth trophoblast. (E,F) Sections of E17.5 CYP19lab-cre Rb−/LoxP;Rosa+/LoxP placentas (E) and E13.5 Tpbp-cre;Rb−/LoxP;Rosa+/LoxP placentas (F) stained with H&E. (G,H) Genotypic analyses of embryos derived from CYP19lab-cre;Rb+/− and RbLoxP/LoxP;RosaLoxP/LoxP intercrosses (G), as well as from Tpbp-cre;Rb+/− and RbLoxP/LoxP;RosaLoxP/LoxP intercrosses (H).

Figure 6.
Figure 6.

Loss of E2f3 in Rb-null TS cells suppresses mutant phenotypes. (A) Expression of E2f1–8 in Rb+/+ and Rb−/− placentas was measured by real-time RT–PCR assays using primers described in Supplementary Figure S3. (B) Placental sections from embryos with the indicated genotypes were stained with H&E. Note that loss of E2f3 suppressed the formation of large clumps of dysplastic trophoblasts. (C) Genotypic analysis of double-knockout embryos derived from crosses between Rb+/− and either E2f+/− or E2f−/− mice.

Figure 7.
Figure 7.

Conditional deletion of Rb and E2f3 in TS cells. (A) Placental tissue sections with the indicated genotypes were stained with H&E and micrographed at low (top panels) and high (bottom panels) magnification. (B) Similar sections as in A were processed for immunohistochemistry using Ki67-specific antibodies and counterstained with hematoxylin; top and bottom panels show micrographs at low and high magnification, respectively. (C) Genotypic analysis of embryos derived from crosses between CYP19-cre;Rb−/LoxP;E2f3LoxP/LoxP;Rosa+/LoxP and Rb−/LoxP;E2f3LoxP/LoxP;Rosa+/LoxP mice.

Figure 8.
Figure 8.

Loss of Rb in TS cells partially disrupts fetal development. (A) Analysis of E13.5 fetal tissues from CYP19-cre; Rb−/LoxP;Rosa+/LoxP and control embryos. Apoptosis was measured in the CNS by TUNEL assays, erythrocyte maturation in peripheral blood smears was monitored by staining with Giemsa, and the presence of macrophages in the liver was assessed by immunostaining with F4/80-specific antibodies and counterstaining with hematoxylin. Original magnifications are indicated for each panel and insets were 40×. (B) Quantification of proliferation and apoptosis of the indicated tissues as determined by BrdU- and TUNEL-assays, respectively. Note the presence of a small percentage of TUNEL-positive lens fiber cells present in CYP19-cre;Rb−/LoxP; Rosa+/LoxP embryos that arises as a consequence of occasional cre-expressing cells in the lens driven by this transgene. Hematopoietic differentiation was measured by the percentage of nucleated RBCs in peripheral blood (Blood) and by the amount of macrophages (% surface area) present in fetal livers (Macrophages). (a*) Mann-Whitney U-test, p < 0.05; (b*) Wilcoxon Signed Rank test, p = 0.002.

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