Rb substantially compensates for the double loss of p130 and p107 in adult but not embryonic neural stem cell lineages

Abstract

The Retinoblastoma (Rb) family of pocket proteins (p107, Rb, and p130) controls all aspects of neurogenesis from stem cell activation to long-term neuronal survival in the brain. Previous studies have reported non-overlapping, often complementary, roles for these cell cycle regulators with possibility for functional compensation. Yet the extent to which each protein might compensate for other family members and whether synergistic effects exist during neural stem cell (NSC) lineage development remain unclear. Fong et al. recently revealed that a triple knock-out (TKO) of all pocket proteins results in a transcriptomic switch from NSC quiescence to activation, followed by niche depletion in the adult hippocampus. Here, we investigated whether pocket proteins are equally critical in NSC fate regulation in the adult subventricular zone (aSVZ) and during embryogenesis. We report that TKO of these proteins results in NSC activation coupled to ectopic progenitor proliferation and massive apoptosis, leading to niche depletion and premature loss of neurogenesis inside the olfactory bulb (OB). Notably, a p107-p130 double knockout carrying a single wild-type Rb allele (DKO) substantially rescues the above defects and maintains adult neurogenesis. In comparison, TKO embryos display severe disruptions in all stages of neurogenesis at E14.5, leading to embryonic lethality. Similar defects are detected when any five out of the six alleles of pocket proteins are lost, with only partial rescue of the proliferation defects observed in DKO embryos. The above TKO phenotypes are partially mediated by opposed deregulations in the Notch-Hes signaling pathway in the embryonic versus the adult brain. Such deregulation is linked to opposite changes in E2F3a and E2F3b embryonic gene expressions. Our data identifies Rb as a critical pocket protein in the control and maintenance of adult OB neurogenesis, and uncovers interchangeable, dose-dependent roles for pocket proteins in the control of neuronal differentiation and survival during development.

Fig. 1. aSVZ-NSCs undergo fast activation and niche depletion over time in TKO but not DKO mice.

A Experimental design illustrating tamoxifen treatment and collection periods of adult mice carrying inducible and compound deletion(s) in pocket proteins. Immunohistochemistry (IHC) on brain sagittal sections in the aSVZ at 4wpt and 16wpt in THC (B, B’, E, E’), DKO (C, C’, F, F’), and TKO (D, D’, G, G’) mice. Insets in (B’–G’) show higher magnification images of the boxed regions inside the aSVZ. Note the lateral ventricle’s enlargement and niche depletion in TKO brains at 16wpt. IHC in the corpus callosum (H, I) and striatum (J, K) at 4wpt showing vimentin-positive progeny with astrocyte morphology derived from aSVZ-NSCs, including some progenitors co-expressing YFP and/or Nestin. Insets in (H–J”’) are higher magnification images of the boxed regions. I–K Higher magnification images of dashed boxes in (H”’, K”’). L Quantification of cell counts in the aSVZ showing significant increase in (Nestin + YFP+) and (Nestin + YFP + GFAP+) cell populations in TKO and DKO compared with THC at 4wpt, followed by gradual decrease and stem cell pool depletion in TKO brains (but not DKO) by 16wpt. Cx cortex, LV lateral ventricle, aSVZ adult subventricular zone. Scale bars, (B–G’): 100 µm, (H–K): 50 µm. Error bars, mean ± SD. Unpaired 2-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. n = 3 biological replicates.

Fig. 2. Ectopic progenitor proliferation and massive neuroblast migration in TKO brains are rescued in DKO mice.

IHC on brain sagittal sections in THC (A–E”’, J–N”’), DKO (B–G”’, K–P”’), and TKO (C–I”’, L–R”’). Inset panels show higher magnification images of dashed boxes in the aSVZ and rRMS. Note the ectopic progenitor proliferation and neuroblast migration inside the dorsal cortex, corpus callosum, and striatum in TKO but not DKO mice at 4wpt compared with THC. Quantification of cell counts in the aSVZ (S) and rRMS (T). Compared with THC, TKO mice show a robust increase in (YFP + DCX+), (YFP + Ki67+) and (YFP + DCX + Ki67+) cell populations at 4wpt followed by gradual decrease, then loss of most dividing progenitors and migratory neuroblasts by 16wpt. DKO mice maintain a strong level of AN compared with THC at 16wpt. Scale bars, 50 µm. Error bars, mean ± SD. Unpaired 2-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. n = 3–4 biological replicates.

Fig. 3. OB neurogenesis is lost in TKO mice by 16wpt but is preserved in DKO mice.

IHC in the granule cell layer -GCL- (A–F”) and the periglomerular layer -GL- (G–L”) showing YFP+ and NeuN+ newborn neurons inside the OB at 4wpt and 16wpt across genotypes. Insets in panels show higher magnification images of the boxed regions in each layer. Quantification of cell counts inside the GCL (M) and GL (N) showing a steady accumulation of newborn neurons inside the OB layers in THC and DKO mice compared with a sharp decline in the level of neurogenesis in TKO brains between 4wpt and 16wpt. Scale bars, 50 µm. Error bars, mean ± SD. Unpaired 2-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. n = 3 biological replicates.

Fig. 4. Loss of OB neurogenesis is due to severe survival defects in TKO mice.

IHC on brain sagittal sections in the aSVZ (A–D”) and rRMS (E–H”) in THC versus TKO mice. Insets show higher magnification images of the boxed regions shown in each region (red; AC-3 staining, and triple staining). Note the massive cell death detected in TKO brains compared with THC at 4wpt and 8wpt. Quantification of cell counts inside of the aSVZ (I) and rRMS (J). TKO mice exhibit extensive cell death in both regions compared with THC and DKO mice. The majority of (YFP + AC-3+) cells co-express DCX in TKO mice, highlighting massive death in neuroblasts. ‡, Triple cell counts were not performed at 16wpt. Scale bars, 50 µm. Error bars, mean ± SD. Unpaired 2-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. n = 3–4 biological replicates.

Fig. 5. TKO embryos display a sharp reduction in brain size and exacerbated proliferation defects in the telencephalon.

A Compared with WT embryos, TKO embryos display significant size reductions in the whole brain and the OBs, which are estimated to be around ~30% and ~85–90%, respectively. B Quantification of Hoechst-positive cells in the dorsal cortex (DC) at E14.5, showing the most prominent decrease in cell density in TKO embryos compared with the other genotypes. IHC on brain sagittal sections in the developing DC in WT (C–C”’), RbKO (D–D”’), DKO (E–E”’), and TKO (F–F”’) embryos. Insets in (C”’–F”’) are higher magnification images of boxed areas in (C”–F”). Note the ectopic proliferation in TKO and RbKO embryos inside the intermediate zone (IZ) and cortical plate (CP) (Ki67+ cells in red) compared with THC and DKO. VZ; ventricular zone, SVZ; subventricular zone. G Quantification of cell counts inside of the different cortical layers of the DC. Scale bars, 50 µm. Error bars, mean ± SD. Unpaired 2-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. n = 3–4 biological replicates.

Fig. 6. DKO embryos rescue the cell cycle exit defects but not the premature differentiation and survival defects observed in TKO embryos.

IHC on sagittal brain sections in WT (A–A”), RbKO (B–B”), DKO (C–C”), TKO (D–D”) embryos at E14.5. A”–D” Higher magnification images of boxed areas in (A–D’). There is delayed cell cycle exit in RbKO and TKO embryos, as marked by several scattered PH3+ cells inside the IZ and CP compared with DKO and THC embryos. Dotted lines in A–D' mark the border between the SVZ and the IZ. Quantification of Sox2+ cells (E) and PH3+ cells (F) in the DC at E14.5. Note the significant decrease in Sox2+ population in all genotypes compared with WT embryos. G–J”’ IHC in the DC showing DCX expression that is clearly confined to the IZ and CP in WT (G) and RbKO (H) embryos, but extended into the SVZ in DKO (I) and TKO (J) embryos. The latter two genotypes also show strong NeuN expression that is largely overlapping with DCX and AC-3, indicating severe differentiation and survival defects. Dotted lines in G'''–J''' mark the lower border of DCX expression in each genotype. K Quantification of cell counts in the DC at E14.5, confirming massive cell death of neuroblasts/neurons in DKO and TKO embryos. A very low number of or no AC-3+ cells were detected in WT and RbKO mice. L Western blot analysis showing 3.9-fold increase in Bax protein expression in TKO versus WT embryos at E14.5. Scale bars, 50 µm. Error bars, mean ± SD. Unpaired 2-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. n = 3–5 biological replicates.

Fig. 7. Abnormal Hes1 and Hes3 expressions in the telencephalon in DKO and TKO embryos.

IHC on brain sagittal sections in WT (A–A”’, E), RbKO (B–B”’, F), DKO (C–C”’, G), and TKO (D–D”’, H) embryos showing Hes1 and Hes3 staining throughout the DC in the latter two genotypes only. A'–D”’ Higher magnification images of boxed areas shown in (A–D). The majority of Hes-positive cells co-express AC-3 (but not Ki67; data not shown). I Quantification of cell counts inside the DC at E14.5. A very low number of or no Hes1+ cells were detected in WT and RbKO mice. Scale bars, 50 µm. Error bars, mean ± SD. Unpaired 2-tailed Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001. n = 3 biological replicates.

Fig. 8. Opposed deregulations in Hes5 and Rbpj transcript levels in the embryonic versus adult brain upon loss of pocket proteins.

In situ hybridization with antisense probes for Hes5 (A–D’, I–J’) and Rbpj (E–H’, K–L’) performed on embryonic (A–H’; E14.5) and adult (I–L’; 4wpt) brain sagittal sections. A’–L’ are higher magnification images of black boxes shown in (A–L). Hes5 and Rbpj mRNA transcript levels are significantly downregulated in the VZ-SVZ in TKO, RbKO, and DKO compared with WT embryos during development. The levels of both transcripts are, however, strongly upregulated in the aSVZ, Cx, and striatum in TKO mice compared with THC mice. M qRT-PCR results performed on cDNA derived from embryonic forebrain of TKO (n = 3) versus WT (n = 4) embryos at E14.5 and showing downregulated E2F3b and E2f4 expressions as opposed to upregulated E2F3a expression. Data is normalized to the 18S gene expression at internal. Legend as in Figs. 1 and 5. Scale bars, 50 µm. n = 3–4 biological replicates.