Presenilins are required for maintenance of neural stem cells in the developing brain
© Kim and Shen; licensee BioMed Central Ltd. 2008
Received: 07 November 2007
Accepted: 08 January 2008
Published: 08 January 2008
The early embryonic lethality of mutant mice bearing germ-line deletions of both presenilin genes precluded the study of their functions in neural development. We therefore employed the Cre-loxP technology to generate presenilin conditional double knockout (PS cDKO) mice, in which expression of both presenilins is inactivated in neural progenitor cells (NPC) or neural stem cells and their derivative neurons and glia beginning at embryonic day 11 (E11). In PS cDKO mice, dividing NPCs labeled by BrdU are decreased in number beginning at E13.5. By E15.5, fewer than 20% of NPCs remain in PS cDKO mice. The depletion of NPCs is accompanied by severe morphological defects and hemorrhages in the PS cDKO embryonic brain. Interkinetic nuclear migration of NPCs is also disrupted in PS cDKO embryos, as evidenced by displacement of S-phase and M-phase nuclei in the ventricular zone of the telencephalon. Furthermore, the depletion of neural progenitor cells in PS cDKO embryos is due to NPCs exiting cell cycle and differentiating into neurons rather than reentering cell cycle between E13.5 and E14.5 following PS inactivation in most NPCs. The length of cell cycle, however, is unchanged in PS cDKO embryos. Expression of Notch target genes, Hes1 and Hes5, is significantly decreased in PS cDKO brains, whereas Dll1 expression is up-regulated, indicating that Notch signaling is effectively blocked by PS inactivation. These findings demonstrate that presenilins are essential for neural progenitor cells to re-enter cell cycle and thus ensure proper expansion of neural progenitor pool during embryonic neural development.
During mammalian neural development, neural progenitor cells (NPCs) divide and populate the progenitor pool in the ventricular zone (VZ) of the developing telencephalon . There are two phases of neurogenesis. The first phase spans from embryonic day 8.5 (E8.5) to 10.5, during which neural progenitor population expands. The second phase commences around E11.5, reaches peak by E15.5, and then diminishes by E17.5, generating most of the cortical neurons through eleven cell cycles [2–6]. During the first phase, neural progenitor cells are columnar, connecting the pial surface and the apical surface, and predominantly undergo symmetric cell division to expand rapidly the self-renewal founder progenitor cells [6, 7]. During the second phase, neural progenitors in the ventricular zone predominantly undergo asymmetric cell division to generate self-renewal daughter progenitor cells that remain in the cell cycle and committed postmitotic neural precursor daughter cells that will ultimately differentiate into neurons or glia [3, 7–10]. Notch receptors are required for neural progenitor cell proliferation [11–13]. In the cell cycle of this asymmetric cell division, at the transition from S phase to M phase, the dividing neural progenitor cells displace their nuclei and subsequently migrate from the basal membrane to the apical surface of the neuroepithelium, a phenomenon termed interkinetic nuclear migration (INM) [14, 15].
Presenilin-1 (PS1) and presenilin-2 (PS2) are the major causative genes of familiar Alzheimer disease (FAD) . Presenilin (PS) is an essential component of the γ-secretase complex, and Notch is one of the physiological substrates of γ-secretase . Notch is one of the most important regulators of neural development in organisms from insects to mammals [18–20]. During mammalian neural development, activation of the Notch signaling pathway is involved in the maintenance of neural progenitor identity and the decision of cell fate . Presenilin is required for Notch signaling during neural development, though expression of Hes1, one of the Notch downstream effector genes, is not suppressed by inactivation of PS1, whereas expression of Hes5 is effectively suppressed . The germ-line deletion of PS1 in mice results in perinatal lethality, cerebral hemorrhages and impairment of neurogenesis, likely due to down-regulation of Notch signaling [22, 23]. Although no phenotypes have been observed in PS2-null mice , earlier and more severe developmental defects are found in PS1/PS2 double knockout mice than in PS1 single knockout mice . This finding suggests functional redundancy between PS1 and PS2 in embryonic development. When PS1 inactivation is restricted to NPC and NPC-derived neurons and glia beginning at ~E11 during the second phase of neurogenesis, the NPC-PS1 cKO mice can live up to 2–3 months postnatally. These mice harbor defects in neuronal migration, cortical lamination and radial glial development with milder loss of the progenitor cells .
Here we investigate that role of both presenilins in neural development, especially in the maintenance of neural progenitor or stem cell population. We employed a Nestin-Cre transgene to circumvent the early embryonic lethality associated with the germ-line deletion of both PS1 and PS2 loci and to restrict PS inactivation to NPCs and NPC-derived neurons and glia. Using this conditional double knockout mouse model, we found that presenilins are required for neural progenitor cells to re-enter cell cycle and for the expansion of neural progenitor population during embryonic development. Precocious exit of cell cycle observed in PS cDKO mice is accompanied by disruption of interkinetic nuclear migration and is followed by premature differentiation into postmitotic neurons. Blockade of expression of Notch downstream target genes, Hes1 and Hes5, in PS cDKO mice may underlie or contribute to the failure of neural progenitor cells to re-enter cell cycle. Thus, presenilins play an essential role for NPCs or neural stem cells to retain their self-renewal capacity to expand the progenitor population.
Gradual loss of PS1 transcripts in the developing PScDKO brain
To determine the spatial and temporal course of PS1 inactivation, we performed in situ hybridization analysis on the neural progenitor cell (NPC)-restricted PS cDKO mice, which were generated by crossing floxed PS1 mice with the Nestin-Cre transgenic mice in the PS2-/- background. We compared the PS1 mRNA expression in PS cDKO (fPS1/fPS1; Nestin-Cre; PS2-/-) embryos and littermate controls (PS2-/-) using a probe specific for PS1 exons 2 and 3. Consistent with our previous report on the analysis of spatial and temporal inactivation of PS1 in NPC-PS1 cKO mice , we found a marked reduction of PS1 mRNAs in PS cDKO embryos at E11.5. At E12.5, no significant signal of PS1 mRNAs was detected in the developing telencephalon of PS cDKO embryos. By E13.5, the PS1 mRNA was no longer detectable throughout the brain (data not shown).
Severe reduction of neural progenitor cells in PScDKO mice
Disrupted interkinetic nuclear migration of neural progenitor cells in PScDKO mice
Premature exit of the cell cycle in neural progenitor cells of PScDKO mice
Suppression of Notch signaling in the PScDKO embryonic brain
The role of presenilins in the second wave of neurogenesis was not studied previously. The regulation of the interkinetic nuclear migration and asymmetric cell division of the neural progenitor cells in the neocortical development was unclear. Here we demonstrate that neural progenitor cells lacking both presenilins fail to maintain their stem cell identity and self-renewal capacity, resulting in depletion of the progenitor population. The interkinetic nuclear migration of neural progenitor cells is also disrupted in the absence of presenilins and is accompanied by the failure of reentering the cell cycle, which leads to precocious neuronal differentiation. Notch signaling was effectively suppressed by inactivation of both presenilins. Together, these findings have uncovered a critical role of presenilins in the cell cycle regulation of neural progenitor cells and the proper expansion of the progenitor population.
Presenilins in the maintenance of neural progenitor cells through the cell cycle regulation
Germ-line deletion of the PS1 gene results in precocious neuronal differentiation and the loss of the neural progenitor cells as early as E10.5 . Conditional inactivation of PS1 in neural progenitor cells of NPC-PS1 cKO mice resulted in milder depletion of the progenitor cells, perhaps due to the later onset of PS1 inactivation in PS1 cKO mice compared to PS1-/- mice . In the current study, we circumvented the early embryonic lethality (~E9) exhibited by PS1/PS2-/- mice and restricted inactivation of both PS1 and PS2 to NPCs and NPC-derived cells. Neural progenitor cells in NPC-PS cDKO mice are depleted quickly between E12.5 and E15.5, a duration of about 6.5 cell cycles (the length of the total cell cycle at E13 is about 11 hours, see Figure 5), to only 17.2% of the control (Figure 1G).
Despite the fact that interkinetic nuclear migration has been a hallmark of vertebrate neural progenitors for seven decades , the mechanism underlying INM is not well understood. Currently, little is known about the molecular mechanisms controlling this unique mode of nuclear movement. Here we found that presenilins are required for the regulation of interkinetic nuclear migration (Figures 2 and 3). It was already severely disrupted in PS cDKO mice at E13.5, when the total number of neural progenitor cells was not yet altered. The abnormal location of M- and S-phase nuclei was also accompanied by the failure of neural progenitor cells reentering the cell cycle and the precocious neuronal differentiation (Figures 3 and 4). Therefore, we conclude that presenilins are required for the control of the INM and for the maintenance of daughter cells in the cell cycle, and that the failure of these processes leads to the depletion of self-renewable progenitor cell population.
Mechanisms of the cell cycle regulation by presenilins
Presenilin may regulate the cell cycle through its downregulation of β-catenin in a γ-secretase-independent manner, as previously reported [33, 34]. The transgenic overexpression of β-catenin in neural progenitor cells promoted these cells to reenter cell cycle, leading to the expansion of NPC population and the enlargement of the developing brain . If presenilins negatively regulate β-catenin in the developing brain, the elimination of PS in this study may be expected to activate the Wnt signaling by releasing free β-catenin, a scenario similar to the transgenic overexpression of β-catenin in the brain . However, NPCs lacking PS exit the cell cycle rather than reenter the cell cycle, resulting in depletion of progenitor cells. In addition, we saw no spinal cord abnormality in our NPC-PS cDKO mice (WYK and JS, unpublished data), whereas a previous report found elevated nuclear β-catenin in PS1-null mice and enhanced cell division in the spinal cord . Therefore, at least for the developmental stages we have studied, the cell cycle dysregulation in the PS cDKO mice is unlikely through β-catenin.
A large number of substrates for presenilin-mediated γ-secretase activity have been reported . Loss of the enzymatic activity may contribute the dysregulation of cell cycle in the PS cDKO mice Notch is one of the physiological substrates of γ-secretase and is required for the proliferation and survival of NPCs or neural stem cells [11, 13, 37, 38]. The severe depletion of neural progenitor cells in NPC-PS cDKO mice in the current study is consistent with the notion that Notch signaling is important for maintaining the NPC population. Hes5 expression is reduced in Notch1-null mutants and PS1-null mice, whereas Hes1 expression is not affected in these mutant mice [12, 13, 22]. Nevertheless, Hes1 is a primary Notch/CBF1 target [39, 40] and promotes the cell cycle by suppressing p27Kip, a CDK inhibitor . Expression of both Hes1 and Hes5 is significantly decreased in PS cDKO mice, suggesting that the presence of either PS1 or PS2 is sufficient for Hes1 expression. When Hes1 and Hes5 were both mutated, complete loss of neural stem cells was observed . This suggests that the more efficient blocking of Hes1 and Hes5 expression in PS cDKO mice may be the main cause underlying the severe depletion of NPCs. However, substantial levels of Hes1 and Hes5 were still observed in PS cDKO mice, suggesting the existence of presenilin-independent mechanisms for the regulation of Notch signaling [43, 44] or Notch-independent regulation of Hes1 expression [45, 46].
Notch signaling is important for the regulation of symmetric versus asymmetric cell division of neural stem cells [47, 48]. The precise control of this symmetric/asymmetric cell division is essential not only for the nervous system to reach its mature size but also for its proper composition [10, 49–51]. Recent reports have shown that two negative modulators of Notch signaling, Numb and Numblike, are important for the maintenance of neural progenitor cells in both earlier stages  and later stages [53, 54] of neurogenesis in mouse forebrain development. The function of Numb/Numblike in neural progenitor cells is still unclear, because two very similar studies reported opposite results for NPC maintenance; one found more NPCs , whereas the other found fewer NPCs . The conclusion of our current study that PS and perhaps Notch signal is required for NPCs to remain in the cell cycle is consistent with the result of the report by Li et al. . Because the numb/numblike mutant mice also showed depletion of neural progenitor cells at early stages of neurogenesis , it is not yet clear regarding the function of Numb/Numblike in the maintenance of NPC population. Further detailed study is warranted to clarify the functional relation of these molecules in NPC maintenance.
Presenilins in brain hemorrhages
One of the surprising phenotypes of PS cDKO mice is the severe hemorrhage in the entire brain. In PS cDKO embryos, hemorrhages are first seen at E14.5 in small numbers of the embryos and by E15.5 spread to the entire brain in all embryos. Nakajima et al.  suggested a new function of PS1 in the endothelial cell of blood vessels based on the study using PS1 null mice. In the NPC-restricted PS cDKO mice, PS1 is apparently not eliminated in the mesoderm-derived blood vessel endothelial cells [26, 56]. Furthermore, cerebral hemorrhages were not found in NPC-restricted PS1 cKO embryonic brains . These findings argue against the cause of the hemorrhage in PS cDKO being the possible leakage of the Nestin-Cre transgene expression in endothelial cells. The generation of all cell types in the cortex occurs in temporally distinct, albeit overlapping, phases – neurons are generated first (peaking at E14 and finishing perinatally), followed by astrocytes (starting at E13, peaking at P1, finishing by P10), and then oligodendrocytes (starting at E17 and continuing until adulthood) [57, 58]. Owing to the severe and almost complete depletion of neural progenitor cells between E12.5 and E15.5, the PS cDKO brain may not generate enough astrocytes. Because astrocytes regulate formation of the blood-brain barrier through direct interaction with endothelial cells or by secreting the growth factors, the absence of astrocytes may result in defects in the formation of the blood-brain barrier [59–62]. The related mutant mice (numb/numblike cDKO and PS1 cKO) that harbor later and milder depletion of neural progenitor cells have not shown cerebral hemorrhages. This suggests that the early depletion of the neural progenitor cells followed by the loss of astrocytes may be the cause of the hemorrhage in the PS cDKO brain.
The generation of neural specific PS1 cKO mice using Nestin-Cre transgenic and floxed PS1 mice [63, 64] was described previously . NPC-PS cDKO mice were generated by crossing the NPC-PS1 cKO mice to the PS2-/- background. Specifically, NPC-PS cDKO (fPS1Δ/fPS1;Nestin-Cre;PS2-/-, fPS1Δ represents a germ-line deletion of the floxed PS1 locus) mice used in the study were generated by crossing male fPS1Δ/+;Nestin-Cre;PS2-/- mice with female fPS1/fPS1;PS2-/-. Since the floxed PS1 locus is linked to the Nestin-Cre transgene on the same chromosome, we were able to obtain NPC-PS cDKO (fPS1Δ/fPS1;Nestin-Cre;PS2-/-) and littermate control (fPS1/+; PS2-/-) embryos at equal ratio. Animal breeding and experiment protocols were in accordance with the National Institute of Health Guidelines for use of live animals and approved by the Harvard Medical Area (HMA) Standing Committee of Animals.
Preparation of embryos and brain sections
After embryos were fixed in 4% paraformaldehyde at 4°C for 1.5 h (E11.5) or 2 h (E12.5 and later stages), they were cryoprotected and embedded in OCT (Sakura Fine Technical) for frozen sections or dehydrated and embedded in paraffin for paraffin sections. For the S-phase labeling, the pregnant mice were injected intraperitoneally with BrdU/PBS (70 mg/kg body weight; Sigma) 30 min before the dissection. For the cell cycle exit experiments, BrdU was injected into the pregnant mice 24 h before the dissection.
Immunostaining and in situ hybridization
For immunostainings, sections were blocked with a blocking solution containing 1% BSA, 3% goat serum, and 0.1% Triton X-100 for 3 h at room temperature and incubated with the desired primary antibodies overnight at room temperature. The following primary antisera or antibodies were used: anti-TUJ-1 antibody (1:400, Babco), anti-phospho histone 3 (1:100, Santa Cruz Biotechnology). For immunohistochemistry, the secondary antibody labeled with biotin (1:400, Vector Labs) was incubated, and the ABC complex (Vector Labs) incubation followed. The staining was performed using the DAPI kit (Vector Labs) or AP Purple (Roche). For the fluorescence staining of the tissues, the primary antibody-treated sections were incubated with the appropriate Alexi Fluor 488- or Alexi Fluor 594-conjugated secondary antibodies (1:300, Molecular Probes) for 2 h at room temperature. Images were viewed on a Zeiss confocal laser scanning microscope and analyzed by using LSM 510 software. In situ hybridization of brain sections was performed as described previously  using the digoxygenin-labeled riboprobes. The Hes1, Hes5, and Dll1 riboprobes were described previously . All statistical analyses of the counted cells were performed using Microsoft Excel software.
Interkinetic nuclear migration and cell cycle reentering assay
For S-Phase nuclei staining, the BrdU injected pregnant mice were dissected and the embryos were harvested at E13.5 or E14.5. The frozen sections were stained with an anti-BrdU antibody for S-Phase labeling. The pictures of stained slides were taken and the VZ were divided into 11 bins. The S-phase nuclei in each bin with a width of 500 μm were counted, and the numbers of the nuclei and their positions were shown in the diagram. The M-phase nuclei, which were stained with an anti-phospho-histone 3 antibody, were counted in dorsolateral corte x sections with a width of 750 μm. The portion of cells that re-enter cell cycle were calculated as following: The number of cells labeled with both BrdU and Ki67 (yellow, re-entered cell cycle) was divided by the total number of BrdU stained cells (green, BrdU+/Ki67- and yellow, BrdU+/Ki67+) 24 h after BrdU pulse labeling.
Cell cycle parameters calculation
Analysis of cell cycle kinetics in the cortex using cumulative BrdU-labeling procedures were performed as described previously  with slight modification. Briefly, BrdU pulses (70 mg/kg body weight) were given to pregnant dams every 2 h for up to 8 h. Under these conditions, all nuclei passing through the S-phase will be labeled. Dams at the age of E13.5 were sacrificed 0.5, 1.5, 2.5, 4.5 and 8.5 h after the first injection. The embryos were fixed for 2 h in 4% paraformaldehyde, paraffin-embedded and processed to reveal BrdU immunoreactivity. The proportions of cells in the proliferative zone that were BrdU labeled were counted in 400 μm wide strips throughout the telencephalon.
Labeling index (LI; labeled cells as a proportion of total cells) was calculated for the ventricular zone after the sections were stained with an anti BrdU antibody. After the staining, the labeling index at each time point was calculated as the number of BrdU-positive nuclei divided by the total nuclei count in the ventricular zone. The resulting data were then used to determine the time of BrdU labeling needed to reach the maximum value of BrdU-stained nuclei, which corresponds to the total cell cycle length subtracted by the length of S phase (Tc-Ts).
Quantitative real-time RT-PCR
At E13.5, cortexes were collected for the preparation of total RNA using Tri reagent (Sigma). The cDNA was synthesized using the Superscript II first-strand cDNA synthesis system (Gibco BRL). Oligonucleotide primers were designed using Primer Express software 1.0 (Applied Biosystems). The specificity of primers was determined by gel electrophoresis of PCR products to ensure a single band with predicted size. Quantitative PCR reaction was performed using SYBR Green on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Relative quantification of gene expression was performed using the comparative CT method according to manufacturer's recommended protocol (User Bulletin #2, ABI PRISM 7700 Sequence Detection System, Perkin-Elmer). Levels of target mRNA were normalized using 18S rRNA. The change of the expression of a target gene in the PS cDKO embryonic brain relative to the littermate control was calculated as: Fold change = 2-(ΔCT, Tg-ΔCT, control).
The authors thank Drs. Beglopoulos and Wines-Samuelson and other members of the Shen laboratory for invaluable help and discussions.
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