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Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells

Nature Neuroscience volume 18, pages 657–665 (2015)Cite this article

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Abstract

The mechanism by which adult neural stem cells (NSCs) are established during development is unclear. In this study, analysis of cell cycle progression by examining retention of a histone 2B (H2B)-GFP fusion protein revealed that, in a subset of mouse embryonic neural progenitor cells (NPCs), the cell cycle slows between embryonic day (E) 13.5 and E15.5 while other embryonic NPCs continue to divide rapidly. By allowing H2B-GFP expressed at E9.5 to become diluted in dividing cells until the young adult stage, we determined that a majority of NSCs in the young adult subependymal zone (SEZ) originated from these slowly dividing embryonic NPCs. The cyclin-dependent kinase inhibitor p57 is highly expressed in this embryonic subpopulation, and the deletion of p57 impairs the emergence of adult NSCs. Our results suggest that a substantial fraction of adult SEZ NSCs is derived from a slowly dividing subpopulation of embryonic NPCs and identify p57 as a key factor in generating this embryonic origin of adult SEZ NSCs.

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Figure 1: Slowly dividing NPCs in the embryonic brain.
Figure 2: Emergence of slowly dividing NPCs during embryogenesis.
Figure 3: H2B-GFP-retaining cells in the young adult SEZ.
Figure 4: Young adult NSCs retain H2B-GFP.
Figure 5: Greater p57 expression in slowly dividing embryonic NPCs than in other NPCs.
Figure 6: Conditional p57 deletion reduces slowly dividing embryonic NPCs.
Figure 7: Conditional p57 deletion reduces young adult SEZ NSCs and their progeny.
Figure 8: High p57 expression contributes to the maintenance of NPCs' undifferentiated state.

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Acknowledgements

We thank K. Tyssowski for editing the manuscript, F. Calegari (Center for Regenerative Therapies Dresden (CRTD)) for reagents, H. Sugizaki, C. Koga and J. Takeuchi for FACS analysis, A Shitamukai, F. Matsuzaki and Y. Sasai for comments and members of the Gotoh laboratory for discussion. This work was supported by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency.

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Author notes

  1. Yusuke Hirabayashi

    Present address: Present address: Department of Neuroscience, Columbia University, New York, New York, USA.,

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Shohei Furutachi, Hiroaki Miya, Tomoyuki Watanabe, Hiroki Kawai, Norihiko Yamasaki, Yujin Harada, Yusuke Hirabayashi & Yukiko Gotoh

  2. The Hakubi Center, Institute for Virus Research, Kyoto University, Kyoto, Japan

    Itaru Imayoshi

  3. Echelon Biosciences, Salt Lake City, Utah, USA

    Mark Nelson

  4. Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

    Keiichi I Nakayama

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Contributions

S.F. designed the study, conducted the experimental work and wrote the manuscript. H.M., T.W., H.K., N.Y. and Y. Harada conducted the experimental work. I.I. generated Nestin-NLS-mCherry mice. M.N. provided 9TB-Dox. K.I.N. generated p57-floxed mice. Y. Hirabayashi supervised the project. Y.G. supervised the project and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Confirmation of the reversibility of H2B-GFP mRNA induction.

(a) Expression of the H2B-GFP transgene was induced at E9.5, and the embryos were examined at E10.5 or E16.5. At E10.5, the telencephalon of Rosa-rtTA/TRE-mCMV-H2B-GFP embryos was dissected and assayed for the level of H2B-GFP mRNA. The telencephalon of E10.5 Rosa-rtTA/TRE-mCMV-H2B-GFP embryos without Dox injection was examined as a negative control. At E16.5, the telencephalon of Rosa-rtTA/TRE-mCMV-H2B-GFP embryos was dissected, and LRCs (the top 10% fraction for H2B-GFP level) and non-LRCs among NPCs (CD133+CD24– population) were isolated by FACS for quantification of H2B-GFP mRNA. (b) Data are means ± SD (n = 3 experiments). Two-tailed Student’s t test; P < 0.001 ((+)Dox E10.5 versus (–)Dox E10.5; (+)Dox E10.5 versus (+)Dox E16.5 LRC; (+)Dox E10.5 versus (+)Dox E16.5 Non-LRC), P = 0.6 ((+)Dox E16.5 LRC versus (+)Dox E16.5 Non-LRC). ***P < 0.001; n.s., not significant (P ≥ 0.05).

Supplementary Figure 2 The distribution of H2B-GFP label-retaining cells in the E17.5 mouse brain.

(a) 9TB-Dox was injected at E9.5 to pregnant mice and brain sections of Rosa-rtTA/TRE-mCMV-H2B-GFP embryos were prepared at E17.5. (b) Immunohistochemistry with an antibody against GFP (green) at different rostrocaudal levels. Nuclei were stained with Hoechst (blue). The boxed regions correspond to the scheme in Supplementary Fig. 3d. Asterisks indicate choroid plexus.

Supplementary Figure 3 The distribution of H2B-GFP label-retaining cells in ventricular zone subregions of the E17.5 mouse brain.

(a) Pregnant mice were injected with 9TB-Dox at E9.5, and brain sections of Rosa-rtTA/TRE-mCMV-H2B-GFP embryos were prepared at E17.5. (b and c) Staining of sections for GFP (green), Pax6 (red, b) and Gsh2 (red, c). Nuclei were stained with Hoechst (blue). The boxed regions in the left panel are shown at higher magnification in the right panels. LRCs (arrowheads) were observed in the ventricular zone of the telencephalon. (d) The distribution of H2B-GFP-retaining NPCs in ventricular zone subregions at different rostrocaudal levels. Ventricular zone subregions are defined as follows: Pax6++Ascl1– as neocortex (NCX), Pax6+Ascl1+ as dorsal lateral ganglionic eminence (dLGE), Pax6–Ascl1+ (lateral) as ventral lateral ganglionic eminence (vLGE), and Pax6–Ascl1+ (medial) as septum (SE). H2B-GFP-retaining NPCs were distributed throughout the Ascl1+ LGE but were enriched in the Pax6+Ascl1+ dLGE. Data are means ± SEM (n = 3 animals). The scheme corresponds to the boxed regions in Supplementary Fig. 2b. Scale bars: 100 μm in (b and c, left) and 25 μm in (b and c, right).

Supplementary Figure 4 Overlapping distributions of H2B-GFP retention and EdU retention.

(a) Pregnant mice were injected with 9TB-Dox at E9.5 and with EdU (four times at 3-h intervals) at E10.5. Brain sections of Rosa-rtTA/TRE-mCMV-H2B-GFP embryos or pups were prepared at E17.5 or P14. (b) Sections for native fluorescence of GFP (green) and EdU staining (red) at E17.5 (top) and P14 (bottom). Nuclei were stained with Hoechst (blue). Asterisks indicate the choroid plexus. (c) Quantitative analysis of H2B-GFP- and EdU-retaining cells in SEZ subregions at P14. Data are means ± SEM (n = 3 animals). Scale bar: 20 μm.

Supplementary Figure 5 Lack of Dcx and NG2 expression in H2B-GFP label-retaining cells in the ventricular zone of the E17.5 mouse brain.

(a) Pregnant mice were injected with 9TB-Dox at E9.5, and brain sections of Rosa-rtTA/TRE-mCMV-H2B-GFP embryos were prepared at E17.5. (b) Staining for GFP (green) and Dcx (red). Nuclei were stained with Hoechst (blue). The boxed region in the left panel is shown at higher magnification in the right panel. (c) Staining for GFP (green) and NG2 (red). Nuclei were stained with Hoechst (blue). Scale bars: 25 μm in (b, left and c) and 7.5 μm in (b, right).

Supplementary Figure 6 FACS strategy used to isolate the embryonic and postnatal NPCs.

(a) Structure of the Nestin-NLS-mCherry transgene. (b) Sections of the forebrain of E13.5 Nestin-NLS-mCherry transgenic mice. The mCherry fluorescence (red) was localized to the ventricular zone. (c) FACS profiles for isolation of NPCs at various time points after H2B-GFP induction. NPCs (boxes in right panels) were defined as CD133+CD24– population (E10.5) or CD133+mCherry+ population (E13.5, E15.5, E17.5 and P3). (d) FACS profiles of negative control cells at E10.5 (left panel) and E15.5 (right panel). (Related to Fig. 2)

Supplementary Figure 7 The distribution of H2B-GFP induced at various time points.

(a) Pregnant mice were injected with 9TB-Dox at E9.5 (one time), E14.5 (two times at 8-h interval), or E17.5 (two times at 8-h interval), and brain sections of Rosa-rtTA/TRE-mCMV-H2B-GFP animals were prepared 1 day after the injection at E14.5 or E17.5 (E15.5 or E18.5, respectively) or at P14 or P28. (b and c) Immunohistochemistry with an antibody against GFP (green). Nuclei were stained with Hoechst (blue). The boxed regions in (b) are shown at higher magnification in the insets. The successful induction of H2B-GFP by 9TB-Dox at E9.5 and E14.5 was confirmed by its distribution in the neocortex at P14 (c). UL, upper layer; DL, deep layer of the neocortex. Asterisk indicates the choroid plexus. (d) Immunohistochemistry with antibodies against GFP (green) and GFAP (red). Nuclei were stained with Hoechst (blue). Note that when 9TB-Dox was administered at E17.5, only a small fraction (31.7 ± 6.0%, mean ± SEM, n = 3 animals) of SEZ-NSCs (GFAP+Sox2+apical process+ cells) retained H2B-GFP at P28, probably as a result of the slowly cycling embryonic subpopulation having already slowed down the cell cycle by E17.5. Indeed, the labeling by H2B-GFP among cells in the ventricular zone was only partial at E18.5 when 9TB-Dox was administered at E17.5. By contrast, most NPCs in the ventricular zone were H2B-GFP positive at E15.5 when 9TB-Dox was administered at E14.5. Strikingly, 92.0 ± 2.2% (mean ± SEM, n = 3 animals) of GFAP+Sox2+apical process+ SEZ-NSCs in the lateral wall of the SEZ retained H2B-GFP at high levels even at P28 when Dox was administered at E14.5, consistent with the notion that most of the slowly dividing embryonic progenitors slow down the cell cycle between E13.5 and E15.5 and contribute to the major population of adult SEZ-NSCs. Scale bars: 50 μm in (b and d) and 200 μm in (c).

Supplementary Figure 8 H2B-GFP-retaining cells in the young adult brain.

(a) Transient H2B-GFP expression was induced at E9.5 and then allowed to become diluted in dividing cells until postnatal or young adult stages. (b) Distribution of H2B-GFP LRCs in the SEZ at different rostrocaudal levels at P28. Shown images are immunohistochemistry with antibodies against GFP (green) and S100β (red). (c) Immunohistochemistry with an antibody against GFP (green) of P28 Rosa-rtTA/TRE-mCMV-H2B-GFP mouse (left) and its negative control (TRE-mCMV-H2B-GFP, right) brain sections. (d) Immunohistochemistry with an antibody against GFP (green) of P14 Rosa-rtTA/TRE-mCMV-H2B-GFP mouse with Dox (left) or without Dox (right) injection at E9.5. Nuclei were stained with Hoechst (blue). Asterisks indicate choroid plexus. Scale bars: 100 μm in (b and c) and 50 μm in (d).

Supplementary Figure 9 H2B-GFP- and BrdU-retaining cells express p57 at a high level.

(a) Pregnant mice were injected with 9TB-Dox at E9.5, and brain sections of Rosa-rtTA/TRE-mCMV-H2B-GFP embryos were prepared at E17.5. (b) Immunohistochemistry with antibodies against GFP (green) and p57 (red). Nuclei were stained with Hoechst (blue). Arrowheads indicate LRCs expressing p57 at a high level. (c) Pregnant mice were injected with BrdU (four times at 3-h intervals) at E10.5, and brain sections of embryos or pups were prepared at E17.5, E18.5, P0, and P10. (d) Immunohistochemistry with antibodies against BrdU (green) and p57 (red). Nuclei were stained with TO-PRO-3 (blue). The boxed regions in the upper panels are shown at higher magnification in the lower panels. Scale bar: 25 μm.

Supplementary Figure 10 Effects of p57 overexpression.

(a) In utero electroporation (IUE) was performed at E14.5 with expression plasmids for GFP alone (control) or for GFP and p57, and brain sections were subjected to immunohistochemistry at E18.5 (b) or P4 (d). (b) Immunohistochemistry with antibodies against GFP (green), Sox2 (blue), and PCNA (red). Arrowheads indicate GFP+ cells in the ventricular zone. (c) Quantification of the data in (b). Data are means ± SEM (n = 4 and 5 animals for control and p57, respectively). Two-tailed Student’s t test; P = 0.0072. (d) Immunohistochemistry with antibodies against GFP (green), Sox2 (blue), and Dcx (red). (e) Our model. Slowly dividing embryonic NPCs are an embryonic origin of adult NSCs, and p57 is responsible for their emergence. Scale bars: 25 μm in (b) and 20 μm in (d). **P < 0.01.

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Furutachi, S., Miya, H., Watanabe, T. et al. Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells. Nat Neurosci 18, 657–665 (2015). https://doi.org/10.1038/nn.3989

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