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, 27 (19), 6706-17

Deletion of Shp2 in the Brain Leads to Defective Proliferation and Differentiation in Neural Stem Cells and Early Postnatal Lethality

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Deletion of Shp2 in the Brain Leads to Defective Proliferation and Differentiation in Neural Stem Cells and Early Postnatal Lethality

Yuehai Ke et al. Mol Cell Biol.

Abstract

The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.

Figures

FIG. 1.
FIG. 1.
Deletion of Shp2 leads to early postnatal lethality. (A) Immunoblot analysis of Shp2 protein contents in whole-brain lysates from the control and mutant animals (Shp2F/+::Cre2/+ at P4 and Shp2F/F::Cre1/+ at P0), with anti-Erk1/2 blotting as loading control. (B) Early postnatal lethality of mutant mice: Shp2F/F::Cre1 mutants [red, n = 12 (males plus females)], male Shp2F/F::Cre2 mutants (pink; n = 8) and female Shp2F/F::Cre2 mutants (orange; n = 12). (C) Growth retardation of surviving Shp2F/F::Cre2 mutant mice. (Left panel) One representative pair of littermates at P10. (Right panel) Growth curve. F/F::Cre/+, male, pink; female, orange; F/+::Cre/+, green; and F/+::+/+, blue. (D) Whole brain from control (CTL) and Shp2F/F::Cre2 mutant (MUT) at P4.
FIG. 2.
FIG. 2.
Impaired corticogenesis. (A) Hematoxylin-and-eosin staining of cerebral cortex at P4. Scale bar, 100 μm. (B) MAP2 expression at P0. Scale bar, 100 μm. (C) EphA4 expression at P0 was examined using in situ hybridization. Scale bar, 100 μm. (D) Calbindin immunostaining at P4. Scale bar, 100 μm. (E to G) Immunostaining for expression of TuJ1 (E) and GFAP (G) in cerebral cortex (E16.5) and NG2 (F) in cerebral cortex (P0). Scale bar, 100 μm. (H) Immunostaining for expression of pH 3 and Ki67 in VZ (E17.5). (I) Significantly reduced numbers of pH 3+ and Ki67+ cells were detected in the Shp2 mutant VZ (pH3, **, P < 0.01; n = 6∼7; Ki67, *, P < 0.05; n = 3; mutant versus control; scale bar, 100 μm). (J and K) Shp2 expression in VZ at E13.5 (J) and subventricular zone at P4 (K). Scale bar, 100 μm. (L and M) Cell apoptosis by TUNEL assay at P4 (L) and P10 (M). Scale bar, 100 μm. PI, propidium iodide.
FIG. 3.
FIG. 3.
Shp2 deficiency results in impaired neurogenesis but modestly enhanced astrogliogenesis. (A) PCR genotyping of the Shp2F allele and the Nestin-Cre transgene. (B) Immunoblot analysis of tissue or cell lysates. Lane 1, freshly isolated cerebral cortex tissue at E14.5; lane 2, embryonic heart at E14.5; lanes 3, 4, and 5, NSC cultures collected at days 5, 7, and 14. (C) Representative images show reduced neural outgrowth and excessive fasciculation of neurites from Shp2-deficient neurospheres at days 3 and 5 (DIV3 and DIV5), compared to controls. Scale bar, 50 μm. (D) Immunostaining of differentiated cells in culture for TuJ1/DAPI and TuJ1/GFAP. Representative images indicate impaired neurogenesis (TuJ1+) at DIV5 (upper panel) but slightly increased proportion of astrogliogenesis (GFAP+) at DIV7 (lower panel) from Shp2-deficient NSCs. Scale bar, 100 μm. (E) Relative proportions of neurons, astrocytes, and oligodendrocytes were determined by counting TuJ1+, GFAP+, and O4+, or CNPase+ cells, respectively, on DIV 5, 7, and 10 and compared with the total number of cells (**, P < 0.01; *, P < 0.05; n = 5). (F) Decreased numbers of TuJ1+ neurons differentiated from Shp2-deficient NSCs compared to controls (*, P < 0.05; n = 5). (G) NSC differentiation in response to various growth factors. NSCs were cultured at 2 × 104 cells/ml in neurobasal/B27 serum-free medium, with a daily supplement of CNTF (100 ng/ml), PDGF-BB(100 ng/ml), LIF (3,000 U/ml), or bFGF (20 ng/ml) or with no supplement. Differentiated cells were detected by immunostaining for TuJ1 and GFAP at day 5. Shp2 deficiency resulted in reduced number of neurons following stimulation with PDGF, LIF, and bFGF (**, P < 0.01; *, P < 0.05; n = 5) but caused a significantly increase in the number of astrocytes following PDGF-BB treatment (*, P < 0.05; n = 12).
FIG. 4.
FIG. 4.
Shp2 is required for NSC proliferation and self-renewal in vitro. (A) Morphological examination shows the reduced number and size of Shp2 mutant neurospheres as compared to controls. Scale bar, 100 μm. (B) Neurosphere assay. NSCs were initially seeded at 5 cells/μl, and the number of neurospheres (NS) was determined after 7 days for primary (1°), secondary (2°), and tertiary (3°) neurospheres (**, P < 0.01; n = 3). 1k, 1,000. (C) Generation of secondary neurospheres. The frequency was expressed as the percentage of secondary neurospheres generated versus the total number of single cells dissociated from primary neurospheres. Self-renewal capacity was defined as the number of secondary neurospheres formed per primary neurosphere (*, P < 0.05; **, P < 0.01; n = 4). (D) Growth curve of NSCs in self-renewal culture. Neurospheres in suspension were gently dissociated into single cells and then enumerated (**, P < 0.01; n = 3). K, 1,000. (E) Growth curve of NSCs in differentiation culture. Single NSCs were seeded on a precoated plate in the absence of bFGF and EGF to induce neuronal differentiation; 2% FBS was added after 24 h to induce astroglial differentiation. Cell proliferation was monitored using the RT-CES system (17). (F) BrdU incorporation assay in NSCs (Nestin+), neurons (TuJ1+ or MAP2+), and astrocytes (GFAP+). (Left panel) Representative pictures of BrdU+ cells in NSCs. (Right panel) Statistical analysis. Scale bar, 50 μm. NSCs: mutant, 18.1% ± 7.5%; control, 54.4% ± 11.2%; **, P < 0.01; n = 5. Neurons: mutant, 15.3% ± 6.1%; control, 32.5% ± 7.1%; *, P < 0.05; n = 3. Astrocytes: mutant, 32.3% ± 4.2%; control, 43% ± 12%; P > 0.05; n = 3). (G) Cell apoptosis (TUNEL) assay in NSCs (Nestin+), neurons (TuJ1+ or MAP2+), and astrocytes (GFAP+). (Left panel) Representative pictures of apoptotic cells in NSCs. Scale bar, 100 μm. (Right panel) Statistical analysis. NSCs: mutant, 12.2% ± 7.2%; control, 8.9% ± 4.6%; P > 0.05; n = 5. Neurons: mutant, 13.7% ± 4%; control, 9.2% ± 1; *, P > 0.05; n = 3. Astrocytes: mutant, 8.3% ± 0.3%; control, 8.9% ± 1%; P > 0.05; n = 3.
FIG. 5.
FIG. 5.
Shp2 relays growth factor signals in NSCs. (A) Signaling in ESCs and NSCs. Cell lysates were prepared from ESCs and NSCs cultured in the presence of LIF (1,000 U/ml) and bFGF (20 ng/ml), respectively, and immunoblotted for Shp2, β-actin, p-AKT and AKT, p-C/EBP and C/EBP, p-Src and Src, p-Erk and Erk, and pY-Stat3 and Stat3. Band densities (means ± standard errors) are shown for the relative changes (fold) by setting the control level to 1 (n = 3 to ∼6). **, P < 0.01, n = 6; *, P < 0.05, n = 3; mutant versus control. (B) Growth factor signaling. Significant differences were detected in the number (left) and colony size (right) of neurospheres (NS) in response to bFGF or bFGF plus EGF. **, P < 0.01; n = 5∼12; mutant versus control). K, 1,000. (C) bFGF signaling. NSCs were starved and stimulated with bFGF (100 ng/ml) or LIF (1,000 U/ml) for the indicated time periods. Cell lysates were immunoblotted for p-Erk and Erk, p-c-Myc, pY-Stat3 and Stat3, and Shp2 and β-actin. (D) Effects of chemical inhibitors on formation of neurospheres. NSCs in neurosphere cultures were dissociated and seeded at low cell density (1,000 cells/well) with bFGF and EGF plus DMSO (0.05%), PD98059 (25 μM), 10058-F4 (25 μM), or Shp2 inhibitor (25 μM), and neurospheres were enumerated at DIV7. (E) Effects of chemical inhibitors on neuronal/astroglial differentiation. (Upper panel) NSC differentiation under treatment with DMSO (0.05%), PD98059 (25 μM), 10058-F4 (25 μM), or Shp2 inhibitor (25 μM) was assessed by immunostaining with TuJ1 and DAPI at DIV2. (Lower panel) NSC differentiation under treatment with DMSO (0.05%), 10058-F4 (25 μM), and AG490 (5 μM) was evaluated by immunostaining for GFAP and DAPI at DIV4. (F and G) Quantitative analysis of TuJ1+ (F) and GFAP+ (G) cells differentiated under treatment with inhibitors as indicated in panel E. For the percentage of TuJ1+ cells (F), compared to control (Ctl) NSCs with DMSO, a significantly reduced proportion was observed for PD98059 or Shp2 inhibitor (*, P < 0.05; n = 6) and mutants (Mt) with DMSO (**, P < 0.01; n = 6). For the percentage of GFAP+ cells (G) compared to control NSCs with DMSO, there was significantly reduced GFAP+ cells in control cells and the mutant with AG490 treatment (both **, P < 0.01; n = 6).
FIG. 6.
FIG. 6.
Exogenous expression of Bmi-1 partially rescues Shp2 deficiency in NSCs. (A) Immunoblot analysis of Bmi-1 expression levels with anti-Erk serving as loading control. Cell genotyping was reconfirmed by anti-Shp2 immunoblotting. Quantitative data were provided after scanning of band density (*, P < 0.05; n = 8). (B) Exogenous expression of Bmi-1 as determined by immunoblot analysis. Shp2−/− NSCs were infected with MSCV-Bmi-1-EGFP (Bmi1) or control MSCV-EGFP vector (control). (C) Shp2−/− NSCs infected with MSCV-Bmi-1-EGFP exhibited an increased size of neurospheres, compared to that infected with the MSCV-EGFP control virus. Representative pictures were taken 7 days after retroviral infection (scale bar, 100 μm). (D) Enhanced proliferation of Shp2−/− NSCs infected with Bmi-1-expressing retrovirus (EGFP+). Representative pictures were taken 5 days after infection. (E) Shp2−/− NSCs expressing exogenous Bmi-1 exhibited increased size and number of secondary neurospheres (NS). 1K, 1,000. *, P < 0.05; n = 10; **, P < 0.01; n = 3.

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