Deletion of Shp2 in NSCs leads to early postnatal lethality.
In previous experiments, we cloned the murine Shp2
(previously called Syp
) cDNA, using a unique PCR strategy designed to search SH2-containing tyrosine phosphatases (9
). In situ hybridization detected Shp2
expression in the neural ectoderm and nervous system in mouse embryos at E9.5 and later stages, suggesting an involvement of Shp2 in neural development (9
). To gain insights into the Shp2 function during brain development, we have analyzed Shp2
expression profiles in the central nervous system. As shown in Fig. S1A in the supplemental material, immunoblotting with a specific anti-Shp2 antibody detected Shp2 protein in isolated neural stem cells, neurons, and astrocytes. Widespread expression of Shp2 was observed in various parts of the adult brain, including the pituitary gland, olfactory bulb, cerebral cortex, hippocampus, cerebellum, and brain stem (see Fig. S1B in the supplemental material).
To decipher Shp2 functions in CNS development, we generated mutant mice with Shp2 selectively deleted in NSCs by crossing a conditional Shp2
) mouse line (40
), with nestin-Cre
transgenic mice. We used two different transgenic lines of nestin-Cre
mice, dubbed as nestin-Cre1
, originally created in the laboratories of Klein and Kageyama, respectively (16
). Crossing of nestin-Cre1
mice with a Rosa26lacZ-loxP
reporter mouse line shows efficient and widespread DNA recombination mediated by the Cre recombinase in precursors of neurons and glia starting around E10.5 (34a
). The Cre recombinase activity in nestin-Cre1
mice was detected within the developing cortical wall, and in all cortical layers in postnatal animals, albeit absent in the blood vessels and meninges. In nestin-Cre2
expression was detected in the ventricular zone (VZ) of telencephalon and spinal cord of the developing CNS and also in the dorsal root ganglia when examined at E11.5 (16
Homozygous mutant pups (Shp2F/F
, abbreviated as Shp2F/F
) were born at Mendelian frequency for both mouse lines as genotyped at postnatal day 0 (P0) (n
= 31 out of 124 pups for Shp2F/F
= 28 out of 105 pups for Shp2F/F
, from the crossing of Shp2F/+
::Cre/+ X Shp2F/F
mice), suggesting no embryonic lethality. Whole-brain lysates of homozygous, heterozygous, and wild-type control mice were analyzed by immunoblotting for Shp2 protein expression. As illustrated in Fig. , Shp2 protein contents were reduced by approximately 90% in brain lysates of both Shp2F/F
mice at P0 and P4, respectively. The residual 10% of Shp2 protein is likely due to its expression in blood vessels and meninges of the brain, where Cre-mediated DNA recombination did not occur in these two mouse lines, as reported previously (16
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 (more ...)
Notably, both of the mutant mouse lines exhibited a very similar early postnatal lethality phenotype, with the Shp2F/F::Cre1 mice slightly more severely affected than the Shp2F/F::Cre2 animals (Fig. ). Most Shp2F/F::Cre1 animals died at P0 to P4, while a few Shp2F/F::Cre2 mice lived a little longer, up to P10. All the surviving Shp2F/F::Cre2 mice exhibited a decrease in body weight starting at P4 (Fig. ). The growth retardation and postnatal lethality phenotype also appeared gender related, with male mice more severely affected than the females by the Shp2 mutation. The brain size of mutant mice was reduced when examined at P4 (Fig. ). In addition to loss of body weight, we observed development of abnormal behaviors in surviving pups after P4, particularly ataxia and the inability to suck milk. Multiple factors are likely to contribute to the early postnatal lethality phenotype.
Shp2 has a positive role in corticogenesis.
We did not observe a dramatic structural abnormality in the mutant brain. More detailed hematoxylin-and-eosin staining, however, showed that lamination of the cortex was compromised in Shp2-deficient brain (Fig. ). We therefore examined cortical development and neuronal cell differentiation/migration using a variety of molecular markers. First, we investigated the expression of MAP2, a marker for dendrite-extended differentiated neurons. High levels of MAP2 expression were detected in the marginal zone and layer V in the control sections at P0, but the signals were weakened in the mutant (Fig. ). This result suggests a possibly delayed and impaired neuronal differentiation.
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 (more ...)
Next, we checked EphA4 mRNA expression, a marker for layers II and III (Fig. ). As compared to controls at P0, almost no signal was detected in the mutant neocortex, which also implies a defective neuronal differentiation because EphA4 is expressed in postsynaptic regions. Another molecular marker, calbindin, is mainly expressed in layers III and IV. The staining was disorganized in the mutants, showing diffused layers III and IV (Fig. ). We also examined expression of reelin, a marker of Cajal-Retzius cells in layer I, and found no significant difference in localization and expression levels between wild-type controls and mutants (see Fig. S2A in the supplemental material). No difference in Dab1 (E15.5) and p35 (P0) expression was recognized between controls and mutants (see Fig. S2B and C in the supplemental material). We also treated cortical neurons isolated at E16.5 with Reelin in vitro and checked phosphorylation of Dab1 at Tyr185 and Tyr220. No significant difference was detected between controls and mutants (see Fig. S2D in the supplemental material). Together, these results suggest that ablationof Shp2 had influenced neuronal differentiation but not Reelin-induced cell migration during cortical development.
To further investigate the impact of Shp2 deletion on corticogenesis, we examined expression of several cell-type-specific markers in cerebral cortex. Compared to controls, we detected reduced expression levels of neuronal marker (TuJ1) (Fig. ) and oligodendrocyte precursor marker (NG2) in the mutant brain (Fig. ). In contrast, signals for astrocyte marker protein GFAP were enhanced at E16.5 (Fig. ). These observations suggest a defective neurogenesis and oligodendrogenesis in cerebral cortex of the Shp2-deficient brain, while at the expense of neurogenesis, astrogliogenesis appeared slightly enhanced.
Finally, we examined levels of pH3, a marker for metaphase, and Ki67, a proliferative maker, in the VZ of embryonic brain. As shown in Fig. , both pH3 and Ki67 signals were significantly decreased in the mutants in comparison to the control, suggesting impaired proliferation of Shp2-deficient NSCs in the mutant VZ. Consistent to this observation, we detected high levels of Shp2 expression in the ventricular zone at E13.5 and subventricular zone at P4 (Fig. ) in wild-type brain. We also examined the impact of Shp2 deletion on neuronal survival. The terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay detected increasing levels of cell apoptosis in P4 and P10 mutant cerebral cortex (Fig. ). Together, these results suggest that Shp2 promotes progenitor proliferation in corticogenesis.
Shp2 oppositely regulates neurogenesis and astrogliogenesis in vitro.
The high-level Shp2 expression in the VZ suggests important roles of Shp2 in neural progenitor cells. To further investigate Shp2 function in NSC differentiation and multipotency, we isolated NSCs from cerebral cortex at E14.5 and cultured them in the presence of bFGF and EGF in vitro. NSCs were genotyped to identify Shp2 mutant (Shp2flox/flox::Cre/+) and control (Shp2flox/flox) cells by PCR analysis of genomic DNA (Fig. ). Immunoblotting analysis indicated efficient Cre-mediated deletion of Shp2 in cultured NSCs in vitro (Fig. ).
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 (more ...)
Morphological examination indicated dramatically decreased neurite outgrowth in differentiating neurosphere cultures (Fig. ). To precisely evaluate the differentiation capacity, we dissociated the differentiated neurosphere cells and counted numbers of each cell type generated following immunostaining with markers for neurons (TuJ1), astrocytes (GFAP), and oligodendrocytes (O4 or CNPase) at different time points. Analysis of the proportions of TuJ1+, GFAP+, O4+, or CNPase+ cells at days in vitro (DIV) 5, 7, and 10 indicated that Shp2 deficiency severely suppressed neuron and oligodendrocyte differentiation (Fig. ), consistent with the in vivo results as described above. In contrast, we observed a modest increase in the proportion of astrocytes generated from Shp2-deficient NSCs compared to controls (Fig. ). However, the absolute numbers of GFAP+ cells were similar between control and Shp2 mutant cultures (Fig. ), apparently due to the reduced proliferative capacity of Shp2-deficent progenitor cells under the culture condition.
We further examined neurogenesis and gliogenesis from NSCs following treatment with growth factors or cytokines in vitro. As shown in Fig. , there was a significant decrease in the number of neurons from Shp2-deficient NSCs following stimulation with platelet-derived growth factor (PDGF)-BB, leukemia inhibitory factor (LIF), or bFGF. In response to CNTF, Shp2 deficiency resulted in modestly decreased numbers of neurons (P > 0.05; n = 12). However, Shp2 ablation did not suppress gliogenesis in response to CNTF, LIF, or bFGF, and there was a significant increase in the number of GFAP+ cells following PDGF-BB treatment (P < 0.05; n = 12). Collectively, our observations reveal a critical role for Shp2 in neurogenesis and oligodendrogenesis but not in astrogliogenesis. This indicates a cell-type-specific requirement for Shp2 in neural development. The opposite effect of Shp2 deletion on neurogenesis and astrogliogenesis also suggests Shp2 actions in cell fate specification from multipotential progenitor cells.
Shp2 is required for proliferation of NSCs and neuronal progenitors in vitro.
We further investigated a putative role of Shp2 in NSC's self-renewal and proliferation potential in vitro. As depicted in Fig. , differences in the number and diameter of neurospheres derived from control and Shp2−/− mutant NSCs were easily observed under a microscope. To further determine the impact of Shp2 deletion on self-renewal of NSCs, we assayed secondary and tertiary neurosphere formation efficiency. As shown in Fig. , Shp2-deficient NSCs exhibited a markedly decreased capacity to generate neurospheres following serial subcloning, suggesting impaired proliferation and/or self-renewal. Indeed, the self-renewal capacity, defined as the number of secondary neurospheres formed per primary neurosphere, was significantly reduced in Shp2−/− NSCs compared to controls. Thus, Shp2 is required for self-renewal of NSCs in vitro (Fig. ).
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 (more ...)
To determine the proliferative capacity, we measured growth curves of NSCs under culture conditions that favor either self-renewal or differentiation. When compared to controls, Shp2−/− NSCs exhibited a significantly decreased cell proliferation rate under culture conditions favoring self-renewal expansion (Fig. ). Similar proliferative defects of Shp2−/− NSCs were also observed when the stem and progenitor cells were cultured under conditions allowing differentiation into neuronal and astroglial cell lineages (Fig. ). To corroborate these results, we measured BrdU incorporation into cultured neurosphere cells under both self-renewal and differentiation conditions. Consistently, results presented in Fig. show that Shp2 deficiency resulted in significantly reduced proliferation in NSCs and neuronal progenitors (TuJ1+) but not in astroglial cells (GFAP+). Modestly increased levels of cell apoptosis were also detected for Shp2-deficient NSCs and neuronal progenitors compared to wild-type control cells (Fig. ). Taken together, these results indicate a primary effect of Shp2 deletion on cell proliferation in self-renewing NSCs and neuronal progenitors.
Shp2 promotes growth factor signals in NSCs.
Our previous experiments indicate that despite a decreased proliferation rate, Shp2-deficient mouse ESCs exhibit enhanced self-renewal capacity, as reflected by the increased frequency of secondary embryonic body formation (4
). This is in contrast to the observation of impaired self-renewal of Shp2-deficient NSCs as described above. To analyze the molecular basis for distinct functions of Shp2 in ESCs and NSCs, we conducted a comparative analysis of intracellular signaling between these two stem cell types in maintenance cultures (Fig. ). We observed remarkably reduced levels of phospho-Erk1/2 in Shp2-deficient NSCs compared to wild-type controls, but no significant difference was detected between wild-type and Shp2 mutant ESCs. Shp2 deficiency resulted in increased phospho-Stat3 levels in both ESCs and NSCs and significantly decreased phospho-Akt in ESCs but not in NSCs. We also found that the phospho-C/EBP signal was significantly decreased in Shp2-deficient NSCs but not in ESCs. Together, these data suggest cell type-specific regulation of signaling pathways by Shp2, which could account for distinct phenotypes seen between Shp2-deficient ESCs and NSCs.
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 (more ...)
We then explored the possible involvement of Shp2 in relay of signals triggered by specific growth factors in NSCs. As previously reported, there are at least two cell populations in neurospheres—bFGF responsive and EGF dependent NSCs—and both growth factors synergize to promote proliferation (5
). Thus, we examined the impact of Shp2 deletion on NSC responses to bFGF and/or EGF. Shp2 deficiency had a modest inhibitory effect on the number and size of neurospheres generated in the presence of EGF alone (Fig. ). However, a significantly decreased number and size of neurospheres were detected in Shp2-deficient NSCs, as compared to controls, in response to bFGF alone or bFGF plus EGF (Fig. ). This result indicates a primary role of Shp2 in bFGF-dependent NSC proliferation and self-renewal. bFGF has been shown to maintain NSCs by promoting proliferation and suppressing differentiation in vitro and in vivo (26
). Stimulation of NSCs by bFGF leads to rapid activation of Erk, whose suppression blocks proliferation of neural progenitors (19
). We observed that phospho-Erk signals were much lower in Shp2-deficient NSCs than in controls in response to bFGF (Fig. ), suggesting inhibition of bFGF-stimulated Erk activation by Shp2 ablation in NSCs.
To further analyze alterations of bFGF signaling in Shp2-deficient NSCs, we assessed phosphorylation and concomitant activation of c-Myc and Stat3 in response to bFGF. Strikingly, we found opposite activities of Shp2 in modulating c-Myc and Stat3 signaling: Shp2-deficient NSCs exhibited decreased phosphorylation of c-Myc (Thr58/Ser62) but increased phosphorylation of Stat3 (Tyr705) in response to bFGF stimulation (Fig. ). Enhanced Stat3 signaling appeared to be specific to bFGF stimulation, as LIF treatment induced similar levels of phospho-Stat3 in control and Shp2-deficient NSCs (Fig. ). Augmented bFGF/Stat3 signaling may explain the impaired neurogenesis and moderately enhanced astrogliogenesis in Shp2-deficient NSCs, consistent with the literature (2
We also took a chemical biology approach to evaluate the effects of Erk, c-Myc, and Stat3 activities in NSCs’ self-renewal and neuronal/astroglial differentiation. NSCs were treated with a pharmacological MEK inhibitor (PD98059) and c-Myc inhibitor (10058-F4) and were evaluated for self-renewal in neurosphere assay. As shown in Fig. , treatment with Erk or c-Myc inhibitors resulted in a significantly reduced number of secondary neurospheres, indicating decreased self-renewal capacity. We also treated NSCs with PD98059, 10058-F4, and Jak2/Stat3 inhibitor (AG490) and subjected the cells to neuronal/astroglial differentiation. As depicted in Fig. , the MEK inhibitor suppressed neuronal differentiation, whereas c-Myc inhibition had no effect. The Stat3 inhibitor significantly attenuated astroglial differentiation (Fig. ). In addition, an Shp2 inhibitor recently identified in this laboratory (Wu et al., unpublished data) significantly suppressed self-renewal and neuronal differentiation of NSCs (Fig. ), consistent with the Shp2 gene knockout data.
The reduced phospho-c-Myc signal also prompted us to examine the expression level of Bmi-1, a transcription factor required for proliferation and self-renewal of NSCs (22
), as a recent report suggests that c-Myc directly regulates Bmi-1
). Immunoblot analysis showed that consistent with the decreased c-Myc activity, Bmi-1 levels were significantly decreased in Shp2-deficient NSCs compared to controls (Fig. ). To define the significance of decreased Bmi-1 expression, we performed a rescue experiment by infecting Shp2-deficient NSCs with a retroviral vector expressing Bmi-1 (Fig. ). As indicated in Fig. , ectopic expression of Bmi-1 in Shp2-deficient NSCs resulted in increased numbers and size of secondary neurospheres. This result suggests that Shp2 mediates growth factor signals, at least in part via control of Bmi-1 expression, in regulation of NSC proliferation and self-renewal.
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 (more ...)