neuronal differentiation

Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling
 



Ann M. Turnley1, 3, Clare H. Faux1, Rodney L. Rietze1, 2, Jason R. Coonan1 & Perry F. Bartlett1, 2
 

1. The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia

2. Institute for Brain Research, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia

3. Centre for Neuroscience, The University of Melbourne, Victoria 3010, Australia

Correspondence should be addressed to P F Bartlett. e-mail: bartlett@wehi.edu.au







The intracellular mechanisms that determine the response of neural progenitor cells to growth factors and regulate their differentiation into either neurons or astrocytes remain unclear. We found that expression of SOCS2, an intracellular regulator of cytokine signaling, was restricted to mouse progenitor cells and neurons in response to leukemia inhibitory factor (LIF)-like cytokines. Progenitors lacking SOCS2 produced fewer neurons and more astrocytes in vitro, and Socs2-/- mice had fewer neurons and neurogenin-1 (Ngn1)-expressing cells in the developing cortex, whereas overexpression of SOCS2 increased neuronal differentiation. We also report that growth hormone inhibited Ngn1 expression and neuronal production, and this action was blocked by SOCS2 overexpression. These findings indicate that SOCS2 promotes neuronal differentiation by blocking growth hormone–mediated downregulation of Ngn1.


 Differentiation of neural stem cells into neurons or astrocytes in the developing forebrain depends on the interaction of intrinsic and environmental cues. Many intrinsic factors that regulate neurogenesis have been identified, including neurogenic basic helix-loop-helix (bHLH) genes such as NeuroD and Neurogenin1-5. Environmental cues include members of the fibroblast growth factor (FGF)6 and bone morphogenic protein (BMP)7 families, platelet-derived growth factor (PDGF)8, 9 and cytokines that signal through the LIF receptor complex (such as LIF and the cytokine ciliary neurotrophic factor, CNTF)10, 11. Interaction between intrinsic and environmental cues can regulate the expression of neurogenic genes and hence regulate neural stem cell fate12-14. This raises the question of how seemingly similar stem cells that are exposed to the same growth factor can result in different cell fates.



Several of the above growth factors can inhibit neurogenesis under some conditions and promote neurogenesis under others. LIF and related cytokines primarily promote differentiation of neural progenitors into astrocytes and inhibit neurogenesis11, 15, 16, but under some circumstances, LIF also promotes neuronal differentiation10. This suggests that neural stem cells have the capacity to overcome signals that normally inhibit neuronal differentiation, perhaps by differential expression of regulatory molecules. Key regulators of LIF signaling are SOCS molecules. The founding member of the SOCS family, SOCS1, was described by its ability to inhibit signaling by cytokines such as IL6 or LIF17-19. There are at least eight members of the SOCS family (SOCS1–7 and the cytokine-inducible SH2 protein, CIS)20 that have been shown to inhibit Janus kinases (JAK) and signal transducer and activator of transcription proteins (STAT) signaling downstream of a wide variety of cytokines and growth factors. SOCS proteins bind to and block JAK or STAT binding sites on cytokine receptors via SH2 domains; they target signaling proteins to the proteasome degradation pathway via their ’SOCS box’ motif21. SOCS molecules are differentially expressed in a variety of tissues and can be upregulated by a range of cytokines and growth factors. Previously we have shown that SOCS1, SOCS2 and SOCS3 are expressed throughout neural development, with SOCS2 being the most highly expressed22.



Here we show that SOCS family members are differentially expressed in neural progenitor cells, neurons and astrocytes. SOCS2 is expressed only in progenitor cells and neurons, and it directly regulates differentiation of progenitor cells into neurons, both in vitro and in vivo. Furthermore, whereas LIF regulates SOCS2 expression in neural progenitor cells, SOCS2 does not appear to regulate LIF signaling. Instead, we found that growth hormone (GH) inhibited neuronal differentiation of neural progenitor cells and that SOCS2 blocked this inhibition. Finally, we propose that GH inhibits neurogenesis by regulating Ngn1 expression.
 


 
 

Results

SOCS2 is differentially expressed in neural cells Previously we have shown by in situ hybridization and northern blot analysis that SOCS1, SOCS2 and SOCS3 are expressed in the developing nervous system22. For a more detailed analysis of which cell types expressed SOCS2, we prepared cultures of embryonic day 10 (E10) neuroepithelial cells, cortical astrocytes and cortical neurons for northern blot analysis. SOCS2 expression was found in neuroepithelial cells and neurons, but not in astrocytes (Fig. 1a and b). In contrast, SOCS1 and SOCS3 were expressed in neuroepithelial cells and astroglial cultures, but not in neuronal cultures (Fig. 1a and b).



LIF receptor signaling upregulates SOCS2 E10 neuroepithelial cells were incubated with growth factors and cytokines for 24 hours before northern analysis of Socs gene expression. IFN increased SOCS1, SOCS2 and SOCS3 levels, and LIF, CNTF and oncostatin M (OSM) increased the levels of SOCS2 and SOCS3 but not SOCS1 (Fig. 1a). FGF1, FGF2, BMP2, BMP4, BMP6, NT3, BDNF, NGF, EGF, IGF1, GH and somatostatin did not significantly alter Socs gene expression (Figs. 1a and 5g and data not shown).



In neuronal cultures, SOCS2 expression was upregulated by LIF but not by IFN, whereas SOCS1 and SOCS3 remained undetectable (Fig. 1b). In astrocyte cultures, SOCS1 and SOCS3 were upregulated by IFN but not LIF, and SOCS2 expression remained undetectable (Fig. 1b). Thus, in differentiated neural cells, SOCS2 expression seems to be restricted to the neuronal lineage, even after stimulation with cytokines.



SOCS2 is not expressed in multipotent stem cells Although neuroepithelial cultures expressed high levels of SOCS2, it was not clear whether expression was restricted to multipotent neural stem cells, neural progenitors or neurons, as all were present in this culture system. Therefore, we isolated multipotent neural stem cells from adult mouse brain (Methods and ref. 23) and compared SOCS2 expression in these cells with that in mixed stem/progenitor cell cultures that were derived from these sorted cells and maintained as neurospheres24. Using RT-PCR, we found SOCS2 expression in these primary, unpassaged neurospheres and in neurospheres derived from E13 embryos and newborn mice, but not in freshly isolated neural stem cells that showed low levels of peanut agglutinin binding and heat-stable antigen expression (PNAlo HSAlo) (Fig. 1c). Immunostaining of undifferentiated neurospheres showed that almost 100% of the cells expressed the undifferentiated neural cell marker nestin, and that almost none of the cells (<0.01%) expressed neuronal (III-tubulin) or glial (GFAP) markers (ref. 24 and data not shown). Thus, SOCS2 seems to be expressed in proliferative, undifferentiated progenitor cells downstream of the multipotent neural stem cell.
   
 Does SOCS2 regulate neuronal differentiation? The observation that SOCS2 expression was restricted to neural progenitor cells and neurons suggested that it may be involved in neuronal differentiation. Therefore, we examined the effect of overexpression and deletion of SOCS2 on neuronal differentiation in vitro and in vivo.



To directly determine the role of SOCS2 on differentiation of progenitor cells, we examined the ability of neural stem cells propagated as neurospheres24 from mutant (Socs2-/-)25 and wild-type (Socs2+/+) mice to generate neurons, oligodendrocytes and astrocytes. There was no discernible difference in the morphology, growth characteristics or cell death between the mutant and wild-type neurospheres (data not shown).



Neurospheres were differentiated for three days, and the cultures were then fixed and immunostained for neuronal (III-tubulin), astrocyte (GFAP) or oligodendrocyte (O4) markers. All cultures were also stained with the nuclear dye DAPI to determine total cell number and allow the percentage of each cell type to be determined. The percentage of neurons generated by the Socs2-/- neurospheres was markedly reduced (Fig. 2a, b and g, j), whereas the percentage of astrocytes was increased (Fig. 2e, f and h, j) compared to wild-type neurospheres. There was no difference in the percentage of oligodendrocytes generated (Fig. 2c, d and i, j). Similar results were obtained for adult neurospheres (data not shown).



This effect was also seen in primary E10 neuroepithelial cells cultured for 48 hours under conditions that favored proliferation12. Again, the percentage of neurons in the Socs2-/- cultures was reduced by 36% compared with wild-type cultures (16.1  0.8% versus 25.1  1.2%; P < 0.001). Thus, both freshly isolated and cultured progenitors from the forebrain of Socs2-/- mice showed a decreased propensity to generate neurons.

 To determine whether the decrease in neurons generated in vitro by Socs2-/- cells was also reflected in vivo, we counted the number of cortical neurons in Socs2-/- and Socs2+/+ mice. As reported, the brains of adult Socs2-/- mice looked normal overall and were not significantly different in size from the brains of wild-type mice (0.48  0.01 g versus 0.50  0.01 g, respectively; see also ref. 25). Most other organs, as well as overall body weight, were significantly larger in the Socs2-/- mice25. There was no significant difference in cortical thickness (somatosensory cortex) between Socs2-/- (1.10  0.01 mm) and wild-type mice (1.12  0.01 mm). To compare neuronal and total cell numbers in Socs2-/- and Socs2+/+ brains, non-adjacent, serial frozen sections (30 m) were immunostained for the neuronal marker NeuN or the nuclear stain DAPI. Neuronal and total cell numbers were counted, using stereological methods, in random fields in each layer of the somatosensory cortex (layers 2/3, 4, 5 and 6). Neuronal density was decreased by approximately 30% in each layer of cortex (P < 0.0001) in the Socs2-/- mice as compared to wildtype (Fig. 3a, b and e), whereas there was no significant difference in the total cell density (Fig. 3c, d and e). Thus, the decrease in neuronal number in the cortex of Socs2-/- mice may have been compensated by an increase in the glial cell population; a similar finding to that in the in vitro experiments described above.



Overexpression of SOCS2 increases neuron numbers If SOCS2 is important in regulating neuronal differentiation of neural progenitor cells, then overexpression of SOCS2 should increase neuronal differentiation and reverse the observed decrease in neuronal differentiation of Socs2-/- cells. Neural stem cells from wild-type or Socs2-/- mice were transiently transfected with a fusion construct (pCMV–SOCS2–GFP) or a GFP control (pEGFP–C) and then differentiated, immunostained and counted as above. Overexpression of SOCS2 in both wild-type and Socs2-/- neural stem cells resulted in a 2 to 3-fold increase in the percentage of neurons compared with GFP-expressing controls (Fig. 4). In addition, the percentage of neurons in SOCS2-overexpressing Socs2-/- neurospheres was significantly greater (P = 0.01) than that in GFP-expressing wild-type neurospheres (Fig. 4). Thus, SOCS2 seems to directly regulate the ability of a neural progenitor cell to differentiate into a neuron.
 
 GH inhibits neuronal differentiation As members of the SOCS family have been shown to act as inhibitors of growth factor signaling, we considered that the observed regulation of neuronal differentiation could result from SOCS2 inhibition of a growth factor that normally inhibits neurogenesis and/or promotes gliogenesis. Two candidate factors were LIF, which we found upregulates SOCS2 expression in neural progenitor cells, and GH, whose dysregulation has been implicated as a cause of the gigantism observed in Socs2-/- mice25. We therefore examined the effects of LIF and GH on in vitro differentiation of Socs2-/- and wild-type neural stem cells.



As previously reported11, 15, 16, LIF promoted astrocyte production. After 3 days of differentiation, the basal percentage of wildtype astrocytes was 7.08  0.51%, and that for Socs2-/- astrocytes was 11.38  1.24%. In the presence of LIF, however, the percentages were 25.08  2.27% and 41.37  3.88%, respectively, indicating a 3.5-fold increase in astrocytes of both genotypes (P < 0.001). LIF also inhibited neuron differentiation (Fig. 5a). These effects were seen in both Socs2-/- and wild-type neurospheres, and there were no differential effects of LIF between the two genotypes, indicating that SOCS2 does not regulate LIF signaling in neural progenitor cells.



The application of GH inhibited wild-type neuronal differentiation (Fig. 5a) and enhanced astrocyte production (59.7  15.5% increase, P < 0.05, n = 3 combined experiments). Wild-type neurospheres treated with GH were of similar appearance to the Socs2-/- neurospheres shown in Fig. 2. A similar decrease in neuronal differentiation was also seen when GH was added to freshly isolated E12.5 neuroepithelial cells for 3 days (61.78  1.83% neurons under basal conditions; 38.88  2.06% neurons with GH, P < 0.001). However, the effect of GH was observed only in wildtype cells. GH did not further decrease neuronal differentiation in Socs2-/- neurospheres compared to basal conditions (Fig. 5a) and did not significantly enhance Socs2-/- astrocyte production (6.4  3.7% increase). GH had no effect on cell number; the mean number of cells per neurosphere was the same for both genotypes (data not shown). Therefore, GH showed differential effects on neuronal and glial differentiation in Socs2-/- and wildtype neurospheres, indicating that GH signaling was regulated by SOCS2 in these cells.
 


 
  The effect of GH on wild-type but not Socs2-/- neurospheres could be explained by hypersensitivity of Socs2-/- cells to endogenously produced GH, leading to Socs2-/- cells being maximally inhibited even in the absence of additional GH. To examine whether GH was produced endogenously, we added somatostatin, which inhibits GH release from cultured brain cells26, 27, to the cultures. Somatostatin significantly increased neuronal differentiation of wild-type and Socs2-/- cells (Fig. 5b), but the effect on the mutant cells was more pronounced, such that the difference between the two genotypes was not significantly different (P = 0.5) after somatostatin treatment. Similar results were obtained with anti-GH antiserum; the percentage of neurons in wild-type and Socs2-/- neurospheres was increased (Fig. 5b). Co-addition of somatostatin and anti-GH did not produce a further increase in neuronal differentiation, indicating that somatostatin and anti-GH were acting through the same mechanism of decreasing availability of endogenous levels of GH in the culture. A GH dose–response assay showed that Socs2-/- cells were approximately 100 times as sensitive to GH as wild-type cells (Fig. 5c).



To show that SOCS2 was directly responsible for regulating neuronal differentiation in response to GH, wild-type neurospheres were transfected with GFP or SOCS2–GFP and differentiated in the presence of GH. Neurospheres transfected with SOCS2–GFP produced significantly more neurons than did GFP-transfected cells (Fig. 5d), indicating that SOCS2 is a potent inhibitor of GH signaling.



Previous studies have shown extensive expression of the GH receptor (GHR) and its splice variant, the GH binding protein (GHBP)28 in many neuronal and glial cells throughout the brain, postnatally29. Therefore, we examined GHR and GHBP expression in neuroepithelium and embryonic brain, as well as in primary stem cells and cultured neurospheres. Northern analysis showed that GHBP RNA was expressed at E10 and E12 in neuroepithelium, with continued expression in the developing brain (Fig. 5e). Immunostaining of E14 cortex showed that GH was expressed in cells of the ventricular zone (VZ), whereas GHR was expressed predominantly in cells migrating away from the VZ (Fig. 5h). Therefore, both GHR and GHBP are expressed in the developing nervous system. In addition, after fluorescence-activated cell sorting (FACS), RT-PCR for GHR was done on primary adult neural stem cells, their progeny and passaged embryonic and newborn neurospheres. This showed that although the GHR was not expressed in the multipotent stem cells, it was expressed in their progeny (Fig. 5f). GHBP RNA was expressed at similar levels in both Socs2-/- and wild-type neurospheres and was not modulated by addition of growth factors such as insulin, GH or LIF (Fig. 5g), indicating that the effect of SOCS2 on GH signaling was not due to regulation of GH receptor levels in the progenitor population.
 
 GH inhibits neurogenin-1 expression The role of SOCS2 in neural progenitor cells appears to be to block the GH-induced inhibition of neural progenitor cell differentiation into neurons. The predominant signal transduction pathway used by GH is the JAK/STAT pathway, and STAT5 is the major STAT protein activated by GH signaling. SOCS2 inhibits STAT5 activity downstream of GH signaling30, 31, but how this translates to regulation of neuronal differentiation is unknown. We proposed that GH inhibits neuronal differentiation by regulating the expression of a neurogenic bHLH transcription factor (such as neurogenin)5 that interacts with the JAK/STAT pathway. We analyzed Ngn1 expression by western blot analysis in lysates from wild-type and Socs2-/- neural stem cells incubated with GH for 1–4 hours. Expression of Ngn1 was decreased by 50–60% in both wild-type and Socs2-/- cells after the addition of GH (Fig. 6a). Consistent with the hypersensitivity of Socs2-/- cells to GH, Ngn1 levels were significantly lower in mutant cells compared to wild-type cells without the addition of GH (Fig. 6a). Similar findings were obtained when expression of Ngn1 in wild-type and Socs2-/- neurospheres was examined by immunocytochemistry (data not shown). To show that SOCS2 was directly responsible for inhibiting the GH-induced downregulation of Ngn1 expression, wild-type neurospheres were transfected with GFP or SOCS2–GFP as described above and incubated with GH for 4 hours. Ngn1 expression was localized to the nucleus of these cells under basal conditions. After incubation with GH, expression of Ngn1 was downregulated in control but not in SOCS2-transfected cells (Fig. 6b). Ngn2 expression was not detected in neurospheres (data not shown).



Ngn1 expression was also examined by immunohistochemistry in E14 cortex of wild-type and Socs2-/- mutant mice. The number of detectable Ngn1-positive cells in the VZ of Socs2-/- mice was reduced to approximately 50% of wildtype (Fig. 6c and d). In more anterior regions of the cortex, the number of Ngn1-positive cells in the cortical plate was also dramatically lower (Fig. 6c). This may be have been due to a decreased number of Ngn1-positive cells and/or a decreased level of Ngn1 expression below the detection limit of our immunostaining.



Thus, regulation of Ngn1 levels by GH, which is in turn regulated by SOCS2, provides a mechanism for the decreased neuronal production both in Socs2-/- cells and in wild-type neural progenitor cells in the presence of GH.

Discussion

Here we have shown that SOCS2 expression is restricted to progenitor cells and neurons and is intimately involved in regulating neuronal differentiation. Overexpression of SOCS2 in neurospheres resulted in significant induction of neuronal differentiation, whereas deletion of SOCS2 resulted in the generation of fewer neurons and more glia in vitro and in vivo. This decreased neuron:glia ratio is similar to that observed in vitro for neural progenitors that differentiated in the presence of LIF or CNTF11, 15.



What factor does SOCS2 regulate? As the level of SOCS2 expression appears to regulate neural progenitor cell differentiation, two key questions are: What regulates SOCS2 expression? and What does SOCS2 regulate? Growth factors that signal through the LIF receptor complex are likely candidates for both regulating and being regulated by SOCS2. In addition to increasing neuronal differentiation10 and promoting astrocyte differentiation11, 15, 16, we showed that they upregulate SOCS2 expression. SOCS2 has been shown to regulate, albeit weakly, signaling by LIF in some cells32. Adding LIF to neurosphere cultures increased the number of astrocytes while inhibiting neuron differentiation; unlike GH, however, this effect was the same in both Socs2-/- and wild-type mice. Thus, SOCS2 does not appear to be a major regulator of LIF signaling in neural progenitor cells. This finding is in accordance with Socs2-/- mice whose phenotype of gigantism is probably due to dysregulation of GH and/or IGF1 signaling25.



GH may be a major regulator of neuronal differentiation. Although a role for GH in neural progenitor differentiation has not previously been described, GH is expressed in whole fetal rat brain during neural development from E10, with a peak of expression before birth33. Moreover, GH production has been shown in cultured brain cells27, and somatostatin is produced by embryonic cortical neurons34. Together with our present finding that GHR/GHBP is expressed as early as E10 in the mouse brain, these results indicate that GH, modulated by SOCS2, is involved in negatively regulating neuronal differentiation of neural progenitor cells.
 
 
 How does SOCS2 regulate neuronal differentiation? The main role of SOCS2 appears to be regulation of GH signaling. Socs2-/- mice have an enlarged growth phenotype, consistent with hyper-responsiveness to GH signaling25. The overgrowth phenotype of the same line of Socs2-/- mice used in this study requires STAT5b for expression of the phenotype31. Activation of both STAT5a and STAT5b is prolonged in cells from these mice in response to GH, resulting from their inability to effectively downregulate the activation31. Inhibition of STAT5 activation by SOCS2 has also been found in overexpression studies30, 35 and appears to involve SOCS2 competitively binding to the STAT5 binding sites on the GH receptor30, 36.



How does the SOCS2-mediated regulation of STAT5 activation result in the regulation of neurogenesis? One possibility is that GH inhibits expression of genes for neurogenic proteins such as neurogenin, similar to FGF regulation of Notch and Delta to inhibit neuronal differentiation12. Expression of SOCS2 would therefore be required to overcome GH inhibition of neurogenic gene expression. The level of expression of neurogenic genes is important in determining neuronal versus glial cell fate. Neural progenitor cells from Ngn2/Mash1 double knockouts show decreased neuronal and increased astrocyte differentiation of cortical progenitor cells4, whereas overexpression of Ngn1 promotes neurogenesis and inhibits gliogenesis by competing with the JAK/STAT pathway for the transcriptional co-activators CBP and p300, as well as by directly inhibiting STAT activation5. Given that SOCS2 appears to act by regulating STAT5 signaling, it may as a consequence regulate the availability of the JAK/STAT pathway to compete with Ngn1 for CBP and p300. Not only were levels of Ngn1 lower in Socs2-/- cells compared to wild-type in vitro and in vivo, but also addition of GH acutely decreased Ngn1 levels in wild-type cells in vitro by 50–60%, to a level comparable to that in Socs2-/- cells. This provides a potential mechanism for the decreased neurogenesis and increased gliogenesis seen in Socs2-/- cells. Unlike Socs2-/- mice, however, Ngn1 null mice do not have a robust cortical phenotype. This is presumably because of compensation by other bHLH genes such as Ngn2 and Mash1; double knockouts are required before a mutant phenotype is observed4, 37. It is probable that SOCS2 does not affect Ngn1 alone but also affects other bHLH genes, although this remains to be determined.



Our data suggests that SOCS2, through its regulation of GH signaling, regulates levels of Ngn1 expression and thus the ability of a neural stem cell to differentiate into a neuron or an astrocyte. Although we have shown that SOCS2 regulates the biological effects of GH signaling in neural progenitor cells, we also found that only LIF and related cytokines, not GH, regulate SOCS2 expression in these cells. We suggest, therefore, that LIF-like molecules inhibit a progenitor’s response to GH by upregulating SOCS2 expression while simultaneously promoting astrocyte differentiation by STAT3 activation16. SOCS2 would therefore be acting as an integrator of multiple signal transduction pathways and its level of expression would be important in determining final biological outcome of signaling by multiple stimuli.
 

Methods

Animals. C57BL/6 mice were obtained from stocks maintained at the Walter and Eliza Hall Institute (WEHI). Socs2-/- mice were a gift from W. Alexander (WEHI) and were maintained on the C57BL/6 background25. Mice were killed by CO2 asphyxiation. The use of animals was approved by the Royal Melbourne Hospital Animal Ethics Committee and performed in accordance with the principles of the NH and MRC of Australia.



Reagents. The following growth factors were used at the concentrations indicated: LIF (1,000 U/ml; AMRAD, Melbourne, Australia), IFN- (20 U/ml, Pharmingen, San Diego, California), recombinant rat GH (100 ng/ml, GroPep, Adelaide, Australia), BMP4 (50 ng/ml, Genetics Institute, Cambridge, Massachusetts), somatostatin (1 M, Auspep, Melbourne, Australia), EGF (20 ng/ml, BD Biosciences, Bedford, Massachusetts), FGF2 (10 ng/ml, Roche, Sydney, Australia). Rabbit anti-rat GH antiserum (AFP5641801) was obtained through NHPP, NIDDK through the National Hormone & Peptide Program, the National Institute of Diabetes & Digestive and Kidney Diseases and A.F. Parlow and used at a 1:500 dilution. Rabbit anti-GHR was a gift from C. Greenhalgh (WEHI).



Neuroepithelial, neuron and astrocyte cultures. Primary neural stem cells were derived from E10 C57BL/6 mouse neuroepithelial cells as previously described38 and grown in serum-free NS-M medium23 containing growth factors as indicated. Primary cortical neuron and astrocyte cultures were prepared from E17 mouse embryos essentially as previously described39.



Primary neural stem cell sorting and RT-PCR. Neural stem cells from the lateral ventral SVZ of adult mice were isolated by FACS as previously described23. RNA was isolated from these cells, as well as cultured neurospheres, and RT-PCR for SOCS2 and a -actin control was performed using standard procedures. The primer sequences for SOCS2 were: sense 5’-GGAATGGAGCGGACAGGACG-3’, antisense 5’-GTACTCAATCCGCAGGTTAGTC-3’. For GHR: sense 5’AAGTGCGGGTGAGATCCAGACAAC-3’, antisense 5’AGAGCCAAGGGAAGCATCATAAGG-3’. For -actin: sense 5’-CTGAAGTACCCCATTGAACATGGC-3’, antisense 5’CAGAGCAGTAATCTCCTTCTGCAT-3’.
 
 
 Neurosphere culture and differentiation. Neural stem cell lines were derived from the subventricular zone (SVZ) of newborn C57BL/6 wild-type and Socs2-/- mice and passaged as neurospheres as previously described24 in NS-M medium23 containing 20 ng/ml EGF. Cells were differentiated for 3 d with factors as described24.



Neurosphere transfection. Transfections were performed using the Effectene transfection reagent (Qiagen, Germany). The cDNA encoding a SOCS2–GFP fusion protein, a gift from D. Hilton (WEHI), was subcloned into the pCMV5 expression vector (pCMV5–SOCS2–GFP). Dissociated neurospheres were plated at a density of 105 cells per well into uncoated 24-well plates (Nunc, Denmark) and immediately transfected with a control plasmid, pEGFP-c (Clontech, Palo Alto, California) or pCMV5–SOCS2–GFP. The cells were grown in NS-M medium containing EGF and FGF2 for 4 d, by which time 87.5  3% of the resultant neurospheres were derived from transfected stem cells. The neurospheres were then differentiated as above for 3 d.



Analysis of neurosphere differentiation. Neurospheres were differentiated for 3 d and then fixed and immunostained as previously described23. The number of III-tubulin positive neurons, GFAP positive astrocytes or O4 positive oligodendrocytes and the total DAPI cell number per sphere were counted and the percentage of each cell type per sphere was determined. Approximately 30 spheres per condition in each of 3–8 individual experiments were analyzed. Results are expressed as mean  s.e.m. Statistical significance was determined using a two-tailed t-test.



Immunohistochemistry and cell counts of cortex. Adult (3–4 month old) male Socs2-/- and wild-type mice were intracardially perfused with PBS followed by 4% paraformaldehyde, and their brains were removed. Serial coronal cryostat sections (30 m) were cut through the cerebrum. Every sixth section was processed for immunohistochemistry using standard procedures. Sections were incubated with mouse anti-NeuN40 (1:300, Chemicon, Temecula, California) overnight at RT, followed by biotinylated horse anti-mouse IgG (1:500, Vector, Burlingame, California) for 2 h at RT, and then Vectastain A+B reagent (Vector) and DAB. For staining of all cells, the nuclear dye DAPI was added to the sections for 30 min.
 


 
 
 The neuronal and total cell density in layers II/III, IV, V and VI of the somatosensory cortex was determined using the optical dissector method41. Stained cells were counted in 30–60 random 43.2  43.2  30 m sampling boxes in each cortical layer in both hemispheres (n = 4 sections). Results are expressed as cells/mm3 (mean  s.e.m. and P value for n = 3 mice). For statistical analyses, ANOVA with a Scheffe post-hoc test was performed, using Statview 4.0 software (SAS Institute, Cary, North Carolina).



For examination of Ngn1 expression in E14 cortex, 10-m serial paraffin sections of Socs2-/- and wild-type embryos were immunostained for Ngn1 using Rabbit anti-Neurogenin1 (1:500, Chemicon) and secondary reagents as above. We counted all Ngn1-positive cells in a 365-m wide field of the cortical VZ, above the middle of the lateral ventricle, of 6–8 sections at least 100 m apart, from three embryos of each genotype (Socs2-/- and wildtype).



Northern analysis. Total RNA was then prepared from the frozen tissue or cells using the RNeasy kit (Qiagen), and northern analysis was performed as previously described22. Socs1, Socs2 and Socs3 cDNAs were provided by D. Hilton (WEHI). The GHR sequence was obtained from the NCBI Entrez nucleotide database, accession number 6857794, and the GHR probe was derived from a 656 bp RT-PCR product amplified from mouse kidney, using the sense primer 5’AAGTGCGGGTGAGATCCAGACAAC-3’ and the antisense primer 5’AGAGCCAAGGGAAGCATCATAAGG-3’. All filters were stripped and reprobed with a GAPDH probe to check RNA loading levels.



In situ hybridization was performed as previously described22.



Western analysis. Western analysis was performed on lysates of neurospheres using standard methods, run on NuPAGE 4–12% Bis-Tris gels (Invitrogen, Carlsbad, California) and transferred to PVDF membrane. Blots were incubated in rabbit anti-neurogenin-1 (1:500; Chemicon) overnight. After washing, blots were incubated in HRP-linked anti-rabbit IgG (1:10,000; Cell Signaling Technology, Beverly, Massachusetts) for 1 h and washed, and ECL was performed using Supersignal West Pico substrate (Pierce, Rockford, Illinois). Membranes were stripped and reprobed with rabbit anti-p42/p44 MAP kinase (1:1000; Cell Signaling Technology) to assess protein loading levels. Autoradiographs were scanned, and the density of the bands was determined using NIH Image software.



Received 12 August 2002; Accepted 2 September 2002; Published online 7 October 2002.
 

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