The Basic Helix-Loop-Helix Genes Hesr1/Hey1 and Hesr2/Hey2 Regulate Maintenance of Neural Precursor Cells in the Brain
2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês
10.1074/jbc.m300448200
ISSN1083-351X
AutoresMasami Sakamoto, Hiromi Hirata, Toshiyuki Ohtsuka, Yasumasa Bessho, Ryoichiro Kageyama,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoNeural precursor cells proliferate in the ventricular zone while giving rise to neurons of deep layers first, then those of the superficial layers, and lastly, glial cells in the brain. Thus, it is essential to maintain neural precursor cells until late stages of neural development for generation of a wide variety of cell types. Here, we found that the Hes-related basic helix-loop-helix (bHLH) genes Hesr1/Hey1 and Hesr2/Hey2 are expressed in the ventricular zone, which contains neural precursor cells. Misexpression of Hesr1 and Hesr2 by electroporation in mouse brain at embryonic day 13.5 transiently maintains neural precursor cells and thereby increases late-born neurons, which are located in the superficial layers. In contrast, misexpression of the genes at later stages inhibits neurogenesis and promotes generation of astroglial cells. In transient transfection assay with cultured cells, both Hesr1 and Hesr2 inhibit transcription induced by the neuronal bHLH genes Mash1 and Math3. These results indicate that Hesr1 and Hesr2 negatively regulate neuronal bHLH genes, promote maintenance of neural precursor cells, and increase late-born cell types in the developing brain. Neural precursor cells proliferate in the ventricular zone while giving rise to neurons of deep layers first, then those of the superficial layers, and lastly, glial cells in the brain. Thus, it is essential to maintain neural precursor cells until late stages of neural development for generation of a wide variety of cell types. Here, we found that the Hes-related basic helix-loop-helix (bHLH) genes Hesr1/Hey1 and Hesr2/Hey2 are expressed in the ventricular zone, which contains neural precursor cells. Misexpression of Hesr1 and Hesr2 by electroporation in mouse brain at embryonic day 13.5 transiently maintains neural precursor cells and thereby increases late-born neurons, which are located in the superficial layers. In contrast, misexpression of the genes at later stages inhibits neurogenesis and promotes generation of astroglial cells. In transient transfection assay with cultured cells, both Hesr1 and Hesr2 inhibit transcription induced by the neuronal bHLH genes Mash1 and Math3. These results indicate that Hesr1 and Hesr2 negatively regulate neuronal bHLH genes, promote maintenance of neural precursor cells, and increase late-born cell types in the developing brain. Neural development involves the following three sequential processes: expansion of neural precursor cells, neurogenesis, and gliogenesis (1Schuurmans C. Guillemot F. Curr. Opin. Neurobiol. 2002; 12: 26-34Crossref PubMed Scopus (289) Google Scholar, 2Panchision D.M. McKay R.D.G. Curr. Opin. Genet. Dev. 2002; 12: 478-487Crossref PubMed Scopus (172) Google Scholar). In mice, neural precursor cells proliferate without giving rise to neurons until mid-gestation. Then, neurogenesis starts and continues until around birth. Differentiating neurons migrate out of the ventricular zone into the outer regions, forming the cortical plate. During this process, newly born neurons migrate through the layers of older neurons to form more superficial layers, thereby making the inside-out pattern. Gliogenesis starts before birth, but the majority of glial cells such as astrocytes are generated after neurogenesis. It has been shown that these developmental processes are regulated by multiple basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; E (e.g. E13.5), embryonic day; P (e.g. P1), postnatal day; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein-2. genes (3Kageyama R. Nakanishi S. Curr. Opin. Genet. Dev. 1997; 7: 659-665Crossref PubMed Scopus (329) Google Scholar, 4Lee J.E. Curr. Opin. Neurobiol. 1997; 7: 13-20Crossref PubMed Scopus (440) Google Scholar, 5Bertrand N. Castro D.S. Guillemot F. Nat. Rev. Neurosci. 2002; 3: 517-530Crossref PubMed Scopus (1164) Google Scholar, 6Ross S.E. Greenberg M.E. Stiles C.D. Neuron. 2003; 39: 13-25Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). 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Guillemot F. Kageyama R. EMBO J. 2000; 19: 5460-5472Crossref PubMed Scopus (244) Google Scholar, 21Nieto M. Schuurmans C. Britz O. Guillemot F. Neuron. 2001; 29: 401-413Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Interestingly, these phenotypes of neuronal bHLH gene deficiency are similar to those of misexpression of Hes1 and Hes5, which also initially maintain neural precursor cells but later promote gliogenesis (13Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. J. Biol. Chem. 2001; 276: 30467-30474Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Because Hes1 and Hes5 can functionally antagonize neuronal bHLH genes (7Sasai Y. Kageyama R. Tagawa Y. Shigemoto R. Nakanishi S. Genes Dev. 1992; 6: 2620-2634Crossref PubMed Scopus (583) Google Scholar, 8Akazawa C. Sasai Y. Nakanishi S. Kageyama R. J. Biol. Chem. 1992; 267: 21879-21885Abstract Full Text PDF PubMed Google Scholar), it is likely that misexpression of Hes1 or Hes5 leads to the situations similar to neuronal bHLH gene deficiency. Thus, antagonistic regulation between multiple bHLH genes is important for timing the processes of precursor expansion, neurogenesis, and gliogenesis. Despite these extensive studies, mutational analyses of the bHLH genes explain only a portion of the neural development, suggesting that other uncharacterized bHLH genes also should be involved in such processes. For example, although the lack of Hes1 and Hes5 impairs maintenance of neural precursor cells, these cells still proliferate at a slower rate without losing the multipotentiality to differentiate into neurons and glial cells (13Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. J. Biol. Chem. 2001; 276: 30467-30474Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 28Ohtsuka T. Ishibashi M. Gradwohl G. Nakanishi S. Guillemot F. Kageyama R. EMBO J. 1999; 18: 2196-2207Crossref PubMed Google Scholar), which suggests that other genes should be involved in maintenance of this cell type. Recent studies demonstrated that Hes-related bHLH genes, termed Hesr genes (also referred to as Hey/HRT/CHF/HERP/gridlock), are expressed in the developing brain and retina (29Kokubo H. Lun Y. Johnson R.L. Biochem. Biophys. Res. Commun. 1999; 260: 459-465Crossref PubMed Scopus (126) Google Scholar, 30Leimeister C. Externbrink A. Klamt B. Gessler M. Mech. Dev. 1999; 85: 173-177Crossref PubMed Scopus (210) Google Scholar, 31Nakagawa O. Nakagawa M. Richardson J.A. Olson E.N. Srivastava D. Dev. Biol. 1999; 216: 72-84Crossref PubMed Scopus (250) Google Scholar, 32Chin M.T. Maemura K. Fukumoto S. Jain M.K. Layne M.D. Watanabe M. Hsieh C.-M. Lee M.-E. J. Biol. Chem. 2000; 275: 6381-6387Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 33Zhong T.P. Rosenberg M. Mohideen M.-A.P.K. Weinstein B. Fishman M.C. 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Here, we found that Hesr1 and Hesr2 are expressed in the developing brain and that misexpression of the Hesr genes transiently increases neural precursor cells and thereby promotes generation of late-born cell types. Both Hesr1 and Hesr2 efficiently inhibit the neuronal bHLH genes Mash1 and Math3, like Hes1 and Hes5. These results indicate that Hesr genes are negative regulators of neuronal bHLH genes and promote maintenance of neural precursor cells. Northern Blot Analysis—HincII fragment of Hesr1 cDNA and BsrGI/SacII fragment of Hesr2 cDNA isolated from mouse retina were used as probes (35Satow T. Bae S.-K. Inoue T. Inoue C. Miyoshi G. Tomita K. Bessho Y. Hashimoto N. Kageyama R. J. Neurosci. 2001; 21: 1265-1273Crossref PubMed Google Scholar). Poly(A)+ RNA (2 μg) from mouse brains was electrophoresed on a formaldehyde/1.2% agarose gel and transferred to a nylon membrane (PerkinElmer Life Sciences). Membranes were hybridized at 42 °C in 50% formamide, 5× SSC, 5× Denhardt's solution, 0.1% SDS, and 100 μg/ml heat-denatured salmon sperm DNA and washed at 65 °C in 0.1× SSC and 0.1% SDS. In Situ Hybridization—Digoxigenin-labeled antisense RNA probes corresponding to EcoRI-SalI and EcoRI-HindIII fragments of Hesr1 and Hesr2 cDNAs, respectively (35Satow T. Bae S.-K. Inoue T. Inoue C. Miyoshi G. Tomita K. Bessho Y. Hashimoto N. Kageyama R. J. Neurosci. 2001; 21: 1265-1273Crossref PubMed Google Scholar), were synthesized in vitro. In situ hybridization was carried out, as described previously (8Akazawa C. Sasai Y. Nakanishi S. Kageyama R. J. Biol. Chem. 1992; 267: 21879-21885Abstract Full Text PDF PubMed Google Scholar). Briefly, 16-μm-thick cryosections were fixed in 4% paraformaldehyde for 10 min, washed three times with PBS, acetylated for 10 min, and then washed three times with PBS for 5 min each. Sections were hybridized at 65 °C overnight in 50% formamide, 5× SSC, 5× Denhardt's solution containing 200 ng/ml digoxigenin-labeled probes. After hybridization, sections were washed at 65 °C in 0.2× SSC, treated for 1 h in 0.5% blocking solution (Roche Applied Science), and incubated at 4 °C overnight in blocking solution containing anti-digoxigenin-alkaline phosphatase Fab fragments (diluted 1:1000, Roche Applied Science). For color reaction, sections were incubated in 100 mm Tris-HCl (pH 9.5), 100 mm NaCl, 50 mm MgCl2, and 2 mm levamisole containing 0.84 mg/ml nitro blue tetrazolium and 0.66 mg/ml 5-bromo-4-chloro-3-indolyl phosphate. Labeled preparations were imaged using a Carl Zeiss Axiophoto microscope equipped with an AxioCam color CCD camera. In Utero Electroporation—Mouse Hesr1 and Hesr2 cDNAs were inserted into the EcoRI site of pCLIG, which directs expression of test genes as well as of enhanced green fluorescent protein (EGFP) through the internal ribosomal entry site from the upstream long terminal repeat promoter with cytomegalovirus enhancer (15Hojo M. Ohtsuka T. Hashimoto N. Gradwohl G. Guillemot F. Kageyama R. Development. 2000; 127: 2515-2522PubMed Google Scholar). For the introduction of DNA, pregnant mice were deeply anesthetized, and a ventral midline incision was made to perform in utero manipulation as described previously (13Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. J. Biol. Chem. 2001; 276: 30467-30474Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The expression vector (5 μg) in solution containing 0.05% trypan blue as a tracer was injected through the uterine wall with a glass micropipette into the telencephalic vesicles of each embryo. 1 μg of pCL-EGFP was also co-injected with the expression vectors in many cases because EGFP+ cells can be more easily identified. After injection, electroporation (five 50-ms square pulses of 50 V with 950-ms intervals) was carried out as described previously (13Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. J. Biol. Chem. 2001; 276: 30467-30474Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Then the abdominal wall of the pregnant mouse was sutured, and embryos were allowed to develop in utero. The brains were fixed in 4% paraformaldehyde in PBS for 30 min at 4 °C, treated in 25% sucrose in PBS overnight, and embedded in OCT compound. 16-μm-thick cryosections were cut and examined immunohistochemically. In the electroporated region, nearly 100% of the cells initially seem to be transfected. Immunochemistry—Fixed cryosections were washed three times with PBS, preincubated in PBS containing 2% bovine serum albumin and 0.1% Triton X-100 for 30 min, and then incubated with antibodies. The primary antibodies used are as follows: rabbit anti-GFP (diluted 1:1000, Molecular Probes), mouse anti-MAP2 (1:500, Sigma), mouse anti-NeuN (1:500, Chemicon), mouse anti-GFAP (1:400, Sigma), and mouse anti-nestin (1:500, Pharmingen). To detect these antibodies, biotinylated goat anti-rabbit IgG (1:200, Vector), biotinylated goat anti-mouse IgG (1:200, Vector), fluorescein isothiocyanate-conjugated anti-mouse IgG (1:200, Vector), fluorescein isothiocyanate-avidin D (1:1000, Vector), Fluorolink Cy3-labeled anti-mouse IgG (1:400, Amersham Biosciences), and Fluorolink Cy3-labeled streptavidin (1:1000, Amersham Biosciences) were used. TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed as follows. Fixed cryosections were incubated in TdT buffer (30 mm Tris-HCl, pH 7.5, 140 mm sodium cacodylate, 1 mm cobalt chloride) with biotin-16-dUTP (12.5 μg/ml, Enzo) and TdT (100 units/ml, TaKaRa) at 37 °C for 1 h and detected with Fluorolink Cy3-labeled streptavidin. Fluorescently labeled preparations were imaged using a Carl Zeiss LSM510 confocal microscope. For quantitative analyses, labeled cells were counted in the area of 460 × 500 μm2 (Fig. 2) and 460 × 1000 μm2 (Fig. 5). Three independent experiments were performed. On average, 197.2 GFP+ cells were counted for Fig. 2 and 209.4 GFP+ cells for Fig. 5.Fig. 5Misexpression of Hesr1 and Hesr2 at E15.5 increases astrocytes at P5. Electroporation was performed at E15.5, and the fates of the transfected cells were examined at P5. A–C, electroporation of pCLIG. Many of the transfected cells (EGFP+) were located in the cortical plate and expressed the neuronal marker NeuN. A higher magnification of the indicated region is shown in the inset in A. D–I, electroporation of pCLIG-Hesr1 (D–F) and pCLIG-Hesr2 (G–I). Many of the EGFP+ cells were located in the ventricular/subventricular zone and were positive for GFAP expression, suggesting that misexpression of Hesr1 or Hesr2 increases astrocytes. A higher magnification of the indicated region is shown in the insets in E and H. There is no significant difference in the TUNEL+ cell numbers among samples C, F, and I. J, quantification of the transfected cell types examined in sections. Ratios of three independent experiments are shown with S.E. A, astrocytes; N, neurons. Scale bars: 100 μm and 10 μm (insets).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Dissociation of Brain Cells—For quantification of electroporated brain cells, the brain was dissociated as described previously (37Hatakeyama J. Tomita K. Inoue T. Kageyama R. Development. 2001; 128: 1313-1322Crossref PubMed Google Scholar). The brain was incubated with 0.05% trypsin and 20 μg/ml DNase I in Hanks' balanced salt solution lacking Ca2+/Mg2+ for 15 min at 37 °C, and 2.5 volumes of the culture medium containing 10% fetal bovine serum was added. The cells were pelleted, resuspended in Hanks' balanced salt solution containing DNase I, triturated, and plated on polyethyleneimine-coated (Sigma) and fibronectin-coated (Invitrogen) 8-well chamber slides (Nalge Nunc). After incubation for 2 h at 37 °C in 5% CO2, the cells were fixed with 4% paraformaldehyde for 10 min and subjected to immunochemical analysis. Luciferase Assay—The reporter plasmid contained the firefly luciferase gene under the control of the β-actin promoter with seven repeats of the E-boxes (AGGCAGGTGGC) (24Bae S.-K. Bessho Y. Hojo M. Kageyama R. Development. 2000; 127: 2933-2943PubMed Google Scholar). Full-length cDNA fragments of Hesr1 and Hesr2 were cloned into the eukaryotic expression vector, pCI Vector (Promega). Expression plasmids for Mash1, Math3, and E47 were described previously (7Sasai Y. Kageyama R. Tagawa Y. Shigemoto R. Nakanishi S. Genes Dev. 1992; 6: 2620-2634Crossref PubMed Scopus (583) Google Scholar). The luciferase reporter (0.1 μg) and the eukaryotic expression plasmids (0.3–0.4 μg) were transfected with the FuGENE 6 transfection reagent (Roche Applied Science) into C3H10T1/2 cells, which were plated in 6-multiwell plates at a density of 2 × 105 cells/well. 2 ng of the plasmid containing the Renilla luciferase gene under the control of the SV40 promoter, pRL-SV40 vector (Promega), was also transfected as an internal standard to normalize the transfection efficiency. After 24 h, the cells were harvested, and the luciferase activity was measured using a Lumat LB9507 (Berthold). Expression of Hesr1 and Hesr2 in the Brain—Hesr gene expression in the brain was first determined by Northern blot analysis. During the period from embryonic day 15 (E15) to adult, both Hesr1 and Hesr2 are expressed (Fig. 1A), whereas Hesr3 expression is almost undetectable (data not shown). Hesr1 and Hesr2 expression was further examined by in situ hybridization. At E12, both Hesr1 and Hesr2 are expressed in the ventricular zone of the developing brain (Fig. 1, B and C). Hesr1 expression is observed throughout the brain (Fig. 1B), whereas Hesr2 expression is restricted to the mediodorsal region of the brain (Fig. 1C, arrowheads). At E15, Hesr1 and Hesr2 are expressed at a high level in the ventricular zone and at a low level in the cortical plate (Fig. 1, D–G). At this stage, Hesr2 expression is also observed in the ventral region, in addition to the mediodorsal region, of the brain (Fig. 1F). Hesr1 and Hesr2 expression is also observed in the ventricular zone of the ventral spinal cord (Fig. 1, H and I). In contrast, the sense probes did not detect any specific signals (Fig. 1, J and K). These results indicate that Hesr1 and Hesr2 are expressed mainly by undifferentiated cells in the developing brain. Misexpression of Hesr1 and Hesr2 at E13.5 Maintains Neural Precursor Cells at E16.5—To analyze the functions of Hesr1 and Hesr2 in neural development, we misexpressed the genes in the developing mouse brain by using the expression vector pCLIG, which also directs EGFP expression as a marker. The expression vector DNA was injected into the telencephalic vesicles of E13.5 mouse embryos in utero, and electroporation was performed. By this method, the vector DNA was transiently introduced into the ventricular cells on the plus side. Embryos were then allowed to develop in utero. Three days later (E16.5), brains were isolated from the treated embryos, and the fates of the transfected cells (EGFP+) were examined histologically. When electroporation of pCLIG was performed at E13.5, the majority of the EGFP+ cells migrated out of the ventricular zone and were located in the intermediate zone and the cortical plate at E16.5 (Fig. 2, A and D). In addition, those in the cortical plate expressed the neuronal marker MAP2 (Fig. 2, A, B, and C, inset), indicating that many of the pCLIG-transfected cells differentiated into neurons during the period of E13.5–E16.5. In contrast, when electroporation of pCLIG-Hesr1 or pCLIG-Hesr2 was performed at E13.5, the majority of EGFP+ cells remained in the ventricular zone at E16.5 (Fig. 2, H, K, O, and R). Furthermore, these cells were negative for MAP2 expression (Fig. 2 H–J and O–Q) but expressed nestin, a marker of neural precursor cells (Fig. 2, K–M and R–T, insets in M and T). Quantification of each cell type in sections demonstrated that whereas nearly 50% of the pCLIG-transfected cells became MAP2+ neurons, only 10% of the pCLIG-Hesr1- or pCLIG-Hesr2-transfected cells did (Fig. 2V). These results indicate that Hesr1 and Hesr2 inhibit neurogenesis and maintain neural precursor cells during E13.5–E16.5. We also performed TUNEL assay to examine whether cell death is induced by Hesr genes. However, we did not see any significant difference in the TUNEL+ cell numbers (Fig. 2, G, N, and U). Thus, block of neurogenesis by Hesr genes is not due to neuronal death. Misexpression of Hesr2 at E13.5 Expands the Ventricular Zone—Because Hesr genes maintain neural precursor cells in the embryonal brain, we next examined whether the ventricular zone is affected by misexpression. pCLIG-Hesr2 was introduced by electroporation into the lateral wall of the telencephalon at E13.5, and the ventricular zone was examined at E15.5 by the specific markers, Hes5 and Notch2. At E15.5, the endogenous Hesr2 expression was present in the mediodorsal region of the telencephalon (Fig. 3A, arrowheads), whereas the electroporation-mediated ectopic Hesr2 expression was observed at the lateral region (Fig. 3A, arrow, and B). In this region, both Hes5 and Notch2 expression was expanded outwardly (Fig. 3, C–F, arrows), compared with the control brain (Fig. 3, G and H). These results indicate that misexpression of Hesr2 expands the ventricular zone by maintaining excessive neural precursor cells. Misexpression of Hesr1 and Hesr2 at E13.5 Generates Late-born Neurons at P1—We next examined the later effects of misexpression of Hesr1 and Hesr2. The expression vectors were electroporated at E13.5, and the fates of the EGFP+ cells were examined at postnatal day 1 (P1). When Myc-tagged Hesr genes were misexpressed under this condition, Myc expression was already lost at P1 (data not shown), whereas EGFP expression was detectable, probably because EGFP is more stable than Hesr proteins (13Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. J. Biol. Chem. 2001; 276: 30467-30474Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Thus, this experiment allowed us to follow the fates of the transfected cells after Hesr expression was down-regulated. When pCLIG was electroporated, the majority of the transfected cells were found in the cortical plate and expressed the late neuronal marker NeuN in addition to MAP2 at P1 (Fig. 4, A–D). When pCLIG-Hesr1 or pCLIG-Hesr2 was introduced by electroporation, many of the transfected cells were found in both the cortical plate and the ventricular/subventricular zone at P1 (Fig. 4, E, H, I, and L). The cells found in the cortical plate expressed MAP2, whereas those in the ventricular/subventricular zone did not (Fig. 4, H and L). These results indicate that some of the transfected cells, which remained as precursors at E16.5 by misexpression of Hesr1 and Hesr2, differentiated into neurons at P1. However, these transfected cells were located in more superficial layers than those transfected with pCLIG (Fig. 4, A, E, and I), suggesting that misexpression of Hesr1 and Hesr2 delays the birth dates of neurons. Agreeing with this notion, many of the transfected cells still did not express NeuN (Fig. 4, G and K), in contrast to the pCLIG-transfected cells, which mostly expressed NeuN (Fig. 4C). These results indicate that misexpression of Hesr1 and Hesr2 at E13.5 increases late-born neurons at P1. Misexpression of Hesr1 and Hesr2 at E15.5 Increases Astrocytes at P5—The observed effects of Hesr1 and Hesr2, maintenance of neural precursor cells and increase of late-born neurons, are very similar to those of Hes1 and Hes5 (13Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. J. Biol. Chem. 2001; 276: 30467-30474Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Previous studies demonstrated that when misexpressed at later stages, Hes1 and Hes5 promote gliogenesis in the brain (13Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. J. Biol. Chem. 2001; 276: 30467-30474Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). We therefore misexpressed Hesr1 and Hesr2 at E15.5 and examined the cell fates at P5. When pCLIG was introduced by electroporation, many of the EGFP+ cells migrated into the cortical plate and differentiated into NeuN+ neurons (Fig. 5A, inset). In contrast, when pCLIG-Hesr1 or pCLIG-Hesr2 was introduced by electroporation, many of the EGFP+ cells remained in the ventricular/subventricular zone and differentiated into GFAP+ astrocytes (Fig. 5, E and H, insets). Quantification of each cell type in sections demonstrates that whereas 80% of the pCLIG-transfected cells became neurons, the majority of pCLIGHesr1- or pCLIG-Hesr2-transfected cells became astrocytes (Fig. 5J). Again, there is no significant difference in the TUNEL+ cell numbers between these samples, indicating that cell death does not account for the effects by Hesr1 and Hesr2 (Fig. 5, C, F, and I). These results indicate that misexpression of Hesr1 or Hesr2 at E15.5 inhibits neurogenesis and promotes gliogenesis. To quantify the effects of Hesr1 and Hesr2 misexpression with an alternative method, we next performed electroporation at E15.5 and dissociated the brains at P5. Those dissociated cells were attached to the plates and stained with anti-MAP2 and anti-GFAP antibodies. Under this condition, EGFP+-MAP2+ neurons (Fig. 6, A–L, arrowheads) and EGFP+-GFAP+ astrocytes (Fig. 6, M–X, arrowheads) can be clearly quantified. When pCLIG was electroporated at E15.5, more than 60% of the EGFP+ cells differentiated into MAP2+ neurons, whereas only 30% became GFAP+ astrocytes at P5 (Fig. 7). In contrast, when pCLIG-Hesr1 or pCLIG-Hesr2 was electroporated at E15.5, about 60% of the EGFP+ cells differentiated into GFAP+ astrocytes (Fig. 7). Although the exact ratios of neurons and astrocytes determined by this dissociation method are somewhat different from those determined by counting in sections (Fig. 5J), both methods clearly showed the decrease of neurons and increase of astrocytes by Hesr genes. We also performed TUNEL assay to examine whether cell death is induced by misexpression of Hesr1 and Hesr2. However, we did not detect any clear induction or block of cell death (data
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