Hes1 but not Hes5 Regulates an Astrocyte Versus Oligodendrocyte Fate Choice in Glial Restricted Precursors

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1 DEVELOPMENTAL DYNAMICS 226: , 2003 ARTICLE Hes1 but not Hes5 Regulates an Astrocyte Versus Oligodendrocyte Fate Choice in Glial Restricted Precursors Yuanyuan Wu, 1 Ying Liu, 1,2 Edward M. Levine, 1,3 and Mahendra S. Rao 2 * To determine the role of Hes genes in the differentiation process of neuroepithelial (NEP) cells to glial restricted precursor cells (GRPs) and subsequently GRPs to oligodendrocytes and astrocytes, we have examined the effects of Hes1 and Hes5 on glial differentiation. We find that both Hes1 and Hes5 are expressed by GRPs and that Hes1 can drive GRPs to an astrocyte cell fate at the expense of oligodendrocyte differentiation. Overexpression of Hes1 in GRPs results in the up-regulation of the astrocyte markers glial fibrillary acidic protein and CD44 and the down-regulation of oligodendrocyte markers myelin proteolipid protein/dm20, GalC, and CNPase. Transcription factors involved in oligodendrocyte differentiation, such as Nkx2.2, Olig1, and Mash1, are also down-regulated in Hes1-overexpressing cells. The effect of Hes1 on gliogenesis is stage-specific as Hes1 does not direct NEP cells to an astrocytic fate. In contrast to Hes1, Hes5 does not promote astrocyte differentiation. Instead, it inhibits both astrocyte and oligodendrocyte differentiation. Overexpression of Notch1 has an effect on gliogenesis similar to that of Hes1 and the mrna levels of Hes1 are up-regulated in cells overexpressing Notch1, suggesting that Notch1 could be an upstream activator of Hes1. Developmental Dynamics 226: , Wiley-Liss, Inc. Key words: Hes1; Hes5; Notch1; GRP; development; differentiation; promotion; astrocyte; oligodendrocyte Received 1 November 2002; Accepted 19 December 2002 INTRODUCTION Accumulating experimental evidence indicates that stem cells and lineage restricted precursor cells exist in the central nervous system (CNS) and that acquisition of a mature phenotype requires the concomitant interactions of a variety of extracellular signals and intracellular transcription factors (Gage, 2000). In the spinal cord, pluripotent stem cells have been termed NEP cells and have been shown to generate neurons, astrocytes, and oligodendrocytes (Kalyani et al., 1997; Rao et al., 1998). Differentiation is a sequential process with more restricted, but still multipotent, precursors initially being generated followed by more differentiated cells. Two such precursors have been characterized, a neuronal restricted precursor, or NRP cell (Mayer-Proschel et al., 1997), and a glial restricted precursor, or GRP cell (Rao and Mayer-Proschel, 1997; Rao et al., 1998). Other restricted precursors likely exist, including oligoneuronal and neuron astrocyte precursors (reviewed in Weissman et al., 2001), but their lineage relationship to NEP cells remains to be established. An important question in developmental biology is to understand the mechanisms regulating alternative fate choice in a given lineage, and the mechanisms directing those decisions, such as the oligodendrocyte/ astrocyte fate choice in the GRP lineage, are still unknown. The process of fate restriction has been studied in multiple systems and evidence from Drosophila and vertebrates has established a central role for Notch-Delta signaling in cell fate determination (Artavanis-Tsakonas et al., 1995; Lewis, 1996). In Drosophila, binding of Notch to its ligands, Serrate 1 Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, Utah 2 Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, Baltimore, Maryland 3 Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, Utah *Correspondence to: Mahendra S. Rao, Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD raomah@grc.nia.nih.gov DOI /dvdy Wiley-Liss, Inc.

2 676 WU ET AL. Fig. 1. Hes1, Hes5, and Notch1 are expressed in glial lineages. A: In situ hybridization was performed on transverse sections of mouse spinal cords from embryonic day (E) 10 to E15. At E10, the Hes1 expression domain is largely complementary with the Hes5 expression domain (arrows). At E12, both genes are highly expressed in the ventricular zone (arrows). Low levels of Hes1 are also detected outside the ventricular zone in the spinal cord and in non-neural tissues. The expression of Hes5 is only detected in the spinal cord (arrow) and dorsal root ganglia (arrowhead). At E15, the expression of Hes1 is diffuse but relatively strong, while the expression of Hes5 is down-regulated. B: Reverse transcriptasepolymerase chain reaction analysis was performed to examine the expression of Hes1, Hes5, and Notch1 in different purified cell populations (see text for details). RNA from rat postnatal day 0 spinal cord (P0 SC) and E14.5 spinal cords (E14.5 SC) was used as control. Note that both Hes1 and Hes5 are expressed in neuroepithelial (NEP) cells and glial restricted precursor cells (GRPs) but not in oligodendrocytes. Hes1 is also expressed in mature astrocytes, whereas Hes5 is absent in this population. Notch1 is expressed in all cell types but is down-regulated in oligodendrocytes.

3 Hes AND GLIOGENESIS 677 or Delta, induces the activation and translocation of the intracellular domain of Notch to the nucleus. The activated Notch then forms a complex with a transcription factor, Suppressor of Hairless [Su(H)], which in turn activates the Hairy/Enhancer of Split (E(Spl)) repressors to down-regulate neurogenic genes (Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995; Schweisguth, 1995). This process inhibits neurogenesis in Drosophila. The same mechanism is thought to be involved in mammalian neurogenesis (de la Pompa et al., 1997; Eddison et al., 2000). Several Notch homologues have been identified in mammals and several ligands, including Delta-like (Bettenhausen et al., 1995; Dunwoodie et al., 1997; Lendahl, 1998) and Jagged (Serrate; Lindsell et al., 1995, 1996; Shawber et al., 1996a). Homologues of Hairy/E (Spl), Hes1 7, have also been identified with Hes1, 3, and 5 expressed in different regions of the CNS (Sasai et al., 1992; Sakagami et al., 1994; Takebayashi et al., 1995). In the developing CNS, Hes proteins have been shown to be involved in regulating neural differentiation. Hes1 and Hes5 are expressed in the developing ventricular zone and are down-regulated during the process of differentiation (Sasai et al., 1992). In Hes1 and Hes5 knockout (KO) mice, neural stem cells cannot be maintained properly and neurons differentiate prematurely (Ishibashi et al., 1995; Ohtsuka et al., 2001). Overexpression of Hes1 prevents neuronal differentiation in the brain (Ishibashi et al., 1994). These data suggest that Hes genes are critical for the proper timing of neuronal differentiation. Later effects of Notch-delta signaling, however, have been difficult to evaluate in KO mice due to these early effects. Recently, it was noted that Notch and Hes genes might also play a role in regulating glial differentiation. Notch and Hes have been shown to promote the differentiation of Schwann cells (Morrison et al., 2000), radial glia (Gaiano et al., 2000), and Müller glial cells (Furukawa et al., 2000; Hojo et al., 2000). However, these findings contrast with the observations that Notch1 and Jagged1 repress oligodendrocyte differentiation in the optic nerve (Wang et al., 1998) and that overexpression of Hes1/5 in the embryonic telencephalon or Notch1 in P19 cells inhibits neurogenesis but does not affect astrocyte differentiation (Nye et al., 1994; Ohtsuka et al., 2001). It is possible that Notch-Hes signaling may have different effects, depending on the types of glial cell involved or the differentiation stage of the precursor cell. The developing spinal cord is a well-developed model for the study of precursor fates, so it is interesting to know whether in this system Notch-Hes signaling directs astrocyte or oligodendrocyte differentiation and at what stages it can act to regulate gliogenesis. In the present study, we have compared the expression patterns of Hes1 and Hes5 in developing spinal cords by in situ hybridization and by reverse transcriptase-polymerase chain reaction (RT-PCR) using mrna from purified cell types. We find that both Hes1 and Hes5 are expressed in GRPs. Hes1 is also expressed in mature astrocytes, whereas Hes5 is not. By using a retrovirus to overexpress genes of the Notch-Hes signaling pathway in GRPs, we find that Hes1 promotes an astrocytic cell fate at the expense of oligodendrocytes. The effect of Hes1 is stage-specific because Hes1 does not promote the astrocyte fate when overexpressed in NEP cells. In Hes1-infected GRPs, Nkx2.2, Olig1, and Mash1 are downregulated. We also find that Hes5 does not promote an astrocyte fate, but rather inhibits both astrocyte and oligodendrocyte differentiation. Finally, Notch has an effect on GRPs similar to that of Hes1, suggesting that Notch1 could be an upstream activator of Hes1. These observations suggest that Notch1-Hes1 (but not Hes5) signaling plays an important role in regulating the astrocyte versus oligodendrocyte fate choice of GRPs in the developing spinal cord. RESULTS Hes1 and Hes5 Are Expressed in Glial Lineages To identify the expression patterns of Hes1 and Hes5 in the developing spinal cord, in situ hybridization was performed on embryonic day (E) 10 E17 transverse mouse spinal cord sections. mrna of Hes1 and Hes5 was seen at E10, but levels were higher at E12 where the expression appeared largely confined to the ventricular zone (Fig. 1A), which overlaps the GRP domain as identified by the A2B5 immunoreactivity (Cai et al., unpublished observations; Rao et al., 1998). At E15, the expression of Hes1 is diffuse but relatively strong in the spinal cord while the expression of Hes5 is down-regulated (Fig. 1A). Generally, at late Fig. 2. Retrovirus-mediated gene transfection up-regulates Hes1 protein level in Hes1-infected cells, and Hes1 overexpression in glial restricted precursor cells (GRPs) affects cell morphology and reduces cell division. A: GRPs and NIH 3T3 cells were infected with plxsn-myc (six epitopes) -tagged Hes1 or control (alkaline phosphatase [AP]) viruses. Cell lysates were collected from infected cells 3 days later, and the levels of myc-tagged Hes1 protein were examined by Western blots using anti-myc antibody. Single bands of myc-hes1 protein were detected in myc-hes1 infected cells but not in AP-infected cells. Anti -tubulin was used as a loading control. B: GRPs were infected by plxsn-myc-hes1 virus and infected cells were stained by anti-myc and anti glial fibrillary acidic protein (GFAP) antibody 4 days later. Myc-Hes1 fusion proteins were detected in nuclei, and many of the infected cells were GFAP. C: Embryonic day 14.5 rat spinal cord cells were infected by Hes1 virus or AP virus. Infected cells were selected by G418 and replated on poly-l-lysine and laminin-coated dishes at a density of cells/dish (35 mm) and cultured in neuroepithelial cell basal medium in the presence of 20 ng/ml basic fibroblast growth factor. The cells were exposed to 10 M bromodeoxyuridine (BrdU) for 2.5, 5, and 10 hr, fixed, and processed for BrdU staining. This panel shows the staining from a 5-hr BrdU exposure. Fewer BrdU cells were observed in Hes1-infected cells. Note the dramatic change of the cell morphology in Hes1-infected cells. Hes1-infected cells are bigger, flatter, and have larger processes than AP-infected cells. The nuclei of Hes1-infected cells are also bigger than those of AP-infected cells. D: The table summarizes the results of BrdU analysis obtained from serial time points of three independent experiments. Between 500 and 1,000 cells were counted for each experiment.

4 678 WU ET AL. embryonic stages (E14 E17), the expression of Hes1 in the spinal cord is largely ubiquitous without obvious pattern (data not shown), consistent with that described previously (Feder et al., 1993). These expression patterns suggest that Hes1 may be expressed in NEPs, GRPs, and possibly mature astrocytes, whereas Hes5 expression is more transient and confined to early stage progenitors. To further determine whether cells of the glial lineages express Hes1 and Hes5, we examined different glial populations by RT-PCR. cdna from purified cells of different stages were isolated. NEP cells were obtained by dissecting out rat spinal cords at E10.5 (Kalyani et al., 1997). Purified GRPs and oligodendrocytes were obtained by fluorescence activated cell sorting (FACS; see Experimental Procedures section). Astrocytes were obtained by inducing astrocytic differentiation of sorted spinal cord GRPs by treatment with 20 ng/ml bone morphogenetic protein-4 (BMP-4) and 20 ng/ml leukemia inhibitory factor (LIF) for 5 days. RT-PCR analysis showed that both Hes1 and Hes5 mrnas were present at multiple stages of glial differentiation. Hes1 could be detected in multipotent NEP cells, GRPs, and astrocytes. Hes1 expression was downregulated and undetectable in GalC oligodendrocytes, suggesting that Hes1 may play a role in regulating the transition from GRP to oligodendrocyte (Fig. 1B). Hes5 expression was more restricted than that of Hes1. No expression of Hes5 was seen in astrocytes or oligodendrocytes, although Hes5 expression was readily detected in NEP cells and GRPs (Fig. 1B). The presence of Hes1 and Hes5 in GRPs raises the possibility that they both participate in regulating gliogenesis. However, the differential expression patterns of Hes1 and Hes5 in astrocytes suggest that they may function differently in astrocyte differentiation. We also examined the expression pattern of Notch1, as it might be an upstream activator of Hes genes. Notch1 was detected in all cell types, although at different levels (Fig. 1B), suggesting that Notch1 could play multiple roles at different stages as suggested before (Frisen and Lendahl, 2001). Overexpression of Hes1 in GRPs Promotes Astrocyte Differentiation In situ and RT-PCR results demonstrated that Hes1 is expressed by GRPs (Fig. 1), which can generate both oligodendrocytes and astrocytes in vitro and in vivo (Rao et al., 1998; Herrera et al., 2001; Gregori et al., 2002). To determine whether Hes1 regulates glial differentiation of GRPs, a replication-incompetent retroviral construct containing a neomycin-resistant gene was used to overexpress Hes1. To confirm that the retrovirus-mediated gene transfection increases protein expression, we evaluated several commercial Hes1 antibodies. However, none of these antibodies worked in either Western blots or immunostaining. As an alternative, we developed a myc-tagged Hes1 viral construct in addition to the untagged one used in the morphologic and cell differentiation assays below. We infected GRP cells and NIH 3T3 cells with virus encoding myc-tagged Hes1. Western blots show that the myc-hes1 fusion protein is expressed in myc- Hes1 infected but not control alkaline phosphatase (AP) -infected cells (Fig. 2A). Immunocytochemistry shows that overexpressed myc-hes1 proteins are properly localized in the nuclei of infected GRP cells (Fig. 2B). After confirming protein overexpression, we then examined the effect of Hes1 on the morphology and differentiation of GRP cells. E14.5 rat spinal cord cells were infected with Hes1 or AP virus. In E14.5 spinal cord, the majority of the mitotic cells are A2B5 GRPs, whereas most cells in the neuronal lineage have already withdrawn from the cell cycle. The infected cells were selected by G418 for 3 days. Because the retrovirus can only infect mitotic cells, cells surviving drug selection were primarily GRPs. Immunostaining confirmed that 90% of cells were A2B5- immunoreactive and were negative for the neuronal markers ( -III-tubulin and MAPII, data not shown). Cells were subjected to bromodeoxyuridine (BrdU) analysis and immunostaining. RNA was harvested from sister dishes at different time points (day 0 end of G418 selection) and subjected to semiquantitative RT- PCR analysis (see Experimental Procedures section). Hes1-infected cells underwent a dramatic change in cell morphology compared with AP-infected cells. Immediately after G418 selection, most AP-infected cells were small, round, phase bright, and bipolar or multipolar cells with short processes, typical of GRPs morphology. In contrast, Hes1-infected cells were bigger (twofold), flat, phase dark, and had large processes and nuclei (Fig. 2C). Their morphology was reminiscent of the morphology of mature astrocytes generated after bone morphogenetic protein (BMP) or serum treatment of GRPs (Rao et al., 1998). To determine whether Hes1 overexpression altered cell proliferation, BrdU incorporation was compared between Hes1 and control infected cultures in the presence of 20 ng/ml basic fibroblast growth factor (bfgf; Fig. 2C,D). Interestingly, BrdU incorporation was significantly reduced in Hes1-overexpressing cells compared with controls irrespective of the duration of the BrdU pulse (2.5 hr, 5 hr, or 10 hr). At each time point, the rate of BrdU incorporation in Hes1-infected cells was less than half of that in AP control cultures, indicating that Hes1 reduced cell division in GRPs. The change in cell proliferation correlated with the change in cell number (data not shown). The dramatic change in morphology and decrease of cell division in Hes1-infected cells suggest that Hes1 may promote astrocyte differentiation in GRPs. Consistent with this hypothesis, CD44 (Alfei et al., 1999) and glial fibrillary acidic protein (GFAP), two astrocyte lineage markers, were up-regulated as early as 3 days after the cells were cultured in medium with reduced bfgf (see Experimental Procedures section for differentiation culture condition), but in the absence of any astrocytepromoting factor (e.g., BMP or ciliary neurotrophic factor [CNTF]; Rao et al., 1998). In Hes1-infected dishes, 78 4% of cells were CD44-positive and 79 2% were GFAP-positive. In

5 Hes AND GLIOGENESIS 679 Fig. 3. Hes1 promotes astrocyte differentiation of glial restricted precursor cells (GRPs). A: Embryonic day 14.5 rat spinal cord cells were infected by Hes1 or alkaline phosphatase (AP) virus. Infected cells were replated and cultured in reduced basic fibroblast growth factor (bfgf; see Experimental Procedures section for differentiation culture condition) for 3 more days to allow spontaneous cell differentiation. Cells were then stained for CD44 and glial fibrillary acidic protein (GFAP) to detect differentiating astrocytes. Although only 4 1% of AP-infected cells were CD44 or GFAP, 78 4% of Hes1-infected cells were CD44, and 79 2% were GFAP. Note that the seemingly different cell morphology detected by CD44 and GFAP staining is due to the different locations of these two antigens. CD44 is on the cell surface, whereas GFAP is a cytoplasmic protein. B: Cell extracts from infected cells were collected at day 3 or day 6 for Western blots. Expression of GFAP was strongly up-regulated in Hes1-infected cells compared with the AP control. A -tubulin antibody was used as a control for sample loading. C: Total RNA from infected cells was harvested at day 0, 2, 4, and 6 (the end of G418 selection and the start of the culture in reduced bfgf is labeled as day 0) and used in reverse transcriptase-polymerase chain reaction. The mrna levels of GFAP were strongly up-regulated in Hes1-infected cells at each time point analyzed compared with controls. G3PDH (25 cycles) was used as a control. AP-infected dishes, only 4 1% of cells were CD44- or GFAP-positive (Figs. 3, 10). This dramatic difference (P ; unpaired t-test; Fig. 10) between Hes1 and AP infection indicates that Hes1 plays a powerful role in astrocyte differentiation. To provide independent verification of the change in GFAP and to determine whether there was an up-regulation of GFAP message, Western blots and semiquantitative RT-PCR were performed. At all time points analyzed, up-regulation of GFAP was detected at both the protein and mrna levels (Fig. 3B,C). GFAP cells induced by Hes overexpression did not express A2B5 and, therefore, are likely to be Type 1 astrocytes. Overexpression of Hes1 in NEP Cells Does Not Promote Astrocyte Differentiation Hes1 is also expressed in NEP cells (Fig. 1B), suggesting that Hes1 may promote astrocyte differentiation in NEP cells as it does in GRPs. We, therefore, infected NEP cells with Hes1 or AP control retrovirus, cultured the infected cells in differentiation medium (reduced bfgf), and compared the ability of the cells to differentiate into astrocytes. In contrast to what we observed in GRPs, overexpression of Hes1 in NEP cells did not show any obvious effect on astrocyte differentiation. Although we could detect higher levels of Hes1 mrna in the Hes1-infected cells, we could not detect any up-regulation of GFAP by either semiquantitative RT-PCR or immunostaining (Fig. 4 and data not shown). This finding suggests that the astrocyte-promoting effect of Hes1 is specific to GRP cells and is consistent with the effect on neurogenesis but not gliogenesis observed at early stages in the Hes1 KO animals (Ishibashi et al., 1994). Hes1 Acts on GRPs to Inhibit Oligodendrocyte Differentiation GRPs are tripotential cells that can generate two types of astrocytes as well as oligodendrocytes both in vitro and in vivo (Rao et al., 1998;

6 680 WU ET AL. Herrera et al., 2001; Gregori et al., 2002). Our results suggest that Hes1 acts at the GRP stage to promote astrocyte differentiation. To determine whether the promotion of astrocyte differentiation results in a corresponding inhibition of oligodendrocyte differentiation, we examined the effects of overexpressing Hes1 in GRPs cultured under oligodendrocyte-promoting conditions (see Experimental Procedures section). Hes1 inhibited oligodendrocyte differentiation significantly when compared with AP-infected cells. We found that in AP-infected cells, approximately 16 2% were myelin proteolipid protein (PLP) -immunoreactive and 13 4% were GalCimmunoreactive 5 days after the cells were cultured in oligodendrocytepromoting conditions. These numbers are consistent with the extent of oligodendrocyte differentiation observed in other experiments (Rao et al., 1998; Gregori et al., 2002). In contrast, in Hes1-infected cells, there were very few PLP- or GalC-immunoreactive cells ( 1%, Figs. 5A, 10). Similar results were obtained when O4 was used as an early marker for oligodendrocyte differentiation (data not shown), suggesting that inhibition of oligodendrocyte in Hes1-infected cells occurs before the acquisition of O4 immunoreactivity. Western blot (Fig. 5B) and semiquantitative RT-PCR (Fig. 5C) analyses also show that the expression of CNPase and PLP/DM20, two markers of oligodendrocyte differentiation, were significantly decreased in Hes1- infected cells compared with levels in AP-infected cells. During the 5-day culture period under oligodendrocyte-promoting conditions, we did not observe any obvious cell death in either Hes1-infected or control cells. TUNEL assays show that the overall number of apoptotic profiles was very low, and there was no significant difference between control and Hes1- infected cultures (data not shown), suggesting that the lack of oligodendrocytes was more likely due to the inhibition of oligodendrocyte differentiation than the death of differentiating cells. Thus, our results suggest that Hes1 overexpression promotes astrocyte differentiation and inhibits oligodendrocyte differentiation at an early stage of gliogenesis. This suggestion is Fig. 4. Hes1 does not promote astrocyte differentiation of neuroepithelial (NEP) cells. NEP cells from embryonic day 10.5 rat spinal cord were infected by Hes1 or alkaline phosphatase (AP) virus and selected by G418. The cells were replated and cultured under differentiation culture condition. Under these conditions in control cultures, approximately 4% of cells differentiate as astrocytes. Total RNA from infected cells was harvested at day 0, 2, 4, and 6. Whereas the levels of Hes1 mrna were higher in Hes1-infected cells than in controls, the levels of GFAP mrna were largely the same in these two cells. G3PDH (25 cycles) was used as a control. consistent with our hypothesis that Hes1 promotes astrocyte fate at the expense of oligodendrocytes. Overexpression of Hes1 Downregulates Multiple Transcription Factors Implicated in Oligodendrocyte Differentiation Previous data indicate that Hes1 inhibits neuronal fate by repressing the transcription of neuronal determination genes (Ishibashi et al., 1995; Castella et al., 1999). Hes1 may also suppress genes participating in oligodendrocyte generation. Several transcription factors have been proposed to have a positive function on oligodendrocyte differentiation. Nkx2.2 is expressed in GRPs and immature oligodendrocytes (Xu et al., 2000; Soula et al., 2001). Loss of Nkx2.2 leads to a failure of mature oligodendrocyte formation (Qi et al., 2001). Mash1 (Kondo and Raff, 2000), Olig1, and Olig2 (Lu et al., 2000; Zhou et al., 2000, 2001) are expressed in oligodendrocyte precursors and may regulate oligodendrocyte development. To determine whether Hes1 acts to down-regulate the expression of these early oligodendrocyte-promoting factors, we examined the effect of Hes1 on the expression of Nkx2.2, Mash1, and Olig1 by Western blots and RT-PCR. Western blots (Fig. 6A) and RT-PCR (Fig. 6B) show that overexpression of Hes1 dramatically reduced the expression of Nkx2.2, Mash1, and Olig1. The down-regulation of Olig1 by Hes1 is consistent with a recent report describing the absence of oligodendrocytes and ectopic generation of astrocytes in Olig1/2 double mutant mice (Zhou and Anderson, 2002). Taken together, our results suggest that Hes1 may suppress these early oligodendrocyte-promoting transcription factors; this suppression could be a possible mechanism for Hes1 to drive GRPs to the astrocyte fate at the expense of oligodendrocytes. TITLE CASE: OVEREXPRESSION OF Hes1 ALTERS THE EXPRESSION LEVELS OF Hes5 AND Notch1 IN GRP CELLS Previous studies suggest that Hes1 and Hes5 may play very similar roles in neural development (Furukawa et al., 2000; Hojo et al., 2000; Ohtsuka et al., 2001). Evidence also suggests that, like their Drosophila homologues, both Hes1 and Hes5 expression could be activated by Notch1 (Jarriault et al., 1995, 1998; de la Pompa et al., 1997). Hes1, Hes5, and Notch1 may function in the same pathway. We, therefore, examined the expression of Hes5 and Notch1 in Hes1-infected cells. As shown in Figure 7, the levels of Hes1 mrna in Hes1-infected cells were always higher than that in APinfected cells at any time point, indicating that overexpression was achieved.

7 Hes AND GLIOGENESIS 681 Fig. 5. Hes1 inhibits oligodendrocyte differentiation of glial restricted precursor cells (GRPs). A: Embryonic day 14.5 rat cells were infected and selected by G418. Selected cells were replated and cultured in an oligodendrocyte-promoting condition ( platelet-derived growth factor, T3, and reduced basic fibroblast growth factor) for 5 more days. Cells were then stained for the oligodendrocyte marker GalC or proteolipid protein (PLP). Although there were 13 4% GalC cells and 16 2% PLP cells in alkaline phosphatase (AP) -infected dish, there were hardly any GalC or PLP cells in Hes1-infected dishes. B: Protein lysates from infected cells in sister dishes were collected at day 3 or 6 and used for Western blots. Expression of the oligodendrocyte-specific protein CNPase was reduced in Hes1-infected cells. A -tubulin antibody was used as a loading control. C: Total RNA from infected cells was harvested and used in reverse transcriptasepolymerase chain reaction at different time points. The mrna levels of PLP/DM20 and CNPase were down-regulated in Hes1-infected cells compared with controls. G3PDH (25 cycles) was used as a control. Levels of Notch1 and Hes5 were down-regulated in Hes1-infected cells when compared with controls. Down-regulation of Hes5 was detected as early as day 0, suggesting that Hes1 may inhibit the expression of Hes5 directly. Consistent with this, the regulatory region of Hes5 gene has five N box sequences (Takebayashi et al., 1995), and Hes1 can repress transcription by binding to these sites (Sasai et al., 1992). Downregulation of Notch1 was not obvious until day 4, suggesting that the effect may be indirect or result from a feedback inhibition by Hes1 overexpression. These results further suggest that both Hes5 and Notch1 alter astrocyte and oligodendrocyte differentiation. Effect of Hes5 Overexpression on Oligodendrocyte and Astrocyte Differentiation Our results suggest that Hes1 acts at the GRP stage to inhibit oligodendrocyte and promote astrocyte differentiation, as well as to down-regulate Hes5 expression. Like Hes1, Hes5 is expressed in NEP cells and GRPs (Fig. 1) and promotes Müller glial cell fate in the retina (Furukawa et al., 2000; Hojo et al., 2000). To determine whether Hes5 can also promote astrocyte differentiation in developing spinal cord, we overexpressed Hes5 in E14.5 spinal cord cells and compared oligodendrocyte and astrocyte differentiation with that in GFP-overexpressing cells (control). Unlike Hes1-overexpressing cells, which showed dramatic morphologic change (Fig. 2C), cells overexpressing Hes5 did not show an obvious change in morphology (data not shown). BrdU incorporation demonstrated that Hes5 did not have a significant effect on cell division either. No cell death was observed in Hes5-infected cells. Total RNA from infected cells was harvested at day 0, 2, or 6 and used to perform semiquantitative RT-PCR. As

8 682 WU ET AL. expected, Hes5-overexpressing cells always had higher levels of Hes5 mrna when compared with GFPoverexpressing cells (Fig. 8, lane a). Unlike Hes1, Hes5 did not promote astrocyte differentiation. Instead, Hes5 inhibited the differentiation of both astrocytes and oligodendrocytes. RT-PCR showed that levels of both an astrocyte marker (GFAP) and oligodendrocyte markers (CNPase and PLP/DM20) were down-regulated compared with control, although the inhibition was relatively weak (Fig. 8, lane b d). Immunostaining showed that most cells were still A2B5 /GFAP /PLP after 5 days in differentiation culture conditions or in oligodendrocytepromoting conditions (Fig. 10 and data not shown). This finding is in contrast to the results with Hes1 overexpression (Figs. 3, 5). These results suggest distinct but overlapping roles for Hes genes in regulating gliogenesis. Both Hes1 and Hes5 inhibit oligodendrocyte differentiation. Hes1 drives GRPs to become mature astrocytes at the expense of oligodendrocytes, whereas Hes5 likely maintains GRPs at a precursor stage. Effect of Notch1 Overexpression Mimics the Effect of Hes1 Overexpression Fig. 6. Hes1 suppresses genes implicated in oligodendrocyte differentiation. A: Embryonic day 14.5 rat spinal cord cells were infected and selected and then replated in differentiation culture condition. Protein lysates from infected cells were collected at day 3 and 6 for Western blots. The expression of Nkx2.2 and Mash1 were suppressed in Hes1- infected cells. B: Total RNA from infected cells in sister dishes was extracted at different times and used for reverse transcriptase-polymerase chain reaction. The mrna of Olig1 was down-regulated by Hes1. G3PDH (25 cycles) was used as a control. AP, alkaline phosphatase. Our results suggest that Hes1 and Hes5 play different roles in gliogenesis in the developing spinal cord. Other evidence has shown that Hes1 or Hes5 might be regulated by the activation of Notch genes (Jarriault et al., 1995, 1998; de la Pompa et al., 1997) and Hes1, Hes5, or both may be essential for mediating the function of Notch (Ohtsuka et al., 1999). We, therefore, decided to examine the role of Notch1 in regulating glial differentiation. Like Hes1, but unlike Hes5, Notch1 continues to be expressed by astrocytes (Fig. 1B), raising the possibility that Notch1 may affect Hes1 expression more directly. To determine whether activating Notch1 alters Hes1 or Hes5 expression levels and affects astrocyte or oligodendrocyte differentiation, we prepared a virus encoding the mouse Notch1 intracellular domain (mnid), a constitutively active form of Notch1, and used it to infect E14.5 spinal cord cells. AP was used as a control. As shown in Figure 9A, lanes b and c, overexpression of mnid resulted in persistent up-regulation of Hes1. In contrast, Hes5 expression was not affected except at day 0. Like Hes1, mnid also promoted astrocyte differentiation (Fig. 9A, lane d) and inhibited oligodendrocyte differentiation (Fig. 9A, lanes e and f). The RT- PCR results were confirmed by immunocytochemistry. Three days after being cultured under differentiation culture condition, 34 2% of cells were GFAP-positive and 30 5% were CD44-positive in mnid-infected dishes. In contrast, less than 4 1% of cells were GFAP- or CD44- positive in control dishes (P 0.001; unpaired t-test; Fig. 9B and Fig. 10). Like Hes1, mnid also down-regulated the expression of Olig1 (Fig. 9, lane g). This finding suggests that Notch1 may be an upstream activator of Hes1 in gliogenesis and that Notch1 and Hes5 can play different roles in gliogenesis in the spinal cord. DISCUSSION Our results show that both Hes1 and Hes5 are expressed in GRPs and that only Hes1 promotes GRPs to differentiate into astrocytes, whereas both Hes1 and Hes5 inhibit oligodendrocyte differentiation. The effect of Hes1 is stage-specific as Hes1 does not drive NEP cells toward an astrocyte fate. Notch overexpression mimics Hes1 overexpression, suggesting that Notch1 may be an upstream activator of Hes1 in GRPs of the developing spinal cord. Hes1 Promotes Astrocyte Fate at the Expense of Oligodendrocyte Fate We have suggested previously that GRPs are the precursors of both astrocytes and oligodendrocytes and

9 Hes AND GLIOGENESIS 683 However, active forms of Notch1 and Notch3 increase Hes1 expression in adult hippocampus-derived progenitors and can promote these cells to an astrocyte fate (Tanigaki et al., 2001). Therefore, it appears that CNS progenitors need to develop to a certain stage before Notch1/Hes1 signaling can promote the astrocyte fate. It will be very important to identify factors that cooperate with the Notch1/Hes1 signaling pathway to regulate glial differentiation. Fig. 7. The mrna levels of Hes1, Hes5, and Notch1 were altered in Hes1-overexpressing cells. Embryonic day 14.5 rat spinal cord cells were infected by Hes1 or alkaline phosphatase (AP) virus. Infected cells were selected then replated in differentiation culture condition. RNA from infected cells was isolated at day 0, 2, 4, and 6 and used for reverse transcriptase-polymerase chain reaction analyses. G3PDH (25 cycles) was used as a control. The levels of Hes1 mrna were higher in Hes1-infected cells than in controls due to expression from the virus. Hes5 levels were down-regulated by Hes1 as early as day 0 and Notch1 mrna was decreased starting at day 4. that glial differentiation involves progressive restriction in cell fate regulated at multiple stages by distinct factors (Lee et al., 2000). Factors that regulate oligodendrocyte differentiation include Nkx2.2, Olig1/2, Neurogenin3 (Lee et al., 2003), Neuregulin (Vartanian et al., 1999), and PDGF (Fruttiger et al., 1999). We and others have noted that BMPs and CNTF can regulate astrocyte differentiation (Gross et al., 1996; Bonni et al., 1997; Nakashima et al., 2001) and Shh may inhibit astrocyte differentiation (unpublished results). To our knowledge, no transcription factor except Hes1 has been shown to promote astrocyte differentiation and repress oligodendrocyte differentiation. In this study, we have shown that overexpression of Hes1 and active Notch1 promote the differentiation of astrocytes from tripotential glial progenitors (GRPs) and inhibit the differentiation of oligodendrocytes. This result is consistent with previously published reports on the instructive role of Notch in promoting glial differentiation during Schwann, Müller, and radial glial cell differentiation (Furukawa et al., 2000; Gaiano et al., 2000; Hojo et al., 2000; Morrison et al., 2000). Our results extend these observations to suggest that Notch1/Hes1 signaling may bias the fate choice between two glial cell fates, in addition to affecting the neuron astrocyte fate choice (Morrison et al., 2000). Stage- and Cell-Specific Effects of Hes Genes Although our study shows that overexpression of Hes1 in GRP cells powerfully drives the cells to an astrocyte fate, it also shows that overexpression of Hes1 in NEP cells could not promote an astrocyte fate. This finding suggests that the effect of Hes1 on gliogenesis is stage- and celltype specific. NEP cells may not be competent to differentiate into astrocytes in response to Hes1. This result is also consistent with the results from other labs. For example, Hes1 and Hes5 do not promote astrocyte fate in embryonic telencephalic stem cells (Ohtsuka et al., 2001). Mechanism of Action of Hes1 Our study suggests that Hes1 plays a decisive role in driving GRPs to adopt an astrocyte fate. Different molecular mechanisms could explain this activity. First, Hes1 could down-regulate genes in oligodendrocyte pathway, allowing GRPs to differentiate as astrocytes. In this scenario, the increased number of astrocytes in Hes1-overexpressing cells is a consequence of the default differentiation fate of tripotential precursors. In addition, some basic helix-loop-helix (bhlh) transcription factors have been shown to negatively regulate astrocyte differentiation. Neurogenin1 (Ngn1), for example, can inhibit astrocyte development by sequestering CBP/ Smad away from astrocyte specific gene promoters (Sun et al., 2001). Hes1, as a transcriptional repressor, could antagonize these inhibitory effects on astrocyte differentiation and, therefore, increase the percentage of astrocytes in culture. Our results show that Hes1 overexpression inhibits the expression of transcription factors such as Nkx2.2, Olig1, and Mash1, all of which are implicated in the oligodendrocyte differentiation pathway. This finding is consistent with the mechanism of inhibiting oligodendrocyte factors by Hes1 to inhibit oligodendrocyte differentiation and, as a result, bias GRPs to the astrocyte fate. Although we believe that inhibition of oligodendrocyte fate is one possible mechanism of promoting the astrocyte fate, we also believe that this mechanism alone cannot explain all of our observations. The induction of astrocyte differentiation by Hes1 is rapid. By day 3 in low bfgf

10 684 WU ET AL. medium, greater than 70% of the cells differentiated into astrocytes in the absence of any other astrocytepromoting factors. In contrast, it takes at least 5 days for GRPs to differentiate into astrocytes in the presence of BMPs, CNTF, or serum (Rao et al., 1998), and the potency of these factors is less than what we observed for Hes1 overexpression. In addition, we found that, although both Hes1 and Hes5 inhibit oligodendrocyte differentiation, only Hes1 promotes astrocyte differentiation. Thus, the induction of astrocyte differentiation by Hes1 may not be solely due to the inhibition of oligodendrocyte differentiation. Hes1 may directly activate astrocyte-specific gene expression or synergistically interact with other astrocytepromoting proteins. So far, only two families of transcription factor, Smads and STATs, activated by BMPs and CNTF/LIF, respectively, have been shown to promote astrocyte differentiation (Bonni et al., 1997; Nakashima et al., 1999). In future experiments, we would like to explore the possible interactions between Notch/Hes and BMP/Smad or CNTF/ STAT pathways. Notch1 Affects the Fate of GRPs by Means of Hes1 but Not Hes5 Hes1 has been shown to be the downstream target of Notch1 in many systems. However, several observations suggest that Notch1 may not always act through Hes1. First, active Notch1 fragment inhibits muscle cell differentiation without Hes1 up-regulation (Shawber et al., 1996b). Second, the expression of Hes5 but not Hes1 is up-regulated upon Notch activation by Delta in O2A cells (Wang et al., 1998). Third, the total mrna of Hes5 but not Hes1 is down-regulated in targeted mutations of Notch1 and RBP-Jk at E9 mice (de la Pompa et al., 1997). Finally, the expression of Hes1 can also be regulated by factors other than Notch1 (Feder et al., 1993; Issack and Ziff, 1998). These observations suggest that the association of Notch1 and Hes1 may be cell type or stage dependent. Our results show that overexpression of active Fig. 8. Hes5 inhibits both astrocyte and oligodendrocyte differentiation. Embryonic day 14.5 rat spinal cord cells were infected by Hes5 or green fluorescent protein (GFP) virus and selected by G418 then replated in differentiation culture condition. RNA from infected cells was isolated at day 0, 2, and 6 and used for reverse transcriptase-polymerase chain reaction analyses. The levels of Hes5 mrna in Hes5-infected cells were increased compared with GFP controls due to expression from the virus. The mrna levels of both an astrocyte marker (glial fibrillary acidic protein [GFAP]) and oligodendrocyte markers (proteolipid protein [PLP]/DM20 and CNPase) were decreased in Hes5-infected cells compared with GFP controls. Notch1 does have an effect on gliogenesis similar to that of Hes1, and the Hes1 mrna is up-regulated in cells overexpressing Notch1. This finding suggests that, at this particular stage, Notch1 may act by means of Hes1 to promote astrocyte fate at the expense of oligodendrocyte fate. Hes1 and Hes5 Have Different Roles in Gliogenesis in Developing Spinal Cord Our result shows that Hes1 and Hes5 play distinct roles in regulating gliogenesis in the developing spinal cord. Although both proteins inhibit oligodendrocyte differentiation, Hes1 drives GRP cells to become mature astrocytes at the expense of oligodendrocyte fate, whereas Hes5 maintains GRP cells at a precursor stage. This observation is important because previous studies more or less suggest that these two proteins play similar roles in regulating cell fates. Both proteins promote the Müller cell fate in retina (Furukawa et al., 2000; Hojo et al., 2000), and misexpression of either of these genes in telencephalon inhibits neuronal differentiation (Ohtsuka et al., 2001). However, some data also suggest that Hes1 and Hes5 may not always act in the same way. For example, Hes1 null mutant mice die around birth, whereas Hes5 null mutant mice do not show any obvious abnormality (Ohtsuka et al., 1999). When O2A cells are induced to differentiate into oligodendrocytes, the level of Hes5 but not Hes1 decreases (Kondo and Raff, 2000). Our study clearly shows that Hes1 and Hes5 function very differently in regulating gliogenesis in the developing spinal cord. Forced-expression in our experiments by using general mammalian promoters suggests that the protein structures themselves are likely to contribute to their different activities in our system. Although previous studies show Hes1 and Hes5 share three evolutionary conserved regions (bhlh domain, orange domain, and WRPW motif in the carboxyl-terminal region), Hes1 and Hes5 are in fact structurally quite different outside these domains. Whereas the Hes1 protein has 281 amino acids, the Hes5 protein is

11 Hes AND GLIOGENESIS 685 Fig. 9. Notch1 has an effect on gliogenesis like that of Hes1. A: Embryonic day 14.5 rat spinal cord cells were infected by mouse Notch1 intracellular domain (mnid) or alkaline phosphatase (AP) virus, and the infected cells were selected then replated in differentiation culture conditions. RNA from infected cells was isolated at day 0, 2, 4, and 6 and used for reverse transcriptase-polymerase chain reaction analyses. Primers designed specifically for misexpressed mouse NID were used to detect the overexpressed mouse Notch1. Although high levels of mouse Notch1 mrna were detected in mnid-infected cells, no exogenous mouse Notch1 was detected in AP-infected rat cells (lane a). The mrna levels of Hes1 were up-regulated, and the higher levels were maintained until day 6 (last time point examined; lane b). Except for day 0, similar levels of Hes5 expression were seen in control and in mnid cultures (lane c). The mrna levels of glial fibrillary acidic protein (GFAP) were also up-regulated by mnid (lane d). The mrna levels of the oligodendrocyte markers CNPase and PLP/DM20 were down-regulated (lanes e and f). The mrna level of Olig1 was also down-regulated in mnid infected cells (lane g). B: AP- or mnid-infected cells were cultured in reduced basic fibroblast growth factor for 3 days. The cells were stained for GFAP. Although only 4 1% of AP-infected cells were GFAP, 34 2% of mnid-infected cells were GFAP. PLP, proteolipid protein. much shorter, comprising only 166 amino acids. The orange domain WRPW interval in Hes5 (36 amino acids) is much shorter than that in Hes1 (125 amino acids). This finding could contribute to the difference of Hes1 and Hes5, because this region has been shown to be important for the function of E(Spl), as transgenic fly mutants with truncated sequence in this region acts as dominant negative variants (Giebel and Campos- Ortega, 1997). It has also been shown that Her4, a zebrafish homologue of E(Spl), loses its effect on neuronal development when the orange domain WRPW interval is deleted (Takke et al., 1999). The different amino acids in the orange domain could also contribute to the differences between Hes1 and Hes5 activities, because these amino acids are known to confer functional specificity among members of the Hairy/E(Spl) family. Domain swapping experiments in Drosophila showed that the orange domain from E(Spl) m8 cannot substitute for the orange domain of Drosophila Hairy, but the orange domain from frog Hairy can (Dawson et al., 1995). In conclusion, we have shown that both Hes1 and Hes5 play important but distinct roles in regulating astrocyte differentiation. Whereas Hes1 promotes astrocyte differentiation of GRPs at the expense of oligodendrocytes, Hes5 keeps GRPs at a precursor stage. The astrocyte-promoting effect of Hes1 is stage specific as Hes1 does not promote astrocyte fate of NEP cells. Our results strongly suggest that Hes1 is a downstream effector of Notch1 in GRPs. Our finding provides another example of the complicated roles that the Notch- Hes signaling pathway has during development. Further experiments will be aimed to identify the downstream molecular targets of Hes genes in spinal cord gliogenesis. EXPERIMENTAL PROCEDURES Preparation of Retrovirus The rat Hes1 and Hes5 cdna were kindly provided by Dr. R. Kageyama. Hes1 cdna was cut with XhoI and cloned into the viral vector plxsn (Clontech). AP viral construct, plapsn (Clontech), was used as a control for plxsn-hes1. For some experiments, Myc tag epitopes (six copies) from the pcs2-mt vector was cloned to the N-terminus of Hes1 in plxsn vector and the protein expression and distribution were detected with anti-myc antibodies. Hes5 was cut with EcoRI and cloned

12 686 WU ET AL. into viral vector plncx (Clontech). Enhanced green fluorescence protein (GFP, Clontech) cloned into plncx was used as a control for plncx-hes5. The cdna of cytoplasmic domain of mouse Notch1 (mnid), initiating at amino acid 1753 was cut from plasmid Notch-IC/GFP (Furukawa et al., 2000) and cloned into plxsn. The viral constructs were transfected with Fugene6 (Roche) into Phoenix cells, a viral packaging cell line kindly provided by Dr. G. Nolan. The culture medium was replaced with NEP basal medium 24 hr later for supernatant. The viral supernatant was collected twice and stored at 80 C for future use. Culturing and Infection of Spinal Cord Neural Precursor Cells Timed-pregnant Sprague-Dawley rats were obtained from Simonsen. Neural progenitor cells were isolated from the spinal cord as described previously (Mayer-Proschel et al., 1997; Rao et al., 1998). Briefly, NEP cells isolated from E10.5 spinal cord were plated on fibronectin (Sigma) -coated dishes and cultured in NEP basal medium with 10% chicken embryo extract and 25 ng/ml bfgf (Peprotech). E14.5 spinal cord cells were plated on poly-l-lysine (Sigma) and laminin (Gibco/BRL) -coated dishes and cultured in NEP basal medium with 25 ng/ml bfgf. The viral supernatant was added at a ratio of 1:3, 18 hr after plating. The cells were incubated with virus for a period of 8 to 10 hr. The virus-containing medium was replaced with NEP basal medium in the presence of bfgf. G418 (200 g/ml, Sigma) was added into the culture medium 48 hr after infection. G418 selection was carried out for a period of 72 hr until all the cells died in an uninfected control dish. Selected cells after infection were replated on poly-l-lysineand laminin-coated dishes, and the cell numbers were matched with each dish to avoid the effect of cell density on cell differentiation. To induce spontaneous cell differentiation (differentiation culture condition), selected cells were cultured in low concentration of bfgf (5 ng/ml) for 2 days and then placed Fig. 10. Quantification and summary of astrocytic and oligodendrocytic markers expressed in infected cells. The graph summarized the counting of glial fibrillary acidic protein positive (GFAP ) and proteolipid protein positive (PLP ) cells in Hes1, Hes5, Notch1, and control (green fluorescent protein [GFP] and alkaline phosphatase [AP]) -infected embryonic day 14.5 spinal cord cells. To determine the effects of the genes on astrocyte differentiation, infected cells were selected by G418 and cultured under differentiation culture conditions for 3 days. The cells were then stained with anti-gfap, and the positive cells were counted. Both Hes1 and mnid promote astrocyte differentiation compared with AP controls. In contrast, Hes5 inhibits astrocyte differentiation compared with GFP controls. To determine the effects of the genes on oligodendrocyte differentiation, infected cells were cultured in oligodendrocyte-promoting conditions (10 ng/ml plateletderived growth factor, 30 ng/ml T3, and 0 5 ng/ml basic fibroblast growth factor) for 5 days. The cells were then stained with anti-plp antibody, and the positive cells were counted. All three genes inhibit PLP expression. Data are based on three independent experiments in which 500 1,000 cells per experiment were counted for each marker. Statistical significance was determined by unpaired t-test. in medium without bfgf for further differentiation. Under these conditions, only a small percentage of normal GRP cells ( 10%) can differentiate into astrocytes or oligodendrocytes over a long period in culture. Astrocyte markers (GFAP and CD44) were monitored in infected cells everyday for a period of 6 days. To promote oligodendrocyte differentiation (oligodendrocyte-promoting condition), 10 ng/ml human platelet-derived growth factor BB (PDGF, Upstate Biotechnology) and 30 ng/ml 3,3, 5-triiodo-thyronine (T3, Sigma) were added into NEP basal medium. The medium included 5 ng/ml bfgf for the first 2 days. The bfgf was omitted when the medium was replaced. In this oligodendrocyte-promoting condition, approximately 15 20% of GRP cells can differentiate into mature oligodendrocytes within 6 days. A greater proportion of GRP cells can differentiate into oligodendrocyte if cells are cultured for longer time periods (for example, Gregori et al., 2002). Oligodendrocyte markers (PLP and GalC) were monitored in infected cells every day for a period of 6 days. Immunocytochemical Staining For staining of cell surface markers, live cells were incubated in primary antibodies at room temperature for 1 hr followed by three washes with medium. The live cells were then incubated in appropriate secondary antibodies for 15 min. For staining of cytoplasmic and nuclear antigens, the cultured cells were fixed with 2% paraformaldehyde. Fixed cells were incubated in primary antibodies at 4 C overnight. After washes with PBS, the fixed cells were incubated in appropriate secondary antibodies for 15 min. The following antibodies were used: mouse anti-a2b5 (1:5, ATCC), rat anti-plp/dm20 (1:1, Dr. A. Gow), mouse anti-galactocerebroside (GalC, 1:3, Dr. B. Ranscht), mouse anti-nkx2.2 (1:1, Develop-

13 Hes AND GLIOGENESIS 687 TABLE 1. Size, Sequence, and Annealing Temperature for Each RT-PCR Reaction a Name Size (bp) Sequence Annealing temperature ( C) G3PDH acc aca gtc cat gcc atc ac 3 55 G3PDH 3 5 tcc acc acc ctg ttg ctg ta 3 Olig aag gag gac att tcc aga ctt c 3 55 Olig1 3 5 gct cta aac agg tgg gat tca tc 3 PLP/DM /400 5 gac atg aag ctc tca ctg gca c 3 55 PLP/DM cat aca ttc tgg cat cag cgc 3 GFAP gaa acc aac ctg agg ctg gag 3 55 GFAP 3 5 ggc gat agt cat tag cct cg 3 Hes cac gct cgg gtc tgt gct gag agc 3 58 Hes1 3 5 atg cca gct gat ata atg gag 3 Hes gtg gag atg ctc agt ccc aag 3 55 Hes5 3 5 tgt agt cct ggt gca ggc tc 3 CNPase ccg gag aca tag tgc ccg ca 3 56 CNPase 3 5 aaa gct ggt cca gcc gtt cc 3 Rat Notch tca agg ccc gga gga aga agt c 3 61 Rat Notch1 3 5 tca ggg gat ggg gtg agg aag Mouse Notch gga gga cct cat caa ctc aca t3 58 Mouse Notch1 3 5 cct tga ggt cct tag ctt cct t3 a RT-PCR, reverse transcriptase-polymerase chain reaction; PLP, myelin proteolipid protein; GFAP, glial fibrillary acidic protein. mental Studies Hybridoma Bank [DSHB]), mouse anti-rat CD44 (1:25, Dr. L. Sherman), and rat anti-brdu (1: 10, Harlan) were used as crude hybridoma supernatants. Mouse antimyc (9E10, 1:500, Dr. J. Janatova), rabbit anti-gfap (1:500, Dako), and mouse anti-brdu (1:100, Sigma) were used as purified antibodies. Western Blots Infected cells were replated in 100-mm tissue culture dishes coated with poly-l-lysine and laminin. The cells were cultured in NEP basal medium containing reduced bfgf (5 ng/ml). Two or five days later, approximately cells were collected from each dish for Western blotting. Cells were incubated and then dissociated in freshly made RIPA lysis buffer (1% Na-deoxycholate, 0.1% sodium dodecyl sulfate, 1 g/ml pepstatin, 50 M sodium fluoride, 1 M sodium orthovanadate, 20 mm Tris-Cl, ph 8.0, 0.14 M NaCl, 10% glycerol, 2 mm ethylenediaminetetraacetic acid, 1% N-P40, and 0.025% NaN 3 )at4 C for 10 min followed by sonication. Insoluble debris was removed by centrifugation. The concentration of the protein was measured by Bio- Rad protein assay (Bio-Rad). Protein mixtures (35 g) were run on precast gradient Tris-glycine gels (Invitrogen) and then transferred to high-bond ECL membranes. The membranes were probed with anti-myc (1:10,000, Dr. J. Janatova), anti- Nkx2.2 (1:1, DSHB), anti-mash1 (1:100, PharMingen), anti-gfap (1:250, Transduction Laboratories), anti-cnpase (1:250, Sigma), and anti- -tubulin (1:5,000, Sigma). Blots were developed by using ECL reagent (Amersham). Semiquantitative RT-PCR Total RNA was extracted from cells by using Trizol (Gibco/BRL). The concentration of total RNA was assessed by spectrophotometry and adjusted roughly to the same level among samples. First-strand cdna was synthesized by using SuperscriptII (Gibco/BRL) and Oligo (dt) (Gibco/BRL). RT-PCR was carried out in a 30- l reaction mixture. Primers for a housekeeping gene (G3PDH) were used as a control (25 cycles). To ensure that PCR cycles ended before saturation, generally, the cycle number for each primer was first tried at 28 cycles and then decreased or increased 1 4 cycles, depending on the intensity of the initial PCR products. Therefore, each group of samples were run for PCR with at least two or three different cycle numbers and the reaction showing clear bands with the lowest cycle number was shown in the figures. Each overexpression experiment was repeated three times. The result shown in each figure was from one representative experiment. The size, sequence, and annealing temperature for each RT-PCR reaction are listed in Table 1. BrdU Incorporation Assay After G418 selection, infected cells were replated on 35-mm dishes at a density of cells/ dish. The cells were cultured in NEP basal medium with 20 ng/ml bfgf for 12 hr, then exposed to 10 M BrdU for an additional 2.5, 5, or 10 hr. The cells were then fixed and processed for BrdU immunostaining. FACS To isolate A2B5 GRPs, dissociated E14.5 spinal cord cells were plated on poly-l-lysine and laminincoated dishes and cultured in NEP basal medium in the presence of 25 ng/ml bfgf for 2 days. The cells were then incubated with anti-a2b5 antibodies (4.5 g/ml, Chemicon) at room temperature for 1 hr. The unbound antibodies were washed out. The cells were incubated with Cy3-

14 688 WU ET AL. conjugated anti-mouse IgM secondary antibody (1:200, Jackson ImmunoResearch) for 15 min. After washing, the cells were dissociated with trypsin and resuspended at a density of cells/ml in culture medium. FACS was performed with a FACS Vantage flow cytometer (Becton Dickinson Immunocytometry Systems) at 4 C. A2B5 sorted cells were checked by immunostaining and found to have a purity of 95%. To isolate GalC oligodendrocytes, dissociated postnatal day 0 (P0) rat spinal cord cells were cultured in NEP basal medium in the presence of 10 ng/ml PDGF and 30 ng/ml T3 for 5 days. The cells were then stained with anti-galc hybridoma antibody supernatant (1:3) and the GalC cells were sorted as described above. The purity of the cells was 90% and the contamination of GFAP astrocytes was less than 5%. In Situ Hybridization Mouse embryos were fixed in 4% paraformaldehyde at 4 C for approximately 5 to 15 hr, depending on the ages of the embryos. Cryostat sections were cut at m. Digoxigenin (DIG, Roche) labeled antisense RNA probes, transcribed from mouse Hes1 (1 kb) and Hes5 (1.7 kb) cdna vectors, were detected with AP-conjugated anti-dig (Roche). BM purple (Roche) was used as an AP substrate. ACKNOWLEDGMENTS We thank Dr. R. Kageyama for Hes1 and Hes5 constructs, Dr. G. Nolan for the Phoenix cell line, Dr. A. Gow for the PLP antibody, Dr. B. Ranscht for the GalC antibody, Dr. J. Janatova for the 9E10 anti-myc antibody, and Dr. L. Sherman for the CD44 antibody. The Nkx2.2 antibody was obtained from the Developmental Studies Hybridoma Bank (DSHB). We thank T. Mujtaba, J. Stubbs, and H. Xue for excellent technical assistance. We also thank T. Van Raay, B. Arenkiel, Dr. J. Lee, Dr. M. Vetter, and Dr. E. Green for critical reading of the manuscript. M.S.R. received funding from NIA, NINDS, and NIDA. E.M.L. was supported by the Department of Ophthalmology and Geron. 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