Sloppy paired 1/2 regulate glial cell fates by inhibiting Gcm Function

Sloppy Paired 1/2 Regulate Glial Cell Fates by InhibitingGcm FunctionSOMA MONDAL,1,2 STACEY M. IVANCHUK,1,2 JAMES T. RUTKA,1,2,3* AND GABRIELLE L. BOULIANNE4

1The Arthur and Sonia Labatt Brain Tumour Research Center, Hospital for Sick Children, Toronto, Ontario, Canada2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada3Divisions of Neurosurgery, Hospital for Sick Children, Toronto, Ontario, Canada4Program in Developmental Biology, Department of Molecular and Medical Genetics, Hospital for Sick Children,University of Toronto, Toronto, Ontario, Canada

KEY WORDSsloppy paired; Foxg1; BF-1; glial cells missing; glia; neu-rons

ABSTRACTOrganization of the central nervous system during embry-onic development is an intricate process involving a host ofmolecular players. The Drosophila segmentation genes,sloppy paired (slp) 1/2 have been shown to be necessary fordevelopment of a neuronal precursor cell subtype, the NB4-2 cells. Here, we show that slp1/2 also have roles in regu-lating glial cell fates. Using slp1/2 loss-of-functionmutants, we show an increase in glial cell markers, glialcells missing (gcm) and reversed polarity. In contrast, mis-expression of either slp1 or slp2 causes downregulation ofglial cell-specific genes and alters the fate of glial and neu-ronal cells. Furthermore, we demonstrate that Slp1 and itsmammalian ortholog, Foxg1, inhibit Gcm transcriptionalactivity as well as bind Gcm. Taken together, these datashow that Slp1/Foxg1 regulate glial cell fates by inhibitingGcm function. VVC 2006 Wiley-Liss, Inc.

INTRODUCTION

Sloppy paired (Slp)-1/-2 and Foxg1 (formerly known asBF-1) proteins belong to the forkhead domain-containingfamily of transcription factors that play essential rolesduring Drosophila and vertebrate embryogenesis,respectively (Cadigan et al., 1994b; Dou et al., 1999;Grossniklaus et al., 1992; Hacker et al., 1992; Huh etal., 1999; Tao and Lai, 1992). The slp locus encodes twostructurally related genes, slp1 and slp2 (Grossniklauset al., 1992). Together, this family of proteins regulatestranscription directly by binding to DNA targets via theforkhead domain, or indirectly, by binding to and affect-ing the activity of other transcription factors. Slp1 andSlp2 have been shown to function both as transcrip-tional activators and repressors. One of the best-charac-terized roles for Slp proteins is in segmentation duringembryogenesis where Slp1 functions as an activator ofwingless and a repressor of engrailed, thus ensuringproper embryonic segmentation (Cadigan et al., 1994b).

In addition to their role in segmentation, Slp1/2 havealso been shown to regulate neuronal differentiation.Specifically, Slp1/2 act downstream of wingless signalingand are required for the specification of the NB4-2

neural precursor cells (Bhat et al., 2000). During Dro-sophila embryonic development, neurons arise from pre-cursor cells called neuroblasts (NB) or neuroglioblasts;the latter also give rise to glial cells (Bossing et al.,1996; Schmidt et al., 1997). The NB4-2 cells represent asubtype of neuronal progenitor cells. Lineage analysis ofNB4-2 indicates that this precursor gives rise to theRP2 motor neuron, RP2sib, and a number of interneur-ons (Bossing et al., 1996; Chu-LaGraff et al., 1995;Schmid et al., 1999). Loss-of-function mutations in slp1/2 result in formation and specification defects of NB4-2cells, leading to RP2/sib lineage specification failure.Conversely, misexpression of Slp1/2 transforms the fateof NB5-3 precursors to NB4-2, thus generating addi-tional RP2/sib neurons (Bhat et al., 2000). While Bhatet al. reported the importance of Slp1/2 in the genera-tion of a subset of NB cells, the effect on glial cell fateswas not determined.

Here, we investigated the role of Slp1/2 in regulatingglial cell fates during Drosophila embryogenesis. Wefind that embryos lacking slp1 and slp2 show increasednumbers glial cells. Conversely, misexpression of slp1and slp2 in Drosophila embryos during CNS develop-ment leads to a repression of glial cell fates. In efforts toelucidate mechanisms contributing to the regulation ofglial cell fates by Slp1/2, we examined the ability of Slp1and its murine ortholog, Foxg1, to modulate Gcm activ-ity. We find that both Slp1 and Foxg1 can repress tran-scriptional activity of Drosophila and murine Gcm,respectively, in cell culture. Furthermore, we demon-strate that Slp1 binds Gcm and that this interaction isconserved in murine protein orthologs. Taken together,our studies are consistent with a model in which theSlp/Foxg1 family of forkhead domain containing proteins

This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0894-1491/suppmat.

JTR is a CIHR Investigator.

GLB is the recipient of a Canada Research Chair in Molecular and Developmen-tal Neurobiology.

Grant sponsor: Canadian Institutes for Health Research; Grant sponsor: Brainchild.

*Correspondence to: James T. Rutka, The Division of Neurosurgery, Suite 1504,The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, CanadaM5G 1X8. E-mail: [email protected]

Received 8 March 2006; Revised 5 October 2006; Accepted 6 October 2006

DOI 10.1002/glia.20456

Published online 7 November 2006 in Wiley InterScience (www.interscience.wiley.com).

GLIA 55:282–293 (2007)

VVC 2006 Wiley-Liss, Inc.

plays an essential role in regulating glial fates by inhibi-ting Gcm function.

MATERIALS AND METHODSFly Strains

The wildtype strain was Oregon-R. D34B fly strain isnull for both slp1 and slp2; transgenic fly lines hs-slp1and hs-slp2 (kind gifts from Ken Cadigan, University ofMichigan) carrying slp1 and slp2 under the regulationof hsp70 were used for the experiments described. Theenhancer trap insert (CyO P[lArB]A208.1M2) fromwhich D34B was derived was used as a control. Flieswere grown on standard media and maintained at 25�C.Other fly strains used include: L2Pin1/CyO, GFP, Ly/TM6, Tb, Sb, P(TwiLacZ), sca-GAL4, and UAS-gcm(kind gift from Yasui Hiromi, National Institute ofGenetics, Japan).

Embryo Collections and Manipulation

Embryos were collected on grape juice/agar plates at25�C for 1 h. The heat shock and recovery regimen usedwas as follows: embryos were placed at 25�C for 4.5 h,heat shocked for 1.5 h at 37�C, allowed to recover at25�C for 1 h, and then heat shocked again for 1 h. Atthis time, eggs were either collected for analysis ofearly glial determination markers or were left at 25�Cuntil Stage 17 of embryonic development as described(Campos-Ortega and Hartenstein, 1997).

RNA Probe Preparation and FISH Analysis

The coding region of the gcm cDNA was PCR ampli-fied and subcloned into pBS (SK) vector to generatesense and antisense RNA probes. Digoxigenin-labeledRNA probes were generated according to manufacturer’sinstructions (Roche, Mississauga, Ontario). Fluorescentin situ hybridization (FISH) studies were performed asper Hughes and Krause (1998). Signal detection wasperformed using a tyramide signal detection system asper manufacturer’s instructions (Molecular Probes,Eugene, OR).

Immunohistochemistry

Staged and heat shock treated embryos were collected,dechorionated in 50% bleach, and rinsed with water.Embryos were fixed in 50% heptane/50% PEMS-formal-dehyde for 15 min. Methanol/heptane was used for devi-tellinization followed by rinsing in PBS-0.3% TritonX-100 (PBT). Embryos were blocked for 30 min in PBTcontaining 2% normal goat serum (Jackson Biolabs,West Grove, PA.). Both primary and secondary antibo-dies were diluted in blocking solution. Primary antibo-dies used included: rat anti-Slp1 (gift from Dr. K. Cadi-

gan, University of Michigan), anti-Repo (DSHB, IowaCity, IA), rat anti-elav (DSHB), mAb22c10 (DSHB), anti-fasII (DSHB). Embryos were incubated with primaryantibodies overnight and secondary antibodies for 1 h.Embryos were washed in PBT and mounted onto glasscoverslips using mounting solution (2% DABCO in 70%glycerol) and visualized using a Zeiss confocal micro-scope. Image capture was done using LSM software(Zeiss) and final image analysis was performed usingAdobe Photoshop 7.0.

Promoter Assays

A 6xgbs-luciferase vector, carrying six tandemlyarranged Gcm binding sites (kindly provided by M.Wegner, Erlangen University, Erlangen, Germany),wasused for all luciferase assays described. Briefly, COS-7cells were transfected with 50 ng of CMV-b-galactosi-dase (b-gal) plasmid, 100 ng 6xgbs-luciferase reporterconstruct, and varying concentrations of GCM andFoxg1. The total transfected DNA concentration waskept constant (7 lg) for all transfections. Transfectionswere performed in triplicate and all reporter gene quan-tification assays were read in duplicate. Twelve hourspost transfection, cells were lysed and prepared for lucif-erase activity determination using a Luciferase AssaySystem (Promega); samples were read on a luminometer.b-Gal activity was determined using a Galacto-LightPlus kit (Applied Biosystems, Foster City, CA) as permanufacturer’s instructions. Luciferase assay readingswere normalized against b-gal readings to account forvariations in transfection efficiency.

Yeast Two Hybrid Screen

The full-length (m)GCM-2 cDNA was PCR amplifiedand subcloned into pGBT9 vector (Clontech, Palo Alto,CA) to create a GAL4 DNA binding domain (DBD)-mGCM-2 fusion construct. The bait construct was trans-formed into the HF7C yeast cells using a standardLiOAc transformation protocol (Gietz et al., 1992).Expression of the fusion protein was confirmed by wes-tern blotting using an anti-GAL4 DBD antibody (datanot shown). A total of 1 3 108 clones from a human fetalbrain cDNA library (Clontech, Palo Alto, CA) werescreened. DNA isolated from positive clones was sub-jected to DNA sequencing followed by BLAST searchanalysis against all known sequences in GenBank todetermine identity.

DNA Expression Constructs and Transfections

Full-length mGCM-1 and -2 cDNAs were PCR ampli-fied from pBS-mGCM-1 and -2, respectively and sub-cloned into pcDNA-myc/his expression vectors (Invi-trogen, Mississauga, ON.). Regions of interest were PCRamplified and subcloned into the pcDNA-myc/his expres-

283SLP REGULATES GLIAL CELL FATES

GLIA DOI 10.1002/glia

sion vector to generate truncation mutants of mGCM-2.Drosophila gcm was PCR amplified from pBS-gcm andsubcloned into pcDNA-myc/his. pFlag-CMV-Foxg1(pFlag-Foxg1) and pBS-slp1 constructs were giftsfrom Dr. E. Lai (Memorial Sloan-Kettering, NY) andDr. K. Cadigan (University of Michigan, MI), respec-tively. Regions of interest of Foxg1 were PCRamplified and subcloned into the pFlag-CMV vector tocreate desired truncation mutants. Full length slp1cDNA was PCR amplified and subcloned into the pFlag-CMV (pFlag-slp1) expression vector (Sigma-Aldrich,Mississauga, ON).

COS-7 cells were transfected using Fugene transfec-tion reagent (Roche, Mississauga, ON) as per manufac-turer’s instructions.

Coimmunoprecipitation and WesternBlot Analyses

COS-7 cells were lysed in TNTE buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% triton-X 100, 1 mM EDTA)and cell debris was pelleted by centrifugation. Total pro-tein concentration was quantified using BioRad Assayreagent. Four-hundred micrograms of total cell lysatewere incubated with anti-myc tag antibody (UBI, LakePlacid, NY) overnight at 4�C to coimmunoprecipitatecomplexes. Incubation in protein G-sepharose was per-formed for 1 h at 4�C to bind immunocomplexes. Pro-tein-bound sepharose resin was washed in TNTE washbuffer (containing 0.1% triton-X 100) and proteins wereeluted by boiling in Laemmli sample buffer. Proteinswere separated by SDS-polyacrylamide gel electrophor-esis (PAGE).

Glutathione- S-Transferase-TaggedProtein Production

Full length slp1 cDNA was PCR amplified and sub-cloned into pGEX-2T vector (Pharmacia) to generate aglutathione-S-transferase (GST)-tagged Slp1 fusion con-struct (GST-Slp1). pGEX-2T-slp1 was transformed intoDH5a bacteria and induced using IPTG (to 1 mM) for3 h at 37�C. Cells were harvested by centrifugation andlysed in FPLB (50 mM Tris-HCl, 150 mM NaCl, 0.5%triton-X 100, 1 mM EDTA) by sonication. Cell debriswas pelleted and the supernatant was incubated withglutathione-sepharose for 1 h at 4�C. Protein-bound glu-tathione-sepharose resin was washed with FPLB bufferand a portion (5 lL) was reserved for protein quantita-tion. Proteins were eluted by boiling in Laemmli samplebuffer, separated by SDS-PAGE, and visualized byCoomassie Blue staining.

GST-Pull Down Assays

One microgram of GST-tagged Slp1 protein and GST-alone was incubated with 500 lg of total COS-7 cell

lysate carrying transfected myc-Gcm for 1 h at 4�C thenwashed in TNTE wash buffer. Proteins were eluted asdescribed, separated by SDS-PAGE, electrotransferredonto PVDF membrane, and western blotted using anti-myc tag antibody.

Electrophoretic Mobility Shift Assays

Radiolabeling oligonucleotides

A single gbs containing double stranded oligonucleotideprobe was end-labeled ([g-32P]ATP) and used as a probein all electrophoretic mobility shift assay (ESMSA) stu-dies. Briefly, 200 ng of annealed oligonucleotide wereincubated with 103 T4 polynucleotide kinase buffer,60 lCi of [g-32P]ATP, and 1 lL of T4 polynucleotidekinase at 37�C for 90 min after which unincorporatedlabel was removed. The sequence of the oligonucleotideis given below:

� 50-GATCCCG ATGCGGGT GCAGATC-30

� 30-CTAGGGC TACGCCCA CGTCTAG-50

EMSA reactions

COS-7 cell were transfected with appropriate plasmidsto independently express myc-mGCM-2, myc-Gcm, flag-Foxg1, or flag-Slp1. Lysates from transfected cells werecollected as described. Mixtures of appropriate lysatecombinations were used in EMSA analysis. Briefly, 5–10 lg of total lysate were used in 15 lL reactionvolumes containing 4 3 104 cpm of labeled probe (�0.1ng), 60 mM HEPES (pH 7.9), 40 mM KCl, 2.5 mMMgCl2, 4% Ficoll, 0.3 mM EDTA, and 0.3 mM DTT.Reaction mixtures were incubated for 30 min on icebefore separation of DNA-protein complexes on a 5%nondenaturing polyacrylamide gel run in 0.25X Tris-borate EDTA buffer (0.022 M Tris-borate, 0.5 mMEDTA) at 225 V for 3–4 h at 4�C. Gels were dried andexposed to film using an intensifying screen at 280�Cfor 12 h.

RESULTSslp Mutants Exhibit Increased Glial Cell Numbers

Slp1/2 are required for the generation of certain neu-roblast fates in Drosophila. Here, we utilized slp1 andslp2 loss-of-function mutants to determine if Slp 1/2 arealso required to specify glial cell fates. Using D34Bembryos, which are null for both slp1 and slp2 (Gross-niklaus et al., 1992), we examined expression of glialcell markers. As a control, we utilized the enhancer trapinsert (CyO P[lArB]A208.1M2) from which D34B wasderived. FISH analysis, was used to detect expression ofgcm, the earliest glial cell marker (Hosoya et al., 1995;Jones et al., 1995). We find that the pattern of gcmexpression is altered in all of the mutant embryos lack-

284 MONDAL ET AL.


ing slp1 and slp2 (n 5 120) compared to age-matchedcontrols (Figs. 1A–F) or to CyO P[lArB]A208.1M2. More-over, the number of gcm staining cells is markedlyincreased (compare gcm staining in Slp1/2 positiveembryos, A–C to slp1/2 null embryos, D–F). The expres-sion of reversed polarity (Repo), a Gcm target gene thatis specifically expressed in all lateral glia (Akiyama etal., 1996; Xiong et al., 1994), was also examined; likegcm, Repo expression markedly increases in mutantembryos (Figs. 1I,J) compared to age-matched controls(Figs. 1G,H). These data indicate that the absence ofslp1/2 results in abundant gcm and Repo expression,suggesting that slp1/2 may have a role in regulatingglial cell fates.

Misexpression of slp1/2 Results in a Reduction ofGlial Cell Numbers

To further demonstrate a role for slp1/2 in regulatingglial cell fates we examined the effects of slp1/2 misex-pression. Misexpression was achieved using the trans-genic fly lines hs-slp1 and hs-slp2 where the expressionof slp1 and slp2 is regulated by the heat shock protein

70 (hsp70) promoter (Cadigan et al., 1994a). We firstconfirmed that heat shock treatments in transgenic flylines induced expression of Slp1 and Slp2 by performinganti-Slp western blots on lysates prepared from embryosthat were heat shocked between Stages 10 and 11 for 90min and then allowed to recover for 60 min. Anti-Slpwestern blot analysis showed an increase in Slp proteinexpression in treated hs-slp1 and hs-slp2 transgenicstrains compared to ORE-R embryos subjected to identi-cal treatment (data not shown).

During embryogenesis, the expression of gcm is evi-dent in the developing CNS and the peripheral nervoussystem (PNS) between Stages 9 and 15 (Hosoya et al.,1995; Jones et al., 1995). To determine if misexpressionof slp1/2 affected glial cell fates, we used heat shocktreatments to misexpress slp1 and slp2 in the respectivetransgenic fly strains prior to, and during peak Gcmexpression. Specifically, flies were heat shock treated atStage 10 for 90 min, allowed to recover for 60 min, andheat shock treated again for 60 min and recovered foran additional 60 min. Although a single heat shock wasinitially utilized to observe Slp1/2 expression by westernblotting, we found that the addition of a second heatshock during the peak period of gcm expression gave themost consistent responses in vivo. The treated embryoswere examined using both glial and neuronal markers.

Since the earliest glial cell marker is gcm, we exam-ined heat shock treated embryos for gcm transcriptexpression by FISH at Stage 13 (Fig. 2). As expected,there is strong gcm expression in the lateral glial cellpopulation in wildtype embryos where the heat shocktreatment does not appear to have any effect (2A). Bycontrast, in heat shock treated hs-slp2 transgenicembryos, �90% of the embryos displayed a markedreduction in the number of gcm positive cells (2D); andthe remaining 10% hardly expressed any gcm (n 5 125)(2E). Heat shock treated hs-slp1 transgenic embryos

Fig. 1. slp1/2 null embryos show increased numbers of gcm andRepo positive cells. Embryos collected from the D34B fly strain weredouble labeled for gcm by FISH and for Slp (A–F). Wildtype embryoshave normal numbers of gcm staining cells (A–C). In contrast, the cor-responding age-matched slp1/2 null embryos demonstrate a markedincrease in the number of gcm staining cells (D–F). Likewise, doublelabeling for Repo and Slp demonstrates similar results (G–J). Wildtypeembryos expressing both slp1 and slp2 have normal number of Repopositive cells (G, H), however, age-matched mutant slp1/2 null embryosdemonstrate excess numbers of Repo expressing cells (I, J). Scale barrepresents 50 lm. Left is anterior and ventral is downward. All imagesare lateral aspects except for C, F, H, and J which represent ventralviews.

Fig. 2. Misexpression of slp results in reduced gcm expression. Heatshock treated embryos analyzed at Stage 13 for gcm expression byFISH analysis. (A) Wildtype embryos appear to have a regular patternof gcm staining. In comparison, gcm staining in hs-slp1 and hs-slp2embryos is noticeably reduced (B–E). (B, C) Heat shock treated hs-slp1transgenic embryos where the number of gcm positive cells is reducedcompared to ORE-R. (D, E) Treated hs-slp2 embryos demonstrating amarked reduction in gcm positive cells. Scale bar represents 50 lm(Left is anterior).



also displayed a reduction in the number of gcm positivecells; however, the effects were not as marked as thoseobserved in the hs-slp2 population (2B and C), suggest-ing that Slp2 is a more potent repressor of Gcm activityin vivo. Of note, misexpression of either Slp1 or Slp2during maximal gcm expression had no effect on glialcell fates in treated embryos.

Anti-Repo immunostaining on heat shock treatedembryos at Stage 13 was also performed to determine ifslp1/2 misexpression affected its expression (Fig. 3).Repo-specific immunostaining in the hs-slp1 and hs-slp2treated flies ranges from being completely absent, indi-cating a strong effect (ST), to closely resembling ORE-Rflies subjected to identical treatments, indicating a weakeffect (WK). Repo staining appearing between the twodescribed patterns was categorized as an intermediate(INT) effect. While there is positive Repo staining evi-dent in the INT effect population, the intensity of thesignal was reduced compared to the wildtype controls.Repo staining indicated that a greater number of hs-slp2flies (86% ST; n 5 245) failed to express Repo comparedto the hs-slp1 line (65% ST; n 5 210). As a negative con-trol, wildtype embryos were subjected to the identicalheat shock regimen followed by anti-Repo immunostain-ing. Repo expression in ORE-R controls was normal.

During embryogenesis, certain glial cells serve asguidepost cells for proper axonal migration. We utilizedan anti-fasciclin II (fasII) antibody to stain motor axonsin the CNS and PNS following heat shock treatment inembryos (Supplemental Fig. 1). During late stage CNSdevelopment, anti-fasII labels three discrete bundles ofaxons per hemisegment within the longitudinal connec-

tives (Supplemental Figs. 1A,B). As previously shown,the three fasII bundles are disorganized in late stagegcm mutant embryos (Hosoya et al., 1995; Jones et al.,1995). Embryos from hs-slp1 and hs-slp2 transgeniclines subjected to heat shock treatment were collected atStage 17 and immunostained for fasII; the result is asevere disruption in fascicle structure and, in manycases, a complete absence of fascicles (SupplementalFig. 1). As with gcm and Repo staining, the effects of heatshock treatment were considerably more marked in thehs-slp2 transgenic line (Supplemental Figs.1I,J) wherenearly 100% of the embryos demonstrated severelydisrupted fascicles compared to the more varied effects inthe hs-slp1 transgenic line (Supplemental Figs. 1C–H).

Misexpression of slp2 Blocks the Effectsof gcm Overexpression

Thus far we have demonstrated that in the absence ofslp1 and slp2 there is increased gcm and Repo expres-sion. Conversely, we also show that ectopic expression ofslp1 and/or slp2 results in a repression of gcm and Repo.From these data we propose that Slp1 and Slp2 repressGcm activity in vivo. If so, then ectopic expression ofslp1/2 should repress effects of ectopic gcm expression.To test this, we examined the consequences of expres-sing a UAS-gcm transgene using a scabrous-(sca) GAL4driver in the presence or absence of hs-slp2 expression(Fig. 4). In these studies, we examined expression of the

Fig. 3. Misexpression of slp results in reduced Repo expression. Arepresentative ORE-R embryo is shown with typical Repo labeling inthe lateral glial cells. The phenotypes of the treated flies are categor-ized as weak (WK): having Repo staining resembling the ORE-R pheno-type, intermediate (INT): having positive Repo staining with disorga-nized glial patterning, and strong (ST): demonstrating a complete lackof Repo staining. Scale bar represents 50 lm (Left is anterior).

Fig. 4. The effects of ectopic gcm expression can be limited by over-expression of slp2. Transgenic flies that express Gcm under the tran-scriptional control of the sca-GAL4 alongside a heat shock slp2 trans-gene are shown. Embryos collected from transgenic flies resulted in theoverexpression of gcm with concomitant slp2 (in the presence of anapplied heat shock). In the absence of heat shock, embryos ectopicallyexpress gcm and show increased numbers of Repo positive cells (PanelA). Ectopic gcm expression concomitant with misexpression of slp2results in embryos with reduced Repo positive cells (compare Panel Dwith panel A). Panels B and E demonstrate the difference in Slp stain-ing in respective embryos with and without heatshock treatment.Panels C and F are the respective merged images.

286 MONDAL ET AL.


Gcm target, repo and found that misexpression of slp2,coincident with ectopic gcm expression, blocked thenumber of Repo positive cells as compared to the expres-sion of ectopic gcm alone (Fig. 4 compare Panels A andD). This experiment provides in vivo evidence that Slpis able to regulate the transcriptional activity of Gcm.

Slp1 and Foxg1 Repress Drosophila Gcm andmGCM-2 Transcriptional Activity, Respectively

To further investigate whether Slp1/2 can inhibit Gcmtranscriptional activity we performed promoter–reporterassays. Since GCM proteins function as transcrip-tion factors that bind to an octameric consensus (A/G)CCCGCAT DNA sequence (Akiyama et al., 1996;Schreiber et al., 1997), we sought to determine whetherSlp1 could modify the transcriptional activity of Gcm(Fig. 5A). Using an artificial 6xgcm binding site (gbs)promoter-luciferase (luc) reporter construct (Tuerk et al.,2000), we tested the activation capacity of Gcm by meas-uring relative luciferase activity. We find that Drosoph-ila Gcm is a potent transcriptional activator, resultingin a 500-fold induction of the luciferase reporter gene.Furthermore, Slp1 inhibits the transcriptional activationcapacity of Gcm in a dose-dependent manner; a fivefoldreduction in transcriptional activity is seen with theaddition of 0.5 lg of pFlag-slp1 plasmid (compare Col-

umns 3 and 5). The effects of flag-Slp1 on the 6xgbs-lucpromoter–reporter were found to be negligible (Column2) compared to empty vector controls (Column 1).Lysates from all transfected cells were tested by westernblotting analysis using anti-myc and anti-flag antibodiesto ensure appropriate expression of myc-Gcm and flag-Slp1 fusion proteins, respectively (data not shown).

We used promoter–reporter assays to determine if theability of Slp1/2 to repress Gcm transcriptional activityis evolutionarily conserved. The murine orthologs ofSlp1 and Gcm are Foxg1 and mGCM-1/2, respectively(Altshuller et al., 1996; Kim et al., 1998; Tao and Lai,1992). Our results demonstrate a 16-fold activation ofthe reporter gene in COS-7 cells transfected withmGCM-2 together with the 6xgbs-luc reporter construct.The effects of Foxg1 on mGCM-2 transcriptional activitywere then assessed by the additional transfection ofFoxg1 (Fig. 5B). With increasing concentrations ofpFlag-Foxg1 (5 ng to 1 lg), we observe a concomitantrepression in mGCM-2 transcriptional activity (Columns4–7). Overall, there is an eightfold repression in mGCM-2 activity with the addition of 1 lg flag-Foxg1. The flag-Foxg1 fusion protein, on its own, showed no appreciabletranscriptional activity with the 6xgbs-luc reporter con-struct (Column 2) compared to empty vector control(Column 1). Again, lysates from all transfections weretested by western blotting analysis using anti-myc andanti-flag antibodies to ensure that expression of myc-

Fig. 5. Slp1/Foxg1 inhibit Gcm transcriptional activity. A luciferasereporter plasmid, 6xgbs-luc, was transfected into COS-7 cells alongwith the indicated plasmids (all DNA quantities are in lg units). Dataare shown as fold inductions which were calculated by comparing lucif-erase activities of each sample to those obtained from cells transfectedwith reporter plasmid and empty expression plasmids (Column 1 inboth panels A and B). (A) A representative gbs-luciferase assay demon-strating that increasing doses of pFlag-slp1 results in concomitantrepression of Gcm transcriptional activity. Gcm causes a 500-fold induc-tion of reporter gene activity (Column 3). With increasing amounts of

pFlag-slp1, activation is reduced fivefold (Columns 4 and 5). Slp1 alonehas no effect on the gbs-luciferase reporter (Column 2). Note the differ-ence in scale of the y-axes between panels A and B. (B) A representa-tive gbs-luciferase assay demonstrating repressive effects of increasingFoxg1 on mGCM-2 transcriptional activity. mGCM-2 activates the re-porter gene 16-fold (Column 3), however, increasing concentrations ofFoxg1 results in a concomitant decrease in luciferase activity (Columns4–7). Foxg1, on its own, is unable to activate transcription of the lucif-erase reporter (Column 2). [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]



mGCM-2 and flag-Foxg1, respectively, were proportionalto the amount of plasmid transfected (data not shown).

Taken together, these data indicate that Slp1 andFoxg1 can respectively repress Gcm and mGCM-2 tran-scriptional activity. Of interest, we found that FoxG1can repress Gcm activity and similarly, that Slp1 is ableto repress mGCM-2 activity indicating that the repres-sive function is conserved in mice and flies (Supplemen-tal Fig. 2). This repression could occur either by pre-venting Gcm proteins from binding to DNA and activat-ing transcription or, alternatively, by forming a proteincomplex that represses the ability of Gcm to activate

downstream genes. To distinguish between these twopossibilities, we performed electrophoretic mobility shiftassays (EMSAs) using the murine proteins (Fig. 6).These studies confirm that mGCM-2 binds a gbs probeand forms complexes A–E (Fig. 6A, Lane 1). In contrast,Foxg1 has no affinity for the gbs probe (Fig. 6A, Lane6), further demonstrating that Foxg1 does not repressGcm activity by competing for binding to the gbs site ondownstream targets. Using anti-myc antibody, mGCM-2-gbs complexes C–E are supershifted to create a newcomplex, F (Lane 2). In contrast, we did not observe anychange in complexes A and B. It is possible that the mycepitopes in complexes A and B are not accessible to theanti-myc antibody and therefore these complexes areunaffected in the supershift reaction. The addition ofFoxG1 to the mGCM-2:gbs combination does not haveany apparent effect on complexes (Lane 4), neither doesthe addition of anti-myc antibody in Lane 5. However,we found that increasing concentrations of lysate con-taining Foxg1 results in changes in mGCM-2-containingcomplexes (Fig. 6B). That is, with increasing FoxG1,mGCM-2:gbs complexes A and B are enriched in inten-sity, suggesting a stabilization of these complexes (Fig.6B, Lanes 2 and 3). Concomitant with complex A and Bstabilization, there is an apparent depletion of com-plexes C–E. We propose that FoxG1 binding to mGCM-2may be disrupting certain mGCM-2:gbs complexes (C–E)and stabilizing others (A and B). We attempted super-shift analyses using the described conditions andobserve no change. As mentioned above, it is possiblethat the myc-epitope is buried in these complexes (Aand B) thus results in no observable supershift. Alterna-tively, the interaction between mGCM-2 and Foxg1 maysimply be unstable under EMSA conditions. Nonethe-less, our data clearly demonstrate that FoxG1 does notcompete with GCM for binding to target sites on DNAbut can influence mGCM-2:DNA complexes. Taken to-gether with the transcription studies, we propose that

Fig. 6. Foxg1 affects mGCM-2:gbs complexs in EMSA. (A) Lane1shows mGCM-2 bound to gbs probe resulting in complexes A–E. Myc-epitope tagged mGCM-2 is supershifted with anti-myc antibody andresults in the formation of a new Complex, F, while depleting complexesC–E. Complexes A and B appear unaffected (Lane 2). The addition ofexcess cold gbs probe (Lane 3) depletes complexes A–E, indicating pre-sence of mGCM-2. Lane 4 is a combination of mGCM-2 and Foxg1,resulting in no apparent change in complexes. In Lane 5 is a combina-tion of mGCM-2, Foxg1 and anti-myc antibody to supershift any com-plexes containing mGCM-2. There is no added supershift effect withthe addition of Foxg1 (compare with Lane 2). Lane 6 demonstrates thatFoxg1 has no affinity for the gbs probe. Lane 7 represents free probe. Asingle nonspecific band is indicated in all lanes, this band is not com-peted away with excess cold probe. Free probe is indicated. (B) Increas-ing Foxg1 concentrations has an effect on mGCM-2:DNA complexes(Lanes 1–3). In Lane 1, (as shown in Fig. A) equal concentrations oflysate containing mGCM-2 and Foxg1 result in complexes A–E (com-plex E appears faint in this image). Lanes 2 and 3 demonstrate a two-fold and threefold increase in concentration of transfected lysate con-taining Foxg1, respectively. In Lanes 2 and 3, complexes A and Bbecome significantly enriched, suggesting complex stabilization; thehigher complexes (C–E) appear to diminish. The nonspecific band isnoted, as expected, it intensifies with increasing lysate concentration.Free probe is not shown on this image.

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Foxg1 binds to a mGCM-2:DNA complexes therebyrepressing transcriptional activity.

Drosophila Gcm and Slp1 are PartiallyColocalized in the Early Developing Embryo

Thus far our data show that Slp1/2 regulate glial cellfates by repressing the activity of Gcm. For this repres-sion mechanism, one would expect colocalization of Gcmand Slp1/2. We first examined the expression patterns ofgcm and Slp1 in developing Drosophila embryos usingdouble labeling studies. The individual expression pat-terns of gcm, Slp1, and Slp2 have previously been wellcharacterized (Grossniklaus et al., 1992; Hosoya et al.,1995; Jones et al., 1995). FISH was used to localize thegcm transcript together with immunohistochemistry speci-fic for Slp1 on whole Drosophila embryos (Fig. 7). Consist-ent with previous studies, we find that gcm expression isfirst detected in early stage blastoderm embryos (Stage 5)within an anterior ventral region called the procephalicmesoderm that includes the hemocyte anlagen. The

expression of gcm is maintained within hemocyte precur-sors through Stage 11 after which time expression disap-pears from these cells. gcm expression within the neuroec-toderm is first detected at early Stage 9 in progenitor cellsthat will give rise to lateral glia. We initially observe colo-calization of gcm and Slp1 in early blastoderm embryos(Stage 5) within the procephalic mesoderm (Figs. 7A–C).Subsequently, both gcm and Slp1 are coexpressed at Stage10–11 in a subset of cells within the developing neuroecto-derm (Fig. 7D). gcm continues to be expressed in all lat-eral glia, whereas Slp1 expression is no longer detected(Fig. 7E). Given the colocalization results, Slp and Gcmmay directly collaborate to specify neuronal versus glialfates in a few cells/lineages within the neuroectoderm.Moreover, a direct interaction between both proteins islikely to occur within the procephalic mesoderm whereboth Slp1 and Gcm are highly expressed.

Slp1 Binds Gcm In Vitro

To determine a mechanism by which Slp1/2 inhibitsGcm activity within hemocytes and the neuroectoderm,

Fig. 7. Colocalization of gcm and Slp1 in developing wildtype Dro-sophila embryos. Double labeling by FISH analysis for gcm (green) to-gether with immunostaining with anti-Slp1 (red). Asterisked regionsrepresent areas of partial colocalization that are magnified as mergedimages. All images were captured at the same focal plane. Expressionof gcm and Slp1 are shown in early to late stage embryos (Panel A–E).Colocalization of proteins is shown in yellow in the merged panels.Panel A: Staining in early embryos (Stage 5) demonstrates that gcmand Slp1 are colocalized at the anterior region within the procephalicmesoderm (asterisks). In the magnified merged image, an arrow pointsto area of colocalization (yellow). Panel B: Later blastoderm stageshows the regular striped patterning of Slp1 and the continued gcmexpression at the anterior end of the embryo. The boxed area in themerged image is magnified to show an area of partial gcm/Slp1 colocali-

zation. Panel C: Stage 10 embryo showing continued expression of gcmand Slp1 at the anterior region (top magnified merge image) and theinitiation of gcm expression within the segments (at the lateral ends ofthe embryo). There are areas within the segments that show coexpres-sion of gcm and Slp1 (bottom magnified merged image). Panel D: Stage11 embryo showing the striped pattern of Slp1 expression and anincrease in the number of gcm staining cells as the lateral glia differ-entiate. The asterisked areas represent regions of partial gcm/Slp1coexpression within the stripes and in areas adjacent to the stripes inthe neuroectoderm. Colocalization is also apparent in embryonic heartcells. Panel E: A representative Stage 15 embryo showing no detectableSlp1 staining and abundant gcm positivity within the developing lat-eral glial cells. Scale bar represents 50 lm. Left is anterior and down-ward is ventral.



we examined the ability of Slp1 to bind Gcm. Epitope-tagged Drosophila Gcm (myc-Gcm) and Slp1 (flag-Slp1)were used in coimmunoprecipitation analyses and weakassociation between the proteins was found (Fig. 8A). Asan alternative method, we utilized in vitro GST-pull-down assays to confirm the interaction (Fig. 8C). Usingthis approach, we find that myc-Gcm interacts withGST-Slp1 (Fig. 8C, Lane 3). As a negative control inthese assays, we tested binding of Drosophila myc-Gcmto the GST moiety alone and observed no binding (Fig.8C, Lane 4). This assay confirmed that GST-Slp1 canbind Drosophila myc-Gcm.

FoxG1 Binds mGCM-1 and -2 by Yeast Two-HybridAnalysis and Co-Immunoprecipitation

Protein binding studies were also performed using themurine orthologs of Slp1 and Gcm to determine if theinteraction observed between the Drosophila proteins isconserved in mammals. Using a yeast-two-hybrid screen,we had previously identified three independent isolatesof a partial cDNA encoding half of the carboxy region ofFoxg1 as a candidate mGCM-2 binding protein. To verifythe mGCM-2/Foxg1 interaction, we performed coimmu-noprecipitation analyses. These experiments confirmedthat Foxg1 can bind to mGCM-2 in vivo (Fig. 9A).

Additionally, binding between Foxg1 and mGCM-1was tested and confirmed. Figure 9B shows thatmyc-mGCM-1/-2 are coexpressed with flag-Foxg1 in thelysates used to perform coimmunoprecipitation studies.

To determine the region(s) of mGCM-2 and Foxg1 thatcontribute to binding, we created truncations for eachprotein and used them to perform immunoprecipitationstudies (Fig. 10). For mGCM-2, we used the DNA-bind-ing region, known as the gcm-box that spans the amino-terminus of the protein (amino acids 1–263). In our stu-dies, the gcm-box was divided into two separate por-tions, the first spanning amino acids 1–174 (GCM-2174)and the second, 174–263 (GCM-2263). For Foxg1, we gen-erated three truncations: the forkhead domain(Foxg1FKH), a region that lies to the amino-terminus ofthe domain (Foxg1N) and a third truncation containingboth the amino-terminal region and forkhead domain(Foxg1N/FKH). All of the truncated constructs wereexpressed as myc-tagged fusion proteins (in the case ofmGCM-2) and flag-tagged fusion proteins (in the case ofFoxg1). Using immunoprecipitation analyses, we demon-

Fig. 8. myc-Gcm binds flag-Slp1. (A) Myc-Gcm coimmunoprecipi-tates with Flag-Slp1. Lane1 shows expression of flag-Foxg1, Lane2 isan anti-myc immunoprecipitation followed by anti-flag western blottingshowing a band that migrates alongside the flag-Slp1 input indicatingbinding between myc-Gcm and flag-Slp1. Lane 3 demonstrates the lackof nonspecific binding to the resin alone. (B) Panel D represents theinput lysate used in the immunoprecipitation reactions shown in C.Lanes 1 and 2 are anti-myc tag westerns demonstrating expression ofmyc-Gcm and flag-Slp1, respectively. (C) GST-pulldown assay illustrat-ing myc-Gcm: GST-Slp1 binding. Lane 1 contains parental COS-7lysate. Lane 2 represents input from COS-7 cells expressing myc-Gcm.Lane 3 represents binding between myc-Gcm and GST-Slp1. Lane 4demonstrates lack of binding between myc-Gcm and the GST moietyalone. Arrow indicates myc-Gcm fusion protein migrates (50 kDa).

Fig. 9. Foxg1 binds mGCM. (A) myc-mGCM-1/-2 coimmunoprecipi-tate with flag-Foxg1. Lanes 1 and 2 show Flag-Foxg1 coimmunoprecipi-tates with myc-mGCM-1 and myc-mGCM-2, respectively. Lane 3 repre-sents parental COS-7 lysate subjected to identical immunoprecipitationconditions as a negative control. Lanes 4 and 5 are input lanes indicat-ing that flag-Foxg1 is present in the transfected lysates. Lanes 6–8show transfected lysates and COS-7 parental lysates incubated withprotein G beads to rule out nonspecific binding to resin. Arrows indi-cate flag-Foxg1 fusion protein band and immunoglobulin heavy chainband (Ig Heavy). (B) Panel B represents input lysates from cotrans-fected COS-7 cells used to perform coimmunoprecipitations (shown inA) probed with anti-myc antibody to demonstrate the presence of myc-tagged mGCM-1 (�50 kDa) and myc-mGCM-2 (�65 kDa).

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strate that the gcm-box (both regions) binds to theamino-terminus of Foxg1 (Fig. 10A). In comparison,when the N/FKH regions of Foxg1 are expressed, weobserve reduced binding to the gcm-box (Fig. 10B). Thissuggests that the gcm-box binds preferentially to the N-terminus of Foxg1. Consistent with this observation, wedid not detect any binding between the gcm-box and theforkhead region alone (Fig. 10C).

DISCUSSION

Our studies demonstrate a role for slp1/2 in glial cellfate determination during Drosophila embryogenesis. Inthe absence of slp1/2 we find increased numbers of glialcells. Conversely, misexpression of Slp1 or Slp2 duringembryogenesis, results in a marked reduction of glialcell numbers. Furthermore, the effects of gcm misex-pression are rescued by the coincident misexpression of

slp2. Finally, we also show that Slp1 and its mammalianortholog, Foxg1, are able to repress Gcm transcriptionalactivity.

The generation of glia during embryogenesis involvesa series of critically timed events. The fates of all glialcells excluding the midline glia are determined by gcmin Drosophila (Hosoya et al., 1995; Jones et al., 1995).gcm mutants fail to develop nearly all lateral glia andthe presumptive glial cells in these mutant embryos dif-ferentiate into neurons resulting in a marked increasein neuronal number, whereas, gain-of-function muta-tions result in excess glial numbers (Hosoya et al., 1995;Jones et al., 1995). Mammalian Gcm homologs, mGCM-1and mGCM-2, have been identified (Akiyama et al.,1996; Altshuller et al., 1996; Kanemura et al., 1999;Kim et al., 1998). Targeted deletion of mGCM-1 in miceresulted in the failure of placental labyrinth formationand embryonic lethality (Schreiber et al., 2000), whereasdeletion of mGCM-2 led to an absence of parathyroidgland formation. Neither mGCM-1 nor mGCM-2 mutantmice displayed any CNS developmental abnormalities.However, transfection of a human medulloblastoma cellline with Drosophila gcm, induced both astrocyte andoligodendrocyte cell types (Buzanska et al., 2001).Furthermore, embryonic brain derived cells from miceinfected with retroviruses carrying either Drosophilagcm or mGCMs could be induced to express the astro-cyte-specific S100b protein (Iwasaki et al., 2003). Inaddition, when mouse GCM-1 was misexpressed in theDrosophila nervous system, additional glial cells wereobserved (Kim et al., 1998; Reifegerste et al., 1999).Finally, the expression of mGCM in Drosophila can par-tially rescue the loss-of-function phenotype in flies (Kimet al., 1998). Taken together, these results suggest thatmammalian GCM proteins also have the potential toinduce glial cell differentiation.

All Gcm proteins function as transcription factors thatrecognize and bind a consensus octameric DNA sequence(A/G)CCCGCAT (Akiyama et al., 1996; Schreiber et al.,1998). Drosophila Gcm activates genes required for glialdifferentiation including repo (Akiyama et al., 1996;Xiong et al., 1994), loco (Granderath et al., 1999) pointed(Klaes et al., 1994; Klambt, 1993) and gcm itself (Milleret al., 1998). In addition, Gcm activates genes that sup-press neuronal differentiation, including tramtrack(Giesen et al., 1997). Consistent with a role in determin-ing glial versus neuronal cell fates, the expression andactivity of Gcm in Drosophila is tightly regulated, how-ever, the mechanisms involved are less clear. We foundthat Slp1 can inhibit Gcm transcriptional activity. More-over, we show that this function is conserved in the mu-rine protein orthologs. The evolutionary conservation ofthis regulatory network implies a critical role for Slp/Foxg1 in glial cell differentiation and the regulation ofGcm function.

The regulation of transcriptional activity by repressorproteins is a well-established biological mechanism. Onesuch example is the Enhancer of split complex E(spl) inDrosophila (Alifragis et al., 1997; Heitzler et al., 1996;Preiss et al., 1988). E(spl) acts as a repressor of target

Fig. 10. Identifying regions of mGCM-2 and Foxg1 required to med-iate their binding. Lysates from COS-7 cells coexpressing truncationmutants of myc-mGCM and flag-Foxg1 were coimmunoprecipitatedusing anti-myc antibody followed by anti-flag western blotting. TheDNA-binding region of mGCM was separated into two distinct portions;amino acids 1–174 (GCM-2174) and the second, 174–263 (GCM-2263).For Foxg1, the forkhead domain (Foxg1FKH), the region that lies to theamino-terminus of the domain (Foxg1N) and a third mutant containingthe entire amino-terminal region (Foxg1N/FKH), were used. (A) Immuno-precipitation analyses demonstrate that both portions of the gcm-boxbind to the amino-terminus of Foxg1. In comparison, there is reducedbinding between the gcm-box regions and the N/FKH regions of Foxg1(B). There is no detectable binding of the gcm-box to the forkheadregion alone (C). Panel D shows the expression of the mGCM-2mutants in lysates used to perform the immunoprecipitation analyses.



gene expression by binding to the corepressor, Grouchoand effectively acting to negatively regulate the neuralpromoting activities of Achaete and Scute (Giglianiet al., 1996; Grbavec et al., 1999; Paroush et al., 1994).Foxg1 and Slp1/2 have been localized to the nucleusand shown to bind to transcriptional corepressors ofthe Groucho/transducin-like Enhancer of split (TLE)family to repress transcription (Kobayashi et al., 2001;Yao et al., 2001). Here we have shown that Slp/Foxg1function as transcriptional repressors of Gcm duringCNS development. One mechanism by which Slp1/Foxg1 might function as transcriptional repressors isto directly bind to, and repress, Gcm transcriptionalactivity. However, we demonstrate that Slp1/Foxg1 donot bind directly to the gcm-binding site. In contrast,we found that both Slp1 and Foxg1 bind directly toDrosophila Gcm and mGCM-1/-2, respectively, andthat binding represses the ability of Gcm proteins toactivate transcription of downstream reporter genes, invitro. Furthermore, we have also shown that mouseFoxg1 can repress Drosophila Gcm activity and simi-larly, Drosophila Slp1 can repress mGCM-2 transcrip-tional activity suggesting that this repression capacityis conserved across species.

For Slp-mediated repression of Gcm to occur by directprotein–protein binding in vivo, coincident expression ofboth Gcm and Slp in the same cell is required. Duringembryogenesis, the expression of slp gene productsbegins early in development (Stage 4) (Grossniklaus etal., 1992) and continues until the completion of embryo-genesis (Urbach and Technau, 2003). Gcm is initiallyexpressed during early blastoderm development in he-mocyte precursors. Expression is maintained withinthese cells as they develop into plasmatocytes at whichtime expression rapidly disappears (Alfonso and Jones,2002). Consistent with a role for Gcm in hemocyte devel-opment, gcm mutants display defects in both hemocytedifferentiation and migration. We find that Slp1 andgcm are initially colocalized within the hemocyte anla-gen. Whether Slp1/2 can inhibit Gcm function withinthese cells to prevent premature differentiation ormigration of hemocytes remains to be determined.

Approximately midway through embryogenesis, gcmexpression is activated in glial precursor cells. Theexpression in glia begins at Stage 9, peaks by Stage 11,and is completely absent following Stage 15 (Alfonso andJones, 2002; Hosoya et al., 1995; Jones et al., 1995). Theinitial gcm expressing cells are located in the most lat-eral position within the neurogenic region of the embryo.These lateral glioblasts divide symmetrically and themedial cell gives rise to the longitudinal glioblasts whilethe more lateral cell migrates distally to give rise to theperipheral glioblasts. It is in these early gcm expressingcells that we find some overlap with Slp1 expression. Ofinterest, once additional gcm expressing cells appearand increase in number, there is no detectable expres-sion of Slp1. Given these observations, it seems that Slpmay bind to and affect Gcm activity in a limited numberof cells during a narrow window of time that interest-ingly, corresponds to the period of initial gcm expression.

As an alternative mode of repression, Slp1 may bind thegcm promoter and regulate its transcription. Althoughthe ability of Slp1 to bind to the gcm promoter has notbeen examined, we have identified several putative Slp1binding sites (Andrioli et al., 2002; Lee and Frasch,2005) located within regions previously shown to berequired for gcm expression (Ragone et al., 2003).Whether these sites are required to regulate gcm expres-sion and glial cell fates remains to be determined.

To further study the temporal regulation of Gcm bySlp proteins, we induced slp1 and slp2 misexpressionduring early and late periods of Gcm expression. Misex-pression of Slp proteins during early gcm expressionresults in severe reductions in the number of gcm andRepo positive cells. In contrast, misexpression of slp1 orslp2 during peak gcm expression did not result in anydisruption of gcm expression and appeared identical towildtype embryos (data not shown). These data suggestthat gcm is sensitive to the effects of Slp during earlydevelopment of the embryonic nervous system. Theseresults may reflect differences in the levels of gcm pres-ent during early versus later development of glial cells.That is, as the expression level of gcm peaks later inglial cell development, gcm is less sensitive to the re-pressive effects of Slp. Thus, we propose that Slp1/2may function as one of the factors required to regulateearly stages of gcm expression and activity within theneuroectoderm.

Previous studies have shown that Slp1/2 are down-stream of wingless signaling and are required for thespecification of the NB4-2 neural precursor cells (Bhatet al., 2000). The effects on glial cell numbers observedas a result of slp1/2 mutations and misexpression inour studies suggest yet another role for Slp1/2. We pro-pose that Slp1 and Slp2 regulate cell fates in the devel-oping neuroectoderm (Supplemental Fig. 3). The inhibi-tory effects of Slp1/2 on Gcm are strongest at the earlystages of CNS development and this may serve to regu-late the initiation of glial fates coincident with promot-ing neuronal differentiation. The interaction betweenSlp1/2 and Gcm may orchestrate the regulation of neu-ronal and glial cell differentiation within the CNS.Clearly, further studies will be required to identify addi-tional factors that regulate both the activation andexpression of gcm and shed light on the mechanismsunderlying cell fate decisions during embryonic CNS de-velopment.

ACKNOWLEDGMENTS

We are grateful to Dr. K. Cadigan for providing theD34B slp mutant fly line and the hs-slp1 and hs-slp2transgenic fly lines, an anti-Slp1 antibody and the slp1cDNA, Dr. M. Wegner for the 6xgbs-luc plasmid, Dr. T.Hosoya for providing the pBS-gcm, pBS-mGCM-1, andpBS-mGCM-2 constructs, Dr. E. Lai for the pFlag-Foxg1construct, and Dr. Y. Hiromi for the UAS-gcm fly strain.We also thank Drs. W. Trimble and V. Auld for criticallyreading this manuscript.

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Sloppy paired 1/2 regulate glial cell fates by inhibiting Gcm Function