ARTICLE

Drosophila NAB (dNAB) Is an OrphanTranscriptional Co-Repressor Required forCorrect CNS and Eye DevelopmentMark Clements,1 Dianne Duncan,2 and Jeffrey Milbrandt1*

The mammalian NAB proteins have been identified previously as potent co-repressors of the EGR family of zinc fingertranscription factors. Drosophila NAB (dNAB), like its mammalian counterparts, binds EGR1 and represses EGR1-mediated transcriptional activation from a synthetic promoter. In contrast, dNAB does not bind the DrosophilaEGR-related protein klumpfuss. dnab RNA is expressed exclusively in a subset of neuroblasts in the embryonic andlarval central nervous system (CNS), as well as in several larval imaginal disc tissues. Here, we describe the creationof targeted deletion mutations in the dnab gene and the identification of additional, EMS-induced dnab mutations bygenetic complementation analysis. Null alleles in dnab cause larval locomotion defects and early larval lethality(L1–L2). A putative hypomorphic allele in dnab instead causes early adult lethality due to severe locomotion defects.In the dnab -/- CNS, axon outgrowth/guidance and glial development appear normal; however, a subset of eve�neurons forms in reduced numbers. In addition, mosaic analysis in the eye reveals that dnab -/- clones are either verysmall or absent. Similarly, dNAB overexpression in the eye causes eyes to be very small with few ommatidia. Thesedramatic eye-specific phenotypes will prove useful for enhancer/suppressor screens to identify dnab-interactinggenes. Developmental Dynamics 226:67–81, 2003. © 2002 Wiley-Liss, Inc.

Key words: transcription; co-repressor; NGFI-A; NGFI-A binding protein; EGR1; Drosophila; neuroblast; CNS

Received 22 July 2002; Accepted 11 October 2002

INTRODUCTION

The regulation of gene transcriptionis critical to the normal functioning ofcells and to the proper develop-ment of whole organisms. One cor-nerstone in the regulation of geneexpression is transcriptional repres-sion, in which DNA-binding proteinsand their cognate co-repressors pre-clude transcription initiation by RNApolymerase and associated basaltranscription factors. By definition,co-repressors do not bind DNAthemselves, but instead serve as“adaptor” or “linker” proteins be-tween DNA-binding transcriptionfactors and their targets of action. A

growing number of transcriptionalco-repressors have been identified,and their role in regulating transcrip-tion is under intense investigation.The prototype for a eukaryotic co-repressor is the yeast Ssn6p-Tup1pcomplex, which binds various DNA-binding proteins and actively re-presses transcription by means ofmultiple mechanisms (Keleher et al.,1992; Simpson et al., 1993; Cooper etal., 1994; Kuchin et al., 1995; Wahiand Johnson, 1995; Hanna-Rose andHansen, 1996). A Drosophila Tup1phomologue, Groucho, has beenidentified as a long range repressor,in that it can effectively shut down

promoter activity, even when teth-ered to DNA several kilobases away(Cai et al., 1996; Chen and Courey,2000; Courey and Jia, 2001). In con-trast, the Drosophila co-repressordCtBP acts as a short range co-re-pressor, specifically blocking tran-scriptional activation by enhancerelements located less than 150 bpaway (Nibu et al., 1998a,b; Keller etal., 2000; Strunk et al., 2001). BothGroucho and dCtBP, as well as theirmammalian orthologs, have beenfound to bind multiple families ofstructurally diverse transcription fac-tors, and both have been found torepress by multiple mechanisms, in-

1Department of Pathology, Washington University, Saint Louis, Missouri2Department of Biology, Washington University, Saint Louis, MissouriGrant sponsor: National Institutes of Health; Grant number: NS40745; Grant sponsor: The Association for the Cure of Cancer of the Prostate(CaP CURE).*Correspondence to: Jeffrey Milbrandt, Box 8118, Pathology Department, Washington University, 660 South Euclid, St. Louis, MO 63110.E-mail: jeff@pathbox.wustl.edu

DOI 10.1002/dvdy.10209

DEVELOPMENTAL DYNAMICS 226:67–81, 2003

© 2002 Wiley-Liss, Inc.

cluding direct binding to compo-nents of the general transcriptionapparatus and the recruitment of hi-stone deacetylase activity to thepromoter (Sundqvist et al., 1998; Chenet al., 1999; Criqui-Filipe et al., 1999;Zhang and Levine, 1999; Courey andJia, 2001; Brantjes et al., 2001). Theseand other transcriptional co-repres-sors have proven essential in regulat-ing multiple aspects of developmentin both Drosophila and mammals.

The mammalian NAB proteins

(NAB1, NAB2) comprise a family ofrecently identified transcriptionalco-repressors whose only knownbinding partners are members of theEGR family of zinc-finger transcrip-tional activators. Each NAB protein ischaracterized by the presence oftwo highly conserved domains: NABconserved domain 1 (NCD1), whichmediates interaction with EGR pro-teins containing a short motif calledthe R1 domain; and NCD2, whichcontains a bipartite-like nuclear lo-

calization signal and which is re-quired for NAB-mediated repression(Svaren et al., 1996; Swirnoff et al.,1998). The NCD2 domain containssequences highly similar to repres-sion motifs found in the previouslycharacterized co-repressors Dr1 andadenovirus E1b 55-kDa, and re-moval or mutation of these se-quences abolishes NAB-mediatedrepression (Swirnoff et al., 1998). Al-though Dr1 directly binds the TATA-binding protein (TBP; Inostroza et al.,

Fig. 1. Sequence of dnab transcript andprotein, with homology comparison tomouse, human, and Caenorhabditis el-egans NAB orthologs. A: The longest dnabtranscript identified is 2.06 kb in length, withan open reading frame encoding a pre-dicted protein of 568 amino acids. Loca-tion of splice sites is indicated with arrows;nucleotides at the splice junctions are boldfaced and italicized. Regions of homologywith mouse/human/C. elegans NAB(CeNAB) conserved domains (NCD) 1 and2 are indicated in bold type and under-lined; NCD1 is more N-terminal, whereasNCD2 is more C-terminal. Locations of mis-sense mutations in the EMS-generateddnabG26 and dnabA23 alleles are indicatedabove amino acid sequence. dnabG26

contains an E98K mutation in NCD1,whereas dnabA23 contains a Q292STOPmutation near the beginning of NCD2. B:Sequence alignment of the NCD1 andNCD2 motifs between dNAB, CeNAB, andthe mouse (m) and human (h) NAB1/NAB2proteins. An E1b 55-kDa homology motif islocated between amino acids 420 and 447in the consensus sequence of NCD2,whereas a Dr1 homology motif is locatedbetween amino acids 456 and 484 in theconsensus sequence. A more N-terminalDr1 homology motif is found in mouse andhuman NAB proteins, but not in dNAB orCeNAB (not shown). C: dNAB NCD1 is 78%identical to the NCD1 domains of mouseand human NAB proteins, whereas dNABNCD2 is 59% identical to the NCD2 do-mains of mouse and human NAB proteins,respectively. Identity was counted if theresidue in dNAB matched the residuefound in at least three of the four proteinsmNAB1, mNAB2, hNAB1, and hNAB2.

68 CLEMENTS ET AL.

1992), interaction assays demon-strate that NAB-mediated repressionmust occur by means of a differentmechanism than interaction withTBP. The NAB proteins have beenidentified as long range co-repres-sors, in that they can repress tran-scription even when tethered toDNA several kilobases away. In ad-dition, tethering experiments inwhich NAB1:Gal4 fusion proteinswere allowed to interact with Gal4responsive promoters have demon-strated that NAB proteins have theability to actively repress transcrip-tion from multiple types of activationdomains (Swirnoff et al., 1998). Thisleaves open the possibility that NABproteins might bind and repressother, as yet unidentified, families oftranscription factors.

Although we understand some as-pects of the molecular mechanismsof NAB-mediated repression, weknow little about the in vivo func-tion(s) of the NAB protein family.NAB2 overexpression in a rat sympa-thoadrenal cell type (PC12 cells)blocks NGF-induced neurite out-growth and maintains these cells ina proliferative state (Qu et al., 1998).This finding suggests that NAB pro-teins might play a role in neuronaldifferentiation generally. In addition,the human NAB proteins have beenimplicated in regulating peripheralnerve myelination, because muta-tions in the NAB-interacting proteinEGR2 have been identified in severalpatients with congenital hypomyeli-nating neuropathies (Botti et al.,1998; Warner et al., 1998; Bellone etal., 1999). At least one of these mu-tations occurs in the domain withinEGR2 (R1 domain) required forNAB1/NAB2 binding. These datasuggest that NAB proteins might alsoserve important functions in glial de-velopment. Still, mice lacking NAB2are viable and exhibit no obviousdefects (Nagarajan and Milbrandt,unpublished data). It remains possi-ble that mouse NAB1 and NAB2 pro-teins are functionally redundant andthat an understanding of their phys-iologic function(s) will require analy-sis of nab1-/-, nab2-/- mice.

One approach to studying thefunction of mammalian genes of in-terest is to characterize the functionof their orthologs in a model genetic

organism, such as Drosophila mela-nogaster or Caenorhabditis elegans.To further our understanding of nabgene function, we have identifiedand characterized a Drosophila NABhomologue, dNAB. Here we de-scribe the dnab gene, which istranscribed in a castor(ming) -de-pendent manner in a subset of em-bryonic central nervous system(CNS) neuroblasts (NBs) late in theirlineages, but not in ganglion mothercells (GMCs) or neurons. dnab ex-pression is reactivated during larvaldevelopment, where it is again tran-scribed in CNS neuroblasts, as well asin several third instar imaginal disctissues. Although it can bind and re-press mammalian EGR proteins,dNAB does not bind the putativeDrosophila EGR-like protein, klump-fuss. We have created targeted mu-tations in the dnab gene and havefound that complete dnab loss offunction causes specific alterationsin a subset of CNS neurons, as well asdefects in larval locomotion andearly larval lethality. Partial loss offunction in dnab causes severe de-fects in adult locomotion and earlyadult lethality. In addition, FLP/FRT-in-duced dnab loss of function in theeye causes clones to be very small orabsent, whereas dnab overexpres-sion early during eye developmentcauses the eyes to be very small withfew ommatidia. These dramatic eye-specific phenotypes will allow futureenhancer/suppressor screens to iden-tify genes that interact with dnab ge-netically, including potential bindingproteins and target genes.

RESULTS

Molecular Analysis of thednab Gene

To study dnab gene function in Dro-sophila, we first isolated dnab cDNAsequences, as described in the Ex-perimental Procedures section. Thednab gene spans approximately 7.0kb at map location 64A12 on the leftarm of chromosome III. It contains sixexons and five introns, and the dnabtranscript encodes a predicted pro-tein of 568 amino acids (Figs. 1A, 4A).The assembled 2.06-kb cDNA se-quence of dnab is likely full-length ornear full-length, as it is approxi-

mately equal in size to the only tran-script seen on Northern analysis andas genomic sequences immediatelyupstream of the 5� end fail to detecttranscript by in situ hybridization(data not shown). A BLAST search ofthe entire Drosophila genome re-veals that dNAB is the only NAB fam-ily protein in Drosophila.

The dNAB protein contains boththe NCD1 and NCD2 motifs, whichare highly conserved between Dro-sophila, C. elegans, mouse, rat, andhuman NAB proteins (Fig. 1B,C).There is 78% homology betweendNAB and murine/human NAB pro-teins in the NCD1 domain, and 57%homology in the NCD2 domain.These are the only two identifiedfunctional domains in this family. Inmouse and human, NAB NCD1 isknown to mediate binding to severalmembers of the EGR family of tran-scription factors, whereas NCD2contains an active transcriptional re-pression domain (Svaren et al., 1996,1998; Swirnoff et al., 1998). Of inter-est, the first of two Dr1 homologymotifs is not present in the NCD2domain of dNAB or C. elegansNAB, suggesting that this domain isdispensable for the repressionfunction of NAB proteins. This resultis in agreement with the findings ofSwirnoff et al., who observed that de-letion of the corresponding regionfrom rat NAB1 did not abrogaterNAB1-mediated repression (Swirnoffet al., 1998).

dNAB Can Bind and RepressMammalian EGR Proteins, butDoes not Bind the DrosophilaEGR-Like Proteinklumpfuss (klu)

The mammalian NAB proteins havebeen shown previously to repress thetranscriptional activity of EGR pro-teins by binding specifically to the R1domain. A single-point mutation inthe R1 domain (I298F) of EGR1 abro-gates binding to mNAB1 and mNAB2(Russo et al., 1993, 1995b). The NCD1domains of both mNAB1 andmNAB2 have been shown to benecessary and sufficient for interac-tion with the EGR1 R1 domain. Wesought to determine whether dNAB,

dNAB: ORPHAN TRANSCRIPTIONAL CO-REPRESSOR 69

like its mammalian counterparts,can bind and repress a mammalianEGR protein containing an R1 do-main by means of the yeast two hy-brid system (Fields and Song, 1989;Fields, 1993; Fields and Sternglanz,1994). To evaluate whether dNAB in-teracts directly with the EGR1 R1 do-main, dNAB NCD1 was cloned as afusion protein C terminal to the GAL4

DNA binding domain in pAS-1(Gal4DBD:dNCD1) and was testedfor interaction in yeast strain AH109with either the rat EGR1 R1 domain(aa 269-304) or R1 domain (I298F)point mutant fused C terminal to theGal4 activation domain in pBM2462(Gal4ACT:rR1 or Gal4ACT:rR1pm, re-spectively). Multiple colonies of dou-bly transformed yeast were selected

on -Leu-Trp drop-out medium, andthe colonies were restreaked onto-His-Leu-Trp drop-out medium to testfor Gal4-mediated activation of theHIS3 reporter gene in AH109. Yeastexpressing both Gal4DBD:dNCD1and Gal4ACT:rR1 were able to growon -His-Leu-Trp medium, whereasyeast expressing Gal4DBD:dNCD1and Gal4ACT:rR1pm were not (Fig.2C). Similar results were obtained inmultiple repeat experiments. Thesedata indicate that the NCD1 do-main of dNAB interacts directly withthe rEGR1 R1 domain in yeast butdoes not interact with the I298FrEGR1 R1 domain point mutant. Inthis respect, dNAB acts similarly tothe mammalian NAB proteins.

The mammalian NAB proteins me-diate dose-dependent transcrip-tional repression by means of theNCD2 domain (Russo et al., 1995a;Svaren et al., 1996; Swirnoff et al.,1998). When rNAB1 and rEGR1 ex-pression constructs are transientlycotransfected into CV1 cells alongwith an EGR1-responsive luciferasereporter construct, EGR1-induced lu-ciferase activity is repressed in adose-dependent manner; similar re-sults are observed for hNAB2 (Fig.2A). To determine whether dNABcan repress transcription mediatedby rEGR1, we tested whether dNABcan repress luciferase activity in thesame assay. Luciferase activity is re-duced in a dose-dependent man-ner when dNAB is coexpressed withrEGR1 (Fig. 2A). These data suggestthat dNAB contains a functionaltranscriptional repression domain,because it represses transactivationby EGR1 in a dose-dependentmanner.

In Drosophila, two EGR-like zinc fin-ger transcription factors have beenidentified: stripe and klumpfuss.stripe is expressed in the embryonicepidermis, and mutants exhibit a dis-ruption in muscle attachment andmyotubule patterning (Frommer etal., 1996). The stripe protein containsa zinc finger DNA-binding domainthat is highly homologous to theEGR1 DBD; amino acids known tomake contact with the EGR consen-sus binding site are completely con-served in stripe. However, it does notcontain an identifiable R1 domain.In addition, stripe expression occurs

Fig. 2. dNAB binds and represses transcription mediated by the mammalian EGR1 proteinbut does not bind the putative Drosophila EGR-like protein klumpfuss. A: In transienttransfection assays in CV1 cells, both rat (r) NAB1 and human (h) NAB2 repress rat EGR1-mediated transcriptional activation of a synthetic promoter in a dose-dependent manner.Similarly, dNAB is capable of repressing rEGR1-mediated transcriptional activation in adose-dependent manner. B: The putative Drosophila EGR-like protein klumpfuss containszinc fingers that are highly homologous to the zinc fingers found in the mammalian EGRproteins and in the related Wilm's tumor protein (WT-1). The black arrows indicate aminoacids that make direct contact with the target DNA sequence. The red arrows indicate anaspartic acid required for stabilization of the contact made by the preceding arginine withguanine 4 of the target DNA sequence. C: The dNAB NCD1 domain interacts with the R1domain of EGR1 but not the with an R1 domain containing the point mutation I293F. Growthon Leu-Trp-His-medium indicates a functional interaction. D: The dNAB NCD1 domain failsto interact with klumpfuss. In this case, the klumpfuss zinc fingers were removed, becauseEGR-like zinc fingers are toxic to yeast.

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in a pattern that does not overlapdnab expression (see below). Thatstripe lacks an R1 domain and that itis not expressed in dnab-expressingcells indicates that stripe is unlikely tobe a dNAB binding partner in vivo.In contrast, klumpfuss is expressedwidely in NBs of the embryonic andlarval CNS, as well as in severalimaginal disc tissues. Mutants exhibitdistal leg defects (tarsal segments3–5 are fused together, as are thetrochanter and femur), loss of bristlesat certain positions, and homeoticchanges in ganglion mother cellfate within certain neuroblast lin-eages of the central nervous system(Klein and Campos-Ortega, 1997;Yang et al., 1997). Klumpfuss alsocontains three EGR-like zinc fingers;amino acids in the mammalian EGRproteins, which are known to makedirect contact with the consensusbinding site, are completely con-served in klumpfuss (Fig. 2B). How-ever, unlike the mammalian EGRproteins, the klumpfuss protein con-tains a fourth zinc finger, making itmore similar to the Wilm’s tumor pro-tein (WT-1) than to the EGR proteinfamily. Klumpfuss also lacks the R1domain found in several mamma-lian EGR proteins. Still, klumpfuss ex-pression in the CNS does overlapdnab expression (see below), indi-cating that it could be a bona fidebinding partner for dNAB in vivo.

To determine whether dNABbinds klumpfuss by means of itsNCD1 domain, a klumpfuss cDNAlacking the zinc finger DNA-bindingdomain was cloned as a fusion pro-tein C-terminal to the GAL4 activa-tion domain in pACT2 (GAL4ACT:kluDZf). The klumpfuss DBD wasremoved in this case, because ex-pression of EGR-like zinc fingers isknown to inhibit the growth ofyeast (Wilson et al., 1992). Interac-tion between GAL4DBD:dNCD1and GAL4ACT:klu�Zf was tested asabove in yeast strain AH109. Yeastexpressing both GAL4DBD:dNCD1and GAL4ACT:klu�Zf failed to growon -His-Leu-Trp medium, indicatingthat the nutritional reporter HIS3 isnot activated (Fig. 2D). These re-sults strongly suggest that dNABdoes not bind klumpfuss by meansof the NCD1 domain, although bind-ing by means of some other portion of

the dNAB molecule cannot be for-mally ruled out. The data are not sur-prising, perhaps, when one considersthat klumpfuss, like stripe, lacks anidentifiable R1 domain. The lack ofother EGR-like proteins in Drosophilastrongly suggests that dNAB interactswith other families of transcription fac-tors in both Drosophila and mammals.

Embryonic and Larval dnabExpression Patterns

To determine the wild-type expres-sion pattern of dnab, we performedin situ hybridizations on embryos andthird instar larvae to detect dnabRNA. In embryos, dnab transcriptappears to be expressed solely in acluster of midline cells and a subsetof neuroblasts (NBs) (Fig. 3A–E); it isundetectable in the peripheral ner-vous system or in any other embry-onic tissue. Neuroblast-specific ex-pression was confirmed by doublestaining for dnab RNA and klumpfussprotein (data not shown). Of inter-est, dnab expression is also absentfrom ganglion mother cells (GMCs)and neurons of the embryonic CNS.Expression begins at stage 11 in mid-line cells, which are most likely mid-line mesectodermal cells, and in oneNB of every hemisegment (Fig. 3A,B).

By stage 13, dnab transcripts areexpressed in 10–12 of the 30 identi-fied NBs of each hemisegment (Fig.3C,D). By stage 14, dnab transcriptlevels begin to decrease, and bystage 16, expression is undetect-able, except in a few cells at thelateral edge of the CNS (Fig. 3E).

The exclusive expression of dnab inonly a subset of NBs of the CNS sug-gests that it might function in deter-mining the “identity” of neuronal lin-eages derived from those NBs afterthe onset of dnab expression. Alterna-tively, dnab might play a more gen-eral role in the differentiation or func-tioning of neurons derived from thesecells. Although dnab RNA is expressedonly in NBs, we cannot preclude afunction for dNAB protein in ganglionmother cells or neurons. This phenom-enon has been observed for severalgenes expressed in the DrosophilaCNS. The RNA for the zinc finger tran-scription factor castor, for instance, isexpressed exclusively in NBs, whereasthe protein perdures and is expressed

in ganglion mother cells and neurons(Kambadur et al., 1998; Mellerick etal., 1992).

dnab expression in third instar lar-vae is observed in both the CNS andin several imaginal discs (Fig. 3K–O).In the larval ventral ganglion, dnabexpression is limited to a subset ofproliferating neuroblasts in the ce-phalic lobes and the thoracic re-gion; expression is excluded from theabdominal segment of the ventralganglion, where proliferative NBsare not observed (Fig. 3K). Whateverits role, dnab likely performs similarfunctions during both embryonicand larval CNS development.

The embryonic and larval CNS ex-pression patterns for dnab are reca-pitulated by lacZ expression in a dnabenhancer trap line, dnabe310 (Fig.3F–J, 3L; see below for enhancer trap-ping technique). �gal staining of thirdinstar larvae containing the dnabe310

enhancer trap also reveals that dnabis expressed, albeit at lower levels, inseveral imaginal discs. In the eye disc,expression is observed in a patternconsistent with expression either in orbehind the morphogenetic furrow(Fig. 3M). Expression in the wing imag-inal disc occurs in the prospectivewing area early in the third larval instar(Fig. 3N). Expression also occurs in theantennal and haltere discs (Fig.3M,O). These imaginal disc expressionpatterns match those observed forthe dnab transcript by in situ hybrid-ization (not shown) and suggest a po-tential role for dnab in imaginal discdevelopment.

Generation of Mutations at thednab Locus

To remove dNAB function, we usedP-element–induced male-specificmeiotic recombination to inducesmall deletions affecting only thednab gene (Preston and Engels,1996; Preston et al., 1996). Four of 26recombinants contained deletionsflanking the retained P element bypolymerase chain reaction (PCR). Todetermine the sizes of these dele-tions, we tested for complementa-tion against the ropG27 allele, whichis a lethal allele of the nearby ropgene (located downstream of thednab transcription unit). Two alleles,dnabe310 and dnab1600, were viable

dNAB: ORPHAN TRANSCRIPTIONAL CO-REPRESSOR 71

over ropG27. These two deletion mu-tants were also viable over the lethaldeletion masxs76 in the masqueradegene, suggesting that neither dele-tion removed the function of eitherrop or mas. To confirm this, the dele-tions in dnabe310 (Fig. 4C) anddnab1600 (data not shown) weremapped molecularly by Southernblot to determine the distal break-point of their deletions. The deletion indnab1600 extends beyond the dnabtranscription unit and into predictedgene CG1299; therefore, it will not beconsidered further in this study.

To our advantage, the deletion indnabe310 spans approximately 1.82kb and removes only the first exonand a portion of the first intron of thednab gene (compare Fig. 4A with B).Southern analysis of BamHI-digestedgenomic DNA from dnabe310/TM3,Sb1 flies confirms the presence of asingle P element in the genome, aswell as the absence of DNA se-quence (including one BamHI site) im-mediately flanking the 3� end of theP element (Fig. 4C). In situ hybrid-ization confirms that the dnabtranscript is completely absent in

dnabe310/dnabe310 embryos,whereas transcripts from the nearbymas and rop genes are present in thewild-type patterns (Fig. 4D–G). Thednabe310 mutation is lethal, and ani-mals die in the 1st to 2nd larval instar.dnabe310/dnabe310 mutant larvaeare less active than wild-type larvaeand appear uncoordinated (Table 1).To determine the lethal phase for thisand all other mutants in this study, wehave used the larval culturing tech-nique of Loewen et al., in which lar-vae are reared on dilute yeast solu-tions in high humidity chambers (seethe Experimental Procedures section;Loewen et al., 2001). This techniquehas been shown previously to extendthe life span of synaptotagmin I mu-tant larvae, which exhibit severe de-fects in locomotion.

An additional advantage of thednabe310 line is that the starting P ele-ment is retained and traps a dnab-specific enhancer(s). Although theoriginal 1483/16 P element insertioncontained lacZ in reverse orientationrelative to the dnab transcriptionunit, the P element is flipped in thednabe310 line, placing lacZ in thesame orientation as the dnab tran-scription unit.

Because we obtained only a sin-gle dnab-specific deletion mutantby means of P-element–induced re-combination, we sought to identifyadditional dnab-specific alleles bytesting the ability of other indepen-dently generated lethal comple-mentation groups that map to the64A-B region of chromosome 3to complement the lethality ofdnabe310. A genetic screen to iden-tify lethal mutations in the 63E-64Agenomic region previously identifiedfive lethal complementation groupsin the region 64A5-64B1,2 (Harrisonet al., 1995). We tested the ability ofthese five allelic groups to comple-ment the lethality of the dnabe310

mutation. Lethal complementationgroups l(3)64Ah and l(3)64Ai containmutations affecting the rop and rep-lication factor C 40kDa (Rfc40)genes, respectively; these are viableover dnabe310. Lethal complemen-tation groups l(3)64Ae and l(3)64Agcontain mutations in unknowngenes; these also complement thelethality of dnabe310. Only the muta-

Fig. 3. dnab is expressed in the embryonic and larval central nervous system (CNS) ofDrosophila, as well as in several imaginal disc tissues. A–E: Detection of dnab transcript byin situ hybridization in the embryonic CNS of wild-type flies. A,B: dnab is expressed inmidline cells and a single pair of bilateral neuroblasts (NBs) at stage 11. C: dnab expres-sion spreads to 4–8 NBs per hemisegment by late stage 12. D: dnab expression peaks in10–12 NBs per hemisegment at stage 13. E: By stage 16, dnab expression is absent in all buta few cells at the lateral edge of the CNS. F–J: Detection of lacZ transcript by in situhybridization in the embryonic CNS of flies containing the dnab enhancer trap dnabe310.lacZ transcript is detected in an identical pattern to dnab transcript; stages are the sameas in A–E. A,F, lateral view; B-E, G-J, ventral view. Anterior is up in all cases. K: dnab transcriptis present in a subset of NBs in the third instar larval ventral ganglion. L: �gal staining revealsa similar pattern of expression for lacZ in the enhancer trap line dnabe310. M–O: �galstaining reveals low-level lacZ expression in imaginal disc tissues of the enhancer trap linednabe310. M, eye-antennal disc; N, wing disc; O, haltere disc.

72 CLEMENTS ET AL.

tions l(3)64AjG26 and l(3)64AjA23

failed to complement the lethalphenotype of dnabe310, providingfurther evidence that the dnabe310

deletion does not affect the func-tion of neighboring genes. We bal-

anced these two alleles over TM3,Kr-GAL4, UAS-GFP, Sb1 chromo-somes to aid in the identification ofhomozygous embryos, and per-formed DNA sequence analysis onPCR-amplified dnab exon regions

from the genomic DNA of pooledhomozygous embryos.

The l(3)64AjG26 allele, here re-named dnabG26, contains a muta-tion causing an E to K change atamino acid position 98 of dNAB (Figs.1A, 4H). Amino acid E98 resides inNCD1 and is completely conservedin C. elegans, Xenopus, mouse, rat,and human NAB proteins. Interest-ingly, this amino acid resides in a re-gion of NCD1, which appears inmammalian NAB proteins to be par-ticularly critical for its interaction withthe R1 domain of EGR proteins.Svaren et al. previously have con-ducted a mutagenesis screen toidentify mutations in NCD1 that dis-rupt interaction with the R1 domain;this screen yielded 21 amino acidchanges, including 2 amino acidsadjacent to the mouse NAB1/NAB2residues corresponding to E98 indNAB. This finding suggests that theNCD1 domain is important in medi-ating protein–protein interactions inDrosophila and that the E98K muta-tion disrupts these interactions. Thel(3)64AjA23 allele, here renameddnabA23, contains a mutation caus-ing a premature stop codon atamino acid Q292 (Figs. 1A, 4H). Thispremature stop removes the entireC-terminal half of dNAB, includingmost of NCD2. Studies on mamma-lian NAB1/NAB2 have revealed thatNCD2 is required for the transcrip-tional repression activity of the NABproteins. Mammalian NAB proteinslacking NCD2 fail to repress EGR-mediated transcriptional activationand also fail to repress EGR-indepen-dent transcription when tethered topromoters with a Gal4 DNA bindingdomain (Swirnoff et al., 1998).

To determine the lethal phase ofeach point mutation in dnab, weanalyzed the development ofdnabG26/dnabG26 and dnabA23/dnabA23 flies. Again, for these anal-yses, larvae were cultured on dilutesolutions of yeast in high humiditychambers (Loewen et al., 2001; seeExperimental Procedures section).dnabG26/dnabG26 flies died as first tosecond instar larvae; larvae ap-peared uncoordinated and initiatedfewer movements than wild-typeflies. In contrast, dnabA23/dnabA23

embryos appeared morphologicallynormal but failed to initiate peristal-

Fig. 4. Molecular mapping of the dnab-null mutation (dnabe310), and locations of pointmutations in EMS-generated dnab alleles dnabG26 and dnabA23. A: Map of dnab genomicregion, revealing the locations of six exons and six introns, as well as the location/orientation of PlacW insertion in the viable 1483/16 line. Bg, BglII; B, BamHI; H, HindIII; C,ClaI; X, XhoI; S, SacI. B: Map of the dnab genomic region in the dnabe310 deletion line. Thedeleted region is shown by grey box. Note the complete removal of exon 1, as well as thereversed orientation of PlacW element in this line. The 5�33� orientation places the lacZtranscription unit in the same orientation as the dnab transcription unit. C: Location ofprobes and BamHi sites used in Southern analysis to map deletion breakpoints in dnabe310.Probe 1 recognizes a 15-kb BamHI fragment, whereas probe 2 recognizes a 3.6-kb BamHIfragment, in dnabe310 genomic DNA. Note the removal of a BamHI site by the deletion. Thedetection of a single BamHI fragment by probes 1 and 2 indicates the presence of only asingle PlacW P element insertion in dnabe310. D: No dnab transcript is detected by in situhybridization in stage 13 dnabe310/dnabe310 embryos. E: masquerade (mas) transcript isdetected in a wild-type pattern by in situ hybridization in stage 14 dnabe310/dnabe310

embryos. The mas gene is located 700 bp upstream of dnab at region 64A12 on chromo-some 3 (not shown). F,G: ras-opposite (rop) transcript is detected in a wild-type pattern byin situ hybridization in early (F) or stage 13 (G) dnabe310/dnabe310 embryos. rop is located13 kb downstream of the dnab transcription unit at region 64A10 on chromosome 3 (notshown). H: Locations of EMS-generated point mutations in dnabG26 and dnabA23 alleles.dnabG26 contains an E98K substitution in NCD1, whereas dnabA23 contains a Q292STOPmutation in NCD2.

dNAB: ORPHAN TRANSCRIPTIONAL CO-REPRESSOR 73

tic movements at the end of embry-ogenesis and, therefore, failed tohatch from the vitelline membrane.The latter result suggested either thatdnabA23 is a stronger allele thandnabG26 and dnabe310 or that addi-tional second-site mutations exist onthe dnabA23 chromosome. To con-trol for the presence of additionalmutations on the dnabe310, dnabG26,and dnabA23 chromosomes, we ex-amined the lethal phase of each al-lele as a transheterozygote over thechromosomal deficiency Df(3L)GN19,whose deletion spans the chromo-somal regions 63E6,9-64B1,2 (Harrisonet al., 1995). As shown in Table 1, thelethal phase for each dnab alleleover Df(3L)GN19 was L1–L2; larvaeagain appeared uncoordinated

and initiated fewer movements thanwild-type. This result suggested thatall three dnab alleles caused lethal-ity during early larval development.

To further characterize the strengthof the dnab alleles, we analyzed thelethal phase of each dnab allele asa transheterozygote over the othertwo alleles (Table 1). Quite surprisingly,dnabe310/dnabA23 and dnabG26/dnabA23 transheterozygotes sur-vived larval development and thevast majority pupated. Most pupaeeclosed, and the adults appearedmorphologically normal in all re-spects. The adults did exhibit groom-ing behavior, but when they tried towalk, they were extremely uncoordi-nated and fell often. If they landedon their backs, a few could right

themselves with considerable diffi-culty. We did not observe the adultflies to fly or jump. Some adults(�20%) were unable to completelyescape the pupal case and be-came trapped there. Adults diedwithin 24–48 hr, presumably froman inability to feed. In contrast, thelethal phase of dnabe310/dnabG26

transheterozygotes was generallyL1–L2; however, a very few (5%)survived larval development andpupated. Pupae appeared morpho-logically normal when examined out-side the pupal case, but dnabe310/dnabG26 pupae never eclosed, al-though weak movements were ob-served.

The above results suggest that thelethality of dnab at any stage is sec-ondary to gross defects in locomo-tion. The results further suggest thatdnabe310 and dnabG26 are eithernull or strong alleles of dnab,whereas dnabA23 is a weak, or hypo-morphic, allele. The embryonic le-thality of dnabA23/dnabA23 homozy-gous embryos might be explainedby the presence of one or more sec-ond-site mutations on the dnabA23

chromosome, particularly in the re-gion 63E6-64B2.

It is particularly interesting that thednabA23 mutation permits develop-ment to adulthood (in combinationwith dnabe310 ), because this muta-tion truncates the dNAB protein andremoves the entire C-terminal half,including most of NCD2. This seemsto indicate that NCD2, which con-tains a known transcriptional repres-sion motif, is dispensable for at leastsome aspects of dNAB function dur-ing development. Furthermore, fliescarrying only a single copy of thednabA23 mutation survive and de-velop normally, suggesting that thetruncated protein produced by thismutation does not act in a dominantnegative manner.

Dnab Function Is Required forProper Formation of eve�

Lateral Neurons (EL) but not forAxon Outgrowth/Targeting orfor Glial Formation

Because dnab is expressed in only asubset of the 30 identified neuro-blasts in each hemisegment, we hy-

TABLE 1. Analysis of Lethal Phase and Behavioral Phenotype for dnabe310,dnabA23, and dnabG26 Loss-of-Function Allelesa

Genotype Lethal Phase Phenotypic Observations

dnabe310/dnabe310 L1-L2d Larvae initiate fewer movements; slowmuscle contractions

dnabA23/dnabA23b Embryonic Morphologically normal; fail toinitiate peristaltic contractionsrequired for hatching

dnabG26/dnabG26b L1-L2 Larvae initiate fewer movements; slowmuscle contractions

dnabe310/Df(3L)GN19c L1-L2 Larvae initiate fewer movements; slowmuscle contractions

dnabA23/Df(3L)GN19 L1-L2 Larvae initiate fewer movements; slowmuscle contractions

dnabG26/Df(3L)GN19 L1-L2 Larvae initiate fewer movements; slowmuscle contractions

dnabe310/dnabA23 Early adult Larval muscle contractions mildlyslower than wild type; pupaeeclose, but adults exhibit tremorand are severely uncoordinated

dnabG26/dnabA23 Early adult Larval muscle contractions mildlyslower than wild type; pupaeeclose, but adults exhibit tremorand are severely uncoordinated

dnabe310/dnabG26 L1-earlyadult

Larvae initiate fewer movements; slowmuscle contractions; �5% pupate;pupae are alive andmorphologically normal, but fail toeclose

aLethal phase analysis was performed using the larval culturing technique ofLoewen et al., in which mutant larvae are separated and reared on dilute yeastsolutions in high humidity chambers; rearing on standard fly food in vialsdramatically increases the incidence of early larval lethality for dnabe310/dnabG26,dnabG26/dnabA23, and dnabe310/dnabA23.bdnabA23 and dnabG26 are the same as 1(3)64AjA23 and 1(3)64AjG26 in Table 1,respectively.cDf(3L)GN19 is a chromosome 3 deficiency that removes DNA sequencesbetween regions 63E6,9 and 64B1,2.dL1-L2, first to second instar larval stages.

74 CLEMENTS ET AL.

pothesized that it may promote theformation of specific neuronal sub-groups. To examine several uniqueneuronal lineages in dnab mutantembryos for defects, we looked spe-cifically at cells expressing even-skipped and engrailed. In the stage15/16 CNS, the homeodomain pro-tein even-skipped (eve) is expressedin approximately 16 CNS neurons perabdominal hemisegment. Medially,pCC and fpCC interneurons and theaCC and RP2 motorneurons eachexpress eve, as do four mediolateralCQ neurons and 8 to 10 eve lateral(EL) neurons (Broadus et al., 1995;Patel et al., 1989; Landgraf et al.,1999). The homeodomain proteinengrailed (en), on the other hand, isexpressed in a larger complement ofcells: three large ventral midline cells

(VUMs), a cluster of dorsal medial(DM) cells, two pair of non-neuronalmedian support (MS) cells, a bilat-eral group of 4–6 posterior interme-diate (PI) cells, a group of 8–10 pos-terior lateral (PL) cells, and two pairof more anterior NH cells (Cui andDoe, 1992).

Although all en� cells are presentin dnabe310/dnabe310 mutant em-bryos (data not shown), a subgroupof eve� neurons develops in re-duced numbers. As shown in Figure 5(compare A,E with B,C and F,G),dnabe310/dnabe310 and dnabG26/dnabG26 embryos are missing 1–4 ELneurons in a subset of abdominalhemisegments per embryo. Be-cause this loss of EL neurons is vari-ably expressed, we quantified theloss by counting the number of ab-

dominal hemisegments containingseven or fewer EL neurons. The val-ues in Table 2 demonstrate thatmore abdominal hemisegments

Fig. 5. eve� neurons form in reduced numbers in the dnab mutant embryonic centralnervous system (CNS). A–D: eve staining of stage 15 CNS in wild-type (wt; A), dnabe310/dnabe310 (B), dnabG26/dnabG26 (C), and dnabA23/dnabA23 (D) embryos. E–H: Higher mag-nification view of representative eve� lateral (EL) neuronal clusters. The dnabe310/dnabe310

(B,F), dnabG26/dnabG26 (C,G) embryos are missing one to four EL neurons in a subset ofabdominal hemisegments per embryo compared with wt (A,E). Wt embryos contain 8–10EL neurons per hemisegment. In contrast, dnabA23/dnabA23 embryos are missing an aver-age of 3.5 EL neurons per abdominal hemisegment, and nearly every hemisegment isaffected (D,H). Results are quantified in Table 2.

Fig. 6. Castor gene function is required forthe appropriate expression of dnab in neu-roblasts of the embryonic central nervoussystem (CNS). dnab transcript is detectedby in situ hybridization in wild-type (wt;A–C), casH23A�1 (D–F), and casH23A�3 (G–I)embryos. A,D,G, ventral view. In the latestage 12 CNS, dnab is expressed in midlinemesectodermal cells and six to eight neu-roblasts (NBs) per hemisegment in wt (A)embryos. dnab transcript is only detectedin mesectodermal cells of casH23A�1 (D)and casH23A�3 (G) embryos at this stage.B,E,H, ventral view. In the stage 13 CNS,dnab is expressed in 10–12 NBs per he-misegment in wt (B) embryos. dnab tran-script is not detected in the thoracic or ab-dominal CNS in casH23A�1 (E) or casH23A�3

(H) embryos at this stage (C,F,I; dorsalview). In contrast, a few NBs in the cephaliclobes (arrows) do express dnab in casH23A�1

(F) and casH23A�3 (I) stage 13 embryos(compare with wt in C). Bilateral staininginside the embryo is nonspecific salivarygland staining observed when full-lengthdnab cDNA is used as probe template; thisnonspecific staining is also observed indnab-null (dnabe310/dnabe310 ) embryos(not shown). casH23A�1 (D–F) and casH23A�3

(G–I) embryos were overstained (24 hr) toaid in the detection of residual dnabexpression.

TABLE 2. Loss of eve� Lateral (EL) Neurons in the Stage 15 CNSof dnab Mutant Embryosa

GenotypeNo. of Hemisegments with�7 EL Neurons

Mean EL Neuronsper Hemisegment

wt (yw) 43/560 (7.68%) 8.1dnabe310/dnabe310 95/518 (18.34%) 7.1dnabG26/dnabG26 108/602 (17.94%) 7.3dnabA23/dnabA23 259/280 (92.50%) 4.6

aCNS, central nervous system; wt, wild-type.

dNAB: ORPHAN TRANSCRIPTIONAL CO-REPRESSOR 75

contain reduced numbers of ELneurons in dnabe310/dnabe310 anddnabG26/dnabG26 embryos vs. wild-type.

The loss of EL neurons is more pro-nounced in dnabA23/dnabA23 em-bryos (Fig. 5D,H). Here 92.5% of ab-dominal hemisegments are affected,and the mean number of neurons lostper hemisegment is 3.5 (n � 280 he-misegments). Because dnabA23 ap-pears by other measures to be a hy-pomorphic mutation in dnab (seeTable 2), we cannot currently explainthe more dramatic loss of EL neuronsin dnabA23/dnabA23 embryos.

In mammals, NAB2 overexpressionhas been shown previously to pre-clude axon outgrowth in culturedPC12 cells and to maintain thosecells in a proliferative state (Qu et al.,1998). In Drosophila, dnab is also ex-pressed in a proliferating preneuralcell type, and strong loss-of-functionmutants exhibit slow, uncoordinatedmovements as early larvae. Wetherefore sought to assess whetherdnab plays a role in axon out-growth/guidance during embryonicCNS development. Within the CNS,the longitudinal, anterior commis-sural, and posterior commissuralfascicles can be labeled withmAbBP102, whereas the peripheralmotor neuron branches that exit theCNS to innervate muscle groups dor-sally along the body wall express theantigen Fasciclin II (FasII). By usingthese markers, we analyzeddnabe310/dnabe310, dnabG26/dnabG26, and dnabA23/dnabA23

embryos for defects in axon out-growth and targeting but foundnone (data not shown).

The human NAB proteins havebeen indirectly implicated in periph-eral glial development; several pa-tients with congenital hypomyelin-ating neuropathies have beenidentified who have mutations inEGR2, a known hNAB1/hNAB2 bind-ing partner (Botti et al., 1998; Warneret al., 1998; Bellone et al., 1999; Tim-merman et al., 1999; Rogers et al.,2000). Therefore, we also examinedglial development in dnab mutantDrosophila embryos by staining forRK2, a glia-specific homeodomainprotein that is expressed in all CNSglia, with the exception of the mid-line glia. In dnabe310/dnabe310 em-

bryos, glia appeared to develop innormal numbers, and migrated ap-propriately to form the longitudinalglial array and exit glia (data notshown).

Castor Function Is Necessarybut not Sufficient to DriveProper dnab Expression inEmbryonic Neuroblasts

To identify regulators of dnab geneexpression, we have screened mu-tants of several NB-expressing tran-scription factors for loss of dnab RNAexpression. Castor is a zinc fingertranscription factor expressed in Dro-sophila NBs and GMCs; castor loss offunction causes reductions in CNSaxonal density and reductions in thenumber of neurons expressing thehomeodomain protein engrailed(Cui and Doe, 1992; Mellerick et al.,1992). Furthermore, castor has beenshown to positively regulate expres-sion of the POU domain transcriptionfactors drifter and I-POU and to neg-atively regulate expression of thePOU domain transcription factorspdm-1 and pdm-2 in the embryonicCNS (Kambadur et al., 1998). Wehave analyzed dnab expression incasH23A�1/casH23A�1 and casH23A�3/casH23A�3 embryos, which containdeletions removing the entire castorgene; dnab expression is affected inthese mutants (Fig. 6). dnab expres-sion in midline cells at stage 11 ap-pears normally (Fig. 6D,G); however,expression fails to spread to NBs dur-ing stages 12/13 (Fig. 6E,H). In thecephalic lobes, only a few cells ex-press dnab (Fig. 6F,I). This finding is incontrast to wild-type, where dnab isrobustly expressed in many cephalicNB (Fig. 6C). These results indicatethat dnab is either a direct or indi-rect target gene of castor. The tem-poral expression patterns of castorand dnab support these conclu-sions. Castor expression first appearsin NBs at stage 10 and spreads to9–10 NBs per hemisegment by latestage 11, the stage at which dnabexpression is first observed (Cui andDoe, 1992; Kambadur et al., 1998).The castor protein has been shownto bind the consensus DNA sequence(G/C)C(C/T)(C/T)AAAAA(A/T). Agenomic region containing the en-tire dnab transcription unit, as well as

10 kb upstream and 10 kb down-stream of dnab was scanned forcastor binding sites. This analysis re-vealed that no consensus castorbinding sites occur in the putativednab promoter region or in dnab in-trons, although at least two closelyrelated sites occur in the putativepromoter region (not shown). It maybe possible that castor directly reg-ulates dnab expression throughthese sites, or through sites in a distalenhancer element. Alternatively,dnab expression might be directlyregulated by the transcription fac-tors encoded by the castor targetgenes drifter, I-POU, pdm-1, orpdm-2. In these scenarios, drifterand I-POU might normally functionas positive regulators of dnab ex-pression, whereas pdm-1 and pdm-2might function as negative regula-tors of dnab expression.

Dnab Gain- or Loss-of-FunctionDisrupts Normal EyeDevelopment

The Drosophila eye consists of ap-proximately 800 precisely patternedlight sensing units, or ommatidia. Toinitially survey whether the dnabgene is essential for Drosophila om-matidial development, we haveperformed both gain- and loss-of-function experiments for dnab in thedeveloping eye.

dnab loss of function in the eye wasachieved by using the FLP/FRT systemto induce somatic recombination be-tween chromosomes bearing muta-tions in the dnab gene and wild-typechromosomes bearing a w� trans-gene. By inspection of gross eye mor-phology, we found that dnabe310/dnabe310 (w-/w-) cell clones failed toform altogether, resulting in a rougheye phenotype and completely w�eyes (Fig. 7B). In contrast, eyes bear-ing dnabG26/dnabG26 cell clonesgrossly appeared normal, althoughthe dnabG26/dnabG26 clones weremoderately reduced in size com-pared with wild-type (Fig. 7C). We didnot analyze dnabA23/dnabA23 eyeclones, due to the possible presenceof closely linked second site mutationson the dnabA23 chromosome. Wealso assessed whether dnab -/- cellclones could be detected in the pres-ence of chromosomes bearing the

76 CLEMENTS ET AL.

Minute mutation Rps174 along with aw� transgene (Fig. 7D,E). Minute mu-tations are dominantly cell prolifera-tion-defective, so they would givednab -/- cells a competitive advan-tage in populating the eye if they arepresent. By using this method, nodnabe310/dnabe310 clones formed,resulting again in a rough eye pheno-type and completely w� eyes (Fig.7D). In contrast, dnabG26/dnabG26

clones formed and appeared grosslynormal, populating greater than 80%of the eye (Fig. 7E).

The absence/reduced size ofdnabe310/dnabe310 eye clones sug-gests that there might be defects incell proliferation or, alternatively, in-creases in cell death during eye de-velopment. That dnabG26/dnabG26

eye clones are capable of prolifer-ating to populate a phenotypicallynormal eye suggests the possibilitythat the dnabG26 allele retains somefunctionality; it may be that evenslight amounts of dnab function aresufficient for normal eye develop-ment.

To complement the loss of func-tion analysis in the eye, we used theGAL4/UAS transgenic system to

overexpress a UAS-dnab transgene.When dnab is expressed under thecontrol of eyeless-GAL4, which per-mits expression very early in the em-bryonic eye primordium (Halder etal., 1995), eyes are completely ab-sent; occasionally a tiny patch ofeye tissue can be seen (Fig. 7G). Amore weakly expressing UAS-dnabtransgene permits development ofextremely small eyes with few om-matidia (Fig. 7H). In contrast, whendnab is expressed under the controlof GMR-GAL4, which initiates expres-sion in cells at the morphogeneticfurrow (Ellis et al., 1993), eyes areglassy from fused facets and havean irregular surface (Fig. 7I). Expres-sion of dnab under the control ofsevenless-GAL4, which expresses be-hind the morphogenetic furrow inthe R7 photoreceptor neuron as wellas several other retinal cells (Sun andArtavanis-Tsakonas, 1997), causes arough eye phenotype with manydark, necrotic patches (Fig. 7J).

The results from the above surveysuggest that dnab has the ability toaffect multiple aspects of eye devel-opment at several developmentalstages. The complete lack of eyes

observed when dnab is expressedunder the control of eyeless-GAL4may be due to an inability of precur-sor cells to proliferate, for instance.The GMR-dnab phenotype mimicsthe phenotype of the glass gene it-self, which causes a complete fail-ure of photoreceptors to form, aswell as aberrant projection of retinalaxons and complete absence of thelarval optic nerve (Kunes et al., 1993;Campos et al., 1995). The sevenless-dnab phenotype, on the otherhand, is reminiscent of the eye phe-notype for Blackpatch mutations,which cause neural degeneration inthe eye and optic lobe of adultbrains beginning at approximately60 hr after pupariation (Duus et al.,1992). These dramatic initial eye-specific phenotypes obviate theneed for more detailed study ofdnab function during ommatidialdevelopment, and should prove in-valuable in identifying dnab-inter-acting genes through enhancer-suppressor screens.

DISCUSSION

Although NAB proteins can bind andrepress the EGR-family of transcrip-tional co-repressors in mammals,dNAB is an orphan transcriptionalco-repressor in Drosophila. dNAB failsto bind klumpfuss, the only EGR-likeprotein expressed in the developingDrosophila CNS. This result is not sur-prising, as klumpfuss lacks the looselydefined R1 domain required bymammalian EGR proteins for NAB1/NAB2 interaction. We sought tocharacterize the function of dnab,the only Drosophila homologue ofthe NAB gene family.

dnab is transcribed in a castor(m-ing)-dependent manner in 10–12neuroblasts per hemisegment, be-ginning at stage 11 near the midlineand spreading laterally to other NBsduring stages 12/13. dnab transcriptis not found in GMCs or neurons ofthe CNS, in the peripheral nervoussystem, or in any other embryonictissue. The onset of dnab expressionin stage 11 NBs coincides temporallywith the last two waves (S4, S5) of NBdelamination from the neurogenicectoderm. dnab expression late dur-ing neuroblast lineage develop-ment, well after each NB has under-

Fig. 7. dnab loss- and gain-of-function in the eye causes dramatic phenotypes. A–E:Mosaic analysis in the Drosophila eye. A: Wild-type eye clones (white) appear normal andconstitute approximately one fourth of total eye tissue. B–E: When dnab -/- clones areinduced by somatic chromosomal recombination using the FLP/FRT system, clones areeither absent (dnabe310/dnabe310, shown in C) or reduced in size (dnabG26/dnabG26,shown in D) compared with wild-type (A). When dnab -/- clones are induced in thepresence of a dominant growth-defective Minute mutation (Rps174), dnabe310/dnabe310

clones are again completely absent, whereas dnabG26/dnabG26 clones appear normaland populate 80% of the eye. F–H: dnab misexpression in the eye using the GAL4/UASsystem. F: eyeless-GAL4; UAS-lacZ eye appears normal, with smooth surface and evenlyspaced ommatidia. G: UAS-dnab expression under the control of eyeless-GAL4 causes anear-complete loss of eye tissue. A very small amount of residual eye tissue does form. H:When a more weakly expressing UAS-dnab transgene is used, small eyes with fewerommatidia form. I: UAS-dnab expression under the control of GMR-GAL4 causes eyes tohave a glassy appearance due to a fusion of facets, as well as a loss of pigmentation. J:UAS-dnab expression under the control of sevenless-GAL4 causes a rough eye phenotypeand the formation of dark, necrotic spots throughout the eye.

dNAB: ORPHAN TRANSCRIPTIONAL CO-REPRESSOR 77

gone multiple rounds of cell divisionand presumably multiple temporaltransitions in transcription factor ex-pression, points to several possibilitiesfor dnab function: (1) dnab may actas a “neuroblast sublineage identitygene,” controlling the cell lineage ofeach NB after its expression com-mences; or (2) dNAB protein func-tion may be required in the ganglionmother cells or neurons to regulatevarious aspects of neuronal differen-tiation. For instance, dNAB functionmay be required for late stages ofaxon outgrowth, synaptogenesis, syn-aptic vesicle recycling, or expressionof genes required for neurotransmittersynthesis. dnabe310/dnabe310 mutantlarvae appear to die secondary tolocomotion defects. This finding is sup-ported by the observation that an ap-parent hypomorphic mutation indnab, dnabA23, permits normal devel-opment as a transheterozygote withnull dnabe310 mutation, but causes se-vere locomotion defects in the adult.These locomotion defects are evenmore severe in the very few dnabe310/dnabG26 flies that pupate; pupae ap-pear morphologically normal but failto eclose, presumably due to paraly-sis. Such locomotion defects havebeen observed most commonly inDrosophila mutants with affected syn-aptic vesicle release or recycling. Syn-aptotagmin 1 (syt) null larvae, for in-stance, are severely uncoordinatedand die during early larval develop-ment. When larvae are cultured un-der more permissive conditions, how-ever, syt mutants can survive toadulthood, where they again exhibitsevere defects in locomotion. Synap-totagmin 1 encodes a synaptic vesi-cle protein required for efficient neu-rotransmitter release (Loewen et al.,2001). Similarly, endophilin A (D-en-doA) mutants die during early larvaldevelopment due to severe locomo-tion defects. D-endoA encodes a pro-tein-containing lysophosphatidic acidacyl transferase activity and a func-tional SH3 domain (Guichet et al.,2002). Mutations in comatose, an-other drosophila gene required forsynaptic vesicle exocytosis, cause lar-val lethality due to reductions in syn-apse size and synaptic branching. Afew larvae do develop to adulthood,but the adults demonstrate seizure-like paralytic behavior. comatose en-

codes the NSF1 (N-ethylmaleimidesensitive fusion-1) protein, which is acytosolic ATPase involved in synaptictransmission (Sanyal and Krishnan,2001). The exact cause of the loco-motion defects in dnab mutants, al-though currently unclear, will certainlybe better understood after analysis ofsynaptogenesis and synaptic functionduring larval development.

Loss of dnab gene function causesidentifiable changes in embryonicCNS development. Although dnabdoes not appear to play a role inglial development or axon out-growth and guidance, dnab genefunction is required for the properformation of eve� lateral neurons inthe CNS; these neurons form in re-duced numbers in dnab mutant em-bryos. These neurons have been pre-viously identified as interneuronswhose axons remain within the CNS(Doe et al., 1988; Bossing et al., 1996;Schmidt et al., 1997). The exactmechanism by which EL neurons arelost is currently unclear. There maybe a cell fate transformation amongthe GMCs giving rise to EL neurons,those GMCs may not undergo celldivision properly or the GMCs them-selves may not form. Further studieswill be required to determine the ex-act cause of EL neuron loss.

Because dnab expression de-pends on the function of the castorzinc finger transcription factor, it re-mains possible that dNAB binds andrepresses castor or one of the POUdomain transcription factors (drifter,I-POU, pdm-1, pdm-2) whose expres-sion is regulated by castor. This phe-nomenon has been observed inmammals, where EGR1 activatestranscription from the NAB2 pro-moter, and NAB2 in turn repressesEGR1 (Svaren, personal communi-cation). According to this model, adNAB binding partner would providea short temporal “burst” of transcrip-tional activity and, in turn, activateexpression of its own co-repressor.Still, it seems unlikely that dNAB inter-acts directly with castor, becausethe castor loss-of-function pheno-type involves losses in axonal densityand the number of engrailed-posi-tive neurons in the embryonic CNS,whereas dNAB loss of function af-fects neither.

The lack of an identified binding

partner for dNAB in Drosophila limitsour ability to further elucidate thephysiologic function(s) of the dnabgene. Although the klumpfuss pro-tein contains DNA-binding zinc fin-gers that are very similar to themammalian EGR zinc fingers, klump-fuss is more closely related to theWT-1 in that it contains an additionalzinc finger and lacks the NAB-bind-ing R1 domain. In addition, klump-fuss affects the development ofsome tissues, such as the leg andbristles, which are obviously not af-fected in dnab mutants; one wouldexpect some correlation in pheno-type, either positive or negative,from two genes that interact. TheDrosophila protein stripe also con-tains an EGR-like DNA binding do-main, but does not possess an iden-tifiable R1 domain and is notexpressed in the CNS. These obser-vations not only indicate the exis-tence of a non-EGR binding part-ner(s) for dNAB, but also suggest thelikelihood that the mammalian NABproteins bind additional families oftranscription factors. Further under-standing of mammalian and Dro-sophila NAB gene function will facil-itate identification of additionalbinding partners by either genetic orphysical interaction screen ap-proaches.

The Drosophila eye provides anexcellent model system for pheno-typic enhancer and suppressorscreens. dnab RNA is expressed inthird instar larval eye-antennal imag-inal discs. As an initial assay to deter-mine whether dnab gene function isimportant for eye development, weanalyzed mosaic eyes containingdnab -/- cell clones, as well as eyesin which dnab was overexpressedby means of the GAL4/UAS trans-genic system. dnab -/- eye clonesare extremely small and few in num-ber. Likewise, eyes in which dnab isoverexpressed under the control ofeyeless-GAL4 are completely ab-sent. When a more weakly express-ing UAS-dnab transgene is used,eyes are extremely small with veryfew ommatidia. These results sug-gest a global role for dnab in theregulation of either cell proliferationor cell death in the developing Dro-sophila eye. In addition, these resultssuggest that enhancer/suppressor

78 CLEMENTS ET AL.

screens in the Drosophila eye willhelp identify genes that interact withdnab genetically, including candi-date binding partners and down-stream transcriptional target genes.

EXPERIMENTAL PROCEDURES

Molecular Analysis andCloning

An initial dnab cDNA fragment wasisolated by Taq PCR using degener-ate oligos in NCD1 and NCD2, re-spectively. An 8- to 12-hr embryocDNA library (Brown and Kafatos,1988) was screened by means ofstandard methods with a 32P-la-beled probe made from the dnabcDNA fragment obtained by de-generate PCR. To isolate the 5� endof the dnab cDNA, 5� rapid amplifi-cation of cDNA ends was performedas follows: 20 cycles of asymmetricPCR (aPCR) using Klentaq (Sigma)were performed on 200-ng embryocDNA library template using a dnab-specific reverse primer (5�-GTTTCG-GTGGCGTTTCGCAGAGA-3�). PCRreaction was digested with EcoRI tocleave all template library inserts at5� end; single-stranded aPCR prod-uct is not digested. After phenol/chloroform purification and ethanolprecipitation, l/20 of reaction wasthen used as template in 30 cyclesof symmetric PCR (sPCR) using nesteddnab reverse primer (5�-CACCGC-CCATTTCCAGCAGGGTGTCGTAGT-3�) and pNB40 vector primer (5�-TAGGTGACACTATAGAATACAAG-3�). The vector primer anneals justoutside the EcoRI cloning site. By usingthis method, Klentaq only extendswhen the vector primer anneals tothe dnab-specific aPCR product; noextension occurs when the vectorprimer anneals to EcoRI-digestedtemplate library DNA. This strategydramatically reduces the amplifica-tion of nonspecific products. Allother molecular methods, includingcloning, PCR screening and South-ern analysis, were performed ac-cording to standard methods (Sam-brook et al., 1989; Ausubel, 2001). Allclones were sequenced in entiretyon both strands by using the Big Dyesequencing kit (Applied Biosystems).

Yeast Two-Hybrid Analysis

Fusions of the R1 domain of EGR1 andEGR1(I293F) point mutant to the GAL4activation domain have been de-scribed elsewhere (Svaren et al.,1996). A fragment encompassing NABconserved domain 1 (NCD1) of dNAB(residues 56-134) was fused to theGAL4 DBD by subcloning them intoEcoRI/BamHI-digested pAS-1 (Clon-tech). A klumpfuss fragment (residues1-590) lacking the zinc finger DBD wasfused to the GAL4 activation domainby subcloning into BamHI-digestedpACT2 (Clontech). These constructswere transformed by standard meth-ods (Ausubel, 2001) into yeast strainAH109 (Clontech). All experimentswere performed in triplicate.

mRNA and Protein Localization

Digoxigenin-labeled dnab probeswere made from one of two dnabcDNA fragments (nt334-1295; nt169-1200). In situ hybridization was per-formed essentially as described pre-viously (Tautz and Pfeifle, 1989), withslight modification (I. Duncan, per-sonal communication). The follow-ing antibodies and concentrationswere used for protein localization:mAbBP102 (1:10; Klambt and Good-man, 1991); mouse anti-Fasciclin II(1:10; Vactor et al., 1993); rat anti-RK2 (1:1,000; Campbell et al., 1994);mouse anti-engrailed (1:10; Patel etal., 1989); and mouse anti-eve (1:10;Brown et al., 1997). Immunocyto-chemistry and �-galactosidase stain-ing were performed as describedelsewhere (Rushton et al., 1995; Bo-quet et al., 2000).

Tissue Culture andTransfections

CMV-driven expression vectors forwild-type and mutant (I293F) ratEGR1, rat NAB1, human NAB2, andthe luciferase report construct con-taining two EGR1 binding sites havebeen described previously (Russo etal., 1993; Swirnoff et al., 1998; Boquetet al., 2000). dnab cDNA sequence(nt169-1657), followed by a STOPcodon, was cloned into pCDNA tomake a CMV-driven dnab expres-sion construct. The extreme C-termi-nus is removed, but this region ap-

pears to be nonessential. Alltransfections were performed usingthe calcium phosphate precipitationtechnique essentially as describedpreviously. Transfections were intoCV-1 cells using 20 ng of the EGR1expression plasmids, 500 ng of lucif-erase reporter, 100 ng of a CMV-lacZreporter, and indicated amounts ofNAB expression plasmids. The lucif-erase activity of each sample wasnormalized to the �-galactosidaseactivity from the lacZ reporter. Back-ground activity from a transfectionwith the reporter alone was sub-tracted from activities obtained withcotransfected EGR1 or EGR1.

Genetics and Fly Stocks

To induce deletions in the dnabgene, we identified a placW P-ele-ment insertion near the dnab locusby PCR screening of available un-mapped third chromosome insertionstrains. The 1483/16 insertion (Sal-zberg et al., 1997; kindly provided byDr. D.M. Glover, University of Cam-bridge, UK) resides 333 bp upstreamof the putative dnab transcriptionalstart site and is a viable insertion. Byusing a CyO, �2-3 transposase chro-mosome II, along with an emcD,rhove-1, rs2, st1, bulD chromosome III,we mobilized the 1483/16 insertionand assayed for recombination be-tween 1483/16 and the dominantvisible emcD marker in male prog-eny. In accordance with previous re-ports, we obtained a recombinationrate of 1.04% (n � 2,504); 14 of 26recombinants were in the directionof emcD. Of these, four containedflanking deletions, but only one al-lele, dnabe310, contained a smalldeletion affecting only dnab bySouthern analysis.

Complementation analysis wasperformed between the dnabe310

deletion mutant and masxs76 (Muru-gasu-Oei et al., 1995), ropG27, andDf(3L)GN19, as well as alleles from thecomplementation groups l(3)64Ae,l(3)64Ag, l(3)64Ai, and l(3)64Aj (Harri-son et al., 1995). l(3)64AjA23 andl(3)64AjG26 failed to complementdnabe310 lethality and containedpoint mutations in dnab coding se-quence. They, therefore, are desig-nated dnabG26 and dnabA23 in thisstudy. FLP/FRT analysis in the eye was

dNAB: ORPHAN TRANSCRIPTIONAL CO-REPRESSOR 79

done using either ey-FLP, GMR-lacZ;P[w�,piM]75C, neoFRT80B or ey-FLP,GMR-lacZ; Rps174, P[w�,piM]75C,neoFRT80B stocks (Xu and Rubin, 1993;Newsome et al., 2000) along withdnabe310, neoFRT80B or dnabG26,neoFRT80B chromosomes. UAS-dnabflies were made by cloning dnabcDNA sequence (nt169-1657), fol-lowed by a STOP codon into theEcoRI site of pUASt (Brand and Perri-mon, 1993); transgene injectionswere performed by standard meth-ods. The following mutant and trans-genic lines were also used: casH23A�1

and casH23A�3; eyeless-GAL4; seven-less-GAL4; and GMR-GAL4 (FlyBase,1999; Mellerick et al., 1992). All thirdchromosome mutations were bal-anced over TM3, Kr-GAL4, UAS-GFP,Sb1 to aid in the identification of ho-mozygous or transheterozygous em-bryos and larvae (which are GFP-negative; Casso et al., 1999, 2000).dnab mutant chromosomes werecleaned of distant second site mu-tations by recombination betweenemcD and h1 markers, respectively.

ACKNOWLEDGMENTSWe thank Ian Duncan, Jim Skeath,Paul Taghert, and Ross Cagan for pro-viding many fly stocks and antibodies,as well as technical assistance. Wealso thank the Bloomington StockCenter and Developmental StudiesHybridoma Bank for making fly stocksand antibodies easily available.

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