Evidence for a Direct Functional Antagonism of the Selector Genes Proboscipedia and Eyeless in Drosophila Head Development

Evidence for a direct functional antagonism of the selector genes proboscipedia and eyeless in Drosophila head development

Corinne Benassayag1, Serge Plaza2, Patrick Callaerts3, Jason Clements3, Yves Romeo4, Walter J. Gehring2 and David L. Cribbs1,* 1Centre de Biologie du Développement-CNRS and Institut d’Exploration Fonctionnelle du Génome, 118 route de Narbonne, Bâtiment 4R3, F-31062 Toulouse Cedex 04, France 2Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland 3Department of Biology and Biochemistry, University of Houston, 369 Science and Research Bldg. 2, Houston TX 77204-5001, USA 4IBCG-CNRS, Université Paul Sabatier, 118 route de Narbonne, F-31062 Toulouse Cedex, France *Author for correspondence (e-mail: [email protected])

Accepted 15 October 2002

SUMMARY

Diversiﬁcation of Drosophila segmental and cellular development. Transient co-expression is detected early identities both require the combinatorial function of after this onset, but is apparently resolved to yield exclusive homeodomain-containing transcription factors. Ectopic groups of cells expressing either PB or EY proteins. A expression of the mouthparts selector proboscipedia combination of in vivo and in vitro approaches indicates (pb) directs a homeotic antenna-to-maxillary palp that PB suppresses EY transactivation activity via protein- transformation. It also induces a dosage-sensitive eye loss protein contacts of the PB homeodomain and EY Paired that we used to screen for dominant Enhancer mutations. domain. The direct functional antagonism between PB and Four such Enhancer mutations were alleles of the eyeless EY proteins suggests a novel crosstalk mechanism (ey) gene that encode truncated EY proteins. Apart from integrating known selector functions in Drosophila head eye loss, these new eyeless alleles lead to defects in the adult morphogenesis. olfactory appendages: the maxillary palps and antennae. In support of these observations, both ey and pb are expressed in cell subsets of the prepupal maxillary primordium of the Key words: Hox, Pax6, proboscipedia, Maxillary palps, Protein- antennal imaginal disc, beginning early in pupal protein, Differentiation

INTRODUCTION vitro contrasts with their highly specific effects in vivo. One source of enhanced Hox specificity derives from their joint Homeobox-containing genes encode transcriptional regulators action with cofactors such as extradenticle and its mammalian involved in many developmental processes including PBX family counterparts that enhance sequence selectivity and specification of unpatterned tissues. These include the highly alter in vivo specificity (Chan et al., 1994; Passner et al., 1999; conserved Hox gene class (Gehring et al., 1994; Manak and Pinsonneault et al., 1997; Rauskolb et al., 1993; van Dijk and Scott, 1994). The Hox genes that specify the segments reiterated Murre, 1994). An important role for stoichiometric along the antero-posterior axis of diverse animals (Lawrence combinations of homeotic proteins with functional partners is and Morata, 1994; McGinnis and Krumlauf, 1992) are viewed supported by observations suggesting that Hox protein levels can as selectors that control morphogenetic processes through the be crucial for their developmental effects (Cribbs et al., 1995; coordinate regulation of batteries of downstream target genes Greer et al., 2000; Roch and Akam, 2000; Smolik-Utlaut, 1990). (Garcia-Bellido, 1975; Garcia-Bellido, 1977). Indeed, the A combinatorial coding model predicts that different Hox genes Drosophila Hox genes encode homeodomain transcription and their partners acting together can specify novel cell or factors whose patterned and overlapping expression leads to segment identities, deploying developmental pathways that are differential activation of target genes in individual segments different to those specified by one gene product acting alone. (Botas, 1993; Graba et al., 1997; Morata, 1993). The Drosophila proboscipedia (pb) gene codes for a Despite the identification of several target genes, important conserved homeodomain protein required for the correct gaps remain in our understanding of the pathway from patterned development of the adult fly mouthparts (Cribbs et al., 1992) Hox gene expression to the development of diverse segmental where two distinct appendages, the labial and maxillary palps, morphologies (Mann, 1995). For example, the poor DNA show a dose-sensitive requirement for pb gene function sequence specificity exhibited by Hox transcription factors in (Kaufman, 1978; Pultz et al., 1988). These appendages derive 576 C. Benassayag and others from the labial and antennal imaginal discs, respectively. expressed normally in the labium and maxillary palps, and ectopically Labial palp differentiation requires the combinatorial action in tissues including the antennae, legs, wings and eyes. PBsy expression of two Hox genes, pb and Scr (Percival-Smith et al., 1997). In in the eyes provokes a dosage-sensitive eye loss without an antenna- contrast, pb appears to be necessary and sufficient for the to-maxillary palp transformation (Benassayag et al., 1997). sy differentiation of maxillary palps, and ectopic PB expression Phenotypically normal males carrying one HSPB element were induces a dose-sensitive transformation of antennae to mutagenized with 35 mM EMS or 7 mM DEB as described (Lewis and Bacher, 1968), then crossed en masse with homozygous HSPBsy maxillary palps (Cribbs et al., 1995). Another consequence of females (eyes slightly reduced). Progeny were screened for dominant ectopic PB expression is a striking dose-sensitive eye loss. The enhancers by selecting females (2× HSPBsy) with strongly reduced observation that PB protein incapable of DNA binding can eyes. Candidate females (presumed genotype: 2× HSPBsy; En/+) were induce this eye loss suggested that context-specific protein- re-crossed with HSPBsy males. Candidates yielding a significant protein interactions are central to this defect (Benassayag et fraction of female progeny with strong eye reduction were retained. al., 1997). We attempted to identify specific protein partners of PB by isolating dominant Enhancer mutations for the PB- Fly strains dependent eye loss. Four such mutations are new alleles of the All stocks and crosses were maintained at 25°C on standard yeast- eyeless gene encoding a D. melanogaster Pax6 homolog. agar-cornmeal medium. Specific fly lines or alleles used were: soPlacZ The Pax gene class encodes proteins with a DNA-binding (Cheyette et al., 1994), dpp-Gal4 (Staehling-Hampton et al., 1994), Paired domain that may be coupled with a class-specific UAS-ey (Halder et al., 1995), ey-GAL4 (Halder et al., 1998), UAS- sy and wt homeodomain (Bopp et al., 1986; Frigerio et al., 1986; Gehring pb 49.1 (Aplin and Kaufman, 1997), HSPB (Benassayag et al., et al., 1994; Manak and Scott, 1994; Strachan and Read, 1994; 1997; Cribbs et al., 1995), pbGAL4 UASLacZ/CyO (constructed by L. Joulia). The eya and so alleles eyaclift1, eya2, so1 and soL31 were from Stuart et al., 1994). Reduced Pax6 function in mammals leads to L. Pignoni and L. Zipursky; eygM3-12 and eygc1 were from Henry Sun; the defective eye structures typical of the dominant congenital ey2 and eyR were as described (Quiring et al., 1994); and Df(4)BA was disorder Aniridia in humans (Ton et al., 1991), or Small eye (Sey) provided by E. Frei and M. Noll (Kronhamn et al., 2002). Specific in mice (Hill et al., 1991). Further roles for Pax6 in mouse genotypes generated for this publication were: UAS-pb; UAS-ey and development are revealed in homozygous mutants lacking nasal solacZ/CyO; dpp-GAL4/TM6B, Hu Tb. structures or showing brain defects (Glaser et al., 1994; Hogan et al., 1988; Schmahl et al., 1993). The spectrum of abnormalities Phenotypic analysis seen in Sey mice thus indicates that Pax6 is required in eye, nose Detailed phenotypic analysis was by light microscopy (Zeiss and brain development. Loss-of-function eyeless mutations in Axiophot) after mounting dissected samples in Hoyer’s medium, or Drosophila lead to reduced or defective eye structures, whereas by scanning electron microscopy (SEM). For SEM analysis, adult flies targeted expression of EY protein induces ectopic eyes (Halder were stored in 95% ethanol until dissection of heads. Preparation and mounting were as described (Benassayag et al., 1997). et al., 1995; Quiring et al., 1994). Recent work has also revealed that apart from this central role in forming the Drosophila eye, Molecular characterization of eyeless mutations eyeless is also required for brain development (Callaerts et al., The molecular characterization of eyeless mutations involved PCR 2001; Kurusu et al., 2000; Noveen et al., 2000). amplification of all the exons from intronic primers, and sequencing To better understand the roles of the eye selector gene eyeless of the cloned products. For a detailed description, see Callaerts et al. (ey) and the basis of its functional relationship with (Callaerts et al., 2001). proboscipedia (pb) in normal development, we analyzed the molecular structures and the mutant phenotypes of the four Histology newly identified eyeless mutations. These alleles code for In situ hybridizations on larvae or on prepupae were performed using truncated EY proteins and are expected to affect all aspects of digoxigenin-labeled probes according to Sturtevant and Bier (as ey function. The mutant phenotypes indicate that eyeless is described at http://www-bier.ucsd.edu/imagdisc.html). Antibody required for adult maxillary palp differentiation. Furthermore, stainings were performed according to Halder et al. (Halder et al., 1998) at the following dilutions: rabbit α ey (1/500), rabbit α pbE9 both EY and PB accumulate in the maxillary primordium (1/175), mouse α eya (1/50), mouse α dac (1/200) and mouse αβ- beginning early in pupal development. We show that PB- gal (1/5000). induced eye loss occurs via post-translational repression of EY- mediated target gene transcription. Furthermore, we detect Pull down experiments direct protein-protein binding in vitro involving the PB GST fusion proteins were produced and purified according to the homeodomain and the EY Paired domain. Direct protein- manufacturer’s specifications (Pharmacia). Quantification of the GST protein binding in a modified yeast two-hybrid analysis is fusion protein was monitored by Coomassie staining to ensure likewise sufficient to diminish or abolish EY transcriptional equivalent amounts of GST fusion proteins in the assay. 35S-labeled activation from a target promoter, sine oculis. These genetic and proteins were synthesized using a coupled in vitro transcription molecular results support a direct functional antagonism translation kit (TNT Promega). The binding conditions were modified between the Hox gene pb and the Pax gene ey in maxillary palp from Chen et al. (Chen et al., 1997): Glutathione resin-bound GST µ development and provide evidence for a functional link proteins were pelleted and washed three times with 500 l of binding integrating eyeless with the position-specific Hox gene network. buffer (20 mM Hepes, 1 mM DTT, 0.1 mM EDTA, 2.5 mM MgCl2, 150 mM NaCl, 0.05% (v/v) NP40, 10% (v/v) glycerol, 100 µg/ml BSA). Labeled proteins were incubated in 0.4 ml binding buffer with approximately 10 µg of bound GST fusion proteins for 2 hours at 4°C. MATERIALS AND METHODS The resin was washed five times with 1 ml of binding buffer, then bound 35S proteins were eluted by boiling for 3 minutes in 25 µl of Genetic screen loading buffer, fractionated by SDS-PAGE and visualized by The X-linked HSPB:2.5 element (and HSPBsy derived from it) is autoradiography (from 15 to 36 hours). We measured bound labeled pb/ey in head development 577 proteins eluted compared with the input by quantitation on a Phospho- of the labial and maxillary palps composing the adult imager. mouthparts. Its selector function in head development is revealed by ectopic expression of PB protein from a pb mini- ‘One and a half hybrid’ experiments gene, HSPB, that induces a reliable dose-dependent One-hybrid experiments were performed using the MATCHMAKER transformation of antennae to maxillary palps (Ant→Mx) one hybrid system (Clontech) according to the manufacturer’s (Cribbs et al., 1995). Ectopic PB also induces a dose-sensitive specifications. The NcoI-SmaI fragment of ey cDNA was subcloned in frame with the Gal4DB in the pAS vector. The PB fragment (amino acids eye loss. We previously screened for PB mutations of sites 128-305) was fused to the GAL4 transactivation domain in the pACT essential for homeotic transformation. A mutant transgene, sy → vector. pAS and pACT are from Durfee et al. (Durfee et al., 1993). HSPB (Arg5 His5 in the homeodomain N terminus) was identified that induces dose-sensitive eye loss even though it Plasmid constructions no longer directs the Ant→Mx transformation (Fig. 1A-C). Plasmids used for GST fusion protein production: pEn24-4 (Bourbon This eye-loss does not require PB DNA-binding ability, et al., 1995) leads to GST-EN fusion protein (EN-374/552) containing suggesting that specific proteins required for eye development HD and adjacent regions. PLS15: pGEXB PBµ4a produces a GST-PB are antagonized by direct contact with PB (Benassayag et al., isoform µ4a (amino acids 126-306) containing HD and adjacent regions 1997). sy (Seroude and Cribbs, 1994). PLS20: pGEXB PB produces the same In a search for such functional protein partners of pb also GST-PB protein carrying the point mutation in the HD transforming involved in head morphogenesis, we screened for dominant Arg5 to His (Benassayag et al., 1997). In vitro transcription or mutations enhancing the observed eye defect. We employed the translation was performed using cDNAs of eyeless, eyes absent, sine sy sy oculis and extradenticle cloned respectively in the following plasmids: mutant transgene HSPB , because PB protein retains the pBSN eyeless (Quiring et al., 1994), pblue eyaI (Bonini et al., 1993), capacity of wild-type PB to induce eye loss but is less toxic for pBSpF3k, and pSp64 ATGexd (van Dijk and Murre, 1994). The flies. Males hemizygous for the X-linked HSPBsy element are truncated EY proteins were generated by deletion of the full-length normal (Fig. 1A), and homozygous females (two transgene eyeless cDNA contained in the pBSN plasmid. To generate a EY∆HD copies) are nearly so (Fig. 1B). In contrast, complete eye loss protein, ends generated by a partial HindIII digestion (position 773) and occurs in females carrying four copies (Fig. 1C). HSPBsy males a complete XbaI digestion of pBSN-eyeless were blunted by filling in were treated with the chemical mutagens EMS or DEB and and the plasmid recircularized. This construct expresses a truncated crossed with homozygous females (two copies of HSPBsy). We protein containing the 245 first amino acids, including the Paired ∆ then selected candidate female progeny (two copies) with a Domain (27 kDa). To obtain the EY PD protein pBSN eyeless was strong eye loss resembling four copies (Fig. 1C). Among digested with NcoI (made blunt by filling in) and BamH1 (made blunt sy sy by exonuclease treatment) and recircularized. This construct generates approximately 80,000 F1 HSPB /HSPB females screened, a protein of 681 amino acids (75 kDa) deleted for 157 amino acids approximately 20 new Enhancer mutations were retained. (between 18-174). To generate the EY∆PB∆HD protein, pBSN eyeless These mutations represent at least five loci, distributed on all was digested with NcoI (making blunt by filling in) and NruII and four chromosomes. Three of the Enhancer mutations were new recircularized. It leads to a protein of 387 amino acids (43 kDa) deleted alleles of Notch (Boube et al., 1998). Four En(sy) alleles for 451 amino acids (18- 468). mapped to the fourth chromosome and were identified as Construction of a pb-GAL4 driver: GAL4 coding sequences were alleles of the eye development gene eyeless based on placed with pb control elements within 6.2 kilobase pair (kb) SalI complementation analyses with three previously characterized fragment harboring pb exons 1 and 2, intron 1, and regulatory eyeless alleles, ey2, eyR and Df(4)BA (Kronhamn et al., 2002; sequences in intron 2 (Cribbs et al., 1992; Kapoun and Kaufman, Quiring et al., 1994). 1995). The circular form of the 6.2 kb fragment subcloned in the plasmid pGEM2 was used for amplification by PCR. One primer, Specific genetic antagonism between pb and ey in situated in exon 1, was oriented upstream and contained restriction PB-induced eye loss sites for BamHI and NotI; the second primer, situated at the 3′ end of JD EH 11 D1Da exon 2, was oriented downstream and contained restriction sites for The strength of the new ey alleles (ey , ey , ey and ey ) NotI and XbaI. The amplified product of 6 kb was digested with NotI, was compared with ey2 and Df(4)BA for its interaction with ligated and transformed into E. coli. This plasmid is deleted for the HSPBsy (Fig. 1D). On crossing HSPBsy/Y; ey*/+ males with 3′ end of exon 1, intron 1 and exon 2. GAL4 coding sequences HSPBsy/HSPBsy females, half the female progeny should be removed from pGATB (Brand and Perrimon, 1993) by digestion with HSPBsy/HSPBsy; ey*/+ and eye loss could attain at most 50%. BamHI and NotI were inserted into the pb plasmid cut with the same In this test, sensitized females heterozygous for the eye-null enzymes. Finally, an EcoRI/SalI fragment containing pb exon 1 as a allele ey2 showed 12% eye loss; similarly, heterozygotes for transcriptional fusion with GAL4 coding sequences, plus known the recently described deletion Df(4)BA (Kronhamn et al., intron 2 regulatory elements, was inserted into CaSpeR4 cut with 2002) showed 13% eye loss. In contrast, most of the new alleles EcoRI and XhoI. This pb-GAL4 plasmid was injected into flies showed a more marked interaction. eyJD, eyD1Da and eyEH together with a plasmid supplying transposase, and transgenic lines sy established by standard methods. The properties of these lines will be interacted similarly with HSPB (Fig. 1D; eye loss between 11 described in detail elsewhere (L. Joulia, H. M. Bourbon and D. L. C., 25 and 29%), although ey was markedly weaker (6%). These unpublished). results suggesting that the eyJD, eyEH and eyD1Da alleles are stronger than ey11 are in general agreement with the results of complementation tests employing ey2, eyR and Df(4)BA RESULTS (Callaerts et al., 2001) (data not shown). Thus three of the new ey alleles interact more strongly with HSPBsy than the Isolation of new eyeless mutations by functional described null alleles. To examine the specificity of the interaction with proboscipedia observed eye loss, we tested for genetic interactions with sine The pb homeotic function is required for correct development oculis (so), eyes absent (eya) and eye gone (eyg) (Bonini et al., 578 C. Benassayag and others

1993; Cheyette et al., 1994; Jun et al., 1998), prominent players immediately after the exon 8, resulting in a protein truncated in the eye differentiation cascade (Fig. 1D) (Halder et al., 1998; by 199 amino acid (Fig. 1E). This molecular characterization Niimi et al., 1999; Zimmerman et al., 2000). None showed a thus confirmed the identity of these Enhancer mutations as ey marked interaction with PBsy (Fig. 1D). The remaining alleles that should affect the EY protein itself. In the case of unidentified Enhancer loci from our screen complement eyJD and eyD1Da this point has been validated (Callaerts et al., mutants of all three genes, and thus do not appear to correspond 2001). to characterized members of the eye hierarchy. In conclusion, these results support a specific genetic antagonism between pb eyeless function is required for adult maxillary and and eyeless/Pax6 in programmed eye loss. antennal development To better understand the relationship between PB and EY in The new eyeless alleles encode truncated proteins normal development, we examined the phenotypic effects of ey The eyeless locus encodes a transcription factor with two mutations in the sensitized HSPBsy genetic context. Two copies highly conserved DNA-binding motifs, a paired domain (PD) of the HSPBsy transgene (the sensitizing condition) showed no and a homeodomain (HD) (Callaerts et al., 1997). Two of the marked effect (Fig. 1B). In contrast, pharate adult females with best characterized eyeless alleles, ey2 and eyR, result from 2× HSPBsy and homozygous for eyJD showed strong maxillary insertion of mobile elements within eye-specific regulatory palp and antennal defects (Fig. 2B,C). In some cases, the sequences (Quiring et al., 1994). In contrast, the four new maxillary palp, whose identity is indicated by the distinctive eyeless alleles isolated by their interaction with PB all affect distal bristles, remains adjoined to the antennal appendage the EY protein itself, based on the molecular lesions identified (Fig. 2C, arrowheads). However, differentiation of the by PCR amplification and sequencing of genomic exons proboscis (which likewise depends on pb function) is not (Callaerts et al., 2001) (Fig. 1E). Sequence analysis revealed affected (Fig. 2B, ‘lb’). Thus in the sensitized context, ey that eyJD (EMS-induced) harbors a point mutation and encodes mutations can provoke strong defects of the maxillary and a protein truncated in the homeodomain after the first helix. The DEB-induced eyD1Da removes the C-terminal 156 amino acids (Fig. 1E), whereas for a second DEB-induced allele, eyEH, a 6 bp sequence in exon 4 (positions 423-428 in exon 4) is replaced by a single T nucleotide (Fig. 1F). The resulting frameshift should yield a truncated protein of 115 amino acids deleted at the C-terminal end of the Paired domain (Fig. 1E). ey11 allele harbors an EMS-induced point mutation in the splice acceptor site of intron 8 (Fig. 1F; CAG→CAA). Translation of unspliced mRNA retaining intron 8 is expected to terminate at a stop codon

Fig. 1. ey alleles isolated as dominant Enhancers of eye loss induced by ectopic expression from the HSPBsy transgene. Dose- sensitive eye loss induced by HSPBsy. (A-C) Heads of flies carrying one, two or four HSPBsy copies, respectively. (B) Two copies; slightly reduced eye (arrowhead). (C) Four copies; complete eye deletion (arrowhead). The genetic screen selected for female progeny carrying two HSPBsy copies (B) or an interacting Enhancer locus, which yields an eye loss resembling four copies (C). (D) Strength and specificity of the pb-ey genetic interaction. Four new alleles (eyJD, eyD1Da, ey11, eyEH), two previously isolated ey loss-of-function alleles [ey2 and Df(4)BA], and several alleles of other genes implicated in eye differentiation, were tested for dose-sensitive interactions with HSPBsy. Males harboring one HSPBsy copy and heterozygous for mutant alleles of the eye development genes sine oculis (so), eyes absent (eya), eye gone (eyg) or ey were crossed with homozygous HSPBsy females. Maximal eye loss expected is 50% in the resulting female progeny (harboring two copies of HSPBsy) because half should carry the eye gene mutation. Several allele names are shortened: eyac1 is eyaclift1 (from L. Zipursky), eygM is eygM3-12 (from H. Sun) and eyD1 is eyD1Da (this paper). (E,F). All four newly isolated ey alleles should yield truncated forms of the EY protein. (E) Representations of wild-type EY protein and of the proteins encoded by eyJD, eyD1Da (Callaerts et al., 2001), or predicted based on the sequences of ey11 and eyEH (this paper). HD, homeodomain; PD, paired domain. (F) Sequences of wild- type ey and of the mutant lesions in ey11 and eyEH. Modifications in the mutant sequences are underlined, exon or intron boundaries are indicated by arrows, and a new stop codon by ‘-’. pb/ey in head development 579 antennal appendages. The phenotype of Fig. 2B in which the reciprocal effect of eyJD on appendages that derive from the appendages are poorly resolved, suggests that ey+ may same antennal imaginal disc constitutes evidence for a participate in partitioning imaginal disc cells into antenna and potential role for ey in apportioning the maxillary portion of maxillary palp during morphogenesis. this disc. The defects observed in eyJD homozygotes appeared To confirm a role of eyeless in this process, we removed stronger than in the hemizygous combination, eyJD/Df(4)BA. HSPBsy from the genetic background to examine the effects of Thus, the truncated protein may have a limited antimorphic the new eyeless mutations alone. All four ey alleles appear character not detected in the presence of wild-type protein. recessive in a non-sensitized background as shown for eyJD (Fig. 2A,D), and can be interpreted as loss-of-function EY and PB proteins are both expressed in the pupal mutations in accord with their molecular lesions. All give maxillary primordium homozygous escapers with visible defects, allowing us to Although mutant phenotypes implicated both pb and now ey in compose an allelic series, from weakest to strongest: maxillary development, no gene expression had been detected ey11>eyD1Da>eyEH>eyJD. Analysis of the phenotypes of in the maxillary portion of the antennal disc in the third instar hemizygotes with Df(4)BA led to the same conclusion. larvae. We analyzed PB and EY expression later, during the eyJD homozygotes display eye reduction or loss (Fig. 2G), prepupal stage when maxillary and antennal structures low viability and strong brain defects associated with abnormal evaginate from the eye-antenna imaginal disc (Fig. 3A). Using behavior (Callaerts et al., 2001). Furthermore, a minority of a rabbit anti-PB serum directed against the C-terminal region surviving eyJD homozygotes (10-20%, after outcrossing) show (anti-E9), PB accumulation was detected in the central part of alterations in the size and/or shape of maxillary palps and the maxillary primordium beginning approximately eight hours antennae (Fig. 2G-I). The altered maxillary palps of eyJD after puparium formation, during evagination of the antennal homozygotes still harbor the two characteristic sensilla and maxillary appendages from the composite disc (Fig. 3B). trichodea (Fig. 2H, arrowheads), suggesting that maxillary EY protein as visualized by a rabbit anti-EY serum identity per se is not affected. Reduced, malformed maxillary accumulates in the same primordium and, within palps are often accompanied by enlarged, misshapen antennae discrimination, at the same time (Fig. 3C). This expression (compare Fig. 2E and 2H; 2F and 2I). Similar although weaker appeared to be limited to the borders of the primordium (Fig. defects were likewise detected for eyEH homozygotes, as well 3C, higher magnification), rather than the center as for PB (Fig. as for certain trans-heterozygous combinations with other 3B, higher magnification). Results of tests for co-expression of Enhancer alleles in the sensitized background (not shown). The the two proteins were mitigated: in situ hybridization or immunostaining experiments were inconclusive, whereas available antibodies that gave acceptable signals in this tissue were both rabbit polyclonal antisera. To address whether endogenous pb and ey

Fig. 2. ey and pb collaborate in maxillary palp development. (A-C) Interaction of HSPBsy and eyJD in head development. (A) This frontal view of the head of an eyJD heterozygote shows no marked defects [as for a female harboring two copies of HSPBsy (see Fig. 1B)], whereas the combination 2×HSPBsy; eyJD/+ often leads to complete eye loss resembling the head in Fig. 1C. (B) Frontal view, head of a HSPBsy/HSPBsy; eyJD/eyJD female showing complete eye loss and the fusion of an antenna and maxillary palp (boxed). (C) Detail of B. This female lacks a normally placed maxillary palp associated with the labial palps (lb) (arrow); the maxillary appendage (with sensilla trichodea, arrowheads) is fused to the antenna. (D-F) Profile view of the head of an eyJD heterozygote. (D) This head possesses normal antennae (ant), maxillary palps (mx) and labial palps (lb). (E) Enlargement of the maxillary palps. Arrowheads indicate the two maxillary sensilla trichodea. (F) Enlargement of the antennal segment A3 (ant) and arista (ar). (G-I) Profile view of the head of an eyJD homozygote. (G) Overall reduction in head size, full eye loss, altered antennae (ant) and maxillary palps (mx), but normal labial palps (lb). (H) Higher magnification of the maxillary palps. Arrowheads indicate the two maxillary-specific sensilla trichodea signaling maxillary identity, but the appendage is strongly reduced (compared with E, same magnification). (I) The antennal segment A3 (ant) is malformed, slightly enlarged and extended from the head. The arista (ar) is shortened, curved and thickened at the base, malformed and extended from the head. This image is from a different individual from that shown in G, but the phenotype is equivalent. 580 C. Benassayag and others

Fig. 3. EY and PB expression in the pre-pupal eye antennal disc. (A) Eye-antennal imaginal discs (DIC microscopy) after dissection from pre-pupae 8 hours after puparium formation. Eyes (ey), antennae (ant) and maxillary palps (mx) indicate the three adult structures derived from this compound disc. (B) PB expression revealed with a primary rabbit anti-E9 antibody and a HRP-coupled secondary antibody. Arrow indictaes expression in the maxillary primordium. Higher magnification of the maxillary primordium is shown in the inset. (C) EY expression detected with a rabbit anti-EY. Arrowhead indicates EY expression in the eye disc. Arrow shows nuclear expression in the maxillary primordium. The inset shows higher magnification of the maxillary primordium. (D-G) pbGAL4>UASlacZ and EY expression in eye antenna disc. The lacZ immunodetection reflects PB GAL4 expression. Double immunostaining employed secondary antibodies revealing EY (red) and β-galactosidase (green), and images were analyzed by confocal microscopy. (D,E) Maxillary primordium (boxed) is shown in more detail in F and G, respectively. In F, a small number of cells co-expressing PB and EY are indicated by the yellow arrowheads. The white open arrowheads show cells expressing only PB or EY. E and G are at a later stage, as judged by increased cell number. Cells express exclusively pb lacZ (green arrow) or EY (red arrow). The strong expression of lacZ seen in the antennal disc in D is ectopic expression in the distal antenna (see Kapoun and Kaufman, 1995). This expression is not visible in E. patterns in the maxillary palps may overlap, we utilized a pb- eye loss (Fig. 4A; compare parts 1 and 6), whereas the later- GAL4 mini-gene based on previous descriptions of the pb- acting GMR-GAL4 driver had little effect (not shown). We promoter region (Kapoun and Kaufman, 1995). Using a pb- next examined the effects of PB on the expression of several GAL4 driver insertion to direct β-galactosidase expression (pb- identified genes acting early in the eye cascade (see Fig. 4A). GAL4>UAS-lacZ), we examined the patterns of pb>lacZ and Despite a drastic reduction of the L3 eye disc, early PB eye- ey expression by double-immunofluorescence labeling and expression (dpp-GAL4>UAS-PB) does not eliminate ey confocal microscopy. Early PB expression is limited to a small mRNA accumulation there (Fig. 4A, part 7). In contrast, number of cells in the distal maxillary primordium (Fig. 3D expression of the ey target genes so, eya and dac was strongly and 3F, green). At this stage, EY expression could likewise be reduced or abolished (Fig. 4A, parts 8,9,10, respectively). One detected in a small group of cells partially overlapping those possible explanation is that PB represses the same ensemble of expressing PB (Fig. 3F, yellow arrowheads). Co-expression targets activated by EY. If so, removing pb activity in labial appears to be very limited in the progression of a dynamic discs could lead to expression of these eye genes there. Indeed, pattern (compare Fig. 3F and 3G). In later prepupae, the novel dac expression is detected in the labial discs of pb– larvae expression patterns of pb>lacZ and ey in the maxillary in accord with the appearance of ectopic legs (Abzhanov et al., primordium are adjacent but exclusive (Fig. 3G). Taken 2001). In contrast, no corresponding expression was detected together, these data show a previously undisclosed co-temporal for eya or so in mutant labial discs (compared with endogenous expression of both pb and ey in the maxillary primordium of expression in the eye). These results thus raise an alternative prepupae, and support an ephemeral co-expression of these possibility, namely that PB simultaneously suppresses genes in a small number of cells. This is in agreement with the expression of all three EY targets by antagonizing ey activity. known function of pb in maxillary determination, and with the This could occur via a post-transcriptional mechanism, acting newly established function of ey in this tissue. at the level of EY protein (model, Fig. 4B). To test whether PB represses EY transcription factor activity, PB regulates EY transcription factor activity the GAL4 system was used to direct expression of these proteins, To further characterize the molecular basis of the functional singly or together, in several imaginal discs under a common relationship between the two genes, the epistatic relations of pb driver (dpp-GAL4). EY activity was monitored by formation of and ey were tested. eyeless mRNA accumulates normally in pb– adult eyes, and by the expression of the known target gene sine embryos, third instar larvae or pre-pupae, whereas PB protein oculis (so) (employing an enhancer trap line that fully accumulated normally in ey mutants (data not shown). These recapitulates normal so expression in the eye imaginal disc observations argued against a transcriptional cross-regulation (Cheyette et al., 1994). Targeted PB expression induces eye loss, effected by the nuclear EY and PB transcription factors. abnormalities in legs, antennae and wings (see Fig. 4C, part 1) The mechanism of PB-induced eye loss was examined (Aplin and Kaufman, 1997), and diminished so expression in the employing UAS/GAL4-mediated expression. The eye loss is eye imaginal disc (Fig. 4C, parts 1,2). Targeted expression of EY temporally specific, because drivers expressed early during eye in the same tissues induces supernumerary eyes (Fig. 4C, part 4) differentiation (ey-GAL4, dpp-GAL4) led to substantial or full and so-lacZ expression in several tissues including the antennal pb/ey in head development 581

Fig. 4. PB inhibits EY function post- transcriptionally. (A) Targeted expression of PB in eyes represses the EY regulatory pathway, but does not affect ey transcription. In situ hybridization (parts 2,7), β-galactosidase staining (parts 3,8) or immunostaining (parts 4,5,9,10) were performed on wild-type (parts 2-5) or dpp-Gal4;UAS-PB (parts 7-10) third instar eye antenna imaginal discs. Adult heads of wild-type or dpp-Gal4;UAS-PB are shown in (parts 1 and 6). (B) Model proposed to explain PB-dependent EY inhibition. Ectopic eye induction, and consequently activation mediated by ey, is inhibited by PB (C). Targeted expression of PB, EY or both proteins with dpp-Gal4 driver. PB led to eye inhibition and leg and wing defects (C1), whereas ectopic eyes were induced by EY (C4, arrowhead). On co-expressing EY and PB, ectopic eye formation was abolished (C7). Expression of the so- lacZ enhancer trap line was examined in eye antenna (C2,5,8) or wing discs (C3,6,9). β-Galactosidase was induced by EY (C5,6 arrow) but not by PB (C2,3). Co-expression of PB with EY inhibits so activation in all tissues examined (C8,9, arrow).

or in chimeric fusion forms containing homeodomain sequences from PB or ENGRAILED (EN) (Bourbon et al., 1995). The GST-PB fusion protein harbored a 180 amino acid interval (Fig. 5A) containing the homeodomain, adjacent YPWM motif and a conserved C-motif of unknown function (Cribbs et al., 1992) (also see Materials and Methods). These GST-containing proteins were bound to glutathione- Sepharose affinity beads, then tested for binding to 35S-labelled probe proteins generated by in vitro and wing imaginal discs (arrows, Fig. 4C, parts 5,6). On co- transcription and translation. No binding was detected between expressing EY and PB, both proteins co-accumulate, cell by cell, immobilized GST protein and any of the probe proteins tested as detected by double immunofluorescence labeling and confocal (EXD, EY, SO and EYA) (Fig. 5B, lanes 5-8). In contrast, GST- microscopy (not shown). In contrast, ectopic eye formation was EN fusion protein effectively bound the homeodomain- strongly reduced or abolished (Fig. 4C, part 7) as was expression containing EXD protein (Fig. 5B, lane 13) (van Dijk and of the eye reporter gene so (compare Fig. 4C, parts 8,9 and parts Murre, 1994), but not the homeodomain-containing EY and SO 5,6). Normal eye formation and so-lacZ expression were partially nor the nuclear protein EYA (Fig. 5B, lanes 14,15,16). On restored on co-expressing PB with increased EY (transgenic plus testing with the same probes, efficient binding of GST-PB to endogenous copies), suggesting a stoichiometric relationship for EXD was observed (Fig. 5B, lane 9), as predicted based on the these proteins. This observation is supported by results obtained YPWM motif present in PB and implicated in binding to the with other GAL4 driver lines (not shown). homeotic co-factor EXD (Johnson et al., 1995). On testing the three proteins of the eye cascade, PB binding was detected with In vitro protein binding is mediated by the EY Paired EY (Fig. 5B, lane 12), but not with EYA or SO (Fig. 5B, lanes domain and the PB homeodomain 10,11). These data support direct, specific contacts between EY To ask whether PB protein interacts directly with EY, we first and the PB homeodomain-containing region. tested for stable in vitro binding in ‘pull-down’ experiments. The EY protein contains two DNA-binding domains, a Glutathione S-transferase (GST) protein was produced intact, paired domain (PD) and a homeodomain (HD) (see Fig. 1E). 582 C. Benassayag and others

Fig. 5. PB interacts with EY in vitro and inhibits EY trans-activation activity. (A) The PB and EY proteins. HD, homeodomain; PD, paired domain. Grey boxes represent the conserved motifs (N, YPWM,C) of mammalian HOX-A2/B2 and PB (Cribbs et al., 1992). The region between amino acids 126-306 was fused to GST. (B) GST interaction assays. Aliquots (10 µl) of in vitro translated 35S-methionine-labeled EY (lanes 1,5,12,16), EXD (lanes 3,7,9,13), EYA (lanes 4,8,10,14) and SO (lanes 2,6,11,15) were incubated with glutathione agarose beads containing bound GST (lanes 5-8), GST-PB (126-306; lanes 9- 12) or GST-EN (lanes 13-16). An aliquot (5 µl) of in vitro translation products is shown in lanes (1-4). In this experiment, GST-EN bound 13% of the input 35S-labeled EXD protein, whereas GST-PB bound 10% of input 35S-labeled EY. (C) The interaction requires the EY PD and PB HD. GST ‘pull-down’ assays were performed in the same conditions as in B, with 35S-labeled full-length EY proteins (lanes 5,9) and truncated forms of EY lacking the HD (lanes 6,10), PD (lanes 7,11), or HD and PD (lanes 8,12), incubated with GST-PB (lanes 5-8) or GST-PBsy (lanes 9-12), carrying the homeodomain R5H mutation indicated in A. Aliquots (5 µl) of in vitro translation products are shown in lanes 1-4. (D) Yeast one-and-a-half hybrid experiment. A strain carrying the integrated reporter vector pLacZi containing one copy of the so10 enhancer upstream of the minimal promoter of the yeast iso-1-cytochromeC gene. Vectors were pAS and pACT. pAS-EY, EY encoding vector. pACT-PB (126-306, see in A) is fused to the GAL4 activation domain. Lane 1, control with empty vector; lane 2, transactivation of the enhancer so10 with EY; lane 3, co-expression of EY and PB inhibits EY-dependent so10 transactivation, whereas PB does not activate so10 enhancer.

harboring the Arg5→His mutation can still induce eye reduction, suggesting that this point mutation might favor speciﬁc protein contacts with some context-speciﬁc eye protein compared to wild-type PB HD (Benassayag et al., 1997). The GST-PB and GST-PBsy proteins were therefore compared for their relative binding affinities to full-length or deleted forms of 35S-EY as described above. EY protein binding to PB or PBsy is strongly reduced or undetectable for all forms of EY lacking a PD (Fig. 5C, lanes 7,8,11,12). In contrast, EY forms containing the PD (complete, or deleted for the linker and HD: ∆HD), were bound by PB and PBsy (Fig. 5C, lanes 5,6). Binding of input 35S-EY protein to GST-PBsy was consistently enhanced approximately four-fold compared with GST-PB (see Fig. 5C, compare lanes 5 and 9). A similar difference was obtained on probing with 35S-EY PD (∆HD, lacking the HD and linker; Fig. 5C, compare lanes 6 and 10). These results, coherent with the behavior of PB and PBsy in vivo, support a direct role for the N terminus of the PB HD in binding the EY PD.

Of these, the PD has been shown to interact with other PB inhibits EY-dependent transcriptional activation transcription factors (Bendall et al., 1999; Fitzsimmons et al., in yeast 1996; Jun and Desplan, 1996; Plaza et al., 1997; Plaza et al., To address whether this protein-protein interaction can 2001). To define sequences within EY required for interaction contribute to the inhibition of EY activity in a cell, a ‘one-and- with PB, we tested deleted forms of EY lacking the PD, the a-half’ hybrid assay in yeast was performed to test the effects HD, or the region containing both, for their ability to bind the of PB on EY-mediated activation of a sine oculis (so) GST-PB fusion protein. EY deleted of its HD retains PB- transcription enhancer. The reporter gene harbors the so10 binding capacity comparable to the intact protein (Fig. 5C, lane fragment of 400 bp, an eye-specific element directly regulated 6). In contrast, EY protein deleted for the PD (∆PD), with or by EY (Niimi et al., 1999), upstream of the lacZ reporter. EY without the HD and linker, showed little or no interaction with expressed from the pAS plasmid activates so10-lacZ (Fig. 5D PB (Fig. 5C lanes 7,8). This finding supports a crucial role for lane 2). PB sequences were fused to the GAL4 trans-activation the PD in binding with PB. domain in pACT-PB, using the same PB HD-containing A PB protein entirely deficient for in vitro DNA binding but peptide as for the pull-down experiments (amino acids 126- pb/ey in head development 583

306). The so10 fragment was not activated by the chimeric provoked by ectopic PB, we identified new ey alleles isolated GAL4-PB protein (Fig. 5D, lane 4), suggesting that the so10 as eye loss Enhancers. These mutations reveal a role for ey, and fragment is not directly bound by PB. Although EY activates a potential biological relevance for this Hox-PAX6 interaction, so10-lacZ in these conditions, co-expression with GAL4-PB in the development of the antennal and maxillary sensory protein inhibited EY-mediated activation (Fig. 5D, lane 3). This palps. ey loss-of-function defects in the sensory palps are result cannot be explained by a saturation because of the GAL4 exacerbated by PBsy. The most direct interpretation of the activation domain (‘squelching’), because this domain alone enhanced ey loss-of-function phenotype with HSPBsy is that does not affect EY activation (Fig. 5D, lane 2). Thus in yeast the newly isolated alleles retain a partial function that can be cells as in vitro, our data favor a direct contact with PB that negated by adequate PB levels. The molecular characterization reduces EY-mediated so activation in yeast, consistent with our of these alleles is consistent with this hypothesis, because all observations in vivo. Together, these results strongly support a four alleles should encode truncated proteins that contain most direct interaction between conserved domains of PB and EY or all of the interacting PD. that accounts for functional antagonism in the ectopic context To better understand the in vivo relationship between these of developing adult eye, and in the normal development of the two selector genes, we sought to define the effects of double maxillary palp. mutants for pb and ey. Although homozygotes for pb– or for the new ey mutants showed viabilities of up to 50% compared with heterozygotes, we have never obtained a double mutant DISCUSSION adult for any of the four ey alleles. This result, although suggesting that the double mutant is synthetic lethal, does not We previously observed that ectopically expressed homeotic offer insight into the tissue(s) implicated in this lethality. PB protein, even a form bereft of DNA-binding capacity, can One tissue in which an interaction is clearly indicated from suppress eye development in a dose-sensitive manner our analysis is the maxillary palp primordium, where we (Benassayag et al., 1997). Genetic and molecular results detected a dynamic expression of EY and of PB (directly or via presented here indicate a central role for direct contacts the pbGAL4 driver) during pre-pupal development. Transient between conserved domains of the Hox selector protein PB and early co-expression of PB and EY in pre-pupae was limited to the eye selector EY. One physiological situation where the a small number of cells, whereas later expression appears interaction between pb and ey is likely to be relevant was exclusive. This result can be rationalized in two ways: first, co- identified, based on their genetic interaction, mutant expressing cells might be rapidly eliminated by apoptosis, phenotypes and expression patterns in forming the adult through a coordinate gene-activation process triggered by a antennae and maxillary palps. Hox-Pax dimer; second, co-expression of the EY and PB transcription factors could induce a developmental pathway ey function in maxillary palp development interference resulting in a G1 cell-cycle arrest, as recently The present work has identified a previously unrecognized role suggested by Jiao et al. (Jiao et al., 2001). These possibilities for ey in the development of the maxillary palps and antennae. are not fully exclusive. Indeed, one or both mechanisms could The mutation employed for most of our experiments here, eyJD, serve to refine the boundaries between antennal and maxillary behaves as a strong allele affecting viability, formation of the cell populations within the antennal disc. adult eyes and brain mushroom bodies (Callaerts et al., 2001), but also of antennal and maxillary differentiation. Consistent Multiple roles of eyeless in head development with a late requirement for ey in the antennal disc, ey In vertebrates, Pax6 has multiple known or inferred roles in expression in the maxillary primordium appears in early stages eye, brain and nasal development (Grindley et al., 1995; Quinn of metamorphosis when both eye-antennal discs have fused, et al., 1996). Apart from the fly eye, several groups have and the antennal and maxillary appendages start to evaginate. identified an eyeless function required for development of the After evagination, the maxillary primordium migrates to join mushroom bodies, neural structures important for olfactory the labial disc in forming the adult mouthparts, whereas the perception and learning (Callaerts et al., 2001; Kurusu et al., antennal primordium remains near the eye. When a contiguous 2000; Noveen et al., 2000). Our study describes a specific role epidermal cell layer has been completed the head sac is for EY in concert with PB in the maxillary and antennal abruptly evaginated under the internal pressure (Jürgens and appendages that are derived from the antennal disc and Hartenstein, 1993). Maxillary expression in early stages of pre- constitute the adult olfactory system. A recent analysis of pupal metamorphosis was limited to the boundary between the mutations producing headless flies revealed a new role for maxillary primordium and the antenna, and ey mutant Drosophila Pax6 in head morphogenesis and thereby suggests phenotypes often involved simultaneously reduced palps and a requirement of ey for the development of all structures enlarged antennae. These reciprocal effects are consistent with derived from eye-antennal discs (Kronhamn et al., 2002). A communicating cell populations, suggesting that ey may parallel can be drawn with the work presented here because contribute to a partitioning of the antennal disc permitting the both cases involve mutations truncating the EY protein which establishment of two separate appendages. Further analysis of induces head defects. Interestingly, because these truncated EY this process will require new maxillary-specific markers proteins still contain the PD, we cannot exclude the fact that permitting the fates of these cells to be followed. the phenotypes obtained reflect an allele-specific antimorphic effect of the PD. Taken together, these results strongly suggest pb/ey antagonism in maxillary versus antennal that eyeless, apart from its known role in eye morphogenesis, development may also play multiple other roles in head formation (notably In the present work, starting from a dose-sensitive eye loss for brain and olfactory sensory systems). 584 C. Benassayag and others

The development of olfactory and visual systems has several binding but still able to inhibit eye development; (ii) using this common features in Drosophila. Both systems are derived from mutant in a genetic screen to isolate PB functional partners, we the composite eye-antennal imaginal disc. Moreover, both have isolated four independent eyeless mutations, all of them leading similar signal transduction pathways and appear to share to a shortened EY protein; (iii) genetic interaction tests showed regulatory networks (Carlson, 1996; Gaines and Carlson, 1995; that PBsy-induced eye loss is highly sensitive to levels of ey Smith, 1996). However, when we examined the expression of function but independent of several other eye-determining ey, so, eya and dac in the pre-pupal maxillary primordium, only genes including eyg, eya or so; and (iv) ectopically expressed ey expression was detected. This observation suggests that ey PB interferes with ey activity in the eye imaginal disc by acts there via a distinct combinatorial code of regulatory genes inhibiting so and eya activation without affecting ey compared with eye development. One possibility proposed in transcription or EY accumulation. this study is that ey activity is modulated by other co-factors In conclusion, our results suggest that a specific Hox/Pax or transcription factors whose activity is likely sensitive to PB. interaction between PB and EY is involved in a normal In this light, it is worth noting that other Enhancer mutations developmental process defining the boundary between the isolated in our screen also similarly affect maxillary palps, antenna and maxillary palp. More generally, the formation of singly or in combination with ey. It will be of fundamental such protein couples could afford a sensitive and delicate measure interest to better understand the molecular basis for how a of the balance of Pax6 level, permitting a finely tuned integration single protein might function in multiple, distinct networks. to generate distinct transcriptional outputs during development.

HOX-PAX6 interaction as a general regulating We thank Fabienne Pituello for her critical reading of the mechanism manuscript, Laurent Joulia for providing a pbGAL4 line and Henry The ey mutants studied here were identified as dominant Sun, Larry Zipursky, Laura Pignoni, Erich Frei, Markus Noll, Uwe enhancers of pb-induced eye reduction. Consistent with the Walldorf and the Indiana University Drosophila Stock Center for various stocks and reagents used in the course of this study. The antagonism observed in vivo, EY and PB proteins interact monoclonal antibodies directed against Eya protein (developed by S. directly in vitro, via the EY Paired domain and the PB Benzer and N. Bonini) and Dac protein (developed by G. M. Rubin) homeodomain. This interaction with PB that renders EY used in this work were obtained from the Developmental Studies unable to activate its downstream target genes can be extended Hybridoma Bank developed under the auspices of the NICHD and to other homeotic genes because Antp, Scr, Ubx, abdA and maintained by The University of Iowa, Department of Biological AbdB repress eye development while increasing apoptosis in Sciences, Iowa City, IA 52242, USA. We thank Agnès Lepage for her the eye disc, and their protein products likewise interact in vitro expert technical assistance; Bruno Savelli for his generous gifts of with EY protein (Plaza et al., 2001). This suggests a time and competence in our ongoing war against the computer; and combinatorial interaction of homeodomain-containing proteins Thom Kaufman for sharing helpful discussions and unpublished data. (Hox and Pax) to specify a given body segment (Plaza et al., Two eyeless alleles were identified by undergraduate students during a fourth-year genetics course at the Université Paul Sabatier, 2001). An inhibition through physical association has been Toulouse, France (eyJD: Jenny Durrieu de Madron; eyEH: Emma Hill). proposed between Pax6 and En-1 during eye development in This work benefitted from the ongoing support of the Centre National quail (Plaza et al., 1997), and between Pax3 and Msx1 for de Recherche Scientifique (CNRS) and grants from the Association muscle development in chicken (Bendall et al., 1999). pour la Recherche sur le Cancer (ARC) and from Retina France. C. B. Moreover, a similar inhibitory mechanism involving a Hox benefitted from the crucial and timely support of the Fondation pour protein HD has recently been reported in vertebrates la Recherche Médicale (FRM) in the form of a short-term fellowship. (Abramovich et al., 2000; Kataoka et al., 2001); in contrast, S.P. was supported by an EMBO long-term fellowship. physical interaction with Hox-B1 protein led to increased Pax6 activity in Hela cells (Mikkola et al., 2000), raising the possibility that additional context-dependent partners modulate REFERENCES the action of Hox-Pax combinations to generate functional diversity. Based on our genetic and molecular data, we propose Abramovich, C., Shen, W. F., Pineault, N., Imren, S., Montpetit, B., that variations on a PD-HD interface can serve to mediate Largman, C. and Humphries, R. K. (2000). Functional cloning and characterization of a novel nonhomeodomain protein that inhibits the combinatorial or hierarchical functional relationships among binding of PBX1-HOX complexes to DNA. J. Biol. Chem. 275, 26172- Hox and Pax genes in normal development. 26177. The recent paper of Jiao et al. proposed that ectopic Abzhanov, A., Holtzman, S. and Kaufman, T. C. (2001). The Drosophila expression of Hox and Pax (and various other) transcription proboscis is specified by two Hox genes, proboscipedia and Sex combs reduced, via repression of leg and antennal appendage genes. Development factors in the eye induces a generic developmental pathway 128, 2803-2814. interference (Jiao et al., 2001). This cellular process was Aplin, A. C. and Kaufman, T. C. (1997). Homeotic transformation of legs to proposed as a general surveillance mechanism that eliminates mouthparts by proboscipedia expression in Drosophila imaginal discs. most aberrations in the genetic program during development Mech. Dev. 62, 51-60. and evolution, but is not a consequence of specific protein Benassayag, C., Seroude, L., Boube, M., Erard, M. and Cribbs, D. L. (1997). A homeodomain point mutation of the Drosophila proboscipedia collaboration. However, the existence of such a mechanism protein provokes eye loss independently of homeotic function. Mech. Dev. does not preclude the possibility of more specific interactions 63, 187-198. such as we describe. The results presented here appear to favor Bendall, A. J., Ding, J., Hu, G., Shen, M. M. and Abate-Shen, C. (1999). a specific role for discrete protein-protein interactions rather Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors. Development 126, 4965-4976. than an indirect interference mechanism. Indeed, (i) by Bonini, N. M., Leiserson, W. M. and Benzer, S. (1993). The eyes absent analysing the residues of PB protein involved in its homeotic gene: genetic control of cell survival and differentiation in the developing function, we identified a PBsy protein with diminished DNA Drosophila eye. Cell 72, 379-395. pb/ey in head development 585

Bopp, D., Burri, M., Baumgartner, S., Frigerio, G. and Noll, M. (1986). Mouse small eye results from mutations in a paired-like homeobox- Conservation of a large protein domain in the segmentation gene paired and containing gene. Nature 354, 522-525. in functionally related genes of Drosophila. Cell 47, 1033-1040. Hogan, B. L., Hirst, E. M., Horsburgh, G. and Hetherington, C. M. (1988). Botas, J. (1993). Control of morphogenesis and differentiation by HOM/Hox Small eye (Sey): a mouse model for the genetic analysis of craniofacial genes. Curr. Opin. Cell Biol. 5, 1015-1022. abnormalities. Development 103 Suppl, 115-119. Boube, M., Seroude, L. and Cribbs, D. L. (1998). Homeotic proboscipedia Jiao, R., Daube, M., Duan, H., Zou, Y., Frei, E. and Noll, M. (2001). cell identity functions respond to cell signaling pathways along the proximo- Headless flies generated by developmental pathway interference. distal axis. Int. J. Dev. Biol. 42, 431-436. Development 128, 3307-3319. Bourbon, H. M., Martin-Blanco, E., Rosen, D. and Kornberg, T. B. (1995). Johnson, F. B., Parker, E. and Krasnow, M. A. (1995). Extradenticle protein Phosphorylation of the Drosophila engrailed protein at a site outside its is a selective cofactor for the Drosophila homeotics: role of the homeodomain enhances DNA binding. J. Biol. Chem. 270, 11130-11139. homeodomain and YPWM amino acid motif in the interaction. Proc. Natl. Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means Acad. Sci. USA 92, 739-743. of altering cell fates and generating dominant phenotypes. Development 118, Jun, S. and Desplan, C. (1996). Cooperative interactions between paired 401-415. domain and homeodomain. Development 122, 2639-2650. Callaerts, P., Halder, G. and Gehring, W. J. (1997). PAX-6 in development Jun, S., Wallen, R. V., Goriely, A., Kalionis, B. and Desplan, C. (1998). and evolution. Annu. Rev. Neurosci. 20, 483-532. Lune/eye gone, a Pax-like protein, uses a partial paired domain and a Callaerts, P., Leng, S., Clements, J., Benassayag, C., Cribbs, D., Kang, Y. homeodomain for DNA recognition. Proc. Natl. Acad. Sci. USA 95, 13720- Y., Walldorf, U., Fischbach, K. F. and Strauss, R. (2001). Drosophila Pax- 13725. 6/eyeless is essential for normal adult brain structure and function. J. Jürgens, G. and Hartenstein, V. (1993). The terminal regions of the body Neurobiol. 46, 73-88. pattern. In The Development of Drosophila Melanogaster (ed. M. B. and A. Carlson, J. R. (1996). Olfaction in Drosophila: from odor to behavior. Trends Martinez-Arias), pp. 687-746. Cold Spring Harbor: Laboratory Press. Genet. 12, 175-180. Kapoun, A. M. and Kaufman, T. C. (1995). A functional analysis of 5′, Chan, S. K., Jaffe, L., Capovilla, M., Botas, J. and Mann, R. S. (1994). The intronic and promoter regions of the homeotic gene proboscipedia in DNA binding specificity of Ultrabithorax is modulated by cooperative Drosophila melanogaster. Development 121, 2127-2141. interactions with extradenticle, another homeoprotein. Cell 78, 603-615. Kataoka, K., Yoshitomo-Nakagawa, K., Shioda, S. and Nishizawa, M. Cheyette, B. N., Green, P. J., Martin, K., Garren, H., Hartenstein, V. and (2001). A set of Hox proteins interact with the Maf oncoprotein to inhibit Zipursky, S. L. (1994). The Drosophila sine oculis locus encodes a its DNA binding, transactivation, and transforming activities. J. Biol. Chem. homeodomain-containing protein required for the development of the entire 276, 819-826. visual system. Neuron 12, 977-996. Kaufman, T. C. (1978). Cytogenetic analysis of chromosome 3 in Drosophila Cribbs, D. L., Benassayag, C., Randazzo, F. M. and Kaufman, T. C. (1995). melanogaster: isolation and characterization of four new alleles of the Levels of homeotic protein function can determine developmental identity: proboscipedia (pb) locus. Genetics 90, 579-596. evidence from low-level expression of the Drosophila homeotic gene Kronhamn, J., Frei, E., Daube, M., Jiao, R., Shi, Y., Noll, M. and proboscipedia under Hsp70 control. EMBO J. 14, 767-778. Rasmuson-Lestander, A. (2002). Headless flies produced by mutations in Cribbs, D. L., Pultz, M. A., Johnson, D., Mazzulla, M. and Kaufman, T. the paralogous Pax6 genes eyeless and twin of eyeless. Development 129, C. (1992). Structural complexity and evolutionary conservation of the 1015-1026. Drosophila homeotic gene proboscipedia. EMBO J. 11, 1437-1449. Kurusu, M., Nagao, T., Walldorf, U., Flister, S., Gehring, W. J. and Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Furukubo-Tokunaga, K. (2000). Genetic control of development of the Lee, W. H. and Elledge, S. J. (1993). The retinoblastoma protein associates mushroom bodies, the associative learning centers in the Drosophila brain, with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555-569. by the eyeless, twin of eyeless, and Dachshund genes. Proc. Natl. Acad. Sci. Fitzsimmons, D., Hodsdon, W., Wheat, W., Maira, S. M., Wasylyk, B. and USA 97, 2140-2144. Hagman, J. (1996). Pax-5 (BSAP) recruits Ets proto-oncogene family Lawrence, P. A. and Morata, G. (1994). Homeobox genes: their function in proteins to form functional ternary complexes on a B-cell-specific promoter. Drosophila segmentation and pattern formation. Cell 78, 181-189. Genes Dev. 10, 2198-2211. Lewis, E. B. and Bacher, F. (1968). Method of feeding ethyl methane- Frigerio, G., Burri, M., Bopp, D., Baumgartner, S. and Noll, M. (1986). sulphonate to Drosophila males. Dros. Inf. Serv. 43, 193-194. Structure of the segmentation gene paired and the Drosophila PRD gene set Manak, J. R. and Scott, M. P. (1994). A class act: conservation of as part of a gene network. Cell 47, 735-746. homeodomain protein functions. Development Suppl., 61-77. Gaines, P. and Carlson, J. R. (1995). The olfactory and visual systems are Mann, R. S. (1995). The specificity of homeotic gene function. BioEssays 17, closely related in Drosophila. Braz. J. Med. Biol. Res. 28, 161-167. 855-863. Garcia-Bellido, A. (1975). Genetic control of wing disc development in McGinnis, W. and Krumlauf, R. (1992). Homeobox genes and axial Drosophila. Ciba Found. Symp. 161-182. patterning. Cell 68, 283-302. Garcia-Bellido, A. (1977). Homeotic and atavic mutations in insects. Am. Mikkola, I. I., Bruun, J. A., Holm, T. and Johansen, T. (2000). Zool. 17, 613-629. Superactivation of Pax6-mediated transactivation from paired domain- Gehring, W. J., Affolter, M. and Burglin, T. (1994). Homeodomain proteins. binding sites by DNA-independent recruitment of different homeodomain Annu. Rev. Biochem. 63, 487-526. proteins. J. Biol. Chem. 7, 7. Glaser, T., Jepeal, L., Edwards, J. G., Young, S. R., Favor, J. and Maas, Morata, G. (1993). Homeotic genes of Drosophila. Curr. Opin. Genet. Dev. R. L. (1994). PAX6 gene dosage effect in a family with congenital cataracts, 3, 606-614. aniridia, anophthalmia and central nervous system defects. Nat. Genet. 7, Niimi, T., Seimiya, M., Kloter, U., Flister, S. and Gehring, W. J. (1999). 463-471. Direct regulatory interaction of the eyeless protein with an eye- specific Graba, Y., Aragnol, D. and Pradel, J. (1997). Drosophila Hox complex enhancer in the sine oculis gene during eye induction in Drosophila. downstream targets and the function of homeotic genes. BioEssays 19, 379- Development 126, 2253-2260. 388. Noveen, A., Daniel, A. and Hartenstein, V. (2000). Early development of the Greer, J. M., Puetz, J., Thomas, K. R. and Capecchi, M. R. (2000). Drosophila mushroom body: the roles of eyeless and dachshund. Maintenance of functional equivalence during paralogous Hox gene Development 127, 3475-3488. evolution. Nature 403, 661-665. Passner, J. M., Ryoo, H. D., Shen, L., Mann, R. S. and Aggarwal, A. K. Grindley, J. C., Davidson, D. R. and Hill, R. E. (1995). The role of Pax-6 (1999). Structure of a DNA-bound Ultrabithorax-Extradenticle in eye and nasal development. Development 121, 1433-1442. homeodomain complex. Nature 397, 714-719. Halder, G., Callaerts, P., Flister, S., Walldorf, U., Kloter, U. and Gehring, Percival-Smith, A., Weber, J., Gilfoyle, E. and Wilson, P. (1997). Genetic W. J. (1998). Eyeless initiates the expression of both sine oculis and eyes characterization of the role of the two HOX proteins, Proboscipedia and Sex absent during Drosophila compound eye development. Development 125, Combs Reduced, in determination of adult antennal, tarsal, maxillary palp 2181-2191. and proboscis identities in Drosophila melanogaster. Development 124, Halder, G., Callaerts, P. and Gehring, W. J. (1995). New perspectives on 5049-5062. eye evolution. Curr. Opin. Genet. Dev. 5, 602-609. Pinsonneault, J., Florence, B., Vaessin, H. and McGinnis, W. (1997). A Hill, R. E., Favor, J., Hogan, B. L., Ton, C. C., Saunders, G. F., Hanson, model for extradenticle function as a switch that changes HOX proteins from I. M., Prosser, J., Jordan, T., Hastie, N. D. and van Heyningen, V. (1991). repressors to activators. EMBO J. 16, 2032-2042. 586 C. Benassayag and others

Plaza, S., Langlois, M. C., Turque, N., LeCornet, S., Bailly, M., Begue, A., Seroude, L. and Cribbs, D. L. (1994). Differential effects of detergents on Quatannens, B., Dozier, C. and Saule, S. (1997). The homeobox- enzyme and DNA-binding activities of a glutathione S-transferase-homeo- containing Engrailed (En-1) product down-regulates the expression of Pax- domain fusion protein. Nucleic Acids Res. 22, 4356-4357. 6 through a DNA binding-independent mechanism. Cell Growth Differ. 8, Smith, D. P. (1996). Olfactory mechanisms in Drosophila melanogaster. Curr. 1115-1125. Opin. Neurobiol. 6, 500-505. Plaza, S., Prince, F., Jaeger, J., Kloter, U., Flister, S., Benassayag, C., Smolik-Utlaut, S. M. (1990). Dosage requirements of Ultrabithorax and Cribbs, D. and Gehring, W. J. (2001). Molecular basis for the inhibition bithoraxoid in the determination of segment identity in Drosophila of Drosophila eye development by Antennapedia. EMBO J. 20, 802-811. melanogaster. Genetics 124, 357-366. Pultz, M. A., Diederich, R. J., Cribbs, D. L. and Kaufman, T. C. (1988). Staehling-Hampton, K., Jackson, P. D., Clark, M. J., Brand, A. H. and The proboscipedia locus of the Antennapedia complex: a molecular and Hoffmann, F. M. (1994). Speciﬁcity of bone morphogenetic protein-related genetic analysis. Genes Dev. 2, 901-920. factors: cell fate and gene expression changes in Drosophila embryos Quinn, J. C., West, J. D. and Hill, R. E. (1996). Multiple functions for Pax6 induced by decapentaplegic but not 60A. Cell Growth Differ. 5, 585-593. in mouse eye and nasal development. Genes Dev. 10, 435-446. Strachan, T. and Read, A. P. (1994). PAX genes. Curr. Opin. Genet. Dev. 4, Quiring, R., Walldorf, U., Kloter, U. and Gehring, W. J. (1994). Homology 427-438. of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia Stuart, E. T., Kioussi, C. and Gruss, P. (1994). Mammalian Pax genes. Annu. in humans. Science 265, 785-789. Rev. Genet. 28, 219-236. Rauskolb, C., Peifer, M. and Wieschaus, E. (1993). Extradenticle, a regulator Ton, C. C., Hirvonen, H., Miwa, H., Weil, M. M., Monaghan, P., Jordan, of homeotic gene activity, is a homolog of the homeobox-containing human T., van Heyningen, V., Hastie, N. D., Meijers-Heijboer, H., Drechsler, M. proto-oncogene pbx1. Cell 74, 1101-1112. et al. (1991). Positional cloning and characterization of a paired box- and Roch, F. and Akam, M. (2000). Ultrabithorax and the control of cell homeobox- containing gene from the aniridia region. Cell 67, 1059-1074. morphology in Drosophila halteres. Development 127, 97-107. van Dijk, M. A. and Murre, C. (1994). Extradenticle raises the DNA binding Schmahl, W., Knoedlseder, M., Favor, J. and Davidson, D. (1993). Defects speciﬁcity of homeotic selector gene products. Cell 78, 617-624. of neuronal migration and the pathogenesis of cortical malformations are Zimmerman, J. E., Bui, Q. T., Liu, H. and Bonini, N. M. (2000). Molecular associated with Small eye (Sey) in the mouse, a point mutation at the Pax- genetic analysis of Drosophila eyes absent mutants reveals an eye enhancer 6-locus. Acta Neuropathol. 86, 126-135. element. Genetics 154, 237-246.