Dominant Drop Mutants Are Gain-Of-Function Alleles Of

Developmental Biology 233, 380–393 (2001) doi:10.1006/dbio.2001.0229, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE Dominant Drop Mutants Are Gain-of-Function provided by Elsevier - Publisher Connector Alleles of the muscle segment homeobox Gene (msh) Whose Overexpression Leads to the Arrest of Eye Development

Brian A. Mozer1 Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the DrMio allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the DrMio mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins. © 2001 Academic Press Key Words: Drosophila; msh; Drop; eye development; gain-of-function mutation; morphogenetic furrow.

INTRODUCTION Gain-of-function alleles represent another important class of dominant mutations. In heterozygotes, dominant negative Dominant mutations in Drosophilia melanogaster have gain-of-function mutations can inactivate the wild-type gene been a favorite subject for genetic study, because of their through a titration mechanism and are useful tools for reverse dramatic phenotypes and tissue specificity. Haploinsuffi- genetics (Herskowitz, 1987). ciency accounts for a significant fraction of dominant muta- Genetic analysis of compound eye development in the fly tions resulting in a range of phenotypes from subtle patterning has identified important regulatory genes involved in di- defects [i.e., Notch; notch wings (Mohr, 1919); Ubx, enlarged verse aspects of pattern formation, including neuronal haltere (Lewis, 1978)] to lethality [dpp (Gelbart et al., 1985)]. specification, cell proliferation, and transcription (see recent review, Thomas and Wassarman, 1999). Dominant 1 Current address: Laboratory of Biochemical Genetics, Building gain-of-function mutations affecting eye development have 36, Room 1C23, NHLBI, NIH, Bethesda, MD 20892. E-mail: led to the identification of genes which are required for [email protected] early pattern formation [Glazed, an allele of wingless (Brun-

0012-1606/01 $35.00 Copyright © 2001 by Academic Press 380 All rights of reproduction in any form reserved. Drop Gain-of-Function Mutation in Drosophila 381 ner et al., 1999)] or cell type differentiation [Ellipse, an MATERIALS AND METHODS allele of the epidermal growth factor receptor (Baker and Rubin, 1989); sevenmaker, an allele of map kinase (Brunner Drosophila Strains et al., 1994); Bar, a mutation in a homeodomain transcrip- The dominant Dr1 and DrMi alleles are described (Lindsley and tion factor (Higashijima et al., 1992)] or that encode essen- Zimm, 1992; Tearle et al., 1994). DrL2 is a ␥-ray-induced allele and tial structural components of all cells [i.e., Glued, a gain- was a gift from E. B. Lewis. Df(3R)KE deletes part of the PTP99A of-function mutation of dynactin (Fan and Ready, 1997)]. gene and distal DNA in the 99A7-10 cytogenetic region (Kai Zinn, Finally, dominant mutations have also identified genes personal communication). l(3)CB24 is a P-element-induced lethal which are not required for eye development, but whose (Hamilton et al., 1995). The msh allele used for complementation ems misexpression leads to its perturbation. For example, the tests with Dr revertants was ltt (Buescher and Chia, 1997). Failure of complementation was observed by the absence of Sbϩ mutation Irregular facets is a dominant gain-of-function ϩ Hu (genotype Drrevertant/lttems) progeny from the cross of females kruppel, allele of a gene encoding a zinc finger transcription Dr/TM6b, Tb Sb with ltt/TM6b, Tb Hu males. The lethal phase of factor required for embryonic patterning but not in eye most of the transheterozygotes was prior to the third larval instar (Carrera et al., 1998). as judged by the absence of Tbϩ larvae among the progeny. Two Dominant alleles at the Drop (Dr) locus were originally ethylmethanosulfonate (EMS)-induced stg alleles, 7M and 7B69, identified because of their severe effect on compound eye used for complementation tests with DrL2, and all other chromo- development but are also associated with recessive pheno- somes used in this study are described in detail on FLYBASE. types including patterning defects in eye, bristle defects, and lethality. Previous genetic analysis of Dr alleles sug- Isolation of Revertant Derivatives of DrMio gested that the mutations affected two closely linked genes, The DrMio chromosome was outcrossed to CantonS females for string (stg) and a distal lethal (Tearle et al., 1994). Dr- two generations and rebalanced prior to mutagenesis. DrMio/TM6b, associated lesions in stg are recessive, affecting postembry- Tb Sb adult males were fed EMS (25 mM in sucrose) as described onic expression in eye which results in defects in cell (Lewis and Bacher, 1968) and crossed to virgin females of the same proliferation and pattern formation (Mozer and genotype. Revertants were recovered over the TM6b balancer Easwarachandran, 1999). In this report, I show that the chromosome and identified by the complete suppression of the dominant Dr mutants are gain-of-function alleles of the dominant eye phenotype. Approximately 100,000 progeny were scored and 13 revertants were identified. With the exception of the muscle segment homeobox (msh) gene, which encodes a R53 and 61 alleles, reversion did not effect the genetic interaction putative transcription factor containing a homeodomain of the Dr mutant chromosome with stg alleles. The R61 revertant (Gehring, 1987) and is required for patterning the embry- is a deletion that removes genes both proximal (stg) and distal (ca) onic nervous system (Buescher and Chia, 1997; Isshiki et to the 3S18 insertion. The R53 allele complemented stg7M and thus al., 1997) and musculature (Nose et al., 1998). Insertion of a appears to have lost the stg-associated lesion on the Dr chromo- 3S18 retrotransposon upstream of the msh promoter was some. P-element-induced revertants of DrMio were isolated in the male implicated as the cause of the dominant eye defect in the ϩ 8 Mio Mio progeny of the cross females Xˆ X; yw, P[lacZ, w ] ;Dr /TMS, Sb Dr mutation. Genetic and molecular analysis of recessive ϩ ⌬2-3 with males yw; TM3, Ser y /Sb. Of ϳ15,000 progeny scored 2 derivatives of Dr (revertant alleles) suggests that overex- wϩ revertants (P10 and P19) with suppressed eyes were identified. pression of msh results in the dominant eye phenotype. The P19 revertant chromosome contained two P-element inser- Intragenic suppression of the dominant Dr eye phenotype tions, one at 99A and a second which was subsequently removed by was associated with either the generation of loss-of- recombination. function mutations in msh (lethal class) or the deletion of the upstream retrotransposon (viable class). Expression of Generation of Dr Mosaic Eye Discs 3S18 encoded transcripts in wild-type retinal precusors Mio Mio[P}10 cells suggests that ectopic expression of msh in the mutant Mosaic eye discs containing patches of Dr and Dr cells were generated by inducing the somatic excision (loss) of the P is the result of transcriptional activation mediated by element. Approximately forty discs were dissected from third- cis-regulatory sequences in the retrotransposon. For the instar Tbϩ larvae of the genotype DrMio[P]10/Sb⌬2-3 from the cross of strongest Dr alleles, the severe reduction of the adult retina females w; DrMio[P]10/TM6b Tb Hu with males Sb ⌬2-3/TM6b, Tb is the result of developmental defects in the third larval Hu. instar eye imaginal disc. In the DrMio mutant, most retinal precursor cells fail to differentiate and undergo apoptosis as Staining Procedures a consequence of the arrest of the morphogenetic furrow. Gal4-mediated ubiquitous expression of msh in wild-type Immunostaining of eye imaginal discs was performed as de- eye imaginal disc cells was sufficient to elicit the furrow scribed (Mozer and Benzer, 1994). Primary antibodies used were MAB22C10 (Fujita et al., 1/50 dilution) and monoclonal anti-␤-Gal msh arrest phenotype, while overexpression of behind the (Promega; 1/50 dilution). Enhancer trap insertions were crossed furrow resulted in eye roughening. These observations into the Dr mutant background and assayed in dissected eye suggests that ectopic expression of msh in retinal precursor imaginal discs. The following reporters were used: dpp:lacZ [line cells ahead of the furrow interferes with its progression. BS3.0 (Blackman et al., 1991)] and C93, a P-element insertion in the

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 382 Brian A. Mozer promoter of the hairy gene (Mozer, Choi, and Benzer, unpublished). RESULTS Wingless expression was monitored histochemically using the wg:lacZ insertion (Kassis et al., 1992). Cell death in the DrMio eye Dominant Dr Alleles Reduce Ommatidial Number disc was assayed using fluorescence microscopy by staining un- without Affecting Cell-Type Specification fixed tissue with acridine orange as described (Bonini et al., 1993). ϳ In situ hybridization was performed as described (Mozer and The wild-type eye (Fig. 1A) consists of 700 ommatidia, Easwarachandran, 1999) using digoxigenin-labeled stg cDNA or each containing eight photoreceptor neurons and additional Mio msh genomic clones. For assaying the expression of the 3S18 nonneuronal cells. The eye of the Dr mutant (Fig. 1B) is element, digoxigenin-labeled probe derived from the rescued plas- severely reduced in size, containing Ͻ30 ommatidia; how- mid pR10E was used. ever, sectioning of the retina revealed no patterning defects (data not shown). Similarly the eye of a novel dominant allele, DrL2, isolated by Ed Lewis (Fig. 1C), exhibits a mild Molecular Biology reduction in eye size but is otherwise normal. Most Dr mutants are semilethal alleles of stg (Mozer and Plasmid rescue of the P10 revertant was carried out as described Easwarachandran, 1999; Tearle et al., 1994). Likewise, the (Mozer and Benzer, 1994). DrMio[P]10 genomic DNA was digested DrL2 mutant is also a loss-of-function allele of stg, as it separately with EcoRI and BglII and fragments of 3.0 (pP10E) and failed to complement the lethality of stg7B and stg7M. 12.0 kb (pP10G) were cloned. Southern analysis revealed the presence of a repetitive sequence in the clones, which was shown to be homologous to the 3S18 retrotransposon family by sequence Dr Dominant Mutations Are Gain-of-Function analysis and Blast search. Genomic DNA surrounding the insertion Alleles of msh was cloned by chromosome walking using a ␭ library of wild-type Loss-of-function alleles can frequently be recovered as genomic DNA and initially screening with a nonrepetitive frag- intragenic suppressors (revertants) of a dominant gain-of- ment derived from the pR10G plasmid. Northern blot analysis and function mutation following secondary mutagenesis. A screening cDNA libraries from embryo and adult head and eye previous study of the Dr mutants suggested that the domi- imaginal disc identified two transcription units on the proximal nant eye phenotype is not due to a gain-of-function muta- side of the insertion site in addition to msh. Genomic sequence of tion in stg, but rather identifies a second distal gene (Tearle the cloned region is available from GenBank, and analysis of the 35-kb region upstream of the msh transcription start site using the et al., 1994). However, genetic analysis of the distal gene in Genefinder program failed to identify other open reading frames. the Dr revertants was complicated by the presence of an Likewise, Blast searches with translations of the sequence in all six additional lethal lesion in stg on the mutant chromosomes. reading frames failed to identify statistically meaningful homology In order to identify the affected gene and the consequence of to known proteins in the data base. genetic reversion, EMS-induced revertants of DrMio were isolated and tested for complementation with Df(3R)KE, a chromosomal deletion which removes the distal 99A region. The deficiency complements all stg mutant pheno- Overexpression of msh Using the Gal4 System types, including the recessive bristle and eye defects asso- Ectopic expression of msh during eye development was achieved ciated with Dr mutant alleles (Mozer and Easwarachandran, using the Gal4 system (Brand and Perrimon, 1993). Two strains, m1 1999). Thus, the phenotype of Dr/Df(3R)KE heterozygotes and m6 (Isshiki et al., 1997), homozygous for the UAS:msh inser- reveals the allelic state of the distal gene independent of stg tions, were crossed to the following driver-bearing strains: ey:Gal4/ mutant effects. CyO, h:Gal4, dpp:Gal4; L/CyO, hsp70:Gal4, and gmr:Gal4/CyO Of 13 EMS-induced revertants of DrMio tested, 8 (R7, 11, (for more information on these strains see http://flybase.bio. 54, 55, 60, 61, 62, and 65) were found to be lethal in indiana.edu). Adult progeny heterozygous for both transgenes were combination with Df(3R)KE, while 5 revertants (R10, 21, identified by the absence of the Cy marker and examined for eye 22, 53, and 59) and the parental DrMio chromosome were defects. Ubiquitous expression was induced following a 1-h heat viable. Thus, reversion of the DrMio dominant eye pheno- shock by incubating culture vials containing hsp70:Gal4/ϩ; UAS: type is most often coincident with lethality, but can also msh/ϩ third-instar larvae at 37°C for1hinawater bath. Following a recovery period of4hatroom temperature, the eye imaginal discs occur by a second mechanism which has no recessive effect. were dissected and immunostained. Eye imaginal discs from larvae Dr revertants were crossed with representative alleles of of the genotype hsp70:Gal4/ϩ; UAS:LacZ/ϩ subjected to the same two previously identified lethal complementation groups experimental conditions served as control. uncovered by Df(3R)KE: l(3)CB24 and msh. All Dr revertants were viable in combination with l(3)CB24. In contrast, 8 Dr revertants were lethal in combination with the msh allele lttems, identifying the distal gene affected in the Scanning Electron Microscopy 1 revertants as msh. Two additional dominant alleles, Dr L2 Adult flies were affixed with silver paste to specimen holders and Dr , were subjected to complementation tests with ems Mio 1 and dried overnight in dessicant prior to sputter coating. Adult eyes Df(3R)KE and ltt . Like Dr , the Dr allele was viable in L2 were viewed in a scanning electron microscope as described (Ren- trans to Df(3R)KE. In contrast, the Dr allele was lethal in franz and Benzer, 1989). combination with both chromosomes, suggesting that DrL2

TABLE 1 regulated in the third larval instar eye disc by the morpho- Summary of Genetic Analysis of Drop (Dr) Mutations: Allelism genetic furrow, which sweeps across the disc in a posterior- with Muscle segment homeotic (msh) to-anterior direction (Ready et al., 1976). Previous studies have suggested that the DrMio allele blocked the differentia- Allele Mutagen Eye phenotype tion of retinal cells (Renfranz and Benzer, 1989) and be- Dominant gain of function longed to a class of eye mutants in which progress of the DrMio Mustard gas Severe reduction morphogenetic furrow was arrested (Heberlein et al., 1993). Dr1 (?) X ray Severe reduction The effect of dominant Dr mutations on retinal morpho- Revertants (DrMio) genesis was examined in detail using markers with regional Drr7,11,54,55,60,61,62, and 65 EMS wt expression in the eye imaginal disc, an antibody specific for 1 lethal neurons, and acridine orange to detect cell death. In a R10,21,22,53,59 Dr EMS wt wild-type disc in which the furrow had traversed to ϳ50% viable1 (Fig. 2A), Mab22C10 labels multiple rows of differentiating DrP10 P element wt DrP19 P element Partial reduction ommatidia, revealing the anterior-to-posterior gradient Loss of function (youngest to oldest) of neuronal differentiation behind the DrL2 ␥ ray Partial reduction furrow (Thomlinson and Ready, 1987). At higher magnifi- lttems(Beuscher et al., 1999) EMS wt cation, immature five-cell preclusters immediately behind the furrow are readily distinguishable from mature clusters Note. Drop (Dr) dominant mutants and their recessive deriva- containing eight neurons at the posterior (data not shown). tives used for this study. Intragenic revertants of DrMio were r P In contrast, five-cell preclusters were not observed in the isolated using EMS (Dr ) or P elements (Dr ) as the mutagen and DrMio eye disc; instead only a single row of mature omma- identified by the suppression of the adult small eye phenotype tidial clusters was detected using MAB22C10 (Fig. 2B). (details of the mutagenesis described under Materials and Meth- ods). The majority of recessive DrMio revertants induced with EMS The reduction of cluster number and absence of imma- Mio are loss-of-function alleles of msh as they failed to complement the ture clusters in the Dr eye disc suggest that the mutation lethality of lttems. The dominant alleles DrMio,Dr1, and DrL2 are blocks progression but not initiation of the furrow. Signal- homozygous lethal; however, only DrL2 was lethal in complemen- ing from Decapentaplegic (dpp), a member of the TGF-␤ tation tests with the msh allele (lttems). These observations suggest superfamily, and its downstream components is required that DrMio is a gain-of-function allele of Msh. The Dr1 allele may for both initiation and progression of the morphogenetic also be a gain-of-function msh allele, based on prior genetic furrow (Burke and Basler, 1996; Chanut and Heberlein, characterization (Tearle et al., 1999) and molecular criteria (this L2 1997; Pignoni and Zipursky, 1997; Wiersdorff et al., 1996). paper). The Dr mutation is a loss-of-function allele of msh, but Expression of dpp RNA in the third-instar eye imaginal disc the similarity of the dominant eye phenotype to that of the extant is restricted to cells within the furrow and can be monitored Dr alleles suggests it may also be a gain-of-function allele. from the lacZ reporter construct, BS3.0 (Blackman et al., 1991). In the wildtype, lacZ driven from the reporter is restricted to a stripe of cells spanning the furrow and the is also a loss-of-function allele of msh. This genetic analysis lateral margins (Fig. 2C). In DrMio eye disc, lacZ expression (summarized in Table 1) demonstrates that Dr mutations is detected only in the lateral margin cells (Fig. 2D). Thus, are alleles of msh: DrMio and Dr1 alleles are simple gain-of- the block of furrow progression in the mutant is associated function mutations while most revertants of DrMio are loss with the absence of dpp expression. Wingless (wg) protein of function. The DrL2 mutation is likely both a loss- and a antagonizes dpp to negatively regulated furrow initiation gain-of-function allele of msh. from the eye disc margins (Ma and Moses, 1995; Treisman and Rubin, 1995), and ectopic expression of wg inhibits furrow initiation from the posterior (Treisman and Rubin, The DrMio Mutation Blocks Early Progression of the 1995). To ascertain if the defect in dpp expression in the Dr Morphogenetic Furrow and Is Associated mutant is due to ectopic wingless, lacZ expression from a with Cell Death Mio wingless reporter (Kassis et al., 1992) was assayed in a Dr The anlage of the fly retina is the eye antennal imaginal mutant background and was indistinguishable from wild- disc. Embryonic eye antennal disc progenitor cells undergo type (data not shown). Thus, the furrow arrest in the Dr continuous rounds of cell division during the first and mutant is not associated with altered wingless expression. second larval instars. Retinal morphogenesis and cell-type The morphogenetic furrow arrest in the DrMio eye disc specification begin at the third larval instar stage and may be a direct effect of the mutation or a secondary continue through late pupal development. Thus, the re- consequence of events anterior to the furrow. Two markers duced eye size in the dominant Dr mutants could be the of anterior precursor cells, the enhancer trap C93 and stg, result of a decrease in early proliferation, arrest of eye were assayed in DrMio eye discs. The stripe of stg RNA formation during morphogenesis, or subsequent loss of expression ahead of the furrow in the wild-type eye disc ommatidia during pupation. (Fig. 2E) was absent in the DrMio disc (Fig. 2F). In contrast, Retinal patterning and cell proliferation are coordinately the DrMio mutation did not affect lacZ expression from the

FIG. 1. Eye phenotype of dominant Dr mutant alleles. Scanning electron micrographs of the compound eye of wild-type (A) and two different dominant Drop (Dr) alleles, Mio (B) and L2 (C). The shape of the mutant eyes resembles an inverted drop. The Mio allele has fewer than 30 ommatidia, while the reduction of the retina caused by the L2 mutation is less severe. (Original magniﬁcation 200ϫ).

C93 enhancer trap insertion in cells anterior to the furrow absent in the mutant disc. These observations suggest that (Fig. 2H). However, the expression of lacZ in cells within in the DrMio mutant the initial defect in the eye disc is ahead and behind the furrow in the wild-type disc (Fig. 2G) was of the furrow; all subsequent defects (absence of dpp expres-

FIG. 2. A defect in the progression of the morphogenetic furrow results in the failure of cluster formation and retinal precursor cell death in the DrMio eye disc. Expression of cell-type-specific markers during eye development in third-instar larval eye discs from wildtype (A, C, E, G, I) and DrMio (B, D, F, H, J). (A, B) Neuronal differentiation behind the furrow revealed by immunostaining with MAB22C10. In the wild-type disc (A) multiple rows of developing ommatidia are labeled. Clusters containing fewer than eight neurons are visible at higher magnification immediately posterior to the furrow (data not shown). In the DrMio eye disc (B) only a single row of differentiating clusters are labeled, and no immature clusters are detected, suggesting a defect in the progression of the morphogenetic furrow. (C, D) Dpp: LacZ expression in the morphogenetic furrow detected by immunostaining. In the wildtype (C) lacZ expression from the BS3.0 reporter is observed within all cells in the furrow and at the dorsal and ventral margin of the disc. In the DrMio eye disc (D) lacZ expression is detected only at the margins. (E, F) Expression of string (stg) anterior to the furrow assayed by in situ hybridization. (E) In the wild-type disc, stg RNA is detected in a band of retinal precursor cells immediately adjacent to the furrow and in additional single cells farther anterior. (F) The stripe of stg expression ahead of the furrow is absent in the Mio mutant. (G, H) Expression of lacZ from the C93 enhancer trap reporter detected by immunostaining. C93 expression in retinal precursor cells anterior to the furrow is unaffected by the mutation. In contrast, the expression of lacZ within the furrow and in additional posterior cells observed in the wildtype (G) is absent in the DrMio eye disc (H). (I, J) Acridine orange (AO) staining of apoptotic cells. In the wild-type eye disc (I) the fluorescence of AO-stained cells indicates the normal level of cell death. In the DrMio mutant (J), extensive fluorescence in the posterior region of the eye disc reveals massive cell death associated with the absence of furrow progression. Original magnification 200ϫ, except (B), 400ϫ. For all images eye disc anterior is to the left. The position of the furrow in wildtype is indicated with an arrow (except I). FIG. 3. Generation of eye mosaics using a P[lacZ, wϩ] insertion in the Dr locus. Two P-element-induced revertants were isolated as X to autosomal jumps into the Dr locus (see Materials and Methods for details). (A) Suppression of the DrMio eye phenotype in the P10 revertant is sensitive to transposase. Wild-type eye of a w; DrMio[wϩ]P10 adult (left). Transposase-induced loss of the P element results in a mosaic eye containing patches of wϪ cells which are mutant for dominant Dr. Reduced eye of an w; DrMio[wϩ]P10/P[ryϩ, ⌬2-3] mosaic (right). (B) Expression of lacZ from the P10 insertion in the third-instar eye imaginal disc visualized by immunostaining with anti-␤-Gal antibody. LacZ expression in the DrMioP[wϩ]P10 eye disc initiates ahead of the furrow, is down-regulated within the furrow, and is also detected in differentiating clusters. (C) Autonomy of dominant Dr mutant clones in mosaic eye discs. Expression of a neuronal marker and lacZ in a w; DrMio[wϩ]P10/P[ryϩ, ⌬2-3] mosaic eye disc coimmunostained with MAB22C10 (black) and anti-␤-Gal (brown). (D) Arrest of the furrow and the reduction of cluster formation in a Dr mutant clone (marked by the absence of lacZ) is independent of a normal furrow and neuronal differentiation in neighboring wild-type tissue.

FIG. 4. Molecular genetics of Dr mutants. EcoRI restriction site map of 70 kb of genomic DNA encompassing the Dr locus. The positions of insertions and other rearrangements associated with the dominant Dr mutations and their revertant derivatives are drawn above the restriction map. The insertion of a 3S18 retrotransposon was found 27 kb upstream of the start of the msh transcript (as defined by Ishicki et al., 1997) in the DrMio allele. In the Dr1 mutant, genomic Southern analysis revealed an insertion and an additional polymorphism mapping farther proximal to msh (data not shown). Disruption or deletion of the 3S18 element was associated with intragenic suppression of the dominant small eye phenotype in some of the revertant alleles. Cloning and sequence analysis revealed that the breakpoint of two P-element-induced revertants was within the 5Ј long terminal repeat (LTR) of the 3S18 retrotransposon. Differences in the precise position of the breakpoint within the LTR as well as the orientation of the P element could account for the partial (P19, not shown) or complete (P10, Fig. 3A) suppression of the Dr eye phenotype. Among the EMS-induced revertants of the DrMio, Southern analysis (data not shown) revealed that the 3Ј end of the 3S18 element is deleted in the viable revertants R10, R21, R22, and R53 (all mshϩ) and in one lethal revertant R61 (mshϪ). E*—polymorphic site in Mio. (E)—polymorphic site in TM6B. sion, failure of cluster formation) are a secondary consequence Insertions of the P[lacZ, wϩ] element frequently detect of the inhibition of morphogenetic furrow progression. local enhancer sequences resulting in tissue-specific expres- Cell death is often associated with eye development mu- sion of lacZ. In the DrMio[wϩ]P10 eye disc, lacZ is detected in a tants and is also required for normal eye development in stripe of retinal precursors anterior to the furrow and Drosophila (Wolff and Ready, 1991). To assess if cell death subsequently within more posterior cells within the furrow contributes to retinal loss in the dominant Dr mutants, and in differentiating clusters (Fig. 3B). In DrMio[wϩ]P10/P[ryϩ, third-instar eye discs were stained with acridine orange (AO). ⌬2-3] third-instar larvae, somatic loss of the insertion In the wildtype (Fig. 2I) the small number of AO-positive cells results in mosaic eye discs made of Drϩ and Dr dominant reveals a low level of cell death. In contrast, AO stains large cells. Thus, the absence of lacZ expression (in the P10 domains in the DrMio eye disc (Fig. 2J), indicating extensive cell expression domain) serves to mark Dr dominant mutant death, primarily in the posterior region. These observations clones. The autonomy of the furrow defect of the DrMio suggest that retinal precursor cells may undergo cell death if mutant was assayed by mosaic analysis in eye discs coim- eye morphogenesis is blocked at an early step. munostained with anti-␤-Gal and Mab22C10. In all mosaic discs, cluster formation was always significantly increased within posterior patches of lacZ-positive cells. In discs with Autonomy of Morphogenetic Furrow Defect in Ϫ large patches of lacZ cells (example shown in Fig. 3C), DrMio Mosaic Eye Discs cluster formation was blocked to a level observed in DrMio Two P[lacZ, wϩ]-induced revertants of DrMio were identi- eye discs (for comparison see Fig. 2B) despite the adjacent fied by complete (P10) or partial (P19) suppression of the wild-type tissue. These observations demonstrate that the dominant eye phenotype. Both somatic and germ-line loss dominant mutation acts autonomously. of the insertion from the P10 chromosome resulted in the recovery of the dominant eye phenotype, demonstrating Molecular Genetic Analysis of Dr Mutants that the suppression is mediated by the insertion. The suppressed eye of a w; DrMio[wϩ]P10 adult is pigmented due to Genomic DNA flanking the P element in the DrMio[wϩ]P10 the presence of the wϩ marker in the P element. Adult eyes revertant was cloned using plasmid rescue. DNA sequence of w; DrMio[wϩ]P10/P[ryϩ, ⌬2-3] are reduced in size and mosaic analysis revealed that the P element had inserted at one end for wϩ (Fig. 3A) due to the somatic loss of the P element. of a 3S18 retrotransposon (Davis and Judd, 1995). The

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. Drop Gain-of-Function Mutation in Drosophila 387 insertion site of the P element in the partial revertant rH96 (Isshiki et al., 1997) was absent from the retinal field DrMio[wϩ]P19 mapped to a similar position in the retrotranspo- (Fig. 5A), but otherwise identical to the pattern of expres- son, but in the orientation opposite to that of DrMio[wϩ]P10. sion of msh RNA revealed by in situ hybridization. In the Using nonrepetitive sequences from the rescued plasmids DrMio eye antennal disc, ectopic msh RNA was not detected, as the starting probe, a chromosome walk was undertaken presumably due to its low abundance in the mutant cells. spanning ϳ70 kb. The positions of Dr mutant alleles within LacZ expression from the P10 insertion could reflect the the chromosome walk are diagrammed in Fig. 4. The transcriptional activity of genes within the flanking DNA insertion site of the 3S18 retrotransposon associated with or mirror the expression pattern of the retrotransposon. In the DrMio mutation maps ϳ27 kb upstream of the 5Ј end of situ hybridization of eye imaginal discs with digoxigenin- the msh gene as defined by P-element insertion (Isshiki et labeled probes specific for 3S18 sequences revealed that the al., 1997). Genomic Southern analysis of the Dr1 allele retrotransposon is expressed in retinal precursor cells revealed that the mutation is associated with an insertion within the furrow (Fig. 5C). Thus, lacZ expression from the and additional rearrangements 35 kb upstream (Fig. 4, data P10 insertion reflects in part the distribution of 3S18- not shown). encoded RNAs. Ectopic expression of msh in the DrMio eye Molecular analysis of the DrMio mutation and the disc likely is the result of transcriptional activation of the P-element revertants suggests that an intact 3S18 retro- msh promoter by cis-regulatory sequences present within transposon is required for the dominant eye phenotype. the retrotransposon that direct its expression in retinal These observations suggest that a second mechanism of precursor cells. intragenic suppression occurs by disruption or deletion of sequences within the repetitive element. In order to test Effects of Gal4-Mediated Overexpression of msh on this hypothesis, digests of genomic DNA from the EMS- Eye Development induced revertants were analyzed by Southern blot (Fig. 4 for position of probe relative to genomic DNA map; South- Genetic and molecular analysis of the Dr mutants sug- ern data not shown). A wild-type genomic probe detects a gested that ectopic expression of msh accounts for the 3.5-kb EcoRI fragment present in the parental DrMio DNA dominant eye phenotype; however, attempts to detect msh but absent in wildtype, corresponding to the 3Ј breakpoint expression within retinal precursor cells in the mutant of the 3S18 insertion. In four viable (R10, 21, 22, and 53) and discs failed. Using the GAL4-UAS system (Brand and Per- one lethal revertant (R61), this fragment is missing, suggest- rimon, 1993), msh expression was ectopically induced in ing that deletion or loss of the 3S18 retrotransposon is undifferentiated retinal precursor cells using the eyeless, associated with phenotypic reversion of the Dr dominant hairy, and dpp drivers; in differentiating cells behind the eye phenotype. furrow using the gmr driver; or in all cells (hsp70 driver) at the third larval instar stage. Strains harboring the UAS:msh transgene were crossed to the appropriate driver (see Mate- Expression of the 3S18 Element and msh in Third- rials and Methods for details) and the effects of msh Instar Eye Antennal Discs overexpression on eye development were examined in the Genetic analysis of DrMio revertants revealed that inacti- progeny. vation of msh in cis results in the suppression of the Gal4-dependent msh overexpression driven by either the dominant eye phenotype. This observation suggests that eyeless or the dpp driver did not adversely affect viability expression of a mutant form of msh or ectopic expression of nor eye development; however, a small fraction of UAS: msh during eye development can account for the gain-of- msh/ϩ; ey:Gal4/ϩ flies exhibited mild eye roughening (data function phenotype. The embryonic expression of msh has not shown). In contrast, h:Gal4-dependent expression of been thoroughly described (D’Alessio and Frasch, 1996; msh resulted in lethality prior to the third-instar stage so Lord et al., 1995) and the requirement for msh in the the effects on eye development could not be determined. development of embryonic mesoderm (Nose et al., 1998) Similarly, expression of msh driven by GMR:Gal4 resulted and the central nervous system (Isshiki et al., 1997; Bue- in lethality prior to eclosion; however, rare escapers exhib- scher and Chia, 1997). During imaginal development msh is ited severe roughening of the compound eye (Fig. 6E). expressed in the wing disc (D’Alessio and Frasch, 1996), but Ubiquitous expression of msh resulted in complete le- its pattern of expression in the eye anntenal disc has not thality as UAS:msh/ϩ; hsp70:Gal4/ϩ adult flies were never been reported. Using an antisense digoxigenin-labeled RNA recovered following a single heat shock at the third-instar probe, the pattern of expression of msh RNA in third-instar larval stage. To analyze the effects of ectopic expression of eye imaginal discs from wildtype and DrMio mutants was msh during development, eye discs from experimental and determined. In the wild-type eye antennal disc, msh RNA control larvae were immunostained with MAB22C10 after was not detected in the retinal precursor cells, but rather heat shock and subsequent 4 h recovery. In all UAS: was restricted to a band of cells within the peripodial lacZ//ϩ; hsp70:Gal4/ϩ control eye discs (Fig. 6A), eye membrane on the ventral margin spanning part of the development proceeded normally as judged by the onset of antennal disc and to the anterior eye disc (Fig. 5B). Like- neuronal marker expression in the five-cell preclusters wise, lacZ expression from the msh enhancer trap insertion, adjacent to the morphogenetic furrow as well as the more

mature clusters at the posterior of the disc. In contrast, ϳ25% (N ϭ 20) of the UAS:msh/ϩ; hsp70:Gal4/ϩ experimental discs (Fig. 6A) revealed an arrest of the morphogenetic furrow as evidenced by the reduction of the number of labeled rows of clusters and the absence of immature clusters near the furrow (Fig. 6B). Most of the affected discs were mosaic with regard to the morphogenetic furrow arrest, presumably due to local variation in the levels of msh overexpression. However, in some cases overexpression resulted in an early and complete arrest of the furrow (Fig. 6C); these discs exhibited only a single row of mature clusters, closely resembling the eye disc phenotype of the dominant Dr mutations.

DISCUSSION

I have provided genetic evidence that Dr dominant mutations are gain-of-function alleles of msh. Reversion of the dominant eye phenotype to wildtype is most frequently associated with the cis-inactivation of the msh gene, suggesting overexpression as the mechanism causing the gain of function. Southern analysis revealed the presence of insertions upstream of the msh transcription start site in two Dr alleles. Molecular cloning and sequence analysis identified the DrMio insertion as a 3S18 retrotransposon. In some revertant alleles disruption or deletion of the retroviral element was coincident with the intragenic suppression of the dominant eye phenotype. In wild-type eye imaginal discs the 3S18 element but not the msh gene is expressed in retinal precursor cells. These observations suggest a mechanism for ectopic expression in which transcriptional activation of the msh promoter is mediated by eye-specific cis-regulatory sequences in the retrotransposon. In the DrMio mutant the development of most photoreceptor cells is blocked due to the early arrest of morphogenetic furrow progression in the eye imaginal disc. Likewise, ubiquitous expression of msh in wild-type eye imaginal discs driven by a heat-shock promoter blocked progression of the furrow. Thus, the genetic data and the results of overexpression demonstrate that msh is both necessary and sufficient for the morphogenetic furrow arrest and defects in eye development in the Dr mutants. In vertebrates, retroviral insertions can result in the overexpression of proto-oncogenes such as c-myc (Corcoran et al., 1984) and wnt-1 (Nusse and Varmus, 1982), contrib- FIG. 5. Expression of msh- and 3S18-encoded RNAs in eye disc. uting to the uncontrolled growth of tumor cells. Retrotrans- (A) Histochemical staining for lacZ from the msh enhancer trap poson insertions have been implicated as the cause of a insertion rH96 (Isshiki et al., 1997) in third-instar eye imaginal large number of different spontaneous mutations in D. disc. (B) Expression of msh transcripts in wild-type third-instar melanogaster (Arkhipova and Ilyin, 1992). The preference eye imaginal disc. The expression of msh in the eye antennal disc was restricted to a narrow band of epithelial cells at the disc edge and is absent from the retinal field. (C) Distribution of 3S18-encoded RNAs in a wild-type third-instar eye imaginal disc revealed by in situ hybridization. The retrotransposon is expressed at high levels in retinal precursor cells within a stripe Expression of the 3S18 RNAs overlaps the distribution of lacZ overlapping the furrow. Additional robust expression was de- expression from the P element in the Mio revertant P10, whose tected in a ring pattern in the antennal portion of the disc. onset is anterior to the furrow (D).

FIG. 6. Effects of msh overexpression on eye development. Ubiquitous expression of msh in third-instar eye imaginal discs directed by a heat-shock promoter arrests progression of the morphogenetic furrow. Neuronal differentiation in the developing photoreceptor clusters was visualized by MAB22C10 immunostaining of eye imaginal discs ubiquitously expressing lacZ (A) or msh (B, C). In hsp70:Gal4/ϩ; UAS:LacZ/ϩ eye discs (A), the anterior-to-posterior gradient of youngest to oldest clusters is evident behind the furrow. Note the absence of mature clusters immediately posterior to the furrow. In the hsp70:Gal4/ϩ; UAS:msh/ϩ eye discs (B) the arrest of the furrow was evident by the reduction in cluster number and the absence of immature clusters. Note that in this example furrow progression was unaffected in part of the disc. (C) Early and complete arrest of the furrow in an hsp70:Gal4/ϩ; UAS:msh/ϩ eye disc. Note that only a single row of clusters has formed, a phenotype similar to that observed in the DrMio mutant (Fig. 2B). Expression of msh behind the furrow causes a rough eye phenotype. (D) Scanning electron micrograph of compound eye of wildtype. Note the regular repeating array of ommatidia and interspersed bristles. (E) Scanning electron micrograph of the compound eye of a gmr:Gal4/ϩ; UAS:msh/ϩ adult. Overexpression of msh in all cells behind the furrow results in severe defects in the structure of the exterior eye, including missing interommatidial bristles and the absence of the corneal lens from most ommatidia. Eye imaginal discs are oriented anterior to posterior, right to left; arrow indicates position of furrow. Original magniﬁcation 800ϫ for A and B, 1600ϫ for C, and 200ϫ for D and E.

of mobile elements to insert into regulatory regions of the some suppressed the dominant eye phenotype. Disruption target genes results in negative or positive effects on tran- of cis-regulatory sequences that mediate eye-speciﬁc ec- scription, including silencing, overexpression, or misex- topic expression of msh could account for the intragenic pression. Expression of retrotransposon RNAs is remark- suppression. ably tissue speciﬁc (Ding and Lipshitz, 1994) and for some A curious genetic feature of the Dr mutations is the classes of insertion has been shown to be mediated by presence of an additional closely linked lesion in the stg enhancer sequences in the long terminal repeat (LTR) promoter resulting in defects in expression in eye (Mozer (Mozer and Benzer, 1994). Insertion of a P element into the and Easwarachandran, 1999). The genomic region upstream 5Ј LTR of the 3S18 retrotransposon in the DrMio chromo- of the msh transcription start appears to be a frequent target

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 390 Brian A. Mozer of DNA insertions, one of which was identified as a 3S18 al., 1997), often resulting in missing muscle or neurons. element. Transposase-induced excision of P-element inser- The analysis of the dominant Dr mutants and the effects of tions is often associated with local deletions around the msh overexpression demonstrate that the loss of retinal insertion site and is used as a tool to generate loss-of- tissue is a consequence of cell death. Although ectopic msh function mutations (Tower et al., 1993). Likewise, local expression in eye may induce apoptosis indirectly, the hopping of a retrotransposon at the stg locus into the msh mechanism remains undefined but could involve the tran- promoter could account for the coincident generation of the scriptional activation of cell-death-promoting genes or the Dr dominant phenotype and the loss of cis-regulatory repression of antiapoptotic regulators. sequences in stg required for expression in eye. In vertebrates, msh homologs (msx genes) constitute a Retrotransposon-induced misexpression of cut (Awasaki three-member family encoding transcriptional regulators et al., 1994) or overexpression of Bar (Tanda and Corces, that are expressed in diverse tissues during development 1991), both of which encode homeodomain-containing pro- (Davidson, 1995). Overexpression studies in vertebrates teins, result in defects in eye development. Similar effects suggest that the genes are downstream of cell signaling on eye development were observed following ubiquitous molecules that mediate inductive interactions between overexpression of the homeodomain protein rough, result- developing tissues such as epidermal induction in frogs ing in the arrest of the morphogenetic furrow and the loss of (Suzuki et al., 1997), limb bud formation in chick (Ferrari et dpp transcription (Heberlein et al., 1993). Likewise, this al., 1998), and tooth (Bei and Maas, 1998) and bone devel- study strongly suggests that the early arrest of the morpho- opment in mammals (Liu et al., 1999). Depending on the genetic furrow in the Dr mutant eye disc (which is also gene, organism, and tissue, overexpression of msx affects associated with defects in dpp expression) is the result of the regulation of cell proliferation and cell-type differentia- ectopic msh. tion and can lead to apoptosis. Mutations in two msx genes The absence of stg expression in the DrMio eye imaginal have been reported, a knockout mouse lacking the msx1 disc suggests that msh overexpression interferes with gene (Satokata and Maas, 1994) and an msx2 knockout events anterior to the furrow which may be required for its (Satokata et al., 2000) and a single amino acid substitution progression. Furthermore, the results of ectopic expression in the homeodomain of msx2 associated with an autosomal using different Gal4 drivers are consistent with this hypoth- dominant human disease, craniosynostosis (Jabs et al., esis, as msh overexpression behind the furrow (gmr driver) 1993). The three mutations cause defects in craniofacial resulted in a normal-sized rough eye while ubiquitous development and development abnormalities of other ecto- expression (hsp/70 driver) arrested the furrow. The failure of dermally derived tissues. Although a mutation in the msx3 the eyeless driver to illicit the furrow stop phenotype may gene is not reported, its expression in neuronal precursor be the result of lower expression levels or other aspects of cells and the overlap of expression with msx1 and msx2 its spatial and temporal regulation in anterior eye disc cells suggest that the genes encode a potentially redundant that differ from the hsp70 driver. Inhibition of the furrow by function required for nervous system development ectopic msh could involve the transcriptional repression of (Shimeld et al., 1996). genes required for furrow progression or the up-regulation The absence of msh expression during Drosophila eye of those genes that serve as negative regulators. Two HLH development is surprising given the observation of msx1 transcription factors, emc and hairy, have been implicated and msx2 expression in vertebrate eye precursors (Mon- in the negative regulation of morphogenetic furrow progres- aghan et al., 1991). Interestingly, the recent completion of sion (Brown et al., 1995); however, hairy expression (as the fly genome project has led to the identification of a monitored by the C93 enhancer trap) was not affected in the second msh gene (Rubin et al., 2000), so it remains an open DrMio mutant, suggesting that it is not a target of ectopic question if the function of the msx genes in eye is conserved msh. between vertebrates and Drosophila. The high degree of The normal initiation and progression of the morphoge- conservation between human genes and their fly homologs netic furrow is the result of the combined inputs of the sets the stage for using model organisms to better under- hedgehog (hh), dpp, and wg signaling pathways (for review stand the biological basis of human disease. Using simple see Treisman and Heberlein, 1998). The absence of furrow but powerful genetic screens, mutations can be isolated as progression in the Dr mutant eye disc was not associated modifiers of any phenotype caused by a human disease gene with a change in wg expression, suggesting that msh or its homologue. For example, overexpression of human misexpression interferes with hh-mediated transcription of disease genes containing expanded repeats implicated in dpp or some other process. Ectopic msh may interfere with neurodegenerative disorders [SCA3 (Warrick et al., 1998); the function of the early eye patterning gene, eyesabsent huntingtin protein (Jackson et al., 1998)] in the developing (eya), which is required for dpp transcription in the furrow Drosophila eye results in a similar late-onset neurodegen- (Hazelett et al., 1998) and whose absence leads to cell death eration in the fly retina. Mutations in a heat-shock protein (Bonini et al., 1993). were found that suppress SCA3-dependent neurodegenera- Overexpression of msh during embryogenesis perturbs tion in eye (Warrick et al., 1999), suggesting that drugs patterning in both the developing mesoderm (Lord et al., targeted to blocking the expression or function of the 1995; Nose et al., 1998) and the nervous system (Isshiki et human homolog could alleviate the symptoms of the dis-

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. Drop Gain-of-Function Mutation in Drosophila 391 ease. Similarly, the dominant Dr mutants and the msh wingless that, similar to loss of APC, interferes with normal eye overexpression eye phenotype may serve as a useful starting development. Dev. Biol. 206, 178–188. point to begin the search for genes that regulate this Buescher, M., and Chia, W. (1997). Mutations in lottchen cause cell evolutionarily conserved class of molecules and ultimately fate transformations in both neuroblast and glioblast lineages in lead to genes relevant to the treatment of craniosynostosis. the Drosophila embryonic central nervous system. Development 124, 673–681. Burke, R., and Basler, K. (1996). Hedgehog-dependent patterning in the Drosophila eye can occur in the absence of Dpp signaling. ACKNOWLEDGMENTS Dev. Biol. 179, 360–368. Carrera, P., Abrell, S., Kerber, B., Walldorf, U., Preiss, A., Hoch, M., and Jackle, H. (1998). A modiﬁer screen in the eye reveals control This paper is dedicated to the memory of Miriam Mozer. The genes for Kruppel activity in the Drosophila embryo. Proc. Natl. author gives special thanks to Bernard Mozer for his unwavering Acad. Sci. USA 95, 10779–10784. encouragement, Seymour Benzer and Hal Gainer for the early Chanut, F., and Heberlein, U. (1997). Role of decapentaplegic in support of this work, and Ward Odenwald for his help with the initiation and progression of the morphogenetic furrow in the overexpression experiments. I also acknowledge the gifts of ﬂy developing Drosophila retina. Development 124, 559–567. strains and other materials from Ed Lewis, Akinao Nose, Bill Chia, Corcoran, L. M., Adams, J. M., Dunn, A. R., and Cory, S. (1984). Barbara Thomas, Nancy Bonini, Judy Kassis, Jim Kennison, and Murine T lymphomas in which the cellular myc oncogene has Kwang Choi. Additional thanks to Kumi Easwarchandran for been activated by retroviral insertion. 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