Drosophila tufted is a gain-of-function allele of the proneural gene

Copyright 2003 by the Genetics Society of America

Drosophila Tufted Is a Gain-of-Function Allele of the Proneural Gene amos

Eric C. Lai1

Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

Manuscript received September 9, 2002Accepted for publication January 8, 2003

ABSTRACTTufted is a classical Drosophila mutant characterized by a large number of ectopic mechanosensory

bristles on the dorsal mesothorax. Unlike other ectopic bristle mutants, Tufted is epistatic to achaete andscute, the proneural genes that normally control the development of these sensory organs. In this report,I present genetic and molecular evidence that Tufted is a gain-of-function allele of the proneural geneamos that ectopically activates mechanosensory neurogenesis. I also systematically examine the ability ofthe various proneural bHLH proteins to cross-activate each other and find that their ability to do so is ingeneral relatively limited, despite their common ability to induce the formation of mechanosensory bristles.This phenomenon seems instead to be related to their shared ability to activate Asense and Senseless.

ALTHOUGH the nervous system of the fruitfly is programmed cell death, leaving four cells in the maturemechanosensory organ (Fichelson and Gho 2003).quite complex, it is also highly stereotyped. These

characteristics make it an ideal experimental system for There are two subclasses of proneural bHLH proteins.The Ato class includes Atonal (Ato) and Absent solo-understanding basic principles of pattern formation.

Accordingly, studies of how the Drosophila nervous sys- multiple-dendritic (MD) neurons and olfactory sensilla(Amos); Ato controls the development of chordotonaltem is assembled have occupied the collective efforts of

hundreds of developmental geneticists over the de- organs, R8 photoreceptors, and a subset of olfactorysensilla (Jarman et al. 1993, 1994; Gupta and Rod-cades.

The pattern of sensory organs in both the embryo rigues 1997) while Amos regulates the development ofcertain MD and olfactory neurons (Goulding et al.and the adult is prefigured by the spatially patterned

expression and activity of the proneural genes, which 2000; Huang et al. 2000). Members of the ASC class areencoded by genes in the ac-sc complex (AS-C; Alonsoencode basic helix-loop-helix (bHLH) transcriptional

activators (reviewed by Bertrand et al. 2002). Proneural and Cabrera 1988) and include Achaete (Ac), Scute(Sc), and Lethal of scute (L’sc). Ac and Sc are theactivity confers neural potential upon groups of adja-

cent cells referred to as proneural clusters (PNCs). In- proneural proteins for the adult mechanosensory bris-tles (Garcıa-Bellido 1979), while L’sc controls theteractions among PNC cells restrict this potential to

sensory organ precursor (SOP) cells; non-SOP cells of development of the embryonic central nervous systemand musculature (Martin-Bermudo et al. 1991; Car-a PNC usually adopt a nonneuronal fate. This process

is commonly referred to as “lateral inhibition” and is mena et al. 1995). A fourth member of the AS-C, asense(ase), also encodes a protein with an ASC-class bHLHmediated by cell-cell signaling via the Notch receptor

and the products of the neurogenic genes (reviewed by domain. It is not, however, a true proneural protein asit is expressed only by SOPs and not in PNCs; it is likelyArtavanis-Tsakonas et al. 1999). Once stably specified,

the SOP typically undergoes a fixed series of cell divi- involved in sensory organ differentiation (Brand et al.1993; Dominguez and Campuzano 1993). Anothersions to generate the different cells that make up a

mature sensory organ, although nonclonally related transcription factor expressed in SOPs is the zinc fingerprotein Senseless (Sens); it is involved in maintenancecells are recruited into certain types of sensory organs.

In the case of the mechanosensory bristles, divisions of of proneural gene expression (Nolo et al. 2000). Al-though expression of Ase and Sens is normally restrictedthe SOP generate five cells: a socket cell and shaft cell

(which produce structures that are visible from the exte- to SOPs, both exhibit “proneural” activity in that theyinduce the formation of ectopic sensory organs whenrior) and a glial cell, sheath cell, and neuron (which

lie beneath the cuticle; Gho et al. 1999; Reddy and misexpressed.The mechanosensory bristles that cover the exteriorRodrigues 1999). The glial cell subsequently undergoes

of the fly can be observed in live individuals at lowmagnification; thus, mutations that affect their distribu-tion are easily identified. Two general classes of mutants

1Address for correspondence: Howard Hughes Medical Institute, Depart- display extra bristles. Those that compromise lateral in-ment of Molecular and Cell Biology, University of California, 539 LifeSciences Addition, Berkeley, CA 94720-3200. E-mail: [email protected] hibition cause multiple SOPs to emerge from an individ-

Genetics 163: 1413–1425 (April 2003)

1414 E. C. Lai

Cytology: Tft1/� polytene chromosomes displayed a cyto-ual PNC, leading to an increase in bristle density orlogically visible aberration at 36F–37A. The nature of the aber-the presence of tight bristle tufts lacking interveningration was analyzed using a tiling set of 5-kb digoxigenin-

epidermal cells (i.e., Bearded; Leviten and Posakony labeled probes representing 100 kb of DNA from the 36F3–71996). In this situation, extra sensory organs arise from region. A contiguous set of probes hybridized to an additional

band in the 37A region of the Tft1 chromosome, suggestingthe normal complement of proneural clusters. A secondthat the Tft1 aberration involves a duplication and transloca-class of mutant displays sensory organs in wholly ectopiction of material from 36F3–7 to 37A. The proximal limit waspositions, although these are always separated by epider-not determined, but extends a minimum of 75 kb upstream

mal cells. This results from the generation of ectopic of amos. Two nonoverlapping probes 0–5 and 5–10 kb down-proneural clusters, from which singularized SOPs are stream of the amos start site both showed variable, but modest,

amounts of duplicated signal. This suggests that the structurechosen. Most of the phenotypically strong mutations inof this end of the aberration is complex, but terminates inthe latter class correspond to lesions in hairy (h) orthe vicinity of amos.extramacrochaetae (emc; Bridges and Morgan 1923; Mos-

Immunofluorescence: Imaginal discs were processed for im-coso del Prado and Garcıa-Bellido 1984). Both en- munofluorescence as described previously (Lai and Rubincode negative regulators of the proneural proteins: 2001). The following dilutions for antibodies were used in

this study: rabbit �-Scute (1:200, gift of Hugo Bellen), mouseHAIRY is a bHLH protein that directly represses tran-�-Achaete [1:100, Developmental Hybridoma Studies Bankscription of ac, while EMC is an HLH-only protein that(DHSB)], rabbit �-Asense (1:2500, gift of Yuh Nung Han),binds to and inhibits the DNA-binding activity of pro-rabbit �-Amos (1:4000, gift of Andrew Jarman), rabbit

neural proteins (Van Doren et al. 1992, 1994; Ohsako �-Atonal (1:2000, gift of Andrew Jarman), guinea pig �-Sense-et al. 1994). A rarer set of mutants with ectopic PNCs less (1:5000, gift of Hugo Bellen), mouse �-Delta (1:100,

DHSB), mouse �-E(spl)b323 (1:5, gift of Sarah Bray), mousehas been designated Hairy-wing (Hw). Once thought to�-Hindsight (1:50, DHSB), mouse �-Cut (1:100, DHSB),represent a distinct locus in the AS-C, Hw mutants aremouse �-�-galactosidase (1:100, DHSB), rabbit �-�-galactosi-actually gain-of-function alleles of ac and sc that ectopi-dase (1:5000, Cappel), and rabbit �-Neur (1:400; Lai et al.

cally activate neurogenesis (Campuzano et al. 1986; Bal- 2001).cells et al. 1988).

Another mutant with a dramatic ectopic bristle phe-notype is Tufted (Tft; Ritterhoff 1952). Surprisingly, RESULTSit has been little studied since its discovery 50 years ago.

Phenotypic and cellular characterization of Tft : TheIn this report, I characterize the cellular basis of the TftTft locus is defined by a single, viable mutant (Tft 1)phenotype and show that it results from the establish-that displays a large number of ectopic mechanosensoryment of ectopic proneural domains and selection ofbristles, particularly in the postalar, dorsocentral, andextra SOPs. Unexpectedly, Tft neither is dependentscutellar regions of the notum (Figure 1, A and B)upon nor significantly cross-activates expression of Ac(Arnheim 1967; Ritterhoff 1952). I analyzed the ex-and Sc, indicating that it harbors proneural activity forpression of several markers of the SOP fate in this mu-mechanosensory organs. Consistent with this, I presenttant, including Hindsight (Hnt; Pickup et al. 2002),genetic and molecular evidence that Tft is a gain-of-func-Senseless (Sens), neurA101-lacZ (A101), and Asense (Ase).tion allele of the proneural gene amos that ectopicallyI observed extra cells positive for each marker in theactivates mechanosensory neurogenesis. Misexpressionpresumptive posterior notal region of Tft/� wing imagi-of any of the proneural proteins can promote the devel-nal discs (Figure 2, A–H, arrows), demonstrating thatopment of mechanosensory organs, and I provide evi-the Tft phenotype is due to ectopic adoption of the SOPdence that this is not generally due to promiscuousfate. Ectopic SOPs appear to subsequently develop intoactivation of Ac and Sc, but rather to induction of Asenormal sensory organs in Tft, since ectopic mechanosen-and Sens.sory organs contain both sockets and shafts and haveneurons that are functionally connected to the central

MATERIALS AND METHODS nervous system (Ghysen and Richelle 1979).Ectopic bristle phenotypes can generally be classifiedDrosophila stocks: All mutant alleles and transgenic stocks

according to whether they arise from ectopic proneuralutilized in this study have been previously described: Tft1/SM-TM6B (Ritterhoff 1952), In(1)sc10-1/FM7 (Garcıa-Bellido clusters or reflect a failure of lateral inhibition. The termand Santamaria 1978), isoRoi/CyO (Chanut et al. 2002), ato1/ bristle “tufting” is popularly used to refer specifically toTM6B ( Jarman et al. 1994), UAS-ato ( Jarman et al. 1993), a failure of lateral inhibition. Indeed, the presence ofdaKX136/SM-TM6B (Caudy et al. 1988), sensE2/TM6B and UAS-

closely spaced or even adjacent bristles in Tft flies (Fig-sens (Nolo et al. 2000), ac 2.2-kb genomic transgene (Vanure 1B) and the determination of SOPs adjacent to eachDoren et al. 1994), dpp40C6-Gal4/TM6C (Staehling-Hampton

et al. 1994), bxMS1096-Gal4 (Capdevila and Guerrero 1994), other in Tft wing imaginal discs (Figure 2, E–H) togetherUAS-amos (Goulding et al. 2000), UAS-sc (Chien et al. 1996), suggest a defect in lateral inhibition. However, the num-neurA101-lacZ/TM6B (Bellen et al. 1989), m4-lacZ (Bailey and ber of ectopic bristles in the Tft-affected region wasPosakony 1995), hs-Gal4 (Brand and Perrimon 1993), Brd1/

significantly increased in Tft1/�; Brd1/� double hetero-TM6B (Leviten and Posakony 1996), and f, hs-flp; 101E-Gal4(de Celis and Bray 1997). zygotes (Figure 1E), indicating that Tft bristles are still

1415Tufted Is an Allele of amos

MAb323 antibody; Figure 3, B and F, arrows), indicatingthe presence of ectopic proneural clusters. Notably,these results also indicate that the Tft phenotype is notdue to a failure to activate components of lateral inhibi-tion. Surprisingly, I did not observe comparable ectopicexpression of Sc and Ac (Figure 3, C, D, G, and H,arrows), the proneural proteins for the adult peripheralnervous system (PNS). Doubly stained preparationsshowed that ectopic SOPs (as marked by Sens) in theTft background were not generally associated with corre-sponding proneural clusters of Ac expression, althoughAc could sometimes be observed in ectopic SOPs (Fig-ure 3, I and J). This contrasts with what has been shownfor other ectopic bristle mutants such as hairy and extra-macrochaetae, which are associated with ectopic clustersof proneural expression and/or activity (Skeath andCarroll 1991; Van Doren et al. 1992, 1994). In sum-mary, Tft is an unusual extra-bristle mutant: It exhibitsboth ectopic proneural domains and some defect in lat-eral inhibition, and ectopic neurogenesis is not gener-ally accompanied by corresponding Ac/Sc expression.

Tufted harbors proneural activity for mechanosensoryorgans: Simultaneous inactivation of ac and sc, theproneural genes for the adult PNS, results in a nearlycompletely bald fly lacking most mechanosensory or-gans (sc10-1/Y, Figure 1C). The bald phenotype of sc10-1/Y isepistatic to that of most other ectopic bristle mutants,indicating the fundamental role for these genes in estab-lishing adult peripheral neurogenesis. In contrast, theectopic bristle phenotype of Tft was epistatic to sc10-1/Y(Figure 1D); similar findings have been noted previously(A. Garcia-Bellido, personal communication cited inGhysen and Richelle 1979). The ability of Tft to bypass

Figure 1.—The Tft1 adult phenotype. Shown are scanning the requirement for Ac/Sc demonstrates that Tft har-electron micrographs (SEMs) of thoraces from adult malesbors an independent proneural activity. In accord withof the following genotypes: (A) Canton-S, showing the wild-this, ectopic expression of Sens and Hnt was specificallytype pattern of mechanosensory bristles. Inset shows an ante-

rior-central portion of the scutellum that normally lacks sen- maintained in the Tft -affected region of sc10-1/Y; Tft/�sory organs. (B) Tft1/�, displaying many ectopic macrochaetae wing discs (Figure 2, I–L, arrows). Since neurogenicand microchaetae; inset shows microchaetae present on the genes do not exhibit proneural activity, the namescutellum. (C) sc10-1/Y fly is essentially devoid of mechanosen-

“Tufted” is somewhat of a misnomer.sory organs. (D) sc10-1/Y; Tft1/� shows ectopic sensory organsI tested Tft for genetic interactions with other lociin the Tft -affected region. (E) Tft1/�; Brd1/� shows enhance-

ment of the Tft phenotype; Brd1 heterozygotes display only a high in the regulatory hierarchy for peripheral neuro-few extra macrochaetae in the Tft-sensitive region (Leviten genesis. Genetic interactions were not observed withand Posakony 1996). (F) Tft1/dakx136 shows suppression of the either ato or sens, nor was Tft enhanced by increasing acTft phenotype.

dosage using an ac genomic transgene (data not shown).However, Tft was partially suppressed by removal of onecopy of daughterless (da; Figure 1F), which encodes asensitive to lateral inhibition. In addition, many Tft bris-

tles were seen at clearly ectopic locations, including the bHLH heterodimeric partner for proneural bHLH pro-teins. In addition, Tft was previously reported to beanterior-central portion of the scutellum (Figure 1, A

and B) and the metathoracic notum (not shown). This suppressed by Df(1)260-1, a deficiency of the entire AS-C(A. Garcia-Bellido, personal communication cited insuggested the existence of ectopic proneural domains,

which are not characteristic of neurogenic mutants. Campuzano et al. 1985). These interactions suggest thatthe Tft phenotype might be due to altered bHLH ac-I assessed the distribution of proneural clusters using

a number of additional markers and observed both ele- tivity.Tft is associated with an aberration at 36F–37A thatvated and ectopic activity of E(spl)m4-lacZ (as marked

by �-galactosidase; Figure 3, A and E, arrows) and ex- results in misexpression of amos: Tft was previouslymapped to �37A (FlyBase 1998) and shown to bepression of E(spl)bHLH proteins (as marked by the

1416 E. C. Lai

Figure 2.—Tft causes ectopic adoption of theSOP fate in an ac/sc -independent manner. Shownare wing imaginal discs from Canton-S (A–D),Tft 1/� (E–H), sc10-1/Y (I and J), and sc10-1/Y; Tft 1/�(K and L). Ectopic cells express a broad rangeof markers of the SOP fate in the presumptiveposterior notum and scutellum of Tft wing discs(arrows), including Hindsight (Hnt, A and E),Senseless (Sens, B and F), neurA101-lacZ stained for�-galactosidase (A101, C and G), and Asense (Ase,D and H). Ectopic SOPs develop independentlyof the normal proneural genes for the PNS. sc10-1/Yindividuals lack most SOPs (compare brackets inB and J), with the exception of ato-dependentpositions (e.g., I, asterisk); nonsensory aspects ofHnt expression [I, tracheal tubes (T)] or Sensexpression [J, wing margin (WM)] are unaf-fected. In this background, Tft still generates ec-topic SOPs (I–L, arrows), even though most otherSOPs are still absent (L, bracket).

reverted by deficiencies of this cytological region region from which ectopic SOPs are determined in thismutant (Figure 4, H–J).(Wright et al. 1976), a genetic property consistent with

it being a gain-of-function allele. Examination of poly- The findings that Tft specifically misexpresses Amosand interacts genetically with da, which encodes atene chromosomes revealed a complex aberration in-

volving a duplication and translocation of sequences known heterodimeric partner of Amos (Goulding etal. 2000; Huang et al. 2000), suggested that Amos is aat 36F3–7 to 37A (Figure 4, A–D, asterisks; see also

materials and methods and the Figure 4 legend for primary contributor to the Tft phenotype. However, theTft duplication extends a minumum of 75 kb upstreamdetails of this analysis). I noted that the proneural

bHLH-encoding gene amos is located at one end of the of amos and affects several additional genes (Figure 4A).In addition, previous reports have differed on the effi-aberration (Figure 4A); amos is involved in the develop-

ment of embryonic multiple-dendritic neurons and cacy with which ectopic Amos induces the formation ofmechanosensory organs (Goulding et al. 2000; Huangadult olfactory sensilla (Goulding et al. 2000; Huang

et al. 2000). Since directed misexpression of Amos re- et al. 2000). I therefore performed additional misexpres-sion experiments to further substantiate the hypothesissults in the ectopic production of several types of sen-

silla, including mechanosensory organs, it was conceiv- that Tft is an allele of amos.Conditional misexpression of amos efficiently initiatesable that Tft is due to misexpression of Amos.

I tested this hypothesis by staining wild-type and Tft peripheral neurogenesis and induces mechanosensoryorgan formation: Although prolonged misexpressiontissue for Amos, which is not normally expressed in

the wing disc (Figure 4E). Ectopic Amos was indeed of amos using drivers such as dpp-Gal4 and bxMS1096-Gal4resulted in pupal lethality, I was able to characterizeobserved in the presumptive posterior notal region of

the Tft wing disc (Figure 4F) as well as at the base of their disc phenotypes in detail using the PNC and SOPmarkers described earlier. Misexpression of Amos withthe haltere disc (data not shown). Consistent with the

genetics of Tft, the domain of ectopic Amos was inde- either driver resulted in massive ectopic expression ofproneural cluster markers such as Delta (Dl), E(spl)m4-pendent of ac/sc (Figure 4G) and included the precise


by Amos (Figure 5J, arrow) served as an additional mea-sure of the identity of many ectopic SOPs as precursorsfor external sensory organs (compare with wild type,inset to J) and contrasted with the activity of the relatedbHLH Ato, which instead represses Cut (Jarman andAhmed 1998). Thus, ectopic Amos efficiently establishesnew proneural domains and strongly induces externalperipheral neurogenesis. Notably, the levels of ectopi-cally produced proteins were generally far greater thanthose of the corresponding endogenous proteins, sug-gesting that the neuronal program was “super-activated”by Amos. In spite of this, Sc was only mildly misexpressedin response to Amos (Figure 7G), and ectopic Ac wasobserved essentially only in the presumptive notum pos-terior (Figure 7J). In fact, some aspects of Ac expressionwere somewhat suppressed by Amos. Taken together withthe observations that the Tft phenotype does not involveAc or Sc, I conclude that the neuronal program initiatedby ectopic Amos is largely independent of the normalproneural genes for mechanosensory neurogenesis.

Unexpectedly, commitment to the SOP fate was notgenerally coincident with expression of Amos on a cell-by-cell basis, in spite of the ease with which Amos in-duced SOP-specific gene expression. I observed thatboth Hnt (Figure 5, K–M) and neurA101-lacZ (Figure 5,N–P) were expressed at low levels or not at all by Amos-expressing cells, while cells that accumulated high levelsof these markers instead had low levels of or lackedAmos. The same observation applied to Tft tissue aswell: Hnt-positive cells in the region displaying ectopicneurogenesis often did not express Amos (Figure 4,H–J). I attempted to assess the autonomy of clones ofAmos-misexpressing cells using a FLP-out Gal4 strategy,but this typically resulted in bald patches in the adult,possibly due to toxicity of high and prolonged expres-sion of Amos. However, in the small number of caseswhere ectopic bristles were formed, they were alwaysAmos�, indicating that induction of sense organs byAmos is likely autonomous (Figure 6C). Consistent withthis, Tft1 was likewise previously determined to be cellFigure 3.—Upregulation and misexpression of downstream

proneural cluster markers in Tft discs. Shown are wing imagi- autonomous in tissue mosaics (Arnheim 1967). There-nal discs from wild type (A–D) and Tft1/� (E–J) stained for fore, the inverse relationship between expression of�-galactosidase (A and E), E(spl)bHLHs stained with Mab323 Amos and SOP markers appears to represent negative(B and F), Scute (Sc; C and G), Achaete (Ac; D and H), and

regulation of Amos in SOPs.Sens � Ac (I and J). Ectopic and increased levels of E(spl)m4dppGal4�UAS-amos pharate adults were occasionallypromoter activity (A and E, arrows) and E(spl)bHLH proteins

(B and F, arrows) are observed in Tft tissue, whereas Sc (C recovered when they were reared at 18�; these individu-and G) and Ac (D and H) expression is only mildly affected. als displayed a large number of ectopic mechanosensorySome ectopic cells that adopt a high level of Ac expression organs (Figure 6, A and B). In fact, the ability of UAS-are seen in well-stained preparations. (I and J) Higher magni-

amos to generate ectopic mechanosensory organs withfication view of the presumptive posterior notum of a Tft1/� disc;this driver was greater than that of other proneuraldouble staining demonstrates that ectopic SOPs (as marked by

Sens; I) are not associated with ectopic clusters of Ac expres- genes, including UAS-ac, UAS-sc, and UAS-ato; only UAS-sion, although ectopic Ac is seen in some SOPs (J). sens was on a par with UAS-amos in this regard (data

not shown). The lethality of Amos misexpression wasreduced by temporal restriction of expression using hs-lacZ, and E(spl)bHLHs (Figure 5, A–E, and data not

shown); of SOP markers such as Hnt, Sens, Ase, and Gal4 and a 6-min heat shock at 38�. As described pre-viously, these animals displayed a variety of ectopic senseNeur (Figure 5, F–I, and data not shown); and also led

to a significant increase in disc size. Induction of Cut organs (Huang et al. 2000). However, I observed pre-

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Figure 4.—Tft1 is associatedwith a duplication and translo-cation in the 36F–37A regionthat results in misexpression ofthe proneural gene amos. (A)Physical map of 100 kb fromthe 36F3–7 region used for cy-tological analysis of Tft1/�polytene chromosomes; themap was modified from Gadflyoutput (Mungall et al. 2003).Selected hybridizations usingthe probes labeled “B,” “C,”and “D” are shown in B–D, re-spectively; the positions of 36E(arrowhead) and 37A (arrow)on the wild-type homolog areindicated. DNA from the re-gion shaded yellow hybridizedto an additional band in thevicinity of 37A, indicating thatthe Tft aberration involves a du-plication and translocation ofsequences from 36F3–7 to 37A.This is most clearly observed inexceptional chromosome fig-ures where the homologs haveseparated (B); a second site ofhybridization is seen on one ofthe two homologs (asterisks).The proximal limit of the aber-ration was not determined andlies to the left of the sequenceanalyzed here. The distal limitterminates in the vicinity ofamos (in red), but its structureis complex. Both probe C (in-cluding the amos transcriptionunit) and a probe includingthe next distal 5 kb of sequence(not shown) showed modestevidence of duplication [C, as-terisks designate full hybridiza-tion; (*) designates partial hy-bridization near 37A]. Thatadjacent but nonoverlappingprobes showed this behaviorsuggests that the distal limit ofthe duplication does not break

cleanly, but has one or more additional anomalies associated with it. The distal limit is therefore shaded in fading yellow. ProbeD is distal to the duplicated region and hybridizes to a single band on each homolog (D, asterisks); aberrant pairing of thehomologs at 37A is still observed. Amos is not expressed in the wild-type wing disc (E) but is ectopically expressed in Tft tissuein the presumptive posterior notal region (F, bracket) in an ac/sc -independent manner (G, bracket). Ectopic SOPs (as markedby Hnt) develop from the Amos-misexpressing region of Tft discs (H–J).

dominant induction of mechanosensory organs and generally attributable to cross-activation of proneuralgene expression, I systematically evaluated the ability ofcampaniform sensilla (Figure 6, D and E). Thus, misex-

pression of Amos efficiently induced the development proneural proteins (Ac, Sc, Ato, Amos, and Sens) toactivate one another when misexpressed using dpp-Gal4of mechanosensory organs, which is consistent with the

Tft phenotype. and appropriate UAS constructs. A subset of these datais shown in Figure 7; the results for Ato and Ac are notLimited cross-activating potential of proneural pro-

teins: Most proneural proteins display a significant shown since their misexpression resulted in only verymild cross-activation, at best, of any of these markersamount of promiscuous activity when misexpressed, in-

cluding a common ability to promote the development when assayed at the third instar.As noted before, misexpression of Amos only mildlyof mechanosensory organs. To assess whether this was


Figure 5.—Directed misexpression of Amos efficiently generates ectopic proneural clusters and SOPs for external sensoryorgans. Shown are discs from dppGal4�UAS-amos individuals, with the exception of B, which is wild type (wt). In general, theAmos-misexpressing discs are shown at a slightly lower magnification relative to the wild-type disc due to their overgrowthphenotype. Misexpression of Amos in a stripe along the anterior-posterior border (A) results in a similar stripe of ectopicproneural clusters, as marked by Delta (Dl, B and C), m4-lacZ (stained for �-galactosidase, D) and E(spl)bHLHs (E), as well asstrong overcommitment to the SOP fate, as marked by Hnt (F), Sens (G), Ase (H), Neur (I), and Cut (J; dR, dorsal radius).Inset to J shows a wild-type dR stained for Cut for comparison; only a small number of cells are normally labeled. Wild-typeexpression patterns for all other markers are shown in Figures 2 and 3. (K–P) Amos is downregulated following adoption of theSOP fate. The same disc is doubly stained in A and F; the boxed region is shown at higher magnification in K–M. Note that ahigher level of Hnt (green) is found in cells that do not express Amos (red). (N–P) Cells that strongly express Amos (red)express low levels of �-galactosidase (green) in the neurA101-lacZ background, while the cells that express the highest levels of�-galactosidase do not express Amos.

activates Sc (Figure 7G) and Ac (Figure 7J), even though 7, S–U). Bearing in mind that one cannot infer directit strongly induced Ase (Figure 7M) and Sens (Figure regulatory relationships from these experiments, it ap-7P). Interestingly, Amos also strongly induced Ato, al- pears that the ability of all of these proneural proteinsthough primarily only in the wing pouch region (Figure to activate the mechanosensory organ is probably not due7D, bracket). In contrast, neither Sc nor Sens detectably to activation of Ac/Sc, the normal proneural bHLHs foractivated either Amos or Ato (Figure 7, B, C, E, and F). this process. Rather, it is correlated with their commonOnly Sens induced an appreciable amount of ectopic ability to induce Ase and Sens.Sc (Nolo et al. 2000) and Ac (Figure 7, I and L), al-though this was mostly restricted to the dorsal wingpouch and notal regions of the disc. Since ectopic Sc DISCUSSIONdid not generally induce Ac (Figure 7K; Gomez-Skar-

amos activates ectopic mechanosensory neurogenesismeta et al. 1995), this might reflect independent cross-in Tft : Although the normal function of amos is to initiateactivation of Sc and Ac by Sens. Finally, all three ofthe development of certain embryonic multiple-den-these proneural proteins could ectopically activate Asedritic neurons and adult olfactory sensilla (Gouldingand Sens (Figure 7, M–R), with their rank order ofet al. 2000; Huang et al. 2000), several lines of evidenceeffectiveness being Amos � Sens � Sc. This correlated

directly with their ability to activate neurA101-lacZ (Figure suggest that it is also responsible for generating the

1420 E. C. Lai

Figure 6.—Adult phenotypes caused by misex-pression of Amos. SEMs of (A) wild-type and (B)dpp-Gal4�UAS-amos nota are shown; many ectopicmacrochaetae are seen in the latter. They gener-ally die as pupae but occasionally survive to thelate pharate adult stage; cuticle deformation isdue to its dissection from the pupal case. (C)Phenotype of a small clone of Amos-expressingcells that have been marked with forked. Most suchclones show only a bald patch; however, excep-tional clones also show a tight cluster of forkedbristles, suggesting that Amos induces sensory or-gans cell-autonomously. No instances of clustersof forked� bristles were ever observed. (D) A proxi-mal-central portion of a wild-type wing. The wingblade proper is devoid of mechanosensory bristlesand has only a small number of campaniformsensilla associated with some of the veins (arrow).(E) hsGal4�UAS-amos fly that was exposed to a6-min heat shock at the white prepupal stage;the wing is covered with several hundred ectopicmechanosensory and campaniform sensilla.

dominant Tft phenotype, which consists of a large num- distinct gain-of-function mutants. An even more stun-ning fact is that both Tft and Roi were first describedber of ectopic adult mechanosensory organs.

First, Tft maps to the same cytological location as amos in the same volume of the Drosophila Information Servicein 1952 (Ives 1952; Ritterhoff 1952). The history ofand is associated with a chromosomal duplication and

translocation that affects amos. Second, Tft mutants ec- amos seems thus to be dominated by coincidences.Unusual features of mechanosensory neurogenesistopically express Amos in precisely the same region from

which ectopic SOPs arise in this mutant. Third, Tft is induced by amos: A curious feature of ectopic peripheralneurogenesis induced by Tft or UAS-amos is that it verysensitive to the dosage of da, which encodes an obligate

bHLH cofactor for proneural proteins such as Amos. minimally involves Ac and Sc, the endogenous proneuralproteins for this process. Tft is not suppressed by com-Consistent with this, da similarly suppresses Roi (caused

by misexpression of amos; Chanut et al. 2002) and en- plete inactivation of these proneural genes and is notmodified by an increase in ac dosage. In addition, Schances the phenotype of amos deficiencies (Goulding

et al. 2000; Huang et al. 2000). Fourth, deliberate misex- and Ac are minimally misexpressed in Tft or in directedAmos misexpression experiments, even though all otherpression of Amos phenocopies Tft and very effectively

generates ectopic mechanosensory organs (in addition PNC and SOP markers tested are strongly induced un-der these conditions. The failure of Amos to induce Scto other types of sense organs). Although I cannot for-

mally exclude the contribution of other genes affected or Ac is especially surprising considering the fact thatSens is very strongly induced by Amos, and Sens canby the aberration at 36F–37A, the collected observations

strongly suggest that the Tft phenotype can be satisfacto- ectopically induce Sc and Ac (although only in a subsetof disc cells). A possible explanation for this paradoxrily accounted for by the ectopic expression of Amos.

It should be noted that Modolell and colleagues have is that the high levels of E(spl)bHLH proteins inducedby Amos are responsible for repressing ac and sc (Vancome to similar conclusions regarding Tft and have also

shown that Tft revertants no longer misexpress Amos Doren et al. 1994; Jimenez and Ish-Horowicz 1997),although it is also the case that Sens can induce E(spl)and/or are hypomorphic for amos (Villa-Cuesta et al.

2003, accompanying article in this issue). Taken to- expression (Nolo et al. 2000).Interestingly, expression of SOP markers such as Hntgether, these observations strongly indicate that Tft is a

gain-of-function allele of amos. and neurA101-lacZ was often inversely correlated with thatof Amos on a cell-by-cell level, even though Amos veryThe past year has witnessed not only the simultaneous

and independent characterization of Tft (this work and strongly induces their expression. Since the effects ofTft (Arnheim 1967) and Amos misexpression are auton-Villa-Cuesta et al. 2003), but also the realization that

the classical mutant Rough eye (Roi) is likewise a gain- omous, this probably does not represent nonautono-mous induction or recruitment of SOPs, but rather neg-of-function mutant of amos (Chanut et al. 2002). There-

fore, this relatively recently identified gene, whose origi- ative regulation of Amos. This control seems unlikelyto reside at the transcriptional level in our experimentsnal characterization was also carried out simultaneously

and independently by two laboratories (Goulding et al. involving the Gal4/UAS system, suggesting that Amosprotein might be unstable in SOPs. amos mRNA is also2000; Huang et al. 2000), is affected by two completely


Figure 7.—Cross-regulatory capabilities of selected proteins with proneural activity. All discs contain dpp-Gal4 and UAS-amos(top row), UAS-sc (middle row), or UAS-sens (bottom row). For reference, the misexpressed protein of choice is visualized in A(Amos), H (Sc), and R (Sens); the wild-type expression patterns of most of these proteins are shown in Figures 2–4. EctopicAmos activates Ato in the wing pouch region (D, bracket), very mildly activates Sc (G) and Ac (J), and leads to strong misexpressionof Ase (M) and Sens (P). By contrast, neither Sc nor Sens activates Amos (B and C) or Ato (E and F) in the wing disc. Arrowsin E and F point to normal endogenous Ato expression in the ventral radius (vR); these discs are identical to wild type withrespect to expression of Ato (not shown). Sc negligibly activates Ac (K), while Sens activates both Sc (I) and Ac (L), althoughonly in a subset of Sens-expressing cells. Both Sc and Sens can modestly activate Ase (N and O), and Sc misexpression alsoresults in some ectopic Sens (Q). The proneural strength of these proteins can be rank ordered as Amos � Sens � Sc on thebasis of their ability to activate neurA101-lacZ (S–U). This correlates directly with their ability to activate Ase and Sens (M–R) andwith the strength of their corresponding adult phenotypes (not shown). D and J, E and K, and I and L are from doubly stainedpreparations; M and P are the same as Figure 5, G and H.

apparently rapidly lost from olfactory SOPs (Goulding of a failure to activate lateral inhibition, since E(spl)bHLH expression is strongly induced by Amos. It mayet al. 2000), suggesting that amos is negatively regulated

at multiple levels shortly after commitment to the SOP simply be the case that Amos’ unusually potent pro-neural activity overwhelms or is not very sensitive tofate. This behavior seems to run counter to that of Ac

and Sc, which accumulate to elevated levels in SOPs, lateral inhibition. Another possibility is that inductionof exceptionally high levels of E(spl)m4 (and potentiallybut may be paralleled by Ato, which is downregulated

in maturing chordotonal SOPs (zur Lage and Jarman other Brd family proteins) by Amos might interfere withlateral inhibition, an explanation that might underlie1999). More detailed studies are required to determine

if there are distinct functional consequences of the ap- the strong genetic interaction between Tft and Brd. De-liberate misexpression of Brd family genes is knownparently different regulation of Ato- and ASC-class pro-

teins in SOPs, or if this reflects some trivial difference to compromise lateral inhibition (Leviten et al. 1997;Apidianakis et al. 1999; Lai et al. 2000a,b).in the timing of the differentiation of these different

SOPs. Another explanation might lie in the difference be-tween the types of sensory organs normally controlledThe ability of Tft/amos to induce closely spaced or

even adjacent SOPs and sensory organs suggests that it by ASC and Ato-class proneural proteins. While singleSOPs for mechanosensory organs are chosen from indi-is able to at least partially overcome or bypass lateral

inhibition. This is not simply due to disconnecting a vidual PNCs of ac- and sc -expressing cells, large numbersof SOPs for chordotonal and olfactory sensilla are in-proneural gene from its normal transcriptional control,

at least in the case of the Gal4-UAS experiments, since stead continuously selected from individual zones of ato-or amos-expressing cells (zur Lage and Jarman 1999;misexpression of Ac or Sc by similar means results in

ectopic, but spaced bristles. It is also not a consequence Goulding et al. 2000). It is conceivable that Ato-class

1422 E. C. Lai

proneural proteins possess an inherent ability to induce diary proneural clusters of Ac/Sc. Even Sc fails to sig-nificantly cross-activate Ac (Gomez-Skarmeta et al.the formation of closely spaced sensory organs. Signal-

ing via the epidermal growth factor receptor (EGFR) 1995), although Sc and Ac have largely indistinguish-able DNA-binding properties in vitro (Singson et al.antagonizes N signaling during SOP determination, and

the development of clustered chordotonal organs not 1994). This is not to say that proneural proteins cannotinfluence each other’s expression, since ectopic Amosonly relies upon EGFR signaling but also is strongly

correlated with localized expression of rhomboid and can strongly activate Ato in the pouch region of the wingdisc and in the vicinity of the morphogenetic furrowpresence of dp-ERK (zur Lage and Jarman 1999; Culi

et al. 2001). It will be interesting to determine if settings of the developing eye disc (Chanut et al. 2002). Thisparticular ability might reflect their related DNA-bind-of Amos activity are similarly associated with EGFR sig-

naling and if any mechanistic links can be established ing domains; however, it should be noted that Ato doesnot reciprocally activate Amos (data not shown). Ac, Sc,between expression of Ato-class proteins and activation

of rhomboid transcription and/or phosphorylation of and Sens also fail to activate either Amos or Ato. So ingeneral, there is limited promiscuity in cross-activationMAP kinase.

Specificity of proneural activity: When misexpressed, of proneural bHLH proteins.A common activity of Ato-class and ASC-class bHLHproneural proteins often induce the ectopic differentia-

tion of sensory structures whose development they do proteins is instead their ability to induce Ase and Sensexpression; Sens also induces Ase. As misexpression ofnot normally control. For example, Sc can promote many

aspects of eye development in the absence of Ato; Sc and either Ase or Sens suffices to initiate mechanosensoryorgan development, their activation may be key to pro-Ato can weakly induce the differentiation of MD neurons;

and Amos can promote the differentiation of chordotonal miscuous induction of mechanosensory organs. In prin-ciple, initiation of an Ase/Da-Sens feedback loop mightorgans. Notably, all proneural proteins have the ability to

promote the formation of mechanosensory organs (Jar- be responsible for triggering a mechanosensory-type de-velopmental program. Consistent with this scenario, aseman et al. 1993; Hinz et al. 1994; Goulding et al. 2000;

Huang et al. 2000; Sun et al. 2000). is required for ectopic neurogenesis in Tft although ac/sc are not (A. Garcia-Bellido, personal communica-Several mutually compatible explanations for their

common ability to induce mechanosensory neurogen- tion cited in Campuzano et al. 1985; Villa-Cuesta etal. 2003), and Sens is strictly required for bristle forma-esis have been put forth. First, experiments with Da

suggested that mechanosensory organs might represent tion by ectopic Sc (Nolo et al. 2000). The precise contri-bution of ase to mechanosensory neurogenesis remainsa “default” output for neurogenesis. Since Da is the

heterodimeric partner for all proneural bHLH proteins, in question though, as it is not normally required forthe development of most bristles. In addition, both L’scit is not expected to exhibit a subtype specificity. Never-

theless, misexpression of Da induces only the develop- and Sc can generate ectopic bristles in cells that carrya deletion of the AS-C (and thus lack ase ; Hinz et al.ment of external sensory organs (Jarman and Ahmed

1998). Second, most proneural proteins are known to 1994; Culi and Modolell 1998). It is conceivable thatproneural proteins might generically substitute for aseexhibit a phase of transcriptional auto-activation (Mar-

tinez and Modolell 1991; Van Doren et al. 1992; Culi in these experimental conditions.Dominant gain-of-function alleles in Drosophila: Al-and Modolell 1998; Sun et al. 1998). Although the

binding specificities of the proneural proteins are dis- though they arise infrequently, dominant gain-of-func-tion alleles can produce dramatic phenotypes that aretinct, particularly between the ASC and Ato subclasses,

high levels of ectopic proteins could conceivably result easily identified, even in the course of unrelated studies.This explains why they are generally among the oldestin some direct cross-activation of ac/sc. Once initiated,

auto-activation by Ac and/or Sc might then be responsi- Drosophila mutants known (Lindsley and Zimm 1992).Although in some cases these mutants have been usefulble for subsequent mechanosensory organ formation.

Third, it is known that the transcription factor Sens, reagents for studying various developmental processes,in other cases, their effects are neomorphic and notwhich itself has inherent proneural activity, is down-

stream of both subfamilies of proneural proteins (Nolo informative of normal development. Perhaps for thisreason, dominant gain-of-function mutants have gener-et al. 2000; Frankfort et al. 2001). Thus, it may be

that the activation of a common downstream target by ally been treated with suspicion or even de facto discrimi-nation by Drosophila geneticists. Indeed, many “old”different proneural proteins could explain how they

can all activate mechanosensory neurogenesis. dominant mutants have escaped the attention of Dro-sophila developmental biologists, even though they of-My data do not speak to the first of these explanations.

However, they do suggest that relatively little cross-acti- ten affect processes (such as wing, eye, and bristle devel-opment) that are otherwise the subject of intensevation occurs at the level of proneural bHLH gene ex-

pression. Although Amos is perhaps the strongest in- scrutiny. This has remained true in spite of the fact thatmisexpression studies and systematic gain-of-functionducer of mechanosensory organs among proneural

proteins it does not do so through induction of interme- genetic screens have become de rigeur since the incep-


Apidianakis, Y., A. C. Nagel, A. Chalkiadaki, A. Preiss and C.tion of the versatile Gal4/UAS system in the past decadeDelidakis, 1999 Overexpression of the m4 and m� genes of(Brand and Perrimon 1993; Rørth 1996). the E(spl)-Complex antagonizes Notch mediated lateral inhibition.

Only very recent years have witnessed the molecular Mech. Dev. 86: 39–50.Arnheim, N., 1967 The regional effects of two mutants in Drosophilacharacterization of a number of classical dominant gain-

analyzed by means of mosaics. Genetics 56: 253–263.of-function Drosophila mutants, including Rough eye Artavanis-Tsakonas, S., M. D. Rand and R. J. Lake, 1999 Notch(Chanut et al. 2002), Glazed (Brunner et al. 1999), Lyra signaling: cell fate control and signal integration in development.

Science 284: 770–776.(Nolo et al. 2001), Drop (Mozer 2001), Beadex (MilanBailey, A. M., and J. W. Posakony, 1995 Suppressor of Hairlesset al. 1998; Shoresh et al. 1998; Zeng et al. 1998), and

directly activates transcription of Enhancer of split Complex genesScutoid (Fuse et al. 1999). In these examples, the mutant in response to Notch receptor activity. Genes Dev. 9: 2609–2622.

Balcells, L., J. Modolell and M. Ruiz-Gomez, 1988 A unitaryphenotype appears to be due to the misexpression of anbasis for different Hairy-wing mutations of Drosophila melanogaster.important regulatory molecule (including transcriptionEMBO J. 7: 3899–3906.

factors and a morphogen). Thus, while these mutations Bellen, H. J., C. J. O’Kane, C. Wilson, U. Grossniklaus, R. K.are all neomorphic, they are nonetheless useful in that Pearson et al., 1989 P-element-mediated enhancer detection:

a versatile method to study development in Drosophila. Genesthey identify “interesting” genes. Many other commonlyDev. 3: 1288–1300.utilized dominant mutations—Curly, Tubby, Additional Bertrand, N., D. Castro and F. Guillemot, 2002 Proneural genes

Veins, Blunt short bristle, and Plexate, for example—remain and the specification of neural cell types. Nat. Rev. Neurosci. 3:517–530.to be characterized. A cursory examination of the Fly-

Brand, A. H., and N. Perrimon, 1993 Targeted gene expression as aBase archive reveals �50 other uncharacterized adult- means of altering cell fates and generating dominant phenotypes.dominant Drosophila mutants for which mutant stocks Development 118: 401–415.

Brand, M., A. P. Jarman, L. Y. Jan and Y. N. Jan, 1993 asense is aare publicly available; about one-half of these mutantsDrosophila neural precursor gene and is capable of initiating sensehave been extant for nearly 50 years or more (FlyBaseorgan formation. Development 119: 1–17.

1998). Undoubtedly, this collection of mutants, once Bridges, C., and T. H. Morgan, 1923 The Third-Chromosome Groupproperly analyzed, will be found to affect many other of Mutant Characters of Drosophila melanogaster. Pub. 327, Carnegie

Institute, Washington, DC.interesting genes as well.Brunner, E., D. Brunner, W. Fu, E. Hafen and K. Basler, 1999The history of amos is particularly instructive in this The dominant mutation Glazed is a gain-of-function allele of wing-

context. Its recent discovery relied upon molecular ap- less that, similar to loss of APC, interferes with normal eye develop-ment. Dev. Biol. 206: 178–188.proaches (degenerate PCR and two-hybrid screening)

Campuzano, S., L. Carramolino, C. V. Cabrera, M. Ruiz-Gomez,and specific genetic lesions in amos have yet to be de-R. Villares et al., 1985 Molecular genetics of the acaete-scute

scribed. Nevertheless, the existence of an amos -like func- gene complex of D. melanogaster. Cell 40: 327–338.Campuzano, S., L. Balcells, R. Villares, L. Carramolino, L. Gar-tion had been genetically inferred for many years, since

cia-Alonso et al., 1986 Excess function Hairy-wing mutationscertain neurons persist in embryos mutant for both thecaused by gypsy and copia insertions within structural genes ofAS-C and ato, and much of the olfactory system develops the achaete-scute locus of Drosophila. Cell 44: 303–312.

independently of these proneural genes. Since the de- Capdevila, J., and I. Guerrero, 1994 Targeted expression of thesignaling molecule decapentaplegic induces pattern duplicationsvelopment of all of these neurons is still sensitive toand growth alterations in Drosophila wings. EMBO J. 13: 4459–manipulation of Da and/or EMC levels, this suggested4468.

the existence of an additional proneural bHLH. Loss Carmena, A., M. Bate and F. Jimenez, 1995 Lethal of scute, aof amos function produces phenotypes complementary proneural gene, participates in the specification of muscle pro-

genitors during Drosophila embryogenesis. Genes Dev. 9: 2373–to AS-C; ato mutants, suggesting it is the “missing”2383.proneural gene (Goulding et al. 2000; Huang et al. Caudy, M., H. Vassin, M. Brand, R. Tuma, L. Y. Jan et al.,

2000). Had Roi and/or Tft been studied earlier, amos 1988 daughterless, a Drosophila gene essential for both neuro-genesis and sex determination, has sequence similarities to mycmight have been discovered long ago. In this author’sand the achaete-scute complex. Cell 55: 1061–1067.opinion, the very history of amos justifies the study of

Chanut, F., K. Woo, S. Pereira, T. Donohoe, S.-Y. Chang et al.,other long-neglected dominant mutants. 2002 Rough eye is a gain-of-function allele of amos that disrupts

regulation of the proneural gene atonal during Drosophila retinalI thank Francoise Chanut, Adina Bailey, and Julia Serano for usefuldifferentiation. Genetics 160: 623–635.discussions of this work; Juan Modolell for communications regarding

Chien, C. T., C. D. Hsiao, L. Y. Jan and Y. N. Jan, 1996 NeuronalTft prior to publication; and especially Todd Laverty for performingtype information encoded in the basic-helix-loop-helix domainin situ hybridizations to polytene chromosomes. I also acknowledgeof proneural genes. Proc. Natl. Acad. Sci. USA 93: 13239–13244.

the following for generous gifts of antibodies and fly stocks: Andrew Culi, J., and J. Modolell, 1998 Proneural gene self-stimulationJarman, Yuh Nung Jan, Saray Bray, Hugo Bellen, Francoise Chanut, in neural precursors: an essential mechanism for sense organJames Posakony, Jose de Celis, the Bloomington Stock Center, and the development that is regulated by Notch signaling. Genes Dev.Developmental Hybridoma Studies Bank. I acknowledge the gracious 12: 2036–2047.support of Gerald Rubin and the Damon Runyon Cancer Research Culi, J., E. Martin-Blanco and J. Modolell, 2001 The EGF recep-Foundation, DRG 1632. tor and N signaling pathways act antagonistically in Drosophila

mesothorax bristle patterning. Development 128: 299–308.de Celis, J. F., and S. Bray, 1997 Feed-back mechanisms affecting

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Villa-Cuesta, E., J. de Navascues, M. Ruiz-Gomez, R. Diez del alleles and might represent an evolutionarily conserved functionCorral, M. Domınguez et al., 2003 Tufted is a gain-of-function in appendage development. Proc. Natl. Acad. Sci. USA 95: 10637–allele that promotes ectopic expression of the proneural gene 10642.amos in Drosophila. Genetics 163: 1403–1412. zur Lage, P., and A. P. Jarman, 1999 Antagonism of EGFR and

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Zeng, C., N. J. Justice, S. Abdelilah, Y.-M. Chan, L. Y. Jan et al.,1998 The Drosophila LIM-only gene, dLMO, is muated in Beadex Communicating editor: T. C. Kaufman

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Drosophila tufted is a gain-of-function allele of the proneural gene