Genetics, Vol. 163, 1403-1412, April 2003, Copyright © 2003
Tufted Is a Gain-of-Function Allele That Promotes Ectopic Expression of the Proneural Gene amos in Drosophila
Eugenia Villa-Cuestaa,
Joaquín de Navascuésa,
Mar Ruiz-Gómeza,
Ruth Diez del Corral1,a,
María Domínguez2,a,
José Félix de Celisa, and
Juan Modolella
a Centro de Biología Molecular Severo Ochoa, CSIC and Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Corresponding author:
Juan Modolell, CSIC and Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain., jmodol{at}cbm.uam.es (E-mail)
Communicating editor: T. C. KAUFMAN
 | ABSTRACT |
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The Tufted1 (Tft1) dominant mutation promotes the generation of ectopic bristles (macrochaetae) in the dorsal mesothorax of Drosophila. Here we show that Tft1 corresponds to a gain-of-function allele of the proneural gene amos that is associated with a chromosomal aberration at 36F–37A. This causes ectopic expression of amos in large domains of the lateral-dorsal embryonic ectoderm, which results in supernumerary neurons of the PNS, and in the notum region of the third instar imaginal wing, which gives rise to the mesothoracic extra bristles. Revertants of Tft1, which lack ectopic neurons and bristles, do not show ectopic expression of amos. One revertant is a loss-of-function allele of amos and has a recessive phenotype in the embryonic PNS. Our results suggest that both normal and ectopic Tft1 bristles are generated following similar rules, and both are subjected to Notch-mediated lateral inhibition. The ability of Tft1 bristles to appear close together may be due to amos having a stronger proneural capacity than that of other proneural genes like asense and scute. This ability might be related to the wild-type function of amos in promoting development of large clusters of closely spaced olfactory sensilla.
DEVELOPMENT of the peripheral nervous system of Drosophila starts by the expression of proneural genes in groups of cells of the embryonic ectoderm and the imaginal discs (reviewed in CAMPUZANO and MODOLELL 1992 
; GHYSEN et al. 1993 ; JAN and JAN 1993 ). Proneural genes confer to cells the capacity to become sensory organ precursors (SOPs). However, this neural potential is not realized in all cells of each proneural group, since the proneural genes also promote negative interactions among the cells of each cluster (lateral inhibition) that are mediated by the Notch (N) signaling pathway (reviewed in ARTAVANIS-TSAKONAS et al. 1999 ). This pathway antagonizes a critical step in SOP commitment, namely, the triggering of proneural gene self-stimulation that allows accumulation of large amounts of proneural protein in the cell that becomes an SOP, presumably to implement a neural differentiation program (CULI and MODOLELL 1998 ). The end result of the neural-promoting ability of the proneural proteins and the antineurogenic (proepidermic) action of the N pathway is that only one or a few cells of each proneural cluster become SOPs.
All the known Drosophila proneural genes encode transcription factors of the bHLH family (reviewed in JAN and JAN 1993 ; JARMAN et al. 1993 ; GOULDING et al. 2000 ; HUANG et al. 2000 ). Of the four members of the achaete-scute complex (AS-C), achaete (ac) and scute (sc) are most important for the development of external SOs, like the bristles and sensilla campaniformia and trichoidea of the head, notum, legs, and wings, and a subset of larval SOs (GARCIA-BELLIDO 1979 ; DAMBLY-CHAUDIERE and GHYSEN 1987 ; CAMPUZANO and MODOLELL 1992 ; RUIZ-GOMEZ and GHYSEN 1993 ). asense (ase) is most important for the development of the tergite bristles, the anterior wing margin bristles and another subset of larval external SOs (DAMBLY-CHAUDIERE and GHYSEN 1987 ; MARI-BEFFA et al. 1991 ; BRAND et al. 1993 ; DOMINGUEZ and CAMPUZANO 1993 ; JARMAN et al. 1993 ). In addition, ase is expressed in all SOPs and because of this it has also been categorized as a panneural gene. The fourth member of the AS-C, the lethal of scute (l'sc) gene, is mostly concerned with the development of the central nervous system (CNS; JIMENEZ and CAMPOS-ORTEGA 1979 ; MARTIN-BERMUDO et al. 1991 ). Three other known proneural genes are found outside the AS-C. They are atonal (ato), which promotes development of the chordotonal organs and photoreceptors (JARMAN et al. 1993 , JARMAN et al. 1994 ); amos (absent solo-MD neurons and olfactory sensilla), which is necessary for the development of some embryonic multidentritic neurons and two types of olfactory SOs of the antenna (GOULDING et al. 2000 ; HUANG et al. 2000 ); and daughterless (da), which encodes the bHLH heterodimerizing partner of all the proneural proteins and is a requisite for their function (CAUDY et al. 1988 ; DAMBLY-CHAUDIERE et al. 1988 ; MURRE et al. 1989 ).
The gene Tufted (Tft) is a candidate for another proneural gene or for a regulator of known proneural genes. Tft is known from only a single dominant gain-of-function allele (Tft1) that promotes the development of a large number of extra bristles in the posterior dorsal mesothorax and at the metanotum (LINDSLEY and ZIMM 1992 ). The removal of ac, sc, and l'sc in the Df(1)sc19 does not suppress the Tft1 phenotype, but the additional removal of ase (Df(1)260-1) suppresses it (A. GARCÍA-BELLIDO, personal communication cited in CAMPUZANO et al. 1985 ). Moreover, the overexpression of ase can mimic the Tft1 phenotype (DOMINGUEZ and CAMPUZANO 1993 ). This has led to the suggestion that Tft might be a transregulator of ase or that it might regulate another proneural gene that requires ase for development of extra bristles.
Tft has been genetically mapped to chromosomal position 37A+ (LINDSLEY and ZIMM 1992 ). The recent cloning and characterization of amos (GOULDING et al. 2000 ; HUANG et al. 2000 ), located at chromosomal subdivision 36F2–6/37A1–2, has opened the possibility that Tft may be an allele of amos. Here we show that this is indeed the case. Tft1 appears to be associated with a rearrangement at the 36F chromosomal region that causes amos misexpression in the embryo and imaginal discs. This misexpression accounts for the Tft1 phenotype. Similar conclusions have been reached by LAI 2003 in the accompanying article in this issue.
| MATERIALS AND METHODS |
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Drosophila stocks:
The following stocks were used: ase1, In(1)sc10.1, Tft1, and Df(2)M36F-S6 (described in http://flybase.bio.indiana.edu:82); UAS-sc (PARRAS et al. 1996 ), UAS-ase (BRAND and DORMAND 1995 ), and UAS-amos (GOULDING et al. 2000 ); and Gal4 line C765 (GOMEZ-SKARMETA et al. 1996 ). The SOP cell-specific lacZ reporter transgene neuralized (neu)-lacZ (line A101.IF3) is described in HUANG et al. 1991 . Tft1 revertants TftRM9 and TftRM11 were induced by X rays (3000 rad) on Tft1/CyO males and detected by the loss of the Tft1 dominant phenotype in the F1 generation.
Histochemistry:
Hybridization in situ to detect specific mRNAs in embryos or imaginal disc whole mounts was performed as described by GONZALEZ-CRESPO and LEVINE 1993 , using antisense RNA (amos, ase) or DNA [E(spl)-m8] digoxygenin-labeled probes. Antibody stainings of imaginal discs were as in CUBAS et al. 1991 . Primary antibodies were rabbit anti ß-galactosidase (Cappel), MAb 22C10 (FUJITA et al. 1982 ), anti-Cut (Developmental Studies Hybridoma Bank), and anti-Achaete (SKEATH and CARROLL 1991 ). Secondary antibodies were from Amersham. Polytene chromosomes were prepared as in ASHBURNER 1989 . They were hybridized with biotinylated 
phage clones (ENGELS et al. 1986 ) harboring inserts of the amos genomic region and fluorescently stained with avidin-Cy3 and 4',6-diamidino-2-phenylindole (DAPI).
| RESULTS |
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The Tft1 mutant phenotype:
The extra bristles characteristic of Tft1 heterozygous or homozygous flies appear mainly in the postalar, dorsocentral, and scutellar regions of the dorsal mesothorax (posterior notum; LINDSLEY and ZIMM 1992 and Fig 1, A–C). Ectopic bristles and other sensory organs are also formed in the metanotum in a position dorsal to the halteres (Fig 1F). In contrast to other mutations that also promote formation of extra bristles such as extramacrochaetae (emc) or hairy (BOTAS et al. 1982 ), the extra bristles of Tft1/+ or Tft1 homozygous flies can develop very close together, forming tufts, and in some cases several tormogens can be fused to each other (Fig 1B and Fig C, and data not shown). These phenotypes are cell autonomous in mosaics (ARNHEIM 1967 ). In addition, homozygous and heterozygous Tft1 flies have a reduced scutellum, probably due to the change of epidermal cells into a neural fate, and very frequently lack the scutum-scutellar suture.
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Figure 1.
Bristle phenotypes of Tft mutants. Nota of wild type (A), Tft1/+ (B), Tft1/Tft1 (C), ase1;Tft1/+ (D), and In(1)sc10.1; Tft1/+ (E) flies are shown. The In(1)sc10.1 allele lacks ac and sc, but not ase, expression. The clusters of ectopic bristles are largest in Tft1 homozygous individuals (arrowheads), disappear in ase1 mutants, and are independent of ac-sc. Ectopic bristles in the metanotum (F) and in the antenna (G) of a Tft1/+ fly (arrowheads) are shown.
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To discriminate whether ectopic Tft bristles are formed following the same rules as for wild-type bristles, we first carried out a cell lineage analysis of the mutant thorax. Mitotic recombination clones were induced in y1 f36a/+; Tft1/+ larvae, and a total of 15 y f clones were studied in the thorax. In all cases the clones included bristles located at different extant macrochaetae positions and the mosaic borders separated bristles located very close together in the same position. This indicates that the SOPs of the ectopic bristles are singled out, as happens with normal bristles (GARCIA-BELLIDO and MERRIAM 1971 ), from neighboring cells not clonally related; that is, they are not derived from a common precursor.
The singling out of mutant SOPs was directly visualized in imaginal discs using the SOP early marker neu-lacZ (A101) (Fig 2H). We observed that in the presumptive posterior notum, extra SOPs were generated in large numbers and very close together. Moreover, the appearance of ectopic SOPs was sequential and occurred concomitantly with the normal SOPs. Thus, it seems that either the proneural activity at the posterior notum is increased or the normal lateral inhibition mechanisms antagonizing proneural activity are inefficient in that region of the presumptive notum. The contribution of the N signaling pathway was analyzed in genetic combinations of Tft1 and mutations in members of this pathway. The number of ectopic bristles in Tft heterozygous flies was increased by mutations in Delta, the N ligand, and it was reduced in the N gain-of-function Abruptex (Ax) and in Hairless (H) alleles (Table 1). Moreover, the overexpression of a UAS-E(spl)-m8 transgene, which encodes one of the bHLH repressors activated by the canonical N signaling pathway (DE CELIS et al. 1996 ; ARTAVANIS-TSAKONAS et al. 1999 , for review), strongly suppressed the Tft1 phenotype (Table 1). This again indicated that the Tft bristles are sensitive to lateral inhibition. On the other hand, the Tft1 mutant phenotype was also strongly modified in combinations with emc alleles, as a hypomorphic condition (emc1/emcpel) substantially increased the number of extra bristles, and the gain-of-function allele Achaetus (emcAch) almost completely suppressed the ectopic bristles. Since emc directly antagonizes proneural gene function (ELLIS et al. 1990 ; GARRELL and MODOLELL 1990 ), it appears that the proneural capability of some regions of the thorax is greatly increased in Tft1/+ or Tft1 homozygous individuals and that the lateral inhibition pathway, albeit operative, is inefficient in preventing excess SOP singling out from these regions.
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Figure 2.
Expression of amos and of other genes in embryos and wing imaginal discs bearing Tft alleles. Wild type (A), Tft1/+ (B), TftRM9 (C), and TftRM11 (D) stage 11–12 embryos hybridized with an amos probe are shown. Note the expanded domains of amos expression in the germ band of Tft1/+ embryos and their nearly complete disappearance in the TftRM11 revertants. amos expression in Tft1/+ (E), ase1;Tft1/+ (F), and TftRM9 (G) third instar wing imaginal discs is shown. The ectopic expression of amos (arrowheads) does not depend on ase (F) and is absent in the revertant (G). Expression of neuralized-lacZ (H), an SOP-specific marker (HUANG et al. 1991 ), in the presumptive nota of Tft1/+ imaginal discs is shown. Note the emergence of a large number of SOPs in the domain of ectopic amos expression (compare with E). In a wild-type disc, only a few SOPs appear in this region (CUBAS et al. 1991 ). The Tft1-dependent extra SOPs also express ase mRNA (I) and accumulate Ac (J) and Sc (not shown) proteins. Images correspond to homozygous Tft1 wing imaginal discs. In J, the Tft-induced SOPs appear as single cells outside the extant proneural clusters (arrowheads point at two of them). (K and L) Wild-type and Tft1/+ discs, respectively, hybridized with an E(spl)m8 probe. Note the increased expression of E(spl)m8 in the region of ectopic amos expression (arrowhead, compare with E and F), which gives rise to ectopic SOPs (compare with H and J).
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Table 1.
Average number (and standard deviation) of ectopic macrochaetae on heminota of Tft1/+ flies harboring different mutations or overexpressing UAS-E(spl)m8
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amos is misexpressed in Tft1 mutants:
Since the Tft1 mutation genetically maps to chromosomal position 37A+ (LINDSLEY and ZIMM 1992 ), very close to the position of the amos proneural gene (GOULDING et al. 2000 ; HUANG et al. 2000 ), we examined whether amos was expressed in wing and haltere discs carrying the Tft1 allele. This was the case, as amos mRNA was detected in the dorsocentral, postalar, and scutellar regions of the presumptive notum and in the postnotum (Fig 2E). Ectopic amos mRNA was also found in equivalent regions of the third instar haltere disc (not shown). In contrast, amos was not expressed in wild-type wing and haltere discs (GOULDING et al. 2000 and our unpublished data). The location of amos misexpression correlated well with the position where ectopic bristles develop in the adult (Fig 1, A–C) and where the ectopic SOPs arise in the wing disc (Fig 2H). The expression of the E(spl)-m8 gene was increased in this region (Fig 2K and Fig L), consistent with an ability of ectopic Amos, similarly to Ac and Sc in the extant proneural clusters of this disc (DE CELIS et al. 1996 ; ARTAVANIS-TSAKONAS et al. 1999 ), to stimulate N signaling.
In the wild type, amos is also expressed during pupation in the distal part of the leg discs, which correlates with the tarsal claws, and in three semicircular bands of the developing third segment antennal wild-type disc, which allows proper development of the antenna's olfactory sensilla (GOULDING et al. 2000 ). amos expression in the antennal disc of Tft1 mutants appeared wild type, consistent with an essentially normal arrangement of olfactory sensilla in Tft1/+ antennae (not shown). However, the presence of an occasional sensory bristle (Fig 1G), similar to bristles that appear when amos is overexpressed in the antenna (GOULDING et al. 2000 ), suggested that amos might also be mildly overexpressed in this region.
In wild-type embryos, amos is expressed during stages 9–12 in a lateral, small group of cells in each of the thoracic and abdominal segments, in addition to a few groups in the cephalic segments (HUANG et al. 2000 ). This pattern is gradually restricted to two or three single cells (Fig 2A) that later will differentiate into dorsal bipolar (dbp) or dorsal multidendritic (dmd) neurons of the dorsal cluster of the peripheral nervous system (PNS; HUANG et al. 2000 ; see also Fig 3A). In contrast, in Tft1/+ embryos, amos expression was expanded into a relatively large region of each segment (Fig 2B). This ectopic expression was not refined to a more restricted pattern and it was associated with the generation of extra neurons in the dorsal cluster (compare Fig 3A with 3B). Occasionally, the dorsal bipolar neuron was duplicated (Fig 3D). Staining of these embryos with anti-Cut antibody, which labels SOPs of the external sensory organs, their descendants including an associated md neuron, and some additional md neurons (BREWSTER and BODMER 1995 ), showed a small increase in the number of Cut-positive cells in the dorsal cluster (not shown).
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Figure 3.
Neuronal defects in the PNS of Tft mutants. Stage 15–16 embryos were stained with 22C10 antibody. (A–C) Dorsal neuronal cluster of Tft1, wild-type, and TftRM11 embryos, respectively, oriented anterior to the left and dorsal to the top. Arrowheads point to the cluster of the fourth abdominal segment. Note the increase in the number of neurons in the Tft1 dorsal cluster and its decrease in the TftRM11 embryo, as compared to the wild type. (D–F) Focus on the position of the dorsal bipolar neuron of Tft1, wild-type, and TftRM11 embryos, respectively. This is duplicated in a segment of a Tft1 embryo and is absent in most segments of TftRM11 embryos (arrowheads).
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Ectopic amos expression is removed in Tft1 revertants:
Revertants of Tft1 devoid of extra bristles on the notum were obtained by X-ray mutagenesis. TftRM9 was homozygous viable and was associated with an inversion with a breakpoint in the vicinity of chromosomal subdivision 37A (M. ASHBURNER, personal communication). The absence of notum ectopic bristles correlated with the absence of amos expression in third instar wing discs (Fig 2G). The embryonic expression during stages 9–12 reverted to that of the wild type (Fig 2C) and the pattern of neurons in the embryonic PNS was essentially normal (not shown). Another revertant, TftRM11, was homozygous lethal. No ectopic expression of amos was detected in TftRM11/+ third instar wing discs (not shown). In embryos homozygous for TftRM11, amos expression was strongly reduced, as compared to that in the wild type (Fig 2D), the Tft1 ectopic neurons were eliminated, and the dorsal cluster had significantly fewer neurons than in the wild type. According to the morphology of the remaining neurons, the reduction appeared to affect dmd neurons (Fig 3C) and, clearly, the dorsal bipolar neuron (Fig 3F). This phenotype is similar to that described for RNAi experiments directed to remove amos function (HUANG et al. 2000 ) and suggests that TftRM11 is a hypomorphic allele of amos. This was verified by complementation tests using the deficiency Df(2)M36F-S6, which eliminates amos, and is homozygous lethal (HUANG et al. 2000 ). We found that TftRM11, which poorly expresses amos, did not complement Df(2)M36F-S6, but TftRM9, which expresses amos at approximately wild-type levels, did complement it (not shown). In summary, the above data indicate that the Tft phenotype is caused by the ectopic expression of amos and that removal of this expression is sufficient to revert the phenotype. Overexpression experiments with an amos transgene reinforced this conclusion (see below).
amos misexpression produces extra SOs:
It is known that the misexpression of amos produces ectopic sensory organs, the type of which depends on the site and the time of misexpression. Thus, in embryos, amos misexpression produces ectopic md neurons and other types of neurons (HUANG et al. 2000 ). In the antenna, it produces extra olfactory sensilla and, occasionally, other types of sensory bristles. And outside the antenna, it can give rise to different types of sensory organs (GOULDING et al. 2000 ).
We compared the Tft phenotype with that caused by amos misexpression. We used different GAL4 lines to drive UAS-amos in the notum of Drosophila. These misexpressions induced embryonic or pupal lethality with most of the GAL4 drivers used (ap-Gal4, tsh-Gal4, pnr-Gal4, MS1096, and en-Gal4). With C765, which drives a fairly ubiquitous expression in all imaginal discs (GOMEZ-SKARMETA et al. 1996 ), pharate flies were obtained at 17°. Their bodies were covered with many sensory organs of different types (Fig 4C, Fig F, Fig H, and Fig I). These were mostly macrochaetae on the head (not shown) and on the notum (Fig 4C), so that the latter resembled the notum of a Tft1 fly (Fig 1C). Large numbers of sensilla campaniformia appeared in the proximal wing (Fig 4H) and a mixture of bristles of diverse types and sensilla campaniformia (with occasional sensilla of unclear types) in the rest of the wing (Fig 4I). Thus, amos overexpression produced different types of sensory organs depending on the site.
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Figure 4.
Phenotypes of expression of UAS-sc, UAS-ase, or UAS-amos in wing disc derivatives. Transgenes were driven by C765-Gal4, which promotes ubiquitous expression in the wing disc (GOMEZ-SKARMETA et al. 1996 ). (A and D) Expression of UAS-sc at 25° promotes few extra bristles on the notum (compare with wild-type notum in Fig 3A) and wing (arrowheads point at some ectopic bristles). (B and E) Expression of UAS-ase at 25° promotes generation of many more ectopic bristles. (C and F) Expression of UAS-amos at 17° gives rise to even more ectopic sensory organs. Only pharate individuals could be recovered. (G) Detail of a wing expressing UAS-ase shown at higher magnification reveals numerous ectopic bristles and sensilla campaniformia (arrowheads). (H and I) Details of selected regions of F (squares) at higher magnification. Note the large number of sensilla campaniformia that develop in the proximal part of the wing (H). In more distal parts (I), bristles of different types are more abundant than sensilla campaniformia. As in Tft1 flies, extra bristles arise in contiguous positions.
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We also compared the proneural capacity of UAS-amos with that of UAS-ase and UAS-scute by using the C765 driver. Under our conditions, UAS-amos (Fig 4C, Fig F, Fig H, and Fig I) was a much stronger inducer of ectopic sensory organs than UAS-ase (Fig 4B, Fig E, and Fig G), and UAS-ase was stronger than UAS-sc (Fig 4A and Fig D).
The Tft notum bristles require ase but not ac and sc:
It has been reported that the Tft phenotype is not dependent on the presence of the ac and sc genes, but it is suppressed by the further removal of the ase gene (A. GARCÍA-BELLIDO, personal communication cited in CAMPUZANO et al. 1985 ). We verified these data (Fig 1D and Fig E) and examined the expression of these proneural genes in Tft1 wing discs to analyze possible regulatory interactions between them and amos. ac, sc, and ase were ectopically expressed in the area where the Tft1 SOPs appear, but their expressions occurred only in single, isolated cells (Fig 2I and Fig J). Not even in younger discs did expression of these genes in that area occur in a proneural-like cluster (not shown). The isolated cells were most likely SOPs, since these three genes are expressed in singled-out bristle precursor cells due to self-stimulatory loops (CULI and MODOLELL 1998 ). The pattern of expression of ase in single cells, as opposed to the homogeneous large domain of ectopic amos, suggests that it is the presence of Amos that triggers the expression of ase and not vice versa. Indeed, the ectopic expression of amos was not removed in Tft1/+ discs simultaneously mutant for the null ase1 allele (Fig 2F). Taken together, these results indicate that the ectopic expression of amos in a relatively large area of the presumptive notum creates an oversized proneural cluster from which many SOPs emerge. These then express ac, sc, and ase, similarly to the SOPs of other external sensory organs (CUBAS et al. 1991 ; SKEATH and CARROLL 1991 ; BRAND et al. 1993 ; DOMINGUEZ and CAMPUZANO 1993 ). Moreover, amos needs the panneural function of ase to single out SOPs from this proneural cluster. We also found that the expression of amos is normal in ase1 embryos and that in ase1 adults the olfactory SOs of the antenna appear unaffected (not shown). This further indicated that the expression of amos did not depend on ase and that this gene is dispensable for the generation of the olfactory SOs.
Tft1 is associated with a small rearrangement of the amos chromosomal region:
Salivary polytene chromosomes from Tft1 larvae appeared essentially normal in the region surrounding the reported cytological position of amos (36F2–6/37A1–2, not shown). However, by using a probe that contained the amos structural gene and 
11 kb of the flanking 5' DNA, we observed two hybridization sites, close to each other, in Tft1 chromosomes (Fig 5A). In contrast, wild-type chromosomes showed a single signal in this region (Fig 5B). Hybridization to Tft1/+ chromosomes (Fig 5C) showed that the distal signal, located in subdivision 36F (a region that is frequently puffed) was shared by the wild-type and Tft1 chromosomes. The proximal signal, specific of the Tft1 chromosome, was located in subdivision 37AB. Similar images of heterozygous Tft1/+ chromosomes were obtained with a probe that contained amos and 15 kb of DNA downstream of it (not shown). The simplest explanation of these data is that a duplication of at least several kilobases, which includes the amos gene, has occurred in the Tft1 chromosome. Consistent with this interpretation, genomic Southern blots hybridized with an amos-specific probe (consisting of the amos structural sequences) showed stronger signals in Tft1 DNA than in the control wild-type DNA (not shown).
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Figure 5.
Hybridization in situ of amos genomic DNA to Tft1/Tft1 (A), wild-type (B), and Tft1/+ (C) salivary gland chromosomes. The focus is on the chromosomal 36F/37AB region. Distal is to the left. Red shows hybridization signals on the DAPI-stained chromosomes (light blue). See explanation in text.
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| DISCUSSION |
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Tft1 is a gain-of-function allele of amos:
The Tft1 mutation was isolated by Ritterhouse in 1952 (LINDSLEY and ZIMM 1992 ). Since then, several studies have aimed at genetically characterizing the nature of this mutation (ARNHEIM 1967 ; TOKUNAGA 1967 ; SUR et al. 1995 ). WRIGHT et al. 1976 induced deficiencies in a Tft1 chromosome and obtained reversions of the Tft1 phenotype, which suggested that the Tft1 mutation corresponded to a gain-of-function allele. GHYSEN and RICHELLE 1979 integrated the Tft1 mutation in their chaetogen model and proposed that some of the regulatory circuitry that controls bristle development would be bypassed in these mutants. They also observed that the Tft1 bristles were completely normal, including the presence of underlying neurons that made functional contacts with the CNS. Our data indicate that, indeed, Tft1 corresponds to a gain-of-function allele of the proneural gene amos, which is misexpressed in specific regions of the embryo and the larva. Thus, we detect expanded domains of amos expression in the lateral regions of stage 11–12 embryos and ectopic expression of amos in proximal/posterior regions of the third instar wing and haltere imaginal discs. Within these areas of expression, ectopic PNS neurons develop in the embryo and tufts of external sensory organs (macrochaetae in the notum and smaller bristles in the metathorax) appear in the adult. Moreover, we have obtained revertants of the Tft1 mutation and these remove both the extra neurons and sensory organs and the ectopic expression of amos. One of these revertants, TftRM11, is in fact a hypomorphic allele of amos and should be renamed amosRM11. Furthermore, overexpression of a UAS-amos transgene in the wing imaginal disc mimics the Tft1 phenotype by producing extensive tufts of macrochaetae. This indicates that, in the Tft1 discs, the sole misexpression of amos is sufficient to trigger the development of the ectopic macrochaetae. Finally, we detect the presence of a modification in the chromosomal region 36F/37AB, the cytological location of amos and Tft1. Although the precise nature of this chromosomal aberration has not been determined, the results are compatible with a modification of sequences in the vicinity of the amos structural gene, most likely a chromosomal duplication, that places amos and/or the duplicated gene under the control of novel cis-regulatory sequences.
Interestingly, Rough eye (Roi) is another gain-of-function allele of amos that shows misexpression of this gene in the developing eye (CHANUT et al. 2002 ). This misexpression disrupts the regulation of the eye proneural gene ato, which in turn leads to an irregular distribution of R8 cells. In addition, amos misexpression induces excess production of the Hedgehog signaling molecule and the irregular recruitment of other photoreceptor cells. Together, these defects lead to the rough eye phenotype.
The Tft1 bristles require ase for development:
The observation that the Tft1 phenotype was suppressed by the Df(1)260-1, which removes the whole AS-C (and additional genes), but not by the Df(1)sc19, which removes only the distal part of the AS-C, suggested that within the proximal part of the AS-C or in its neighborhood there was a genetic function necessary for the generation of the Tft1 bristles (A. GARCÍA-BELLIDO, personal communication cited in CAMPUZANO et al. 1985 ). We have verified the nondependence of the Tft1 phenotype on the ac and sc proneural genes by showing that the In(1)sc10.1, null for both of these genes (LINDSLEY and ZIMM 1992 ), does not eliminate the Tft1 bristles. In addition, we have identified the genetic function necessary for generating these bristles as the proneural gene ase. Indeed, the ase1 mutation [Df(1)sc2] removes 17–18 kb of AS-C DNA (CAMPUZANO et al. 1985 ) and ase is the only gene found within this interval (CAMPUZANO et al. 1985 ; ALONSO and CABRERA 1988 ; GONZALEZ et al. 1989 ). Df(1)ase1 almost completely suppresses the Tft1 phenotype. ase has been categorized as both a proneural and a panneural gene (BRAND et al. 1993 ; DOMINGUEZ and CAMPUZANO 1993 ). It is expressed in all SOPs of the external sensory organs, but it is dispensable for many of them, like most if not all of the notum macrochaetae. Since in flies lacking ase the olfactory sensilla develop normally (E. VILLA-CUESTA, unpublished data), it is clear that amos does not always require ase to generate sensory organs. It has been suggested that ase might be required to reinforce the proneural potential of other genes in places where sensory organs arise close to each other, as at the anterior wing margin, where ase complements ac and sc (DOMINGUEZ and CAMPUZANO 1993 ). This increased proneural function might be necessary to overcome strong lateral inhibition mediated by the Notch pathway. However, if this were the reason for the ase requirement for the Tft1 bristles, one would expect that the removal of ase would lead to a decrease in the density of bristles, rather than to their almost complete elimination. Possibly the levels of amos expression at the Tft1 wing disc are insufficient to provide enough proneural function for notum macrochaetae development, or alternatively these sensory organs have a strict requirement for a proneural gene of the AS-C type to develop. Carefully controlled misexpressions of amos in an AS-C- background may help resolve this alternative.
Relative proneural capacity of amos, ase, and sc:
We have compared the capacity of UAS-amos, UAS-ase, and UAS-sc to generate external sensory organs in the wing disc derivatives. By using the C765 driver, which promotes ubiquitous but not overly strong expression in this disc (GOMEZ-SKARMETA et al. 1996 ), we managed to recover adult flies. On the notum and wings, the three transgenes induced development of essentially the same types of sensory organs, namely, mostly macrochaetae on the notum, similarly to the Tft1 phenotype, and chaetae and sensilla campaniformia on the wing (Fig 4). However, UAS-amos was much more effective than UAS-ase, and UAS-ase was more effective than UAS-sc. In contrast, others have reported that UAS-sc was more effective than UAS-amos in inducing bristle development on the wing blade (HUANG et al. 2000 ). In that study, ectopic expressions were limited to a time interval after puparium formation and the absolute number of ectopic sensory organs recovered with UAS-amos was much smaller than that recovered under our experimental conditions. Since the number of sensory organs generated depends on the levels of proneural gene expression and the developmental stage (RODRIGUEZ et al. 1990 ), this discrepancy is not surprising. Evidently, we cannot rule out that part of the high efficiency of our UAS-amos transgene might be due to it being very strongly expressed. A conclusive demonstration of the proneural potential of the different proneural proteins will require a precise determination of their levels of accumulation.
Still, in the wild type, amos is able to generate large groups of contiguous sensory organs, namely, the antennal olfactory sensilla. The Tft1 mutant, as well as our experimental conditions of UAS-amos expression, reproduces this ability and generates large numbers of contiguous sensory organs, although these are either macrochaetae on the notum or sensilla campaniformia at the proximal part of the wing (Fig 4). Hence, this ability does not seem restricted to a specific tissue (the antenna), but seems more dependent on the particular proneural gene being expressed and on the levels of its expression. Note that strong and generalized overexpressions of ac or ac plus sc in some Hairy-wing mutants (Hw1 and Hw49c) do not give rise to contiguous bristles (CAMPUZANO et al. 1986 ; BALCELLS et al. 1988 ; LINDSLEY and ZIMM 1992 ), as amos does in Tft1 mutants. Conceivably, amos might be able to generate closely spaced bristles by being relatively inefficient at inducing Notch-mediated lateral inhibition. However, mutations in members of the N signaling pathway that decrease or increase signaling tend, respectively, to potentiate or suppress the Tft1 phenotype, indicating that lateral inhibition is still functional among the cells expressing amos ectopically. Moreover, the expression of E(spl)-m8, an effector of the N pathway, is enhanced in the region where amos is ectopically expressed, indicating the activity of the pathway. Hence, we suggest that amos can give rise to closely spaced sensory organs due to a strong proneural potential. Consistently, we find that the Tft1 phenotype is very sensitive to an excess of function of emc, a direct antagonizer of the proneural function (ELLIS et al. 1990 ; GARRELL and MODOLELL 1990 ). Furthermore, amos does not require ase to generate the large number of packed olfactory sensilla on the antenna. In contrast, ac and sc, with presumably weaker proneural activities, do require ase to give rise to the densely packed bristles of the anterior wing margin (DOMINGUEZ and CAMPUZANO 1993 ).
Neuronal specificity of amos:
It has also been reported that UAS-amos is able to generate, on the notum and wing, sensilla morphologically similar to the olfactory ones of the antenna (GOULDING et al. 2000 ). Under our experimental conditions, most sensilla generated by UAS-amos were similar to those typical of each region, that is, macrochaetae on the notum, thin and stout bristles near the anterior wing margin, sensilla campaniformia similar to those of vein L3 on the wing blade, etc. On the wing blade were also many small bristles, some of which might have a resemblance to olfactory sensilla. The dorsal cluster of Tft1 embryos showed a slight increase in the number of Cut-positive cells, some of which could correspond to extra external sensory organs and their associated dmd neurons, and a larger increase in the total number of neurons, which suggests that the ectopic amos induced development of more than one kind of neuron. Similarly, HUANG et al. 2000 showed that UAS-amos promoted differentiation of md neurons in the ventral cluster, chordotonal neurons in the lateral region, and an unspecified type of neuron in the dorsal cluster. However, we cannot conclude that amos, by itself, can induce many kinds of sensory organs/neurons. Upon emergence, amos-induced SOPs may activate other proneural genes like ac, sc, and ase. This is indeed the case for the Tft notum macrochaetae, and we have found that ase is essential for their development. Thus, examination of the sensory organs that arise upon expression of each individual proneural gene in the absence of all the others will be necessary to unveil their contribution to the identity of sensory organs (see BRAY 2000 for further discussion on this topic).
| FOOTNOTES |
|---|
1 Present address: Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom. 
2 Present address: Instituto de Neurociencias, Universidad Miguel Hernández, 03550 San Juan, Alicante, Spain.
| ACKNOWLEDGMENTS |
|---|
We are grateful to S. Campuzano, J. L. Gómez-Skarmeta, and colleagues of our laboratory for suggestions and constructive criticism of the manuscript; to A. García- Bellido, J. Moscoso del Prado, and T. R. Wright for advice; to M. Ashburner for cytological examination of Tft revertants; and to A. Jarman for providing reagents and stocks. Predoctoral fellowships from Ministerio de Ciencia y Tecnología to E.V.-C. and from Comunidad Autónoma de Madrid to J.d.N. and R.D.d.C. are acknowledged. Grants from Dirección General de Investigación Científica y Técnica (PB90-0085 and PB98-0682) and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa are acknowledged.
Manuscript received August 21, 2002; Accepted for publication November 11, 2002.
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ABSTRACT
MATERIALS AND METHODS
