endogenous genes, homogeneous but limited expression of sc promotes .... the anterior part (awb) but progressively decays in the posterior part (pwb, panel C).
The EMBO Journal vol. 1 1 no.9 pp.3385 - 3393, 1992
The extramacrochaetae gene provides information for sensory organ patterning
Pilar Cubas and Juan Modolell Centro de Biologfa Molecular, Consejo Superior de Investigaciones Cientificas and Universidad Aut6noma de Madrid, Canto Blanco, 28049 Madrid, Spain
Communicated by J.Modolell
The Drosophila adult epidermis displays a stereotyped pattern of bristles and other types of sensory organs (SOs). Its generation requires the proneural achaete (ac) and scute (sc) genes. In the imaginal wing disc, the anlage for most of the thoracic and wing epidermis, their products accumulate in groups of cells, the proneural clusters, whose distribution prefigures the adult pattern of SOs. These proteins then induce the emergence of SO mother cells (SMCs). Here, we show that the extramacrochaetae (emc) gene, an antagonist of the proneural function, is another agent that contributes to SO positioning. In the wing disc, emc is expressed in a complex and evolving pattern. SMCs appear not only within proneural clusters but also within minima of emc expression. When one of these spatial restrictions is eliminated, by ubiquitously expressing ac-sc, SMCs still emerge within minima of emc. When in addition, the other spatial restriction is reduced by decreasing emc expression, many ectopic SMCs emerge in a relatively even spaced and less constant pattern. Thus, the heterogeneous distribution of the emc product is one of the elements that define the positions where SMCs arise. emc probably refines SMC (and SO) positioning by reducing both the size of proneural clusters and the number of cells within clusters that can become SMCs. Key words: achaete/extramacrochaetae/pattern formation/ scute/sensory organ determination
Introduction In Drosophila, the set of bristles and other types of cuticular sensory organs (SOs) constitutes a classical model to study pattern formation. On the notum of the fly, two types of bristles appear: large bristles (macrochaetae), which appear in fixed number at constant positions, and small bristles (microchaetae), which are evenly distributed but whose number and position vary slightly. Each SO is generated by a sensory mother cell (SMC) that undergoes two differential divisions (Bodmer et al., 1989; Hartenstein and Posakony, 1989). The four progeny cells differentiate into the components of the SO. In the case of the notum macrochaetae, their SMCs appear during the third instar larva and early pupa stages in precisely defined positions of the wing imaginal disc (Cubas et al., 1991; Huang et al., 1991; Skeath and Carroll, 1991). Their distribution prefigures the adult macrochaetae pattern. Thus, the
generation of SMCs at specific sites largely explains macrochaetae positioning. How, within the imaginal discs, are the positions of the SMCs specified? The achaete (ac) and scute (sc) proneural genes, whose protein products confer on cells the ability to become SMCs, are instrumental to this process (reviewed in Ghysen and Dambly-Chaudiere, 1988, 1989; Campuzano and Modolell, 1992). In wild type imaginal discs, ac and sc are co-expressed in groups of cells (the proneural clusters), which define the areas where SMCs arise (Romani et al., 1989; Cubas et al., 1991; Skeath and Carroll, 1991). ac/sc expression in these clusters is thought to be controlled by a complex set of cis-regulatory sequences, which respond to local combinations of transcriptional regulators (prepattern) present in imaginal discs (Ruiz-Gomez and Modolell, 1987; Leyns et al., 1989) and by self- and crossstimulatory interactions between these genes (Martinez and Modolell, 1991; Skeath and Carroll, 1991). A fixed number of cells from each cluster (one or a few) become SMCs and prevent, by cellular interactions (reviewed in Simpson, 1990), similar determination of neighbouring cells. In mutants with expanded proneural clusters, extra SMCs appear in new positions (Cubas et al., 1991; Skeath and Carroll, 1991; our results). Thus, although the primary function of ac and sc is to promote neural determination, their spatially restricted expression helps define SMC positioning. However, there appear to be additional topographical cues for positioning SMCs. In the absence of the ac-sc endogenous genes, homogeneous but limited expression of sc promotes development of a small number of notum macrochaetae (Rodriguez et al., 1990). Although this generalized expression should not restrict SMC determination to specific sites, these SOs still appear in wild type positions. This indicates that cells at the sites where SMCs emerge have an increased ability to respond to the proneural effects of ac-sc and become SMCs. Such capacity to respond is unevenly distributed in the disc and may help SMC positioning in the wild type. This conclusion is supported by the facts that in wild type discs, SMCs arise in extremely reproducible positions within proneural clusters and that some clusters of ac - sc expressing cells do not give rise to SMCs (Cubas et al., 1991). A candidate for modulating the responsiveness of cells to proneural function is the extramacrochaetae (emc) gene (Botas et al., 1982), a negative trans-regulator of ac and sc (Moscoso del Prado and Garcia-Bellido, 1984a). The EMC, AC and SC proteins have a helix-loop-helix (HLH) dimerizing domain, but only AC and SC contain an adjacent basic region necessary for DNA binding and transcriptional activation (Villares and Cabrera, 1987; Ellis et al., 1990; Garrell and Modolell, 1990; for a review see Garrell and Campuzano, 1991). AC and SC would activate genes implementing the neural developmental pathway. EMC would interfere with this AC-SC proneural function by 3385
P.Cubas and J.Modolell
sequestering these proteins in complexes uncapable of effective interaction with DNA (Van Doren et al., 1991). In addition, emc insufficiency promotes increased accumulation of SC, which suggests that EMC negatively affects ac-sc transcription by interfering with ac and sc self- and cross-stimulation and/or with other possible HLH activators (Cubas et al., 1991; Martinez and Modolell, 1991; Skeath and Carroll, 1991). However, these effects of emc appeared to have limited topographical relevance in view of the reported ubiquitous presence of the emc RNA (Ellis et al., 1990; Garrell and Modolell, 1990) and to the fact that quantitative heterogeneities in the distribution of this RNA had not been detected. Therefore in this context, we have re-examined the emc mRNA distribution in the imaginal wing discs, the anlagen for most of the thorax epidermis and the wings. We show that the distribution of this RNA is actually heterogeneous. SMCs arise in places where emc expression is minimal. Moreover, under conditions of generalized ac-sc expression, SMCs are still determined within minima of emc RNA. emc distribution is independent from the expression of ac-sc. This and other data suggest that the heterogeneous distribution of EMC protein is one of the factors that defines the capacity of cells to become SMCs and therefore provides information that acts in SMC positioning simultaneously with that provided by the proneural genes.
Results emc RNA is heterogeneously expressed in imaginal
wing discs The distribution of the emc RNA was determined in early, mid and late third instar larvae wing discs by in situ hybridization with a digoxigenin labelled emc probe (Figures lA-C). In contrast to an earlier study performed with a radioactively labelled probe hybridized to disc sections (Garrell and Modolell, 1990), a heterogeneous and evolving distribution of emc RNA was detected. Thus, in the presumptive notum, the differences progressively increase and the central area with minimal expression becomes broader as the disc grows. In the wing pouch, mRNA accumulated in a band approximately two cell diameters wide that coresponded to the presumptive wing margin (Figure lA). At the presumptive anterior wing margin, the expression was progressively reinforced and became flanked by two bands of minimal accumulation, while at the posterior margin, the signal decreased with increasing disc age (Figures lB and C). A stripe of cells, which became broader with age and ran along the posterior side of the presumptive third vein (see below), accumulated increasing amounts of emc RNA and partially split into two stripes in the oldest discs (Figures IC and F, arrowed). An additional adjacent band, bordering anteriorly the presumptive third vein, appeared shortly before puparium formation (Figures IC and F). SMCs preferentially emerge in regions with low levels of emc mRNA To analyse whether there is a relationship between emc expression and the emergence of SMCs, we carried out in situ hybridizations with discs from transformant strain AJOJ-IF3, in which SMCs were visualized shortly after singling out by their accumulation of ,B-galactosidase (Cubas et al., 1991; Huang et al., 1991; Skeath and Carroll, 1991).
3386
In the presumptive wing margin, the SMCs of the recurved bristles appeared in the two valleys of emc expression that ran along both sides of the narrow ridge of strong emc expression [Figures 2A (arrows) and B]. The third vein sensilla SMCs appeared adjacent to the anterior border of the broad stripe flanking this presumptive vein (Figure 2A). In the presumptive notum, the macrochaetae SMCs that became visible during third instar (the ones appearing later were not analysed), arose in regions of low emc expression adjacent to areas of high expression (Figures 2C, E, F and H). Still, the levels of emc RNA varied among the different sites of SMC emergence. For instance, it was substantially higher at the APA and PNP sites than at the PDC and PSA sites (Figures 2C, E and F).
Spatial relationship between emc and ac - sc expression pattern The pattern of ac-sc expression defines the regions of the imaginal disc with proneural potential, that is the regions where SMCs can arise. emc probably antagonizes this potential by both limiting ac-sc expression and reducing the concentration of AC -SC proteins available for proneural function (Ellis et al., 1990; Garrell and Modolell, 1990; Cubas et al., 1991; Van Doren et al., 1991). To gain an insight into how the interactions between these genes relate to SMC positioning, we have compared the emc and ac-sc patterns of expression with the positions where SMCs emerge. Although emc probably interferes with ac-sc transcription, the pattern of AC -SC accumulation was not complementary to that of emc RNA distribution (compare Figures lA-C with Figure 3A). Thus, the position of the distinct ac-sc expressing cell clusters seems not to be imposed by the distribution of emc product. However, EMC probably helps control the level of ac - sc transcription and the size of the clusters, since its insufficiency results in the expansion and reinforcement of at least the presumptive notum clusters and its overexpression in the opposite effects (Cubas et al., 1991; Figure 3D). Strong expression of both emc and ac - sc does occur in some areas of the disc. This condition apparently does not permit SMC determination, probably because of the titration of AC -SC proteins by the high amounts of EMC. Examples are the presumptive postnotum and, at least during the third instar, the prescutum (Figures lA-C; Figure 1 of Cubas et al., 1991). The SMCs of the notum microchaetae nevertheless arise in the presumptive prescutum region several hours after puparium formation, which suggests that around the time of pupariation, ac-sc expression in this region is enhanced and/or that of emc is decreased. A similar event occurs within the DC cluster. After the PDC precursor has emerged, strong emc and ac-sc expression co-exist in the anterior part of the cluster (Figures 2F and G, arrows), an area in which the ADC precursor later appears (Figure 2H). When this SMC arises, the emc expression has been extinguished in that area (compare Figures 2F and H). It should also be pointed out that at the time the APA and trl precursors emerge, their ac-sc cluster overlaps with regions of strong and weak emc expression (Figures 2C and D). Although at different times, both SMCs arise among the cells near the anterior (left) side of the ac - sc expressing cluster (Figure 2D; Cubas et al., 1991), where emc expression is lower and consequently the antagonistic action of this gene is reduced. This may help explain the eccentric position of these SMCs within the ac-sc cluster. The patterns of ac-sc
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Fig. 1. Distribution of emc RNA in wild type (Oregon R) imaginal wing discs obtained from larvae of -35-0 h before puparium formation (A-C) and in mutant In(J)sct0l (D), emcPSC (E) and Ach (F) discs. The distribution of RNA becomes more complex with increasing disc age (A-C). Some of the changes are discussed in the text. The generalized accumulation in the presumptive wing blade is maintained at a relatively high level in the anterior part (awb) but progressively decays in the posterior part (pwb, panel C). Strong expression develops in part of the presumptive dorsal wing radius area (dwr) and between the dorsocentral (dc) and scutelar areas (sc), probably comprising the scutelar suture. The presumptive third vein region (v3) has been identified by the SMCs of the sensilla campaniformia characteristic of this vein (Figures 2A and B). At the level of resolution provided by these assays, no significant differences have been observed between the emc RNA patterns of wild type discs and those from the mutants examined, except the overall levels of RNA which were lower (emCP5C) or higher (Ach) than in the wild type discs, as shown by the staining times required to reveal the patterns. Nomenclature for other prospective regions is as follows: awm, anterior wing margin; pwm, posterior wing margin; n, notum; pnt, postnotum; psu, prescutum. All discs are shown with the anterior part towards the left and under the same magnification. For a detailed fate map of the wing disc see Bryant (1975) and Campuzano and Modolell (1992).
and emc expression were complementary at the anterior wing margin (compare Figures 2A and B with Figure 3A), so that the rows of maximal ac - sc expression overlap with minima of emc. This complementarity probably helps define the precise rows of the SMCs of the recurved bristles. emc RNA distribution is not affected by the presence of AC-SC proteins and SMCs Genetic data have suggested that AC -SC proteins act as repressors of emc function (Garcia-Alonso and GarciaBellido, 1986). To investigate whether this antagonistic effect of AC -SC occurs at the level of emc transcription, we examined the emc RNA distribution in discs of the strain
In (J)scl° l, a double mutant for ac and sc in which no functional AC and SC proteins are synthesized (Campuzano et al., 1985; Villares and Cabrera, 1987). The emc RNA distribution was essentially unchanged (Figure 1D). Hence, this pattern seems, at least qualitatively, independent of the presence of AC -SC and the suggested genetic interactionI probably takes place post-transcriptionally. Since In(J)sc'0 discs lack SMCs [excepting those of the stout bristles of the wing margin, which are independent of ac-sc and emerge several hours after puparium formation (Garcia-Bellido, 1979; Hartenstein and Posakony, 1989)], this experiment further shows that the emc pattern is essentially unaffected by the presence of these precursors.
3387
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Fig. 2. SMCs emerge at sites of low emc RNA accumulation; comparison between the distributions of emc RNA and sc products in the neighbourhood of SMCs. Discs of strain A101-IF3 obtained from third instar larvae of different ages were first stained with X-gal, to visualize emerged SMCs (green) and later hybridized with either an emc (A-C, E, F and H) or a sc (G) DIG-labelled probe to reveal the pattern of emc or sc RNA accumulation (purple). Alternatively, discs were stained with the help of an anti-SC antibody (D). The SMCs shown appear from - 30 h (APA) to a few hours (ADC) before puparium formation. Except in G, the examples of SMCs have been chosen from the youngest discs that displayed each particular SMC, so that the emc and sc product distributions are as similar as possible to those at the time of SMC emergence. Panel A shows the presumptive anterior wing blade region just after the SMC of the distalmost recurved bristle of the ventral row has emerged (arrowhead). (This SMC is always the first to appear of the two rows of precursors.) Arrows point to the two troughs of low emc expression where the remaining recurved bristle SMCs will emerge, as shown in the older disc depicted in B. Note the position of the L3-2 SMC, corresponding to the middle sensillum campaniformium of the third vein, adjacent to the strong bar of emc expression. It marks the position of the presumptive third vein, anterior to the bar of strong expression. The emerging SMCs for several notum macrochaetae are shown in the following panels: the APA in C, PNP and PSA in E, PDC in F and ADC in H. (PDC in H has already divided.) Arrows in F and G point to the approximate positions where the ADC SMC will later appear. In all cases, SMCs appear within local minima of emc RNA accumulation, usually near a border between high and low accumulation areas. Panels C-H are reproduced at very similar magnifications and in the same orientation as the whole discs of Figure 1. Thus, considering the reproducibility among discs of similar age of the emc RNA and ac-sc RNA or protein distributions and that of the position of SMCs within ac-sc expressing clusters, it is clear that the APA (D) and DC (G) clusters overlap with regions of strong and weak emc RNA accumulation (compare with C and F, respectively). In D, the arrow points to the approximate position where the trl SMC later appears (Cubas et al., 1991). The APA precursor is visible due to its increased accumulation of SC protein.
Positioning of SMCs in the presence of ubiquitous ac - sc expression
The above results show that SMCs are determined within proneural clusters and in regions of low emc expression. Thus, the distributions of AC -SC and EMC proteins seem to impose spatial restrictions as to the sites where SMCs can arise. As a test for this hypothesis, we have examined the SMCs positions in discs without one of these restrictions, namely the regionalized expression of ac-sc. We have used
3388
Hw4 ;A O1.IF3 discs, in which ac-sc are ubiquitously expressed (Figure 3B; Balcells et al., 1988). Many extra SMCs arose in characteristic areas (Figures 3F and G). On the presumptive posterior wing margin, at the same time as on the anterior margin, two rows of SMCs emerged. They flanked the narrow stripe of high emc expression. The gap between the anterior and posterior double rows where SMCs were not determined, corresponds to the thick band of strong expression running alongside prevein 3. SMCs also appeared
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Fig. 3. Emergence of SMCs in discs with generalized expression of proneural products. Upper row shows the distribution of SC protein in wild type (A), male Hw49C (B) and male In(J)sc10 l;SC9RR64 (C) discs, as revealed by anti-SC antibody staining. The different proneural clusters of sc expression are identified in Cubas et al. (1991). Mutant discs in B and C have generalized distributions of SC protein, at a higher level in B than in C (see also Balcells et al., 1988; Rodrfguez et al., 1990). Note that the Hw49C disc has only a few sites of relatively high accumulation of SC, especially in the dorsocentral-scutellar region (arrowheads) and the In(1)sc'0A;SC9RR64 disc just two of them, namely, in the presumptive vein 3 (v3) and the site of the SMCs of the twin sensilla of the wing margin (ts). Individual cell nuclei showing high levels of SC accumulation in A-C correspond to SMCs (Cubas et al., 1991; Skeath and Carroll, 1991). (D) shows the presumptive notum region of an emcpel/emcE12 disc stained with the anti-SC antibody. Note the strong reinforcmeent of SC accumulation in the prescutum-dorsocentral presumptive areas (psu, dc) due to the emc insufficiency. Such over accumulation was never observed in wild type discs (compare with similar regions of the disc in A). (E, F and G) show the emerged SMCs (green) and emc RNA distribution (purple), revealed as explained in the legend to Figure 2, in In(I)sc0o1; SC9RR64;A101.1F31+ (E) and Hw49C;AJOJ.1F31+ (F and G). Under these conditions of generalized sc expression, SMCs still appear in regions of low emc expression, many times near the borders of areas of high expression. In E, the SMCs outside the areas of high sc expression correspond to several dorsal radius sensilla (arrowed) and the PSA macrochaetae. In the discs of this genotype, the most frequently observed SMCs are those for the ADC, PDC, PSA and APA macrochaetae and several sensilla campaniformia of the dorsal wing radius. In F, note the ectopic SMCs of the posterior wing margin (pwm) and those flanking the posterior side of the thick vertical band of emc expression. They probably correspond to extra SOs on vein 4 and accordingly, they are tentatively labelled v4. Other SMCs, which appear close to a fainter band of emc expression, may correspond to SOs appearing on or near vein 5 (empty arrow). In G, an ectopic SMC appear in the dorsocentral region near a band of strong emc expression (arrowhead) that separates the presumptive scutum and scutellum. Other SMC appearing on the presumptive notum correspond to wild type SOs and some of them bear their standard names (Lindsley and Grell, 1968; Garcia-Bellido, 1979). H, a disc from a Hw49C/+ ;emcPe' ;AIOl. 1F31Df(3L)emcE 12 female larva stained with an anti-,3-galactosidase antibody. The low emc expression, together with the generalized expression of ac-sc, results in a great number of ectopic SMCs. Many of them are out of focus and appear as diffuse, brown spots. Camera lucida drawings of discs from different larvae indicate that the pattern of SMCs, especially on the notum (n), varies and that positions are not precisely defined.
at other sites along the posterior side of this band, but not on it. During third instar, a few extra SMCs emerged in the dorsocentral notum region. They appeared within an enlarged cluster of ac-sc expression (Figure 3B), but avoid
an overlapping area of high emc RNA accumulation (Figure 3G). The fact that the distribution of emc RNA in Hw49C
discs is unmodified reinforces the conclusion that this pattern is independent of ac - sc expression. 3389
P.Cubas and J.Modolell
Transformant flies In(J)sc'0 l; SC9RR64 carry an sc gene lacking most of its controlling sequences (Rodriguez et al., 1990). This gene was weakly and homogeneously expressed, except for two clusters of strongly expressing cells in the presumptive wing pouch (Figure 3C). The few SMCs that emerged outside of these clusters, were located within minima of emc expression and in positions apparently similar to those of wild type SMC (Figure 3E; results not shown). As a further test for the above hypothesis, we examined the pattern of SMCs in HW49c1 + ;emcPellemcEF2;A101. lF31 + discs in which the spatial restriction imposed by emc had been reduced by using one of the strongest, albeit hypomorphic, emc viable mutant combinations (GarciaAlonso and Garcia-Bellido, 1988). Although these discs carried a wild type copy of the AS-C, its AC -SC protein distribution should be generalized and specially reinforced in the prescutum-dorsocentral presumptive areas due to the emc insufficiency (Figure 3D; Cubas et al., 1991). Many additional ectopic SMCs emerged, mostly in the presumptive notum and dorsal wing blade and base (Figure 3H). In the notum, the positions of individual SMCs were not precisely defined, since they varied when comparing different discs and it was difficult to identify the SMCs for the wild type macrochaetae. In general, SMCs appeared to be relatively well separated from one another, as if inhibitory interactions between SMCs were involved in generating this pattern. Thus, attenuation of the topographical constraints imposed by the EMC and AC -SC distributions leads to a less precise arrangement of SMCs. This may explain the variable location of many of the adult notum macrochaetae in this and other combinations of AS-C and emc alleles (Moscoso del Prado and Garcia-Bellido, 1984b; our unpublished results). It should be emphasized that even under these conditions of reduced topographical constraints, SMCs failed to appear in many regions of the wing discs. This suggests in agreement with other data (Rodriguez et al., 1990) that additional factors restrict the capacity of the imaginal tissue to develop SMCs. emc expression in emc mutants emc expression was examined in discs obtained from loss (emcPel, emcP5C) and excess (Achaetous, Ach) of function emc mutant larvae. At the level of resolution provided by the DIG stainings, no significant modifications of the wild type pattern were detected, except in the overall levels of transcript (Figures IE and F). These were significantly
reduced in emcPel and emc 5C, both associated with insertions of the P element in the promoter region of the emc gene (Garrell and Modolell, 1990), since staining times required for visualizing the pattern were longer than in wild type discs. Contrarywise, the levels were increased in discs from Ach, a mutant that contains an insertion of the Tirant element within the emc coding region and gives rise to a truncated but active protein (Garrell and Modolell, 1990). These results support the genetic inference that the different emc alleles represent scalar degrees of activity of a single genetic function (Garcia-Alonso and Garcia-Bellido, 1988). Note that in emc lack of function mutants, extramacrochaetae appear on the prescutum and potnotum, regions that are normally devoid of these SOs (Garcia-Alonso and GarciaBellido, 1988). As shown above, strong ac-sc and emc expression overlap in the corresponding presumptive regions of the disc. These observations suggest that the decrease in
3390
the levels of EMC product releases sufficient AC -SC for proneural action.
Discussion In this work we have shown that emc RNA is heterogeneously distributed in wing imaginal discs according to a pattern that evolves as discs mature. As discussed below, this pattern appears to be one of the factors that control the position of SOs by helping define where SMCs arise within the discs. emc helps define SMC positions In imaginal discs, SMCs are singled out within clusters of, cells that express ac-sc (proneural clusters, reviewed in
Campuzano and Modolell, 1992). The AC-SC proteins confer on cells the ability to become neural precursors. Since these proteins belong to the family of HLH transcriptional activators, it is thought that they bind as heterodimers with Daughterless (DA) (an ubiquitously distributed proneural protein) to DNA and activate genes that effect neural differentiation (reviewed in Garrell and Campuzano, 1991). Thus, the pattern of ac-sc expression is one of the determinants for SMC positioning. emc, genetically described as an AS-C repressor (Botas et al., 1982; Moscoso del Prado and Garcia-Bellido, 1984a), encodes an HLH protein that in vitro behaves as a competitive inhibitor of the formation of DNA-binding DA-AC and DA-SC complexes (Van Doren et al., 1991). Assuming a similar action in vivo, EMC should reduce the proneural activity of DA, AC and SC. Moreover, EMC also interferes with ac and sc transcription (Cubas et al., 1991). According to this scenario, the heterogeneous distribution of emc RNA in the wing disc should define a pattern of EMC protein that negatively affects both the transcription of ac-sc and the proneural activity of its products. This double negative modulation would depend on the local levels of EMC and would therefore be exerted to different extents in different parts of the disc. Moreover, given the temporal evolution of the emc pattern, the negative modulation would also vary with time in many regions of the disc. Consequently, the heterogeneous emc expression should help define the position and time of the development of SMCs. The AC -SC proteins could only have a proneural effect when present in amounts sufficient to overcome the threshold imposed by the local EMC concentration. We thus propose that in addition to the clustered distribution of AC-SC, a second agent responsible for SMC positioning is the distribution of peaks and valleys of EMC. According to this model, a weak but homogeneous sc expression, as that in In(1)sc'OA;SC9RR64/SC9RR64 discs, should promote SMCs determination only in regions where AC - SC is not entirely titrated by EMC. This should occur in the valleys of emc transcription and this is precisely where SMCs emerge (Figure 3E). In Hw49C discs, ac-sc expression is ubiquitous and more intense and a higher number of SMCs are determined; they still mainly emerge within minima of emc expression (Figures 3F and G). The fact that a short ubiquitous pulse of sc expression provided by an hsp70-sc chimaeric gene (HSSC) promotes, in the absence of the endogenous ac-sc genes, development of SOs in mostly wild type positions (Rodriguez et al., 1990) may also be explained in part by the emc-imposed topological
emc and sensory organ positioning
restrictions for SMC singling out. Interestingly, the notum macrochaetae most resilient to the excess of emc function in Ach mutants are the same ones that most readily appear in the In(J)sc''1;SC9RR64/SC9RR64 flies (de Celis et al., 1991; Rodriguez et al., 1990; Figure 3 legend). The wild type positions of some SMCs seem also readily explainable by specific features of the distributions of EMC and AC-SC. Thus, in the prospective anterior wing margin, the combination of two parallel ridges of ac - sc expression with two overlapping troughs of emc expression should precisely define the two rows of SMCs that will later originate the wing margin's dorsal and ventral rows of recurved bristles. Similarly, another trough of emc expression may help position SMCs on the presumptive dorsal wing vein 3. In the presumptive notum, the overlap of ac - sc proneural clusters with regions of high and low emc expression may account for the eccentric position within the proneural cluster of some SMCs like those for the APA macrochaeta or trl sensillum trichoideum (Cubas et al., 1991). Also, the overlap of some proneural clusters with regions of high emc expression prevents appearance of SMCs (postnotum, Figure 4A) or at least delays their appearance
A
(DC cluster or prescutum region). It is of interest that controlling the time when SMCs are determined is most important for specifying the type of SO that they will give rise to (Rodriguez et al., 1990). Early determination in the prescutum, promoted by reduced EMC concentrations (emc mutations) or increased sc expression (HSSC), yields macrochaetae while late determination (wild type conditions or HSSC) exclusively gives rise to microchaetae. SMCs tend to single out near borders of maxima versus minima of emc. The steep gradient of EMC concentration at these borders should enlarge the differences in the amounts of AC-SC uncomplexed with EMC between neighbouring cells. These differences may facilitate SMC singling out. Indeed, in the notum, cell mosaics with different doses of ac-sc genes have similar densities of microchaetae, but at the borders, microchaetae mostly appear on territory with the highest number of doses (J.F.de Celis, personal communication). Hence, SMCs are preferentially singled out on this side of the border. This cellular behaviour suggests that the inhibitory effect of a cell on its neighbours (lateral inhibition) is positively related to its AC -SC contents. Therefore cells in contact with others that have lower amounts of AC-SC
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