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Development 121, 3111-3120 (1995) Printed in Great Britain © The Company of Biologists Limited 1995 3111 Genetic determinants of sense organ identity in Drosophila: regulatory interactions between cut and poxn Michel Vervoort1, Daniele Zink3, Nathalie Pujol2, Kathleen Victoir1, Nathalie Dumont1, Alain Ghysen2,4 and Christine Dambly-Chaudière1,4 1Laboratoire de Génétique du Développement and 2Laboratoire de Neurobiologie, Université Libre de Bruxelles, 67 rue des Chevaux, 1640 Rhode-St-Genèse, Belgium 3Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neuenheimer Feld 282, 6900 Heidelberg, Germany 4Laboratoire de Neurogénétique, Unité 432 INSERM, Université de Montpellier II, place E. Bataillon, 34095 Montpellier Cedex, France SUMMARY Two genes involved in defining the type of sense organ have been identified in Drosophila. The gene cut differentiates the external sense organs (where it is expressed) from the chordotonal organs (where it is not); among the external sense organs poxn differentiates the poly-innervated organs (where it is expressed) from the mono-innervated organs (where it is not). Here we show that the expression of poxn in normal embryos does not depend on cut, and that poxn is capable of inducing the expression of cut. We have identified a small domain of the very large cut regulatory region as a likely target for activation by poxn. INTRODUCTION Another proneural gene, atonal (ato), has been shown by a similar LOF/GOF analysis to be responsible for the formation of the ch organs (Jarman et al., 1993). The AS-C and ato genes encode transcriptional regulators (Villares and Cabrera, 1987; Cabrera and Alonso, 1991; Jarman et al., 1993) and are assumed to act by regulating the expression of a battery of target genes. Among the potential targets are genes expressed both in ch and es precursors, which are thought to confer properties shared by all mother cells (Bier et al., 1989). Other potential target genes are expressed only in subsets of mother cells. One such gene is cut (Jack, 1985), which is expressed in the AS-C-dependent precursors and in their progeny, but not in ato-dependent mother cells (Blochlinger et al., 1990). LOF and GOF analysis of cut have demonstrated that the absence of the gene leads to the transformation of es into ch organs (Bodmer et al., 1987) while its ubiquitous expression results in the opposite transformation (Blochlinger et al., 1991). This implies that cut directs the differentiation of es organs. If both the AS-C genes and cut are involved in the determination of es organs, what is the relationship between these genes? The expression of achaete and scute is transient and is turned off before the mother cell divides, well before its progeny will differentiate (Ruiz-Gomez and Ghysen, 1993). It is believed that cut is activated by the AS-C genes, and is maintained by self-activation through the lineage until the daughter cells differentiate (Blochlinger et al., 1991). In other words, the AS-C genes would determine the es specificity by activating cut which itself directs the acquisition of es properties. The gene poxn (Bopp et al., 1989; Dambly-Chaudière et al., 1992) is also expressed in a subset of mother cells, those that A major goal in the analysis of development is to understand how specific cell fates are established. Progress along that line has been made recently in the case of the peripheral nervous system of Drosophila, which comprises different types of sense organs and is particularly well suited for a genetic analysis. In this paper, we will concentrate on three types of sense organs: the mono-innervated external sense organs (mes), the poly-innervated external sense organs (p-es) and the chordotonal internal sense organs (ch). The three types of organs differ in their lineage, morphology and neuronal connectivity, thereby providing a useful system to examine the genetic determinants of these differences. Each sense organ comes from a single ectodermal precursor, the sensory mother cell (Bate, 1978; Hartenstein and Posakony, 1989; Bodmer et al., 1989). The competence of ectodermal cells to become mother cells depends on the expression of ‘proneural’ genes (Ghysen and Dambly-Chaudière, 1989). A first set of proneural genes are those of the achaete-scute complex (AS-C; Garcia-Bellido, 1979). Mutations that result in a loss of function (LOF) of these genes lead to the disappearance of all m-es and p-es organs (Garcia-Bellido and Santamaria, 1978; Dambly-Chaudière and Ghysen, 1987), while a gain of function (GOF) of the AS-C genes promotes the formation of supernumerary external sensory organs (GarciaAlonso and Garcia-Bellido, 1986; Rodriguez et al., 1990). These opposite LOF and GOF phenotypes are typical of ‘selector genes’ which direct developmental choices (GarciaBellido, 1975; Lewis, 1978), and suggest that the AS-C genes are responsible for the formation of external sense organs. Key words: poxn, cut, neuronal identity, peripheral nervous system, Drosophila, neurogenesis, determination genes 3112 M. Vervoort and others give rise to the p-es organs. The LOF/GOF analysis of poxn revealed that embryos deleted for this gene have no p-es organs, while the ubiquitous expression of poxn early during embryogenesis leads to the formation of supernumerary p-es organs (Dambly-Chaudière et al., 1992). The ubiquitous expression of poxn during metamorphosis induces the transformation of adult m-es into p-es organs. This transformation affects not only the external morphology of the organs but also the number of neurones, their projection in the central nervous system and their functional properties (Nottebohm et al., 1992, 1994). Thus poxn is responsible for the establishment of the morphological and functional properties typical of p-es organs. In summary, if a SMC expresses neither cut nor poxn, it will form a ch organ; if it expresses cut but not poxn, it will produce a m-es organ, and if both cut and poxn are expressed, a p-es organ will form. An intriguing result of the GOF analysis of poxn in embryos was that some of the poxn-induced supernumerary organs appear at positions where no m-es organs are found in wildtype embryos, suggesting that these supernumeraries do not originate from m-es precursors. Here we investigate the origin of the ectopic es organs, and demonstrate that the expression of poxn can transform ch into es precursors by inducing the expression of cut. MATERIALS AND METHODS Drosophila strains and crosses KL11.1 and KL11.4 are insertions of the hsp-poxn construct on the third and second chromosome, respectively (Dambly-Chaudière et al., 1992). Df(1)260.1 is a complete deletion of the AS-C (Garcia-Bellido, 1979). ct145 is a null mutation of the cut locus (Jack, 1985; Bodmer et al., 1987). E7 2nd 36 is an enhancer trap insertion in which lacZ is expressed in most of the md neurones (Bier et al., 1989). A3-lacZ is an insertion on the third chromosome of a P element containing 2.7 kb of cut regulatory sequence fused to lacZ (Jack and Delotto, 1995). In order to see the effect of ubiquitous poxn expression in AS-C mutants, KL11.1/TM6 males were crossed with y Df(1)260.1 f / FM6 virgins. In the case of cut mutants, KL11.1 males were crossed with y ct145/+; KL11.1/+ females. The effect of poxn on the pattern of lacZ expression in E7 2nd 36 was assessed in the progeny of KL11.1/+; E7 2nd 36/+ flies; the effect of poxn expression on A3-lacZ expression was observed in the progeny of KL11.4/+; A3-lacZ/+ flies. Heat-shock treatments Heat-shock experiments in the embryo were done as follows: 3 pulses of 10 minutes at 37°C separated by 2 intervals of 5 minutes at 25°C are applied to 4 to 6 hours old embryos that are kept at 25°C before and after the heat shock. This protocol gives a high frequency of supernumerary p-es organs. Heat shocks of pupae were done as described in Nottebohm et al. (1992) and applied between 6 and 12 hours APF. For giant chromosomes immunostaining, third instar larvae were collected, put at 37°C for 30 minutes and left at 25°C for 24 hours before dissection. Staining and mounting 22C10 staining was done as described in Ghysen et al. (1986); antiCut as described in Blochlinger et al. (1990); and anti-Poxn as in Dambly-Chaudière et al. (1992) using the anti-Poxn at a 1:250 dilution. Polyclonal anti-galactosidase was from Cappell and was used at a 1:200 dilution. X-gal staining of whole pupae was done using the method described for embryos by Ghysen and O’Kane (1989), including the heptane-fixative step, except that 2.5% glutaraldehyde was used for fixation instead of formaldehyde. The reaction was allowed to continue for 5 days at 37°C. Cuticule preparations of late embryos were mounted and observed as described in DamblyChaudière and Ghysen (1986). Giant chromosomes Immunolabelling was done using the method of Zink and Paro (1989) using the following conditions: the salivary glands were fixed for 25 seconds, anti-Poxn was used at a 1:10 dilution. In situ hybridisation on giant chromosomes was performed as described in Langer-Safer et al. (1982). RESULTS The ectopic expression of poxn transforms ch organs into es organs The ubiquitous expression of poxn during embryogenesis leads to the formation of an excess of p-es organs (DamblyChaudière et al., 1992). Most of the supernumerary organs result from the transformation of m-es into p-es precursors. Some of these organs, however, appear at positions where no m-es organs are found in the wild type. One possible explanation is that the ectopic organs result from the transformation of internal sense organs. We have therefore examined whether poxn has any effect on the internal sense organs. There are two major types of internal sense organs, ch and multidendritic (md; Bodmer and Jan, 1987). The ch and md neurones can be distinghuished from each other and from the es neurones by using the 22C10 antibody, which recognises an antigen in the cytoplasmic membrane of all sensory neurones (Zipursky et al., 1984): es neurones have a long and thin dendrite that extends to the surface of the embryo; ch neurones have a stubby dendrite that extends under the epidermis and md neurones develop their multiple dendrites much later than the other neurones, after hatching (Bodmer and Jan, 1987). Besides the difference in morphology, ch also differ from other neurones in that ch neurones or their precursor cells often migrate away from their original position while most other neurones are found near the position where their precursors form. For example, the precursors to the five lateral ch organs in the abdominal segments (lch5, Fig. 1A) form dorsolaterally but later migrate to a lateral position (Ghysen and O’Kane, 1989; Bier et al., 1990; Salzberg et al., 1994). This migration event is ch-specific: if the precursors to the lch5 are transformed into es precursors due to the ubiquitous expression of cut, the transformed neurones keep a dorsolateral position (Blochlinger et al., 1991). We have labelled embryos with 22C10 after heat-induced expression of poxn. Since the hsp-poxn chromosomes are homozygous lethal (Dambly-Chaudière et al., 1992), crosses were made between heterozygotes and therefore one half of the progeny is expected to carry one copy of the hsp-poxn construct. In about one half of the embryos, we observe the displacement of the lch5 neurones to a more dorsal position (Fig. 1B, arrow). These neurones have es-like dendrites (Fig. 1C), confirming that they have acquired an es identity. About one fourth of the embryos show a disorganisation of the peripheral nervous system, with a loss of ch neurones and a large excess of es ones. We believe that this phenotype corresponds to the embryos that carry two copies of the heat-shock Regulatory interactions between cut and poxn 3113 construct. The large excess of es neurones in these embryos probably reflects the transformation of many ch and m-es into p-es organs. Fig. 1. Effect of the ubiquitous expression of poxn on sensory neurones. Lateral and dorsal clusters of neurones in the fifth abdominal segment of a wild-type (A) and in a hsp-poxn (B,C) embryo after heat-shock treatment. The arrowheads in A and B point to the v’ch1 chordotonal organ which arises from the ventral cluster. The thick arrow indicates the lateral chordotonal organs (lch5) in A. In the heat-shocked embryo, no neurones are present at the position of the lch5 (asterisk in B), but a new dorsal cluster is present (arrow). These neurones have es-like dendrites that extend into a more superficial focal plane (arrows in C). Anterior is left, dorsal is up. construct, as we do not observe it in experiments where embryos have at most only one copy of the heat-shock poxn can form es organs in AS-C− embryos If the expression of poxn can transform ch into es precursors, one would expect to observe ectopic es organs even in embryos where only ch precursors are present. Embryos deficient for the AS-C genes lack all es organs and most md neurones, but their ch organs develop normally (Dambly-Chaudière and Ghysen, 1987). We have found that the expression of poxn can lead to the formation of external structures typical of es organs in ASC− embryos (Fig. 2C). In the thoracic segments, where the external structure formed by the p-es organ (kölbchen; Hertweck, 1931) is easy to detect even in an otherwise naked embryo, we detected a mean number of 0.2 kölbchen per hemisegment in heat-shocked ASC−; hsp-poxn/+ embryos. Some hemisegments bear up to 2 kölbchen. These kölbchen appear at ventral and dorsolateral positions that correspond to the positions where the ventral and dorsolateral ch precursors arise. Using 22C10 labelling, we observed that some of the ch neurones are morphologically transformed into es-like neurones (not shown). Taken together, these results show that the ubiquitous expression of poxn can lead to the transformation of ch into es organs. In contrast, we have never observed a clear transformation of the few md neurones that remain in AS-C− embryos. Since most md neurones are absent in such embryos, we have used another method to decide whether poxn affects the md neurones. We induced poxn in an enhancer-trap line E7 2nd 36 strain, where lacZ is expressed in all md neurones but not in ch or es neurones (Bier et al., 1989). We did not detect any change in the pattern of lacZ-expressing cells (not shown), thus confirming the lack of effect of poxn on md neurones. Fig. 2. Induction of p-es organs by ubiquitous poxn expression in AS-C− embryos. (A) Ventral view of the third thoracic segment of a wildtype embryo after heat-shock treatment. The arrow points to the ventral kölbchen (poxn-dependent organ), the arrowhead to the Keilin organ (poxn-independent organ). (B) Third thoracic segment of an AS-C− embryo after heat-shock treatment; no sensory organ are present. (C) Third thoracic segment of an AS-C−; hsp70-poxn embryo after heat-shock treatment. The arrow points to a kölbchen in ventral position. This organ is slightly displaced relative to the normal kölbchen (A) and probably arises from the transformation of the ventral ch organ (vch1; Ghysen and Dambly-Chaudière, 1986). 3114 M. Vervoort and others poxn cannot promote es development in the absence of cut In the absence of cut, no es organ is formed (Bodmer et al., 1987). Yet we observed in AS-C− embryos that ch precursors (which do not express cut) can form es organs if they are forced to express poxn. One way to explain this result is that poxn can substitute for cut. We tested this explanation by inducing the ubiquitous expression of poxn in cut− embryos. These embryos are easily distinguished from the cut+ embryos produced in the same cross because their normal complement of external sense organs is deleted. We examined 96 heat-shocked cut−; hsp-poxn embryos containing on the average one copy of the hsp-poxn construct, and did not detect a single external sense organ, either at the positions where the normal es organs should have appeared, or at the positions where transformed ch organs would be expected, or in any other position. By contrast, the cut+ embryos produced in the same cross form about 0.6 supernumerary organ per hemisegment, as reported previously (Dambly-Chaudière et al., 1992). It might be argued that the heat shock is for some reason less efficient in cut− than in cut+ embryos. We could however rule out this remote possibility by relying on another phenotype resulting from the heat-induced expression of poxn. We have noticed that the ectopic expression of poxn modifies the morphology of the antennal organ, a complex sensory structure that does not depend on AS-C and cut. The poxn-induced transformation of this organ is fully observed in heat-shocked cut−; hsp-poxn embryos, demonstrating that poxn is indeed expressed ectopically in these embryos. We conclude that, in the absence of cut, poxn is not capable of imposing an es fate to the ch precursors. poxn expression is independent of cut but depends on AS-C All the data so far suggest that the presence of an active cut gene is necessary for poxn to impose an es fate to ch precursors. Does this conclusion hold true for the normal p-es precursors? It has been observed that p-es organs are absent in cut− mutants (Bodmer et al., 1987). This result is consistent with the hypothesis that poxn requires cut to promote the p-es fate. Alternatively, it might be that the expression of poxn in p-es precursors depends on cut activity. We have therefore asked whether or not poxn is expressed in cut− mutants. Fig. 3A shows the pattern of expression of poxn in cut− embryos. The p-es precursors and their progeny express poxn normally but never give rise to an es organ (as monitored by the absence of any external structures and the presence of morphogically ch-like neurones; Bodmer et al., 1987; our own observations) while they give rise to a poly-innervated organ (M. V. and A. G., unpublished data). We conclude that cut is necessary for poxn to confer their complete fate to the normal p-es precursors. The observation that the expression of poxn does not depend on cut raises the possibility that it is also independent of ASC activity, and relies directly on other positional cues. Fig. 3B shows an embryo homozygous for a complete deletion of the AS-C labelled with the anti-Poxn antibody. The expression of poxn is abolished in these embryos except in the central nervous system (arrowheads). Fig. 3. Expression of poxn in cut− and AS-C− embryos. (A) The distribution of Poxn protein in cut− embryos is indistinguishable from the wild-type pattern (as described in Dambly-Chaudière et al., 1992). The arrows and arrowheads show, respectively, the ventral and dorsal lines of p-es organs. In the second and third thoracic segments, the dorsal p-es organs are displaced laterally (asterisks). (B) In AS-C− embryos, no cell expresses poxn in the peripheral nervous system. Labelling can be observed, however, in the central nervous system (slightly out of focus, empty arrowheads). Fig. 4. Effect of the ubiquitous expression of poxn on cut expression. Lateral (l) and dorsal (d) clusters of three adjacent abdominal segments of a wild-type (A) and of a hsp-poxn (B) embryo after heat-shock treatment are shown. Arrows in B point to two new dorsolateral clusters of cut-expressing cells. The position of these cells corresponds to that of the es-like neurones shown in Fig. 1B). poxn induces cut We have shown so far that the formation of normal and ectopic p-es organs under the control of poxn requires cut. Since the expression of poxn can cause ch precursors to form es organs, one has therefore to postulate that the expression of cut has Regulatory interactions between cut and poxn 3115 -10 0 +10 +20 +30 +40 +60 +50 A3 +70 E C F B wing margin +80 embryonic es organs posterior spiracle malpighian tubules been activated in the transformed precursors. We have examined this hypothesis by observing the pattern of cutexpressing cells after ubiquitous activation of poxn. In conditions that lead to the formation of ectopic es organs, we observe the presence of ectopic cut-expressing cells in the dorsolateral position that is typical of transformed ch organs (Fig. 4). We have confirmed this result by using embryos deleted for the AS-C genes, where the only precursors left are those that do not express cut. Labelling AS-C− embryos with anti-Cut antibodies reveals the presence of Cut product in non-neuronal cells only (Blochlinger et al., 1991), while after ubiquitous expression of poxn, ectopic clusters of cut-expressing cells can be observed at positions consistent with those of the sensory organs seen on the cuticle (not shown). A regulatory sequence of cut can be activated by poxn in ch organs The regulatory region of cut extends over approximately 200 kb. It comprises several independent enhancer regions, each of which directs the expression of cut in different tissues (Fig. 5; Jack et al., 1991; Liu et al., 1991). Some of these regions have been shown to direct the expression of the reporter gene lacZ in the peripheral nervous system (Jack and DeLotto, 1995). Late during embryogenesis, the 2.7 kb fragment called A3 drives lacZ expression in the same subset of larval sense organs that also express cut: in all es organs and in some md neurones, but in none of the ch organs (Blochlinger et al., 1990). We have examined the pattern of lacZ expression at earlier stages of development, at the time that the first mother cells are formed. The first precursors arise as two pairs of cells in each segment, an anterior pair, which gives rise to es organs, and a posterior pair generating ch organs (Ghysen and O’Kane, 1989). The anterior precursors express achaete and cut (Ruiz-Gomez and Ghysen, 1993; Blochlinger et al., 1990), while the posterior ones express only atonal (Jarman et al., 1993). Consistent with the pattern of expression of cut, we found that A3 directs lacZ only in the anterior cells and in their progeny (Fig. 6A). After heatshocking hsp-poxn; A3-lacZ embryos, however, we observe lacZ expression in posterior cells (Fig. 6B). We conclude that the A3 fragment can be activated in ch precursors by poxn. embryonic central nervous system adult es organs tracheae anterior spiracle Fig. 5. Schematic representation of the organisation of the cut locus. The map shows a part of the region upstream of cut. Scale is in kilobases and is based on data from Jack (1985). The large arrow indicates the beginning of the transcribed region. The boxes represent regions which, when fused to lacZ, drive the expression of the reporter gene in defined tissues (Jack et al., 1991; Jack and DeLotto, 1995). The nomenclature of the boxed regions is according to Jack and DeLotto, 1995. The A3 enhancer drives lacZ in adult poxndependent bristles We found that the A3 fragment also directs the expression of the reporter gene in a subset of adult es organs, the p-es organs (chemosensory bristles) and some m-es organs (the campaniform sensilla). The second type of m-es organs, the mechanosensory bristles, do not express the reporter gene (Fig. 7A). The expression of lacZ becomes detectable only at about 40 hours after pupariation formation (APF). This is a relatively late stage in the development of these organs, several hours after all the cells of the lineage have been generated. At earlier stages, we detect no lacZ expression in the imaginal discs, Fig. 6. Expression of lacZ in the second thoracic segment of A3-lacZ and A3-lacZ; hsp70-poxn embryos. (A) The expression of lacZ is observed in the ‘A’ cells (arrowhead), posterior to the tracheal pit (asterisk), as well as in the more ventral ‘V’ cells, which appear slightly later, but never in the posterior ‘P’ cells which appear first. The P cells are located anterior to the next tracheal pit (doubleasterisks) and are ch precursors (Ghysen and O’Kane, 1989). (B) After the ectopic expression of poxn, some P cells express lacZ (arrow). Nomenclature of the cells is according to Ghysen and O’Kane (1989). 3116 M. Vervoort and others Fig. 8. lacZ expression in A3-lacZ; hsp70-poxn pupae after heatshock treatment. (A) Wing disc showing expression of lacZ 30 minutes after a heat shock delivered at 6 hours APF. The arrowheads point to the p-es precursors of the anterior wing margin. Arrows indicate the precursors of the campaniform sensilla (m-es organs). (B) Higher magnification of the wing margin of the same disc. Fig. 7. Expression of lacZ in the tibia of A3-lacZ and A3-lacZ; hsp70-poxn pharate adults after heat-shock treatment. (A) X-gal staining of the tibia of a A3-lacZ pharate adult. Labelling is restricted to cells belonging to the recurved, open-tipped p-es bristles (arrows). (B) X-gal staining of the proximal region of the tibia of an A3-lacZ; hsp70-poxn pharate adult after heat-shock treatment. Many p-es bristles (arrows) are present and are all associated with blue cells. except a very weak expression in the posterior part of the eye disc. The mother cells that will produce chemosensory bristles and campaniform sensilla appear several hours earlier than those that will form mechanosensory bristles, both in the wing (Huang et al., 1991; Hartenstein and Posakony, 1989) and in the leg (Nottebohm et al., 1994). Thus the differential expression of lacZ driven by the A3 fragment could be due to the difference in the time of emergence of the different sensory organs, rather than to the difference in identities. In order to determine whether A3 responds to precursor identity or to stages of pupal development, we have examined the effect of transforming the identity of precursors cells without changing their time of emergence. The precursors of the tibial chemosensory bristles (p-es) are determined at about the time of puparium formation, while the precursors of the mechanosensory bristles (m-es) appear 12 hours later (Nottebohm et al., 1994). Inducing the ubiquitous expression of poxn at 12 hours after puparium formation (APF) transforms the m-es precursors into p-es precursors (Nottebohm et al., 1992), making it possible to form chemosensory bristles from ‘late appearing’ precursors and hence to distinguish whether the A3 enhancer responds to time or to cell type. In the first case, time dependence, we expect lacZ to be expressed only in the normal (early appearing) chemosensory bristles while in the second case, identity dependence, we should see lacZ expression in both normal (early) and transformed (late) chemosensory bristles. The result is shown Fig. 7B: the normal as well as the supernumerary p-es bristles express lacZ. We conclude that the A3-controlled expression of lacZ in the p-es organs is correlated to the identity of these organs and not to their time of emergence. Moreover, this experiment confirms that the A3 fragment contains at least one element that responds directly or indirectly to poxn. Transient activation of A3-lacZ by poxn The expression of A3-lacZ in supernumerary p-es organs is detected 40 hours APF, at the same time when this construct is also expressed in the normal p-es cells. This is long after the ubiquitous induction of poxn. We examined whether poxn might have a more immediate effect on A3 by assessing the expression of lacZ 30 minutes and 60 minutes after induction. As mentioned previously, the A3 fragment drives no detectable expression of lacZ before 40 hours APF, except in the eye. The same is true for heat-shocked pupae bearing only the A3-lacZ construct and for non-shocked A3-lacZ; hsp-poxn pupae: in both cases, lacZ is only weakly expressed in eye discs and not at all in the precursor cells of the other discs. In contrast, when we heat-shocked pupae of the A3-lacZ; hsp-poxn line at 6 hours APF, we detected a transient expression of lacZ in several precursor cells (Fig. 8). These include the cells that will form the p-es bristles of the anterior wing margin and of the legs as well as the precursors of the campaniform sensilla and of chordotonal organs. In addition, lacZ is strongly expressed in the eye disc (not shown). One hour after the heat shock, the expression of lacZ is not detectable any more in the precursor cells, and only occasionally and weakly in the photoreceptors. Regulatory interactions between cut and poxn 3117 Fig. 10. The A3 fragment contains a Poxn-binding site. (Upper panel) Localisation of the insertion site of the A3 fragment in A3lacZ transgenic flies by in situ hybridisation using P-element DNA as a probe. Hybridisation band (arrowhead) is located in 73A. (Lower panel) Immunostaining of A3-lacZ; hsp70-poxn giant chromosomes with anti-Poxn antibody. Arrowhead points to a Poxn-binding site in 73A. Fig. 9. Distribution of Poxn protein after heat-shock treatment. (A) Wing disc, (B) leg disc and (C) salivary glands of A3-lacZ; hsp70-poxn pupae labelled with the anti-Poxn antibody 30 minutes after heat shock. The arrowheads and arrows in A point to the precursors of the p-es organs of the wing margin and to the precursors of the campaniform sensilla of the wing blade, respectively. tified these sites and found that one of them corresponds to the cytological position of the cut locus. This result suggests that cut may be a direct target of poxn. The A3 fragment makes the associated lacZ gene inducible by poxn. If this induction is direct and involves binding of the Poxn product, one might expect the A3 fragment to comprise an immunodetectable Poxn-binding site. We have stained the giant chromosomes of A3-lacZ; hsp-poxn larvae after heat shock and observed a new binding site at the position where the A3-lacZ construct is inserted, 73A (Fig. 10). Poxn binding is never observed at this position in the absence of the A3-lacZ construct insertion. This result indicates that the A3 fragment contains a Poxn-binding site and supports the idea that poxn directly regulates the expression of cut. DISCUSSION The transient expression of lacZ occurs only in some precursor cells including those that already express poxn. One explanation for this restricted activation is the need for specific cofactors present only in these cells. Alternatively, it might result from a non-homogenous distribution of the heat-induced Poxn protein. We examined this hypothesis by immunolabelling discs with anti-Poxn. We found that 30 minutes after the heat shock, Poxn is present in a few cell types only. Besides the p-es precursor cells where it is normally present, Poxn is found in campaniform sensilla precursors, in some photoreceptors and muscle cells, and in the salivary glands cells (Fig. 9). Poxn protein binds to the cut regulatory sequence If poxn activates cut directly, one would expect the Poxn protein to bind to the cut locus on salivary glands chromosomes (Zink and Paro, 1989). As poxn is normally not expressed in the salivary glands, we have used the hsp-poxn construct to induce the expression of poxn in this tissue prior to the immunolabelling. We found that the Poxn product binds reproducibly to about forty sites in the genome. We have iden- The ectopic expression of poxn changes ch into es fate by activating cut The ubiquitous expression of poxn leads to the formation of an excess of p-es organs, mainly arising from the transformation of m-es organs (Dambly-Chaudière et al., 1992; Nottebohm et al., 1992). We have shown here that some of the supernumerary organs come from the transformation of internal sense organs of the ch type. One obvious explanation for the ability of poxn to transform ch into es organ is that poxn can substitute for cut. We observed, however, that in cut− embryos, poxn is normally expressed although p-es organs do not form. Moreover, the overexpression of poxn in cut− embryos does not lead to the formation of es organs. Thus poxn cannot substitute for cut for the formation of es organs. An alternative explanation for the fact that poxn is capable of imposing an es fate to precursors that do not express cut, is that poxn can activate the expression of cut. We have shown that the ectopic expression of poxn results in the presence of Cut product in sensory cells that do normally not express cut, 3118 M. Vervoort and others indicating that poxn can indeed activate cut, either directly or indirectly. Poxn-binding sites on salivary gland chromosomes The gene poxn encodes a protein which contains a putative DNA-binding region (the paired domain) and an activation region (the acidic C-terminal domain), suggesting that it acts as a transcriptional regulator (Bopp et al., 1989; DamblyChaudière et al., 1992). We have identified the binding sites of Poxn on giant chromosomes and found that one of them corresponds to the cytological position of cut. The detection of bound transcriptional regulators on salivary gland chromosomes (Zink and Paro, 1989; DeCamillis and al., 1992; Rastelli et al., 1993) has been shown to identify bona fide targets in several cases, as for example that of Polycomb (Pc). Anti-Pc immunolabelling was observed at the positions of several genes known to be regulated by Pc (Zink and Paro, 1989; Zink et al., 1991). In contrast to Pc, poxn is not expressed in salivary glands and we had therefore to induce its expression using a heat-inducible construct. This raises the question of whether the binding sites that we observe correspond to bona fide targets of poxn. Our reasons to think that Poxn binds to its normal targets when ectopically expressed in salivary gland cells are the following. First, we detected a defined number of sites (forty) at completely reproducible positions. Second, several of the sites correspond to putative targets of poxn (M. V., unpublished observations). These include, besides cut, the gene poxn itself, which is thought to be autocatalytic (C. D-C., unpublished observations), the gene east (Vijayraghavan et al., 1992), which is specifically expressed in the leg chemosensory neurones (M. V., unpublished observations) and a new gene that is specifically expressed in the p-es, but not in the m-es, organs of the embryo (P. Gautier, C. D-C. and A. G., unpublished observations). leads to a transient ectopic expression of A3-lacZ in some es precursor cells, and also at a later stage in the cells of the mechanosensory sense organ. We conclude that poxn acts, directly or indirectly on the A3 fragment to activate the expression of the adjacent reporter gene. Since larvae carrying the A3 fragment inserted on the third chromosome were shown by immunolabelling with anti-Poxn antibody to have a new Poxn-binding site at the position where the fragment is inserted, we conclude that the activation of A3-lacZ by poxn is a direct effect due to the binding of the Poxn protein to one or more sites in the A3 fragment. The response of A3-lacZ to poxn overexpression is restricted to sensory cells, suggesting that activation ofA3 also requires other factors present in these cells. Since the activation of A3 region and the maintenance of Poxn protein after heat-induced expression of poxn are mostly restricted to cells that already express cut, one possibility is that the Cut protein is needed for Poxn-mediated transcriptional activation. This would also explain the very low efficiency of the transformation of ch into es organs by poxn ubiquitous expression, as compared to the high efficiency of the m-es to p-es transformation. The occasional transformation of ch into es may reflect the fact that high concentrations of Poxn product can exert an effect even in the absence of Cut protein, or that cut is transiently expressed in ch precursors. Other functions of poxn do not seem to depend on cut, however. In cut null mutant, the expression pattern of poxn is completely normal. This pattern reveals several features that are dependent on poxn activity, e.g. the number of cells (more than four) and the shift in the position of the thoracic organs relative to the abdominal ones (see Fig. 3). Thus it may be that only a subset of poxn function, among which would be the activation of cut itself, requires both poxn and cut, while another set, including the lineage and migration properties of the p-es organs, would be cut-independent. The complexity of the A3 fragment and the poxn-cut relationship During embryogenesis, A3 activates lacZ expression at all stages of larval es organ development. In contrast, during metamorphosis, A3 is active only at a very late stage of adult es organ formation and only in a subset of these organs, the campaniform sensilla and the chemosensory bristles. We do not know whether the latter pattern has any specific function or role, nor why the embryonic and pupal patterns are so different. One possibility is that the late expression of A3-lacZ in adult sense organ development is related to a difference between larval and adult es organ formation. We note that the patterns of cut expression at the late stages of sense organ development differ in the larva (where the trichogen and tormogen cells express cut at a higher level than the neurone and the thecogen) and in the adult (where all cells express cut at the same level). This suggests that there may be separate controls for cut expression at the latest stage of sense organ development in the embryo and in the pupa, and that the dissimilar behaviour of A3 in these two systems may be related to this difference. The expression of the A3-lacZ construct responds to the expression of poxn at both stages. During embryogenesis, the ubiquitous expression of poxn induces the expression of A3lacZ in ch precursors where this construct is normally never expressed. During metamorphosis, the expression of poxn Functional significance of the poxn-cut relationship cut is expressed in all m-es and p-es organs and in most md neurones (Blochlinger et al., 1990, 1991) while poxn is only expressed in a subset of these cells, the p-es set (DamblyChaudière et al., 1992). The activation of cut in the m-es precursors (and in the md neurones) could depend on the AS-C genes, and be subsequently maintained throughout the lineage by self-activation (Blochlinger et al., 1991). What then could be the meaning of the activation of cut by poxn, since poxn itself is expressed only in AS-C-dependent precursors where cut would be activated anyway? One possible explanation is that the control of cut by poxn is a remnant of an ancestral situation where the attributions and roles of the two genes were different. The p-es bristles of present-day adult flies are bifunctional: they are innervated by one mechanosensory and several chemosensory neurones, and their shaft comprises side by side a closed lumen resembling that found in mechanosensory bristles and an open-tipped lumen where the chemosensory dendrites extend. This raises the possibility that the ancestral es organs were of the p-es type. In that case, poxn may originally have been a major determinant of es organ formation, directing the expression of several other genes (one of which was cut) to control and realise this program of differentiation. The m-es organs may then be considered as incomplete p-es organs, formed by using Regulatory interactions between cut and poxn 3119 a subset of the complete p-es program. Accordingly, cut seems to have only a partial effect on the identity of the sense organs. We have noticed that, in cut mutants, the organs that correspond to the poxn-expressing lineage still contain more than one neurone, a property typical of chemosensory organs (M. V. and A. G.; unpublished observations). Furthemore, even in null mutants, some es organs (about 5%) are not transformed into ch and others are often only partially transformed (68% of the transformed organs lack external structures but do not produce the typical scolopale structures of the normal ch neurones; Bodmer et al., 1987). All this suggests that cut may be primarily a determinant of the external derivatives of the es organs. A second explanation, which is not exclusive with the first one, emphasises the co-operation between cut and poxn. 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