Interactions between the EGF receptor and DPP pathways establish distinct cell fates in the tracheal placodes

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1 Development 124, (1997) Printed in Great Britain The Company of Biologists Limited 1997 DEV Interactions between the EGF receptor and DPP pathways establish distinct cell fates in the tracheal placodes Pablo Wappner, Limor Gabay and Ben-Zion Shilo* Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel *Author for correspondence ( SUMMARY The formation of the tracheal network in Drosophila is driven by stereotyped migration of cells from the tracheal pits. No cell divisions take place during tracheal migration and the number of cells in each branch is fixed. This work examines the basis for the determination of tracheal branch fates, prior to the onset of migration. We show that the EGF receptor pathway is activated by localized processing of the ligand SPITZ in the tracheal placodes and is responsible for the capacity to form the dorsal trunk and visceral branch. The DPP pathway, on the contrary, is induced in the tracheal pit by local presentation of DPP from the adjacent dorsal and ventral ectodermal cells. This pathway patterns the dorsal and lateral branches. Elimination of both pathways blocks migration of all tracheal branches. Antagonistic interactions between the two pathways are demonstrated. The opposing activities of two pathways may refine the final determination of tracheal branch fates. Key words: EGF receptor, DPP, trachea, ERK, branching morphogenesis, Drosophila INTRODUCTION The formation of branched tubular structures is an essential process in the development of all multicellular organisms. Tubulogenesis is needed in vertebrates for the development of the vascular system, lung buds, kidney tubules and mammary glands. In Drosophila, several such structures develop during embryonic and larval stages. One set of tubular structures including the tracheal system and salivary ducts appears to have a common mechanism of induction, mediated by the bhlh/pas protein TRACHEALESS (Wilk et al., 1996; Isaac and Andrew, 1996). Other tubular structures such as the Malpighian tubules arise by a different mechanism (Baumann and Skaer, 1993). One aspect common to all tubular structures is the need to follow a highly stereotyped and elaborate process of migration, to generate the final structure of the organ. Another feature shared by tubular structures that arise simultaneously in different parts of the organism, is the capacity to form a continuous network joining the different clusters. The Drosophila tracheal system is an attractive model organ to study the different phases of branching morphogenesis (reviewed in Manning and Krasnow, 1993). It is initiated as ten tracheal placodes on each side of the embryo. Invagination of the tracheal cells generates the tracheal pit. Several rounds of cell division give rise to the final tracheal cell number of ~80 cells per placode, and all subsequent stages of tracheal differentiation ensue without further cell division. The migration of the tracheal cells forming the different branches is guided by expression of the BRANCHLESS protein, a secreted FGF-like molecule, on ectodermal cells that prefigure the pattern of tracheal migration (Sutherland et al., 1996). Expression of BRANCHLESS is highly dynamic: as migration progresses, BRANCHLESS always leads the migrating tips. The tracheal cells respond to BRANCHLESS by activation of the BREATHLESS receptor tyrosine kinase, an FGF receptor homologue (Glazer and Shilo, 1991; Klämbt et al., 1992). The causal relationship between the expression pattern of BRANCHLESS and directional migration of the tracheal cells was clearly demonstrated. In the absence of BRANCHLESS or BREATHLESS, no tracheal migration takes place, while expression of BRANCHLESS at ectopic sites attracts tracheal cells (Sutherland et al., 1996; Klämbt et al., 1992). However, several issues remain unresolved. From the initial, simultaneous migration of the six tracheal tips at each tracheal pit, the number of cells within each branch is fixed and reflects the final cell number. The dorsal trunk and visceral branch contain ~twenty five cells each, the anterior and posterior lateral trunks six and ten cells, respectively, and the dorsal branch only five cells. We assume that this finely tuned cell number can not be regulated by the BRANCHLESS/BREATHLESS pathway. While this system can provide directionality for the migration of the different branches, it cannot supply the information instructing individual cells within the tracheal pit to join one branch primordium or another. Thus, additional mechanisms are necessary prior to the onset of migration, to specify within the population of pluripotent tracheal cells, the number of cells assigned to each branch. An indication for generating distinct populations of tracheal cells before the onset of migration was provided by the expression pattern of decapentaplegic (dpp) and its type I receptor thick veins (tkv), as well as from the characterization of tracheal phenotypes resulting from mutations in the Dpp

2 4708 P. Wappner, L. Gabay and B.-Z. Shilo signaling pathway. tkv has a localized zygotic expression within the tracheal pits, and the activating signal is provided by the expression of dpp in two stripes abutting the pits. In tkv mutant embryos, specific tracheal branches, namely the dorsal branch and lateral trunks, fail to develop (Affolter et al., 1994). This observation suggested that pre-patterning of the cells within the tracheal pit may take place prior to activation of BREATHLESS. Since the DPP pathway appears to be required only for the development of a subset of tracheal branches, it is interesting to identify signaling pathway(s) which may be responsible, in parallel, for fate determination of the other branches. This work examines the role of the EGF receptor (EGFR/DER) signaling pathway in tracheal morphogenesis and its interaction with the DPP pathway. The EGFR pathway comprises a cassette that is utilized multiple times during development (reviewed in Schweitzer and Shilo, 1997). SPITZ (SPI), the primary ligand which activates EGFR during embryogenesis, is produced as an inactive transmembrane, TGFα-like precursor (Rutledge et al., 1992). SPITZ and EGFR are ubiquitously expressed. The signaling pathway is triggered by a tight regulation of SPITZ processing. Only upon cleavage of SPITZ does the molecule become potent as a secreted ligand (Schweitzer et al., 1995). Processing is regulated by an unknown mechanism, involving two novel transmembrane proteins, RHOMBOID (RHO) and STAR (S) (Bier et al., 1990; Kolodkin et al., 1994; Schweitzer et al., 1995; Golembo et al., 1996). The expression of RHO is tightly regulated, anticipating the activation profile of EGFR and ectopic RHO expression gives rise to deleterious consequences (Ruohola-Baker et al., 1993; Noll et al., 1994; Golembo et al., 1996; Gabay et al., 1997a). RHO was reported to be transiently expressed in the tracheal pits and mutations in rho give rise to tracheal defects (Bier et al., 1990). This work examines the role of the EGFR pathway in defining the fate of tracheal cells, prior to the onset of migration. We show that, in all mutants of the EGFR signaling cassette, the dorsal trunk and visceral branches fail to form, while the dorsal branch and lateral trunks develop normally. This phenotype reflects a cell autonomous effect of EGFR in the tracheal cells and is complementary to the defects observed in tkv mutant embryos. Double mutants of rho and tkv indeed show loss of all tracheal branches. Ectopic activation of the DPP pathway seen in short gastrulation (sog) mutant embryos or following ubiquitous expression in the tracheal pits of DPP or constitutively activated TKV, gives a phenotype similar to the one of spitz group mutants. Conversely, targeted expression of secreted SPITZ in a sensitized tkv/+ background leads to a phenotype reminiscent of tkv mutants. These results indicate that the DPP and EGFR pathways exert antagonistic activities on the tracheal pit cells, as monitored by their migration pattern. We propose a model in which the branch fate of individual tracheal cells results from a balance between the two opposing pathways. MATERIALS AND METHODS Fly strains The following strains were used: flb 2C82, flb 2W74, spi OE92, rho 38, S IIN23, sim H9 (provided by S. Crews), tkv str-ii, put 135 (provided by M. Affolter), rho-lacz R1.1 (provided by M. Levine), sog YP01, sog YH02 (provided by F. M. Hoffmann), btl-gal4 (provided by S. Hayashi), UAS-DER type II, UAS-dpp (provided by F. M. Hoffmann), UASactivated tkv (provided by S. Cohen) and UAS-sSpi. Antibodies and probes Staining was carried out by standard procedures using the following antibodies: rat anti-trachealess (generated against the PAS B domain), rabbit anti-β-gal (Cappel), mouse anti-egfr, and a monoclonal antibody against diphospho-erk (Yung et al., 1997; Gabay et al., 1997a,b) (provided by R. Seger and Sigma Ltd., Israel). Rabbit anti- SAL antibodies (Kühnlein et al., 1994) were provided by R. Schuh. Secondary antibodies (FITC, LRSC or HRP-conjugated) were from Jackson. To visualize fluorescent stainings of the tracheal tree with the confocal microscope (Biorad 1024), a composite of several sections was generated. RNA probes for in situ hybridization were prepared from plasmids containing the kni (provided by H. Jäckle), bnl (provided M. Krasnow) and sog (provided by M. Hoffmann) cdnas. Homozygous mutant embryos were identified by absence of balancer chromosome β-gal staining. tkv, rho double mutants were identified by absence of β-gal staining on both second and third balancer chromosomes. flb, put double mutants were identified similarly, as well as by the overall morphology of the embryos (failure to undergo germband retraction and dorsal closure). RESULTS Pattern of EGFR activation in the tracheal placodes The previously reported expression of RHO in the tracheal pits (Bier et al., 1990) provided the initial indication for the activation of the EGFR pathway in these cells. Double labeling with an anti-trachealess (TRH) antibody marking all tracheal cells, and a rho-enhancer trap line allowed us to follow rho expression at a higher resolution. rho is expressed at the tracheal placode stage, and is restricted to a subset of cells, located in the central and dorsal part of each placode (Fig. 1A,B). How does the restricted expression of rho in the placode correspond to the resulting pattern of EGFR activation? We described recently the capacity to follow in situ, with an antibody recognizing the double phosphorylated form of ERK (dp-erk), the activated state of signaling pathways (Yung et al., 1997; Gabay et al., 1997a,b). It is now possible to examine how the pattern of EGFR activation corresponds to the RHOexpression profile in the tracheal placodes. Prominent dp-erk was detected in the tracheal placodes at stage This pattern is EGFR-dependent, as it was abolished in rho mutants (Fig. 1E,F) as well as in other mutants of the spitz group (Gabay et al., 1997a). Double staining with anti-dp-erk and anti-trh demonstrated that EGFR activation does not extend beyond the placodes (Fig. 1C,D). The dp-erk domain is broader than the region of rho expression, but higher levels are detected in the center of the placode and lower levels toward the periphery. Since RHO is known to regulate SPITZ processing, this pattern probably reflects the diffusion of the secreted form of SPITZ originating within the rho-expressing cells, in the central part of the placode. The fact that both rho expression and ERK activation are confined to the tracheal placodes is an indication that EGFR function is also restricted to the trachea. Tracheal phenotypes of EGFR-pathway mutants Tracheal development was examined in mutants for

3 EGF receptor and DPP interactions in trachea 4709 Egfr/DER/flb and the genes comprising the spitz group. We utilized the structure of the branches at the end of embryogenesis as an indicator for the cell fates induced earlier, prior to migration. Similar phenotypes were identified for flb, spi, rho and Star mutants (Fig. 2). While the number of cells in each tracheal pit appears normal, certain tracheal branches fail to develop, specifically the dorsal trunk and visceral branch are missing or incomplete. Typically, in a few segments (2-4), the dorsal trunk undergoes partial or total migration leading to fusion while, in the rest of the segments, it is absent. The cells that fail to migrate remain clustered at the original position of the tracheal pit (schematized in Fig. 2D). spitz group mutants participate in patterning the embryonic ventral ectoderm (Mayer and Nüsslein-Volhard, 1988). SPITZ processing in the ventral ectoderm is restricted to the midline (Golembo et al., 1996). The secreted form of SPITZ may diffuse laterally, thus generating a gradient of EGFR activation that patterns the ventral ectoderm (Gabay et al., 1997a). To investigate whether the tracheal phenotype observed in spitz group mutants results indirectly from defects in the ventral ectoderm, we monitored tracheal development in single minded (sim) mutant embryos. SIM is associated with the EGFR pathway only in the context of the ventral ectoderm, by promoting the expression of the SPITZ-processing machinery in the midline (Kim and Crews, 1993; Golembo et al., 1996). No tracheal abnormalities were observed in sim mutants (Fig. 2H), providing further evidence that the phenotypes described above for flb and the spitz group reflect an intrinsic defect in the trachea. Subdivisions of tracheal fates in spitz group mutants were also monitored with molecular markers. spalt (sal) mutant embryos show specific defects in migration of dorsal trunk cells (Kühnlein and Schuh, 1996), pointing to an important role of SAL in this process. The sal gene is expressed in a dynamic pattern in embryogenesis. During tracheal migration, SAL expression becomes restricted to the dorsal trunk cells (Kühnlein and Schuh, 1996; and Fig. 3A,B). Expression of SAL was examined in rho mutant embryos. The dorsal trunk failed to form and, concomitantly, expression of SAL was not detected. Occasionally, in a few segments, partial migration of the dorsal trunk cells took place and a parallel expression of SAL was observed (Fig. 3C,D). knirps (kni) was shown to be expressed in the tracheal cells in a complementary pattern to sal. It is excluded from the dorsal trunk cells and expressed in all other branches (Vincent et al., 1997). Expression of kni in rho mutant embryos is normal (Fig. 3E). EGFR function is confined to the trachea branchless (bnl) expression in tissues outside the trachea was shown to provide the essential migration cues (Sutherland et al., 1996). It is possible that failure to form the dorsal trunk in spitz group mutants is a result of altered expression of bnl. However, in rho mutant embryos, expression of bnl is unaffected (Fig. 3F). Importantly, the small clusters of ectodermal cells that express bnl ahead of the migrating dorsal trunk cells appear normal. To confirm that the tracheal phenotypes of spitz group mutants result directly from restricted activity of the EGFR pathway in the trachea, we tried to rescue the mutant phenotype by expressing EGFR in the tracheal cells. The btl-gal4 construct is expressed only in the midline and trachea, according to the normal expression pattern of breathless (Shiga et al., 1996). This driver was used to induce the expression of a UAS-EGFR construct, in the background of the severe flb 2C82 allele. Expression of the construct was detected by pronounced expression of EGFR in the trachea and midline (Fig. 4D). Rescue of the tracheal mutant phenotype was observed; while, in flb embryos, the dorsal trunk completely fails to migrate and fuse, in the embryos with targeted expression of EGFR in the trachea, complete migration and fusion of the dorsal trunk was seen (Fig. 4C). As expected, other defects of the flb phenotype, such as failure to retract the germband, are still observed. This result demonstrates that the role of EGFR in tracheal development is autonomous. The converse experiment, in which a dominant-negative EGFR construct was induced by the same driver, did not give rise to tracheal phenotypes, probably due to insufficient levels of the construct. Fig. 1. Pattern of EGFR activation in the tracheal placodes. (A,B) Expression of RHOMBOID in the tracheal placodes was monitored at stage 10 by following a rho-lacz enhancer trap line (green) and the tracheal cells with anti-trh (red). RHO expression is confined to the central part of the placode. (C,D) Activation of the EGFR pathway in the placode was monitored by the dp-erk antibody (green) and anti-trh (red). While activation is observed in all tracheal cells, it is more pronounced in the central part, corresponding to the domain of RHO expression. Arrows show the dorsal and ventral parts of the tracheal placode, where lower levels of dp-erk are indicated by the red (rather than yellow) color of the cells. No activation is detected outside the placodes (placode margins are dotted), suggesting an autonomous function for DER in the trachea. (E,F) In a rho mutant embryo, only residual levels of dp-erk are detected in the placode. They may correspond already to the activation of BREATHLESS in the tracheal cells.

4 4710 P. Wappner, L. Gabay and B.-Z. Shilo Patterning the trachea by the EGFR and DPP pathways Mutants in the DPP pathway develop a normal tracheal dorsal trunk and visceral branch, but the dorsal branch and lateral trunks are defective (Affolter et al., 1994; Fig. 5). In mutants for the type I receptor, THICK VEINS, the dorsal branch and lateral trunks are missing. In mutants for the type II receptor, PUNT (PUT) the phenotype seems less severe; while the dorsal branch is missing, the lateral trunks are partially or totally developed. This difference between the two mutant phenotypes was attributed to the fact that put has a higher maternal contribution (Letsou et al., 1995). Since the branches missing in spitz group mutants are present in DPP-pathway mutants and vice versa, the phenotypes can be considered complementary. Thus, EGFR patterns the cells that will form the branches migrating along the anteroposterior axis, while the DPP pathway defines the cells in the branches migrating dorsoventrally. In order to determine whether both pathways have an instructive role in determining tracheal cell fates, the phenotype of double mutant embryos for the two pathways was examined. The rationale is that if, on the one hand, one of the two possible directions of migration (dorsoventral versus anteroposterior) represents a default state in tracheal cell differentiation, one of the pathways may only function to modulate the other. In this case, double mutants will show the same phenotype as one of the single mutants. If, on the other hand, the two pathways induce two different cell fates that eventually determine alternative directions of migration, the double mutant embryos should not form any tracheal branches. The phenotype of the double tkv, rho mutants reveals the latter scenario. In these embryos, no patterning of the tracheal system is observed (Fig. 5C,D). The tracheal cells remain clustered in the tracheal pits, very similar to the phenotype observed for btl or bnl mutant embryos. This observation indicates that both the DPP and EGFR pathways have an instructive role in specifying the capacity of tracheal cell subsets to migrate in certain directions and form the different tracheal branches. Antagonistic interactions between the EGFR and DPP pathways The EGFR and DPP pathways determine fates of different subsets of naive tracheal cells. It was therefore interesting to determine whether the pathways have antagonistic effects. In this scenario, it should be possible to manipulate cell fates and migration behavior by changing the relative strengths of the two pathways. First, we tested the consequences of hyperactivating the DPP pathway. short gastrulation (sog) encodes an antagonist of DPP (Francois et al., 1994; Holley et al., 1995). During stage 11, it is expressed as a dorsal stripe abutting the tracheal pits (Francois et al., 1994; Fig. 6A). In sog mutant embryos, hyperactivation of the DPP pathway is expected. Indeed these embryos exhibit a tracheal phenotype reminiscent of the mutants for the EGFR pathway, the dorsal trunk is absent and Fig. 2. Tracheal phenotypes of mutants in Egfr/flb and the spitz group genes. (A) Wild-type embryo at stage 14, stained with anti-trh antibodies. Dorsal trunk is shown by arrowhead, and visceral branch by arrow. (B) Scheme of tracheal branches. DB, dorsal branch; DT, dorsal trunk; VB, visceral branch; LTa, lateral trunk anterior; LTp, lateral trunk posterior. (C) flb 2W74 mutant embryo showing absence of dorsal trunk (arrowhead) and reduced visceral branches (arrow). The dorsal branch and lateral trunks are normal. (D) Scheme of flb tracheal phenotype. Dark circles depict tracheal cells and open circles show positions where tracheal cells are missing. (E) spitz tracheal phenotype. (F) rho tracheal phenotype. (G) Star tracheal phenotype. (H) sim null mutant embryos show no tracheal defects. Arrowheads in E-H indicate presence or absence of the dorsal trunk. Since Sim participates only in patterning the ventral ectoderm by the EGFR pathway, this indicates that the Egfr tracheal phenotype is not an indirect consequence of ventral ectodermal defects.

5 EGF receptor and DPP interactions in trachea 4711 the visceral branch reduced (Fig. 6B). In a complementary experiment, the DPP pathway was uniformly activated in all tracheal cells in wild-type embryos, by using the trachealspecific driver btl-gal4 to induce expression of DPP or activated TKV. In the latter case, phenotypes should reflect a cell-autonomous activity of the DPP pathway in the trachea, since expression of the activated receptor was induced. Again, the dorsal trunk is missing, and the visceral branches are significantly reduced (Fig. 6C,D). Similar results were recently reported by Vincent et al. (1997). In contrast to the Egfr/flb and spitz group phenotypes, the cells that normally form the dorsal trunk do not remain at the position of the tracheal pits following hyperactivation of the DPP pathway. Instead, they join the cells forming the dorsal branch, such that each dorsal branch contains up to twenty cells instead of five. Therefore, the DPP pathway diverted tracheal cells from a dorsal trunk to a dorsal branch fate, in spite of normal signaling by EGFR. This was ascertained by showing that the pattern of EGFR-induced dp-erk in the tracheal placodes remains normal, following hyperactivation of the Dpp pathway (not shown). In a reciprocal experiment, the EGFR pathway was hyperactivated in the trachea by using btl-gal4 to induce the expression of UAS-secreted Spitz. Despite a general elevation in the levels of dp-erk in the tracheal pits and migrating tracheal cells (not shown), no defects in the formation of branches were detected (Fig. 6E). Hyperactivation of the EGFR pathway was subsequently monitored in sensitized embryos where the level of DPP signaling was reduced. Secreted SPITZ was induced by btl-gal4 in embryos heterozygous for a tkv null mutation. These embryos displayed a defective tracheal system, similar to the one observed in homozygous tkv mutant embryos (Fig. 6F). While the dorsal trunk and visceral branch formed normally, the dorsal branch and lateral trunks were missing. Our interpretation is that, when the strength of DPP pathway is reduced, the background is sensitized, allowing hyperactivation of EGFR to recruit cells from the DPP domain into an EGFR-dependent fate. Finally, we reasoned that, if the two pathways are antagonistic, it should be possible to suppress the phenotype of hypomorphic mutations in one pathway by a complementary reduction in the level of signaling elicited by the opposing pathway. put embryos display a hypomorphic phenotype (Fig. 5B) that is less severe than that of tkv, presumably due to residual levels of normal maternal transcripts. flb 2W74 is a temperature-sensitive allele; mutant embryos display an intermediate flb phenotype at 25 C, but the dorsal trunk is always discontinuous (Fig. 2C). In the double mutants, severe general abnormalities were identified due to the combined effects of failure to retract the germband and complete dorsal closure. The put defects in the dorsal branch remain. However, the dorsal trunk is continuous, indicating a suppression of the flb tracheal defect, elicited by a reduction in the level of DPP signaling (Fig. 6G). The rescue confirms the occurrence of antagonistic interactions between EGFR and DPP pathways. DISCUSSION The FGF receptor BREATHLESS is expressed in all tracheal cells (Glazer and Shilo, 1991). Localized presentation of the ligand, BRANCHLESS, is responsible for guiding the direction of migration (Sutherland et al., 1996). Restricted activation of BREATHLESS is not sufficient, however, to determine the precise number of cells that will be allocated to each tracheal branch. This work shows that the EGFR and DPP pathways are essential for inducing complementary cell fates, which will later determine the migration behavior of the cells. In addition, antagonistic interactions between the two pathways may further refine the pattern. Tracheal branch fates are induced by EGFR and DPP prior to the onset of migration EGFR and BREATHLESS are receptor tyrosine kinases essential for normal tracheal morphogenesis. Since activation of ERK is common to both pathways, by following the pattern of dp-erk, it is possible to define directly the time window in which each pathway is activated (Gabay et al., 1997a). EGFRdependent activation of ERK in the tracheal placode precedes the activation by BREATHLESS. Only after EGFR induction is diminished, does a new dp-erk pattern that is induced by BREATHLESS appear (Gabay et al., 1997b). The latter pattern is first detected in broad subdomains of the pit, in accordance with the initial broad pattern of BRANCHLESS expression. As the BRANCHLESS pattern refines towards the onset of branch migration, a corresponding refinement of dp-erk is observed. When migration takes place, dp-erk is restricted to the branch tip cells, immediately adjacent to the local BRANCHLESS source. Tracheal mutant phenotypes for Egfr/flb and the spitz group genes indicate that the EGFR pathway is responsible for patterning the dorsal trunk and visceral branches. The temporally distinct phases in which dp-erk is induced by the EGFR and BREATHLESS pathways, indicate that determination of branch fates by the EGFR pathway precedes the migration of the tracheal cells triggered by BREATHLESS. Tracheal phenotypes of mutants in the DPP pathway demonstrate that this cascade induces the lateral and dorsal branch fates. We assume that these aspects of patterning also precede migration. It has been shown by mosaic analysis that tracheal branch subdivisions are not specified prior to cell divisions in the tracheal placodes (Samakovlis et al., 1996), and may therefore be determined after completion of cell division. Taking into account that TKV is expressed specifically in the pits (Affolter et al., 1994) and RHO is transiently expressed at the placode and pit stages (Bier et al., 1990; Sturtevant et al., 1996), we propose that when the simultaneous migration of the six tracheal branches ensues in each tracheal pit, the fate of the cells has already been determined, with respect to the branch that they will join. Thus, each cell adopts a particular migration behavior, leading to allocation of a fixed cell number in each branch. Several lines of evidence rule out the possibility that the defects in tracheal migration in spitz group mutants, as well as in tkv and put mutants, reflect patterning defects in the ectoderm. Ectodermal defects could in turn lead to abnormal expression of cues guiding tracheal cell migration, e.g. BRANCHLESS. The expression pattern of the limiting elements of the EGFR and DPP pathways (namely RHO and TKV, respectively), strongly suggest that the mutant tracheal defects originate in the tracheal cells. The following observations also indicate that the defects monitored result directly

6 4712 P. Wappner, L. Gabay and B.-Z. Shilo Fig. 3. Expression of branchless and specific tracheal markers in rho mutant embryos. (A,B) Wild-type stage 14 mutant embryo stained with anti-trh (red) and anti-sal (green). Arrow shows SAL expression in the dorsal trunk cells. (C,D) In a rho mutant embryo, expression of SAL is absent. Occasionally, partial migration of dorsal trunk cells is observed, concomitant with expression of SAL (arrow). (E) Expression of kni in a stage 13 rho mutant embryo appears unaffected, in comparison to a wild-type embryo (not shown). (F) Expression of bnl in a stage 13 rho mutant embryo appears unchanged, in comparison to a wild-type embryo (not shown). bnl expression in the cell clusters normally directing dorsal trunk migration (arrow) is unaffected. from tracheal-cell defects. Tracheal phenotypes in spitz group mutants do not coincide with regions of ectodermal defects. The ventral ectoderm is specifically reduced in these mutants, while the tracheal defects are observed in the dorsal trunk and visceral branch. sim mutant embryos affect only patterning of the ventral ectoderm by EGFR and do not lead to defective development of the trachea. Expression of branchless appeared normal in spitz group mutants. Finally, dp-erk is localized to the tracheal placodes and the tracheal phenotype of flb mutant embryos could be rescued by expression of the normal DER protein only in the tracheal cells. With respect to the DPP pathway, again tkv and put mutants do not affect overall cell fates in the ectoderm. Lateral tracheal trunks fail to develop in the ventral region, which is clearly not patterned by the pathway. The ectopic effects of activated TKV could be observed under conditions where it was specifically expressed in the trachea. Finally, expression of branchless was slightly altered in tkv mutant embryos, but not to an extent that could account for the severe tracheal phenotype (Vincent et al., 1997). We have shown by ectopic expression experiments and partial loss-of-function mutations, that antagonistic interactions are taking place between the EGFR and DPP pathways in the tracheal placodes. We propose that subdivisions of tracheal fates that direct the migration behavior may emerge from a balance of the two pathways. Therefore, the relative concentration of the ligands SPITZ and DPP, as well as the relative potency of the two pathways, bring about the final outcome. In a situation where the DPP pathway was ectopically induced in all tracheal cells, additional cells were recruited to other branches, at the expense of the original ones. In mutants for the EGFR or DPP pathways, while subsets of tracheal branches are missing, the number of cells recruited into the normal branches is not altered. Thus, the normal activity of the DPP or EGFR pathway, respectively, is not sufficient to recruit Fig. 4. Autonomous function of EGFR in the trachea. (A) In flb 2C82 mutant embryos, the dorsal trunk (arrow) and visceral branches are not formed, as visualized by anti-trh antibodies (red). (B) Anti-DER staining (green) of the same embryo shows only weak general staining. (C,D) In a similar mutant embryo, which also carries btl-gal4 and UAS- EGFR, prominent EGFR staining can be detected at the sites of btl expression, including the tracheal cells and midline (arrow). Fusion of the dorsal trunk in several segments (arrowhead) can be detected. Other aspects of the Egfr phenotype, e.g. germband retraction, are not rescued by the localized expression of EGFR.

7 EGF receptor and DPP interactions in trachea 4713 Fig. 5. Tracheal phenotypes of mutants in the DPP pathway, and double mutants with rho. (A) tkv null mutant embryos show absence of the dorsal branch and lateral trunks (schematized in E). The visceral branch forms normally, but is less visible since it is in a lower focal plane. (B) put mutants also show absence of the dorsal branch and reduced lateral trunks. (C,D) in double mutants for tkv and rho, the cells remain in the original position of the tracheal pit and do not migrate (schematized in F). It is interesting to note that individual cells are still capable of migrating to their destinations (arrowheads in D), raising the possibility that the EGFR and DPP pathways may be inducing the expression of adhesion molecules that allow the cells in each tracheal branch to follow the leading migrating cell. Fig. 6. Antagonistic interactions between the EGFR and DPP pathways. (A) Expression of sog at stage 11 in a dorsal ectodermal stripe (tracheal pits are encircled). (B) sog mutant embryos lack the dorsal trunk (arrowhead) and visceral branch, and have an extended dorsal branch (arrow). (C) Ectopic expression of DPP in the tracheal pits of wild-type embryos (driven by btl-gal4) antagonizes the activity of the EGFR pathway in the dorsal trunk and visceral branch primordia (arrowhead), and diverts these cells to the dorsal branch (arrow). (D) Similar results were obtained when the expression of activated tkv was induced, demonstrating the cell-autonomous effect in the trachea (Schematized in H). (E) Induction of secreted SPITZ by btl-gal4 did not give rise to any phenotypes. (F) However, a similar induction in the sensitized background of tkv/+ embryos resulted in a tkv-like phenotype, where the dorsal branch was missing (arrow), but the dorsal trunk (arrowhead) and visceral branches formed normally (Schematized in I). (G) In a double mutant for the hypomorphic flb 2W74 and put alleles, rescue of the dorsal trunk (arrowhead), which is discontinuous in flb mutants, is observed (schematized in J).

8 4714 P. Wappner, L. Gabay and B.-Z. Shilo extra cells. It is possible, however, that the antagonistic activities of the two pathways help to define sharper borders between the different subsets of tracheal fates (see below). Model for tracheal patterning The branch fates induced by EGFR and DPP are in accordance with the spatially restricted activation of each pathway. RHO, which is essential for processing of SPITZ, is expressed in the central part of the placode. The dp-erk pattern showed that EGFR activation is more pronounced in the same region. However, since the dp-erk domain was broader than the RHO-expression pattern, it seems that there is a gradient of SPITZ originating in the center of the placode, and declining dorsally and ventrally. Conversely, the TKV receptor is expressed uniformly in the placode but may be specifically activated in the dorsal and ventral parts of the placode by DPP, emanating from stripes abutting those domains. We propose that two opposing gradients of DPP and SPITZ are operating within the placode. The cells in the center of the tracheal placode encounter high concentrations of secreted SPITZ, and low or negligible levels of DPP. Conversely, the cells located at the dorsal and ventral domains of the placode encounter high concentrations of DPP and low levels of secreted SPITZ. Therefore, induction by the EGFR and DPP pathways from opposing directions generates three subsets of cell fates, as shown schematically in Fig. 7. We postulate that these fates confer the capacity to migrate in distinct directions. This scenario is consistent with the phenotypes of the different mutants and with the ones observed upon ectopic expression. In spitz group mutants, the central cells remain unpatterned and fail to migrate, since only the dorsal and ventral cells receive DPP concentrations sufficient to confer the capacity to undergo migration. In contrast, upon ectopic DPP expression, all the cells encounter high ligand concentrations, thus instructing them to follow a dorsal branch migration behavior. The capacity of DPP gradients to induce different levels of receptor activation has been reported for other tissues, such as the wing and leg imaginal discs (Lecuit et al., 1996; Nellen et al., 1996; Goto and Hayashi, 1997). It should be noted that while the EGFR and DPP pathways are essential for patterning all tracheal branches, additional patterning systems may be required. For example, within ventral domain of the placode, it is not clear how the cells are subdivided precisely between the lateral trunk anterior and posterior branches. Similarly, within the EGFR domain, the basis for the distinction between dorsal trunk and visceral branch remains to be investigated. How are tracheal cell fates translated to directional migration? How does the activation of the EGFR or DPP pathways determine the capacity of a given tracheal cell to join one branch or another? We presume that activation of each pathway is translated into the induction of expression of different sets of genes, or post-translational modification of distinct proteins. One possibility is that the BRANCHLESS/BREATHLESS pathway is not sufficient to drive the actual migration of the tracheal cells. Activation of EGFR and TKV may thus participate in the induction of a parallel and essential guiding system. For example, each pathway could trigger the expression of a different receptor, one having a ligand that is expressed dorsally and ventrally, and the other triggered by a ligand expressed laterally. Only the combined activation of BREATH- LESS and one of these putative receptors could result in tracheal migration. Thus, the overlap between two activated receptors, which may initially be fairly coarse, could refine the system to determine more precisely the number of cells allocated to each branch. Another possibility is that the BRANCHLESS/BREATH- LESS system is sufficient to drive the migration of the tracheal cells. However, due to the localized distribution of BRANCH- LESS throughout migration, only the tip cell closest to the ligand source, is undergoing activation. This is indeed corroborated by the pattern of diphospho-erk observed in the migrating tracheal cells (Gabay et al., 1997b). Migration is thus driven by the leading cells, but the capacity of the other cells to follow and the integrity of each tracheal branch, requires specific adhesion between the cells. According to this possibility, the EGFR and DPP pathways do not affect the capacity of the tracheal cells to migrate per se, but rather their ability to be recruited to one branch or another, through specific adhesion interactions. If the latter option is correct, in a situation where both pathways were abolished, some tracheal cells, namely the leading cells in each branch, are expected to retain the capacity to migrate. This seems indeed to be the case, since in tkv, rho double mutants, we observed that single cells detach from the tracheal pits and migrate to the expected dorsal and ventral positions, while the other cells remain in the pit and are unable to follow (Fig. 5D). The involvement of cell adhesion molecules in tracheal morphogenesis is beginning to be characterized. Throughout tracheal migration, the continuity and integrity of the tissue is maintained. One would therefore expect cell adhesion molecules and in particular adherens junction proteins, to play an important role. Recent observations indicate that the D/E CADHERIN-ARMADILLO complex is involved in the formation of specific tracheal branches. Mutants for shotgun, encoding D/E CADHERIN in Drosophila, as well as for armadillo show specific loss of the tracheal dorsal trunk, while the other tracheal branches migrate normally (Uemura et al., 1996). This phenotype is similar to the one observed for Egfr and the spitz group mutants, and suggests a functional connection between the EGFR pathway in the trachea and the formation of adherens junctions. It is well established that D/E CADHERIN interacts with the β-catenin homologue ARMADILLO for the formation of adherens junctions, which are essential for Drosophila embryonic development (Cox et al., 1996). D/E CADHERIN promotes cell adhesion by mediating homophilic interactions through its extracellular domain. To participate in adherens junctions, D/E CADHERIN needs to be activated on its cytoplasmic domain by ARMADILLO with which it forms a complex. ARMADILLO has a dual function and also participates in the wingless signaling pathway. Therefore, ARMADILLO is differentially regulated for each of the two functions (reviewed in Peifer, 1995). ARMADILLO was shown to be phosphorylated on tyrosine, serine and threonine residues. Recruitment of ARMADILLO to adherens junctions may be dependent upon tyrosine phosphorylation (Peifer et al., 1994). In mammalian cells it was indeed shown that such phosphorylation of β-catenin depends upon the EGF receptor

9 EGF receptor and DPP interactions in trachea 4715 Many corollaries between development of the vertebrate vascular system and the Drosophila tracheal network are emerging. Most notably, the extensive involvement of growth factors and their receptors is being uncovered in both systems (reviewed in Folkman and D Amore, 1996). A major difference between the development of the two tissues is that recruitment of endothelial cells to new vessels depends upon cell division, where vascular endothelial growth factor (VEGF) functions both as a mitogen and a trophic factor. In contrast, cell division in the tracheal system terminates prior to the formation of branches and BRANCHLESS is responsible only for guiding migration of the cells. Since the number of cells in each branch is not modulated by cell division, it must be specified prior to the onset of migration, by the EGFR and DPP pathways. Fig. 7. Model for patterning the tracheal cells by the EGFR and DPP pathways. At stages 10-11, the EGFR pathway is activated in the tracheal placode. Due to RHO expression in the central part of the placode and restricted diffusion of secreted SPITZ, stronger activation levels are observed in the central part. In contrast, DPP is expressed on the ectoderm in two stripes abutting the tracheal placode. Thus, higher levels of TKV/PUT activation may be induced in the dorsal and ventral parts of the placode. As a result, two different cell populations are determined in the trachea; the EGFRinduced cells in the central part and the DPP-induced cells in the dorsal and ventral regions, thus generating a Spanish flag pattern. Antagonistic interactions between the EGFR and DPP pathways may also help to generate sharper borders between the different cell populations. When tracheal cell migration begins at stage 12, different tracheal cells will be recruited to different tracheal branches, according to the fate they assumed. It is possible that activation of BREATHLESS by BRANCHLESS induces only the migration of the tip cells (marked by *), and the fate of the other cells would determine which leading tip cell they will join. pathway (Shibamoto et al., 1994; Hoschuelsky et al., 1994). Similar to mammalian cells, the EGFR pathway may phosphorylate ARMADILLO on tyrosine residues, recruiting it into adherens junctions, specifically in the dorsal trunk cells. A different set of adhesion molecules may be regulated by the DPP pathway in the other tracheal branches. Concluding remarks Common features between the different tracheal branches have been described. These collective hallmarks include guidance of migration by the universal BRANCHLESS/BREATHLESS system, and the induction of the same markers (e.g. pointed, pruned or escargot) in subsets of cells within all branches (Samakovlis et al., 1996). 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