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. Author manuscript; available in PMC: 2008 Aug 1.
Published in final edited form as: Dev Biol. 2007 May 3;308(1):30–43. doi: 10.1016/j.ydbio.2007.04.047

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis

Anita Hsouna a,1, Hakeem O Lawal b,1, Iyare Izevbaye b,2, Tien Hsu a,*, Janis M O'Donnell a,*
PMCID: PMC1995089  NIHMSID: NIHMS28375  PMID: 17585895

Abstract

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, we noted interesting and unexpected mutant phenotypes in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. We also demonstrate expression of Punch, Ple, Catsup and dopamine in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, we show that these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally we show that dopamine regulates endocytosis via controlling the dynamin protein level.

Keywords: Drosophila, trachea, dopamine, Punch, pale, Catsup, breathless, endocytosis, GTP cyclohydrolase, tyrosine hydroxylase

Introduction

The development of the Drosophila tracheal system, an extensively branched system of oxygen delivery tubes, models the patterning of vasculature in mammalian angiogenesis, both in cellular mechanisms of directed migratory behavior and in the conserved roles of numerous gene products (for reviews, see Metzger and Krasnow 1999; Affolter and Shilo 2000; Ghabrial et al. 2003). Detailed genetic analysis of Drosophila tracheal development has led to the identification of more than thirty genes (Affolter and Shilo 2000) engaged in patterning this complex structure. In both flies and mammals, the key defining process is the outgrowth of branches, regulated by growth factor signaling pathways. In Drosophila, the fibroblast growth factor (FGF) signaling pathway is utilized repeatedly to pattern the trachea in a stereotyped fashion with branching occurring in distinct stages: the primary and secondary branching forming through a tightly regulated genetic program (Metzger and Krasnow 1999) and tertiary branching arising in response to tissue oxygen demand (Wigglesworth 1959; Jarecki et al. 1999). The tracheal network arises during embryogenesis from laterally and segmentally repeated placodes each of 80-100 cells. Tracheal cells express the FGF receptor encoded by the breathless gene (btl; Lee et al. 1996). These cells migrate outward from a tracheal placodes in response to FGF (Drosophila Bnl, encoded by branchless) emanating from surrounding mesodermal cells (Sutherland et al. 1996). The entire tracheal architecture is formed by cell elongation, migration and rearrangement without further cellular division (Metzger and Krasnow 1999; Affolter and Shilo 2000), making it an ideal system for studying tubular cell migration. Disruption of FGF-FGFR signaling has a direct effect on tracheal tubule formation. Misexpression of bnl from sources other than the abutting mesodermal cells causes migration of tracheal cells to the ectopic source (Sutherland et al. 1996; Ribeiro et al. 2002), while expression of a constitutively active Btl receptor can cause the sprouting of supernumerary branches (Lee at al. 1996).

In the course of examining developmental roles of genes that regulate the biosynthesis of catecholamines, dopamine (DA) in particular, we found that abnormal embryonic tracheal patterning was a common mutant phenotype for these genes (Reynolds and O'Donnell 1987). This is surprising, since more typically, DA serves as a neurotransmitter and neurohormone in Drosophila, as in mammals, although previous studies in both Drosophila and mammals have implicated catecholamines in developmental processes such as embryonic nuclear division and ovarian development (Chen et al. 1994; Neckameyer 1996; Pendleton et al. 1996; Mayerhofer et al. 1997; Neckameyer et al. 2001; Pendleton et al. 2005) such as embryonic nuclear division and ovarian development.

The DA biosynthesis machinery is highly conserved in mammalian and Drosophila systems. DA production requires the tightly regulated collaboration of two enzymatic pathways. The DA pathway itself is initiated by the conversion of tyrosine to L-3-4-dihydroxyl-phenylalanine (L-Dopa), which is subsequently converted to DA (Nagatsu et al. 1964; Axelrod 1971). The rate-limiting step in the pathway, conversion of tyrosine to L-Dopa, is catalyzed by the enzyme tyrosine hydroxylase (TH), which is encoded by pale (ple) in Drosophila (Neckameyer and White 1993). Human and Drosophila TH share 60% amino acid similarity (Neckameyer et al. 2005). TH catalytic activity requires and is regulated by the cofactor, tetrahydrobiopterin (BH4), synthesized via the pteridine pathway (Krishnakumar et al. 2000). The initiating and limiting component of BH4 biosynthesis, and therefore, of DA production, is the activity of the enzyme GTP cyclohydrolase I (GTPCH). The human and Drosophila GTPCH proteins share 80% similarity (McLean et al. 1993). Mutations in the Drosophila GTPCH gene Punch (Pu) result in dose-dependent deficiencies in DA pools (Mackay et al. 1985; Weisberg and O'Donnell 1986; O'Donnell et al. 1989; Krishnakumar et al. 2000). Similarly, mutations in the human GTPCH locus lead to the hereditary diseases hyperphenylalaninemia and Dopa-responsive dystonia (reviewed in Thöny et al. 2000). Both Pu and ple mutations are homozygous lethal during embryogenesis. A third regulatory gene of DA synthesis, Catecholamines up (Catsup), acts as a negative regulator of both GTPCH and TH. The protein encoded by Catsup contains seven predicted transmembrane domains and functions in post-translational modification of both enzymes. Homozygous loss-of-function alleles of Catsup also show developmental defects and die in an allele-dependent fashion, from embryogenesis to pupariation (Stathakis et al. 1999).

Here we demonstrate a role for the DA biosynthesis pathway genes in regulation of tubule migration in Drosophila trachea, and report new findings that the action of these genes is mediated via dopamine by controlling FGFR endocytosis.

Materials and methods

Drosophila strains and genetics

The Pu mutant alleles employed in this study were generated in EMS mutagenesis screens. The genotypes of the strains are: dp cn PuAA17 a px sp/SM1, dp cn PuZ22 a px sp/SM1, and shv b cn PuK5-2 bw/SM1. The genetic characteristics of these strains are reported in Mackay et al. (1985) and Reynolds and O'Donnell (1988); all are homozygous lethal and arrest during late embryogenesis. The homozygous lethal ple2 is a loss-of-function allele recovered in an EMS screen (Neckameyer and White 1993). The homozygous lethal Catsup26 allele is a 600 base pair deletion derived from the mobilization of a 5' P-element insertion in CatsupKO5042 (Stathakis et al. 1999). Pu and Catsup mutants were maintained in a CyO-GFP background by crossing mutant flies into a w; In(2LR)noc[4L]Sco[rv9R], b1/CyO, P{ActGFP}JMR1 balancer chromosome. Mutant fly stocks of awdj2A4 and shi2 were obtained from the Bloomington Stock Center and btlH82Δ3 was a gift from D. Montell (Johns Hopkins University, Baltimore, MA; Lee et al. 1996). awdj2A4 is a single P-element insertion, resulting in a completely null genotype (Krishnan et al. 2001; Dammai et al. 2003). The insertion site has been mapped to the 5′ untranslated region by the Drosophila genome project (Spradling et al. 1999). Both awdj2A4 and btlH82Δ3 are homozygous lethal and are maintained by pairing with the balancer TM3, P{ry+ t7.2 =HZ2.7}DB2 Sb. The DB2 reporter gene shows β-galactosidase (β-Gal) expression in the maxillary segment. shi2 is a temperature-sensitive allele of shibire that encodes Drosophila dynamin (van der Bliek and Meyerowitz 1991). Genetic recombination was used to incorporate 1-eve-1, a transgenic line expressing the lacZ reporter gene in all tracheal cells at all stages from the trachealess gene promoter (Perrimon et al. 1991; a gift from A. Brand, University of Cambridge, Cambridge, UK). Transheterozygous embryos of shi2 or ple2 with 1-eve-1 were generated by standard genetic crosses.

The y w; btl-Gal4; UAS-GFP, UAS-lacZ fly was constructed as a trachea marker strain. The y w; btl-Gal4; UAS-btl::GFP transgenic line expresses a Btl::GFP fusion protein in btl-expressing tissues, including trachea. It was provided to the Hsu lab by T. Kornberg (University of California, San Francisco) and has been characterized previously (Sato and Kornberg 2002; Dammai et al. 2003). Note that a dash (-) is used to denote a promoter/enhancer fusion with a reporter gene while a double colon (::) is used to denote an open-reading-frame fusion. For example, btl-Gal4 designates the yeast Gal4 cDNA downstream of the btl promoter; while UAS-btl::GFP designates a btl/GFP fusion protein coding sequence regulated by the yeast UAS enhancer element. The binary Gal4/UAS transgenic expression system has been described (Brand and Perrimon 1993).

To generate Pu transgenic strains, the wild type Pu open reading frame were cloned into the P-element transformation vector (pUAST) downstream of the UAS enhancer element. The resulting transformation vector was then purified and injected into w1118 embryos by Genetic Services® (Cambridge, MA) and transgenic animals were isolated using established methodology (Spradling and Rubin 1982). Expression of the transgenes in trachea was achieved by mating homozygous strains with btl-Gal4 strains.

Punch rescue constructs w, UAS-Pu; PuZ22/ CyO, P{actGFP}JMR1; +/+ and w; PuZ22/ CyO, P{ActGFP}JMR1; btl-Gal4 were generated separately and crossed. Progeny with the genotype w, UASPu/+; PuZ22/PuZ22; btl-Gal4/+ were then selected as embryos without constitutive (actin) GFP expression and subjected to further analysis.

Antibodies

Rabbit anti-Drosophila GTPCH antibodies (Chen et al. 1994) were purified using Protein Aagarose beads (BioRad, Hercules, CA) and concentrated to 10 mg/ml. A 1:4000 dilution was used for immunohistochemistry (IHC). The rabbit anti-Ple antibody has been described (Neckameyer et al. 2000; Neckameyer et al. 2001) and was used at 1:2,000. Guinea pig anti-Catsup antibody has been described (Stathakis et al. 1999) and was used at 1:2,500 dilution after preadsorption with wild-type embryos. The mouse monoclonal anti-dopamine antibody (AbCam) was used at 1:5000. The mouse monoclonal anti-dp-MAPK (Sigma) was used at 1:500. The mouse monoclonal antibody 2A12 was obtained from the Developmental Studies Hybridoma Bank (DSHB, University of Iowa) and used at a 1:3 dilution. Mouse monoclonal anti-Drosophila dynamin (Shibire) antibody (BD Biosciences) was used at 1:250 for embryos and 1:300 for S2 cells in IHC and 1:2000 in Western blotting. Mouse monoclonal anti-β-Gal (Sigma) was used at 1:3000 for IHC. Rabbit anti-GFP (AbCam) was used at 1:200 for S2 cells in IHC and 1:2000 for Western blotting. Mouse monoclonal anti-Drosophila Crumbs antibody (from DSHB, University of Iowa) was used at 1:4 dilution for IHC.

Western blot of embryonic extracts

Wild type and mutant embryos were collected from grape juice agar plates after 17 hours of egg-laying at 25C° and dechorionated in 50% bleach for 3 minutes. After rinsing in H2O, embryos were sonicated for 8 seconds on ice in extraction buffer (50 mM HEPES, pH7.6, 100 mM KCl, 1 mM EGTA, 1 mM MgCl2, 1% Triton X-100, 10% glycerol) with protease inhibitor (from Roche) and supernatants were collected after centrifugation at 3,000 x g for 20 min at 4 °C. 50 μg of protein extracts were run on 7% SDS-polyacrylamide gel, electro-blotted, probed with anti-Drosophila dynamin (Shibire) antibody at 1:2000 (BD Biosciences), and reprobed with anti-β-actin antibody at 1:10,000 (mouse monoclonal from Sigma). The intensity of the bands was quantified and the relative intensities of the dynamin bands were normalized against the actin loading control using the Kodak Gel Doc II Software.

Immunohistochemistry

Whole-mount immunohistochemistry followed standard procedures (Hsu et al. 2001; Dammai et al. 2003). For fluorescence staining, Alexa Fluor 488- or 546-conjugated secondary antibodies were used (Invitrogen/Molecular Probes) at 1:200 dilution in 2-hr incubation at room temperature. For color staining, biotin-conjugated secondary antibodies (Vector) were used at 1:1000 dilution in 2-hr incubation at room temperature. Color development used the VectaStain reagents following the manufacturer's instruction (Vector).

Pharmacological treatment of embryos

Wild-type and mutant embryos were collected for twenty minutes on fresh grape agar plates, following a 90-min pre-lay. Embryos were washed in 1x PBS and exposed to heptane for twenty seconds after which they were immediately soaked in 1x PBS or 1x PBS containing DA (0.1 M; from Sigma) or 3-IT (2 mg/ml; from Sigma) for 5 min. The dosages were pre-determined empirically to be sub-lethal. The embryos were then transferred to fresh agar plates, placed in a humid chamber, and incubated at 25 °C for 17-24 hr.

Drosophila Schneider 2 (S2) cell culture and treatments

S2 cells (obtained from American Type Culture Collection) were maintained in Schneider's media supplemented with 10% heat-treated FBS (Cambrex). For immunohistochemistry, they were grown on coverslides coated with poly-L-lysine and were transfected using Lipofectamine 2000 (from Invitrogen) with pUC-HygMT-btl::GFP (as described in Dammai et al. 2003). Btl::GFP fusion protein expression was induced with 10 μM CuSO4 for 2 hr. The cells were then treated with buffer, DA (final concentration 0.1 mg/ml) or 3-IT (final concentration 1 mg/ml) for 12 hr, fixed and stained for GFP (rabbit polyclonal from AbCam, used at 1:200) and Drosophila Dynamin (Shibire) (mouse monoclonal from BD Biosciences, used at 1:300). There was limited or no cell death at these drug dosages. S2 cell extracts were prepared and Western-blotted as described previously (Hsu et al. 2001), and probed with anti-GFP (rabbit polyclonal from AbCam, used at 1:2000) and reprobed with anti-Drosophila dynamin (Shibire) antibody (mouse monoclonal from BD Biosciences, used at 1:2000) and with anti-β-actin antibodies (mouse monoclonal from Sigma, used at 1:10,000). The intensity of the bands was quantified using the Kodak Gel Doc II Software.

Results

Punch is expressed in the trachea during the migratory phase of development

Specific expression of Punch in the trachea has been noted (Samakovlis et al. 1996; Chen 1995). However, the correlation between DA synthesis and tracheal development has not been explored. As a first step toward elucidating the role of Punch in tracheal development, we analyzed more closely its temporal and spatial expression patterns. For comparison, the tracheal cells were marked by a transgenic line 1-eve-1 that expresses β-Galactosidase (β-Gal) in all tracheal cells at all stages, under control of the trachealess promoter (Perrimon et al. 1991). Tracheal development starts at the embryonic stage 10 with the formation of 10 placodes on each side of the embryos. These ectodermal placodes start invaginating inward at stage 11 (1-eve-1 in Fig. 1A) and begin to extend branches at stage 12. These branches continue to extend and connect with the neighboring subunits during stage 13 through stage 15, until the stereotypic network of primary and secondary tubules are formed at stage 16. We found that strong Punch expression in the trachea encompasses only the mid-phase of tracheal development. Compared with the pan-tracheal reporter expression in 1-eve-1, Punch expression is transient. It starts weakly at stage 11 and becomes most abundant from stage 12 to stage 14, coinciding with the active phase of tracheal branch migration (Fig. 1A). It fades after stage 15, when tubule extension is complete. To determine more precisely the subcellular localization of Punch, the trachea were double-stained for Punch and the transmembrane protein Crumbs (Crb). Enlarged views of portions of individual tracheal subunits are shown in Figure 1B. At stage 12, Punch in the developing trachea completely overlaps with Crb, indicating localization at or near the cell periphery. The peripheral localization of Punch is maintained through stage 14, in addition to some intracellular staining (red staining; arrowhead in the inset). At stage 15, when tracheal lumen begins to expand, weak peripheral localization of Punch is still detectable (solid arrow in the inset), while staining is also seen in the lumen (empty arrow in the inset). Intracellular and lumen Punch expression becomes barely detectable at stage 16 (data not shown). The significance of lumen staining at stage 15 is not clear at this time but may reflect the role of L-Dopa in cross-linking lumen cuticles (Wright 1987) and that the Punch-containing enzymatic complex may function extracellularly for this developmental role.

Fig. 1.

Fig. 1

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Pu and Catsup are involved in tracheal development. (A) Comparison of β-Gal (directed by the trachea-specific trachealess promoter) and Punch expression patterns in the trachea. β-Gal expression in 1-eve-1 marks all tracheal cells at all stages while Punch is expressed most abundantly in the trachea from stage 12 to stage 14. The insets show enlarged views of individual tracheal subunits stained for Punch. Arrows point to the site from which the dorsal trunk extends. The expression of Punch fades after stage 14. Bars are 50 μm. (B) Close-up views of portions of single tracheal subunits at stage 12, 14 and 15, double stained for Punch (red) and Crb (green). Crb is a transmembrane protein and Punch overlaps with Crb completely in the trachea at stage 12. At stage 14 Punch still shows extensive overlap with Crb in addition to some intracellular localization (arrowhead). At stage 15, peripheral localization of Punch is diminished (arrow) with an increased expression in the lumen (open arrow). Bars are 10 μm. (C) y w embryos were stained with anti-Ple or anti-Catsup antibodies, as indicated. The insets show enlarged views of two tracheal subunits with Ple or Catsup expression. The higher level of general staining indicate that the two proteins are expressed in multiple tissues. Bars are 10 μm. (D) Embryos of indicated genotypes were stained with a tracheal lumen-specific antibody 2A12. Unless specified, all embryos are oriented with anterior to the left and dorsal side up. (a) y w represents wild-type. The major tracheal branch that runs along the length of the embryo, dorsal trunk, is marked (DT). (b-c) Embryos homozygous for two Pu alleles Z22 and K5-2 show disrupted tracheal network. The insets highlight ectopic branches (arrowheads). (d) shi2 temperature-sensitive mutant embryos were collected at 25° for 7 hr and then incubated at 30° for 7 hr. The inset highlights ectopic branches (arrowheads). (e) An awdj2A4 mutant. The inset highlights ectopic branches (arrowheads). (f-g) Two Catsup26 mutant embryos showing moderate and severe phenotypes, respectively. Empty arrows in (f) mark the gaps in the dorsal trunk, while (g) shows almost no branch migration. (h) Ventral view of a btlH82Δ3 mutant embryo showing little branch migration. Bars are 50 μm.

We also detected tracheal expression of the other two DA synthesis pathway components Ple and Catsup (Fig. 1C). It should be noted that the expression of these genes includes strong maternal contributions (Stathakis et al. 1999; Neckameyer et al. 1993; Neckameyer et al. 2000). Thus, although the two antibodies have proved specific (Stathakis et al. 1999; Neckameyer et al. 2000; Neckameyer et al. 2001), immunostaining of the embryos shows general staining in multiple tissues. Nonetheless, above-background staining in the trachea can be observed (Fig. 1C, insets) throughout tracheal development for both proteins.

DA synthesis pathway mutants exhibit tracheal development phenotypes

Specific expression of Punch in the tracheal cells during branch migration suggests a role in tracheal morphogenesis. In our initial examination of the tracheal network, monoclonal antibody 2A12 against a lumen antigen was used (Fig. 1D). Compared to wild-type (y w; Fig. 1Da), Pu mutants show misdirected tubules and ectopic branching, in addition to disruption of the dorsal trunk (Figs. 1Db-c). These phenotypes were generated by multiple alleles and in both heterozygotes and homozygotes. The examples shown are embryos developed through stage 14-15, as judged by well-developed midgut (not shown), in order to avoid secondary effects from early embryonic defects. These ectopic branching phenotypes are similar to those observed in mutants of Drosophila metastasis suppressor Nm23 homolog (awd) and dynamin (shi), which cooperate in internalizing and down-regulating FGFR (Btl) (Dammai et al. 2003; Figs. 1Dd-e). By contrast, mutants of Catsup, which antagonizes Pu, exhibit phenotypes lacking tracheal branches. These lack-of-migration phenotypes of Catsup mutants range from a few gaps in the dorsal trunk (Fig. 1Df) to an almost complete lack of tubule formation (Fig. 1Dg). No Pu-like ectopic branches are observed in these mutants. The Catsup phenotypes are reminiscent of those observed in the btl mutants (see Fig. 1Dh as an example; Lee et al. 1996).

DA synthesis pathway gene functions regulate tracheal cell migration

Lumen staining using 2A12 monoclonal antibody is useful for observing overall structure of the tracheal network, but the cellular behavior underlying the branching defects cannot be discerned. For example, gaps in the dorsal trunk may result from mis-directed tubule migration that fails to connect with the lateral subunits (ectopic migration) or from lack of migration. That is, apparently similar phenotypes can result from opposite cellular behaviors. We therefore combined the mutant genes of interest with the 1-eve-1 transgene that marks all tracheal cells. 1-eve-1 alone showed limited or no tracheal phenotype (Dammai et al. 2003; Fig. 2A). In Pu mutants, ectopic looping and branch migration are observed (Fig. 2B-C). Importantly, in severely disrupted trachea, gaps in the dorsal trunk are always accompanied by mis-directed tracheal cells (arrow in Fig. 2C). These phenotypes are similar to those observed in the shi and awd mutants (Fig. S1) that are known to result in ectopic migration (Dammai et al. 2003). In the most extreme cases there was a complete loss of tracheal network while rounded “run-away” tracheal cells were seen throughout the embryos (Fig. 2D). Note that for these phenotypic analyses, we only documented late-stage embryos with developed body parts such as gut (empty arrowhead in Fig. 2C) or mandible structures (brackets in Fig. 2C and 2D), in order to avoid complications from early embryonic defects. The Pu phenotypes can be rescued by exogenously expressing a UAS-Pu transgene controlled by btl-GAL4 in the trachea (Fig. 2E), even at the suboptimal expression temperature of 18°. Incubation at 25° resulted in a greater extent of rescue (fewer ectopic migration phenotypes) and the appearance of the opposite phenotype (lack of migration). Induction of opposite phenocopies by loss-of-function and over-expression of Pu strongly supports the notion that Pu has a specific dosage-dependent role in regulating tubule migration. These results indicate that the tracheal phenotypes in Pu mutants are the result of defective Pu function in the trachea, not indirect effects from early patterning or from neighboring tissues. The effectiveness of the transgene expression was confirmed by measuring the tyrosine hydroxylase activity in the embryonic extracts (Supplementary Fig. S2). Conversely, the Catsup mutant showed lack-of-migration phenotypes similar to that in btl mutants (Figs. 2F-G and Fig. S1). Note that there are no ectopic tracheal cells emanating from the tracheal tube ends at the gaps of dorsal trunk, indicating that these gaps result from lack of migration, not ectopic migration.

Fig. 2.

Fig. 2

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Tracheal cell migration defects in DA synthesis pathway mutants. The embryos were stained for β-Gal. Unless specified, all mutants are homozygotes marked by the 1-eve-1 transgene that expresses β-Gal under control of the trachealess promoter. All embryos are oriented with anterior to the left and dorsal side up. (A) 1-eve-1 represents wild-type. (B-D) Mutant embryos homozygous for two Pu alleles AA17 and Z22. In inset (Bb), arrows point to ectopically migrating branches and tracheal cells. Sharp arrows point to ectopic looping of the dorsal branches. Bracket marks convoluted dorsal trunk. (Cc) shows severe disruption of the tracheal network with abnormal branching and sprouting. Arrow points to the misguided branch emanating from the truncated dorsal trunk. (Dd) shows a severe Pu mutant; rounded “run-away” tracheal cells can be identified (arrowheads). (E) Rescue of Pu phenotypes by Pu transgene expression in trachea. Wild-type Pu expression in Pu mutant background gives three broad phenotypic classes as indicated. w; PuZ22/PuZ22; btl-Gal4 or w, UAS-Pu; PuZ22/PuZ22; +/+ each gave similar distribution of phenotypes, thus only w; PuZ22/PuZ22; btl-Gal4 was tabulated as the parental Pu mutant. The above two lines were crossed to generate the rescue lines, as described in Materials and Methods. The embryos were incubated at 18° or 25° to control the expression level of the Pu transgene. (F-G) Mild and severe phenotypes of Catsup mutant showing missing branches and stunted branch migration. Note that there is no ectopic branching at or near the gaps in tracheal tubes in these mutant, indicating loss-of-migration. (H) A ple heterozygote showing moderate tracheal phenotypes similar to those in Pu mutants. (Hh) Sharp arrow points to ectopic looping, arrow points to ectopically migrating branches at the breakage point and arrowhead points to ectopically migrating cells. (I) A ple heterozygote showing convoluted tracheal trunk (bracket). Bars are 50 μm.

Interestingly, heterozygous mutants of ple showed similar ectopic migration phenotypes (Fig. 2H) as in Pu mutants. Note that heterozygous ple was examined for tracheal phenotypes because homozygous ple exhibited severe early embryonic defects. Besides ectopic migration phenotypes, we often observed in ple mutants (Fig. 2I), and less frequently in Pu mutants (Fig. 2B), the appearance of convoluted dorsal trunks. Such phenotypes have been linked to defects in secretion and deposition of cuticle chitin in the tracheal lumen (Luschnig et al. 2006; Wang et al. 2006). It is therefore worth noting that L-Dopa, the direct enzymatic product of Pale/TH, is required for cuticle cross-linking (Wright 1987). However, cuticle deposition and lumen formation are initiated at late stage 14. As such, this aspect of the Pu and ple function is temporally distinct from the earlier tubule migration function.

DA directly regulates tracheal cell migration

Since the product of Punch/GTPCH enzymatic function, BH4, is a cofactor of the Pale enzyme (TH), which in turn is a rate-limiting enzyme in the DA synthesis pathway, the similar tracheal phenotypes observed in Pu and ple mutants suggest that DA itself may play a role in modulating tracheal development. Indeed, DA is enriched in the wild-type trachea (Fig. 3A) but is absent in the Pu mutant (Fig. 3B). The pattern is consistent with the enrichment of Pu in tracheal cells (Fig. 1A). We next examined whether DA has a direct role in tubulogenesis. In this assay, a transgenic line expressing a GFP reporter gene driven by the btl enhancer was used to allow for examination of live embryos. The Catsup (lack of migration) and Pu and ple (ectopic migration) phenotypes were reproduced by DA and 3-iodotyrosine (3-IT, a TH inhibitor) treatments, respectively (Fig. 3C-F). Note that the dosages of DA and 3-IT were sublethal as the embryos developed normally into late stages. DA treatment resulted in gaps in the dorsal trunk (empty arrow in Fig. 3D) accompanied by clumps of tracheal cells that did not migrate out of the dorsal trunk (empty arrowheads in Fig. 3D). The clumping of tracheal cells in the dorsal trunk is in contrast to the normal and 3-IT treated samples that show clear and hollow lumenal space (sharp arrows in Figs. 3C and 3E). This phenotype is consistent with reduced tracheal cell migration. By contrast, in 3-IT treated embryos, misdirected branches (arrow in Fig. 3E) and ectopically localized tracheal cells were observed (bracket in Fig. 3E). The mislocalized tracheal cells are rounded and are not organized into tubule structures. All phenotypic classes generated by DA and 3-IT are summarized in Figure 3F. They are consistent with the lack-of-migration (DA treatment) and ectopic migration (3-IT treatment) mutant phenotypes described in Figure 2.

Fig. 3.

Fig. 3

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Tracheal development can be modified with DA and a TH inhibitor in wild-type embryos. (A-B) 1-eve-1 or PuAA17; 1-eve-1 embryos were processed for double-staining with anti-DA (green) and anti-β-Gal (red) antibodies. DA is enriched in the wild-type trachea but absent in Pu mutant. (C-F) y w embryos carrying btl-GAL4 and UAS-GFP transgenes were collected at 25 °C and mock treated (control) or treated with DA or 3-IT. Stage 15-16 embryos were examined under a fluorescence microscope. The GFP reporter gene is expressed in all tracheal cells. All embryos are shown with dorsal side on the left and anterior to the top. (C) A mock-treated embryo displays an orderly patterning of tracheal architecture. The sharp arrow points to well-formed lumen. (D) A DA-treated embryo showing a gap in the dorsal trunk (empty arrow). The enlarged view shows accumulation of non-migrating cells (empty arrowheads) clustered in the dorsal trunk, obstructing the lumen. (E) A 3-IT treated embryo showing ectopic branches (arrow). The enlarged view shows ectopically located and abnormally shaped (rounded) tracheal cells (bracket). The sharp arrow points to a well-formed lumen. (F) Quantification of the above recorded phenotypes (C-E). The drug exposure protocol was optimized to ensure minimal early developmental defects and increased embryo viability. Only embryos surviving past stage 14 were counted. Scale bars in (A-B) are 10 μm; in (C-E) are 50 μm.

DA synthesis pathway regulates internalization of Btl receptor

We have previously shown that awd and shi cooperate in internalizing Btl, thus limiting the chemotactic signaling strength (Dammai et al. 2003). The striking phenotypic similarities between Pu/ple and awd/shi, as well as between Catsup and btl, suggest that DA synthesis pathway components may also modulate the FGFR signaling. If this hypothesis is correct, Pu and ple mutants are expected to increase the FGFR level, resulting in ectopic migration, while in Catsup mutants, the FGFR level should be down-regulated, resulting in reduced migration. Therefore, Pu should exacerbate, and Catsup rescue, the phenotypes of awd and shi; while reduced dosage of FGFR (in btl mutant) should rescue Pu but exacerbate Catsup. Since we have observed that the tracheal developmental functions of these genes of interest are partially haplo-insufficient (Dammai et al. 2003; data not shown), a simple transheterozygous analysis was conducted. All mutant alleles are marked with the 1-eve-1 transgene for visualizing tracheal cells. The severity and characteristics of ectopic migration and lack-of-migration phenotypes are defined in Figures 2 and Supplemental Figure S1. As shown in Table 1, PuAA17 and awdj2A4 heterozygotes showed 84% and 81% mutant embryos with ectopic migration defects, respectively, while combined heterozygous and hemizygous shi2 (progenies of X-linked homozygous shi2 females crossed with wild-type males) showed 90% embryos with ectopic migration phenotypes. Transheterozygotes of PuAA17-awdj2A4 and PuAA17-shi2 (the latter includes hemizygous shi2) resulted in 100% penetrance with greatly enhanced severity. Conversely, btlH82Δ3 and Catsup26 heterozygotes showed 84% and 67% embryos with lack-of-migration phenotypes, respectively, while btlH82Δ3-Catsup26 transheterozygotes resulted in 100% penetrance for the same phenotype and with enhanced severity. When mutations generating opposite phenotypes were combined, as in PuAA17-btlH82Δ3 or Catsup26-awdj2A4 transheterozygotes, the resulting severity of the respective phenotypes was greatly reduced. The same analyses were performed with another Pu allele PuZ22 and they showed the same pattern of genetic interactions. As a validation of the genetic interaction scheme, we combined heterozygous Catsup with either of the two Pu alleles; the transheterozygotes showed improved ectopic or lack of migration phenotypes. In summary, btl allele can rescue different Pu alleles but Pu phenotypes are exacerbated by awd and shi. By contrast, the Catsup phenotype was exacerbated by btl but rescued by awd.

Table 1.

Genetic interactions between DA synthesis and Btl endocytosis pathway genes.

Genotypes n % Normal % Ectopic migration
phenotypea 		% Lack-of-migration
phenotypea
moderate severe moderate severe
PuAA17/+; 1-eve-1/+ 24 16 71 13 0 0
awdJ2A4, 1-eve-1/+ 26 19 69 12 0 0
shi2/+ or Y; 1-eve-1/+ 21 10 66 24 0 0
PuAA17/+; 1-eve-1/awdJ2A4, 1-eve-1 19 0 53 47 0 0
shi2/+ or Y; PuAA17/+; 1-eve-1/+ 17 0 53 47 0 0
btlH82D3, 1-eve-1/+ 25 16 0 0 60 24
Catsup26/+; 1-eve-1/+ 21 33 0 0 67 0
Catsup26/+; 1-eve-1/btlH82D3, 1-eve-1 18 0 0 0 55 45
PuAA17/+; 1-eve-1/btlH82D3, 1-eve-1 22 41 46 0 13 0
Catsup26/+; 1-eve-1/awdJ2A4, 1-eve-1 17 35 36 5 24 0
PuZ22/+; 1-eve-1/+ 21 33 57 10 0 0
PuZ22/+; 1-eve-1/awdJ2A4, 1-eve-1 15 0 46 54 0 0
shi2/+ or Y; PuZ22/+; 1-eve-1/+ 16 0 44 56 0 0
PuZ22/+; 1-eve-1/btlH82D3, 1-eve-1 20 35 35 5 25 0
Catsup26/PuAA17 21 57 38 0 5 0
Catsup26/PuZ22 19 74 26 0 0 0
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a

As defined in Figures 2 and S1. Moderate phenotype is defined as identifiable defects in tubule structures while the overall tubular network is intact. Severe phenotype is defined as lack of overall tracheal network due to disruption of organized tubule movement (ectopic migration) or severe blockade of migration.

To examine whether dopamine synthesis mutants affect Btl/FGFR signaling, we examined the activity of MAP kinase (MAPK), the main transducer of FGFR signaling, within the tracheal cells. In wild-type (1-eve-1), activated MAPK (doubly-phosphorylated MAPK, dp-MAPK), is detected only in the migrating tip cells (arrowheads in Fig. 4A). In Pu mutants (Fig. 4B and 4C), there is significantly elevated levels of dp-MAPK in all tracheal cells, concomitant with ectopic migration phenotype. By contrast, in Catsup mutant, dp-MAPK is not detected in all tracheal cells, especially at the stunted tracheal tube ends (empty arrows in Fig. 4D). Therefore, the Pu ectopic migration phenotype can be correlated with the increased MAPK activity, while the Catsup lack-of-migration phenotype is consistent with reduced MAPK activity.

Fig. 4.

Fig. 4

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MAPK activation levels in DA synthesis pathway mutants. 1-eve-1, PuAA17; 1-eve-1, or Catsup26; 1-eve-1 embryos were processed for double staining with anti-dp-MAPK and anti-β-Gal antibodies. In wild-type embryos (A), dp-MAPK is detected in the migrating tip cells (arrowheads). In Pu mutants (BC), dp-MAPK is over-expressed in all tracheal cells. In Catsup mutant embryos (D) dp-MAPK is not detectable in the tracheal cells, especially not in the stunted tube ends (empty arrows).

The observed genetic interactions between Pu and endocytic pathway mutants (awd and shi) raised the interesting prospect that DA synthesis pathway may also be involved in Btl/FGFR endocytosis in tracheal cells. To test this model, we first examined the expression of a Btl::GFP fusion protein expressed in the trachea. This fusion protein has been used in previous studies and has been shown to be functional (Sato and Kornberg 2002; Dammai et al. 2003). In the wild-type (1-eve-1) embryo without treatment, there is localization of Btl::GFP at the cell periphery as well as localization in intracellular vesicles (Fig. 5A). With DA treatment, there is a significant reduction in membrane Btl::GFP and a concomitant increase in the Btl::GFP-containing vesicles, both in size (arrowheads in Fig. 5B) and in number (Fig. 5D). By contrast, when the embryos are treated with 3-IT, there is an increased level and a more defined appearance of the peripheral Btl::GFP (i.e. sharper outlines of the tracheal cells; arrows in Fig. 5C) and a decrease in intracellular vesicular Btl::GFP (Fig. 5D).

Fig. 5.

Fig. 5

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Dopamine promotes internalization of Btl receptor. (A-C) Embryos of transgenic flies btl-GAL4; UAS-btl::GFP were treated with buffer (control), DA or 3-IT and observed for Btl::GFP fusion protein expression. Anterior is to the left and the dorsal side is up. All images were acquired using identical settings to ensure accurate quantification. Middle panels show enlarged views of the indicated areas in top panels. Arrows in the middle panels point to the position of three cells further enlarged in the bottom panels. (A) A stage 15 embryo shows normal Btl-GFP distribution at plasma membrane and some cytoplasmic localization in vesicles. (B) A stage 15 embryo treated with DA. The level of membrane Btl-GFP is greatly reduced. (C) A stage 15 embryo treated with 3-IT. Btl::GFP shows more pronounced surface localization. (D) Quantification of vesicle numbers per cell in DA and 3-IT treated vs. control embryos. To avoid cell- and position-specific variations, GFP-containing vesicles are counted in cells at the same position of multiple embryos. Error bars show standard error (control, n=7 individual cells; 3-IT-treated, n=9 individual cells; DA-treated, n=8 individual cells; each data set was acquired from a minimum of 4 representative embryos). The 3-IT and DA treated embryos display significantly reduced and increased number, respectively, when compared to untreated control. ** denotes statistically significant differences as compared to the control (p< 0.05).

To better quantify the response of Btl-GFP expression levels to drug treatments, we repeated the assays in cultured Drosophila cells (Fig. 6A). In untreated S2 cells stably transfected with the btl::GFP vector, the Btl::GFP fusion protein is present mostly in the cytoplasm with some surface localization. DA treatment resulted in reduction of Btl::GFP while 3-IT treatment resulted in increased accumulation of Btl::GFP in the cell periphery. Changes in total Btl::GFP levels upon drug treatments were confirmed by Western blotting (Fig. 6B, top panels). Taken together, it appears that DA can promote internalization and down-regulation of Btl while blocking DA synthesis results in over-accumulation of Btl.

Fig. 6.

Fig. 6

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DA promotes endocytosis of Btl/FGFR in S2 cells. S2 cells were transfected with plasmids expressing Btl::GFP fusion protein, treated with the indicated agents and either (A) double stained for GFP and Shi/dynamin or (B) processed for Western blotting. (A) Without drug treatment (control), Btl::GFP fusion protein is mostly internal with some surface localization. These cells also express endogenous Shi/dynamin. DA treatment resulted in significant reduction in Btl::GFP level and an increase in Shi/dynamin level as well as more pronounced peripheral localization of Shi/dynamin (arrows). 3-IT treatment resulted in increased Btl::GFP level near the cell periphery and concurrent reduction in Shi/dynamin level. (B) Western blot for Btl::GFP (using anti-GFP antibody) and Shi/dynamin in lysates from the cells treated as in (A). The blot was reprobed for β-actin as a loading control. The numbers under the Btl:GFP and dynamin protein lanes indicate the relative protein levels (normalized to the β-actin controls).

DA regulates the level of dynamin

The results so far indicate that the function of Pu in tracheal morphogenesis is mediated through the action of DA, which in turn promotes internalization and turnover of the chemotactic receptor Btl/FGFR. To further explore the role of Pu and DA in regulating the endocytic pathway, we first examined the expression of Shi/dynamin in DA or 3-IT treated cells. As shown in Fig. 6A (middle panel), DA treatment resulted in increase of Shi/dynamin levels in S2 cells with pronounced peripheral localization (arrows), while 3-IT caused a dramatic reduction. The change of dynamin expression levels is confirmed by Western blotting (Fig. 6B, middle panels). We next examined whether this regulatory event occurs in the trachea (Fig. 7A). In wild-type (1-eve-1) embryos, Shi shows enriched expression in tracheal cells, and within the tracheal cells, Shi is abundant at the cell periphery. DA treatment results in a slight enrichment of the peripheral localization in tracheal cells as well as in the neighboring non-tracheal cells. In Pu mutants, Shi expression is diffused and reduced. DA treatment rescued the Shi protein expression level in the Pu mutant. Importantly, we show that DA treatment could also rescue the tracheal defects in Pu mutants (Fig. 7B). To confirm that the Shi/dynamin expression is regulated by the DA synthesis pathway components in vivo, we examined the Shi/dynamin protein levels in the embryonic extracts from different mutants. As shown in Figure 7C, the two Pu mutants show 40-60% reduction in Shi/dynamin. The ple2 allele, a hypomorph, show 23% reduction while the Catsup26 allele show an 83% increase.

Fig. 7.

Fig. 7

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Dynamin level is regulated by DA synthesis pathway components in the trachea. (A) Drosophila embryos of the genotypes 1-eve-1 (representing wild-type) or PuAA17; 1-eve-1 (Pu) were double stained for β-Gal and dynamin. 1-eve-1 and Pu mutant embryos were treated with either H2O or DA. Anterior is to the left and dorsal side is up. Images are zoomed-in view of 2 tracheal subunits. In untreated wild-type (1-eve-1) trachea, dynamin is enriched at the cell peripheries. DA treatment (1-eve-1 + DA) slightly increased peripheral localization of dynamin in wild-type in both tracheal and neighboring cells. In the Pu mutant, dynamin level is reduced. DA treatment (Pu + DA) restored the level of dynamin. (B) PuAA17; 1-eve-1 embryos were treated with either H2O or DA (DA). Tracheal phenotypes were examined by staining for β-Gal. DA treatment rescued the Pu phenotype. (C) Total embryonic protein extracts from the indicated strains were Western-blotted for Shi/dynamin and reprobed for β-actin. Numbers under the protein lanes are the relative levels of dynamin normalized to the β-actin controls.

Discussion

DA regulates cell migration

In this report, we demonstrate that genes involved in DA biosynthesis also regulate tracheal cell migration, and that this function is mediated by DA. This is unexpected since DA is normally associated with neuronal function. However, we believe this novel developmental function is not fortuitous because of the strong and highly specific expression of Punch in tracheal cells during the migratory phase of tracheal development (Fig. 1). In addition, the ectopic migration tracheal phenotypes in Pu/GTPCH mutants can be rescued by expressing a Pu/GTPCH transgene in developing trachea using a btl promoter, demonstrating the trachea-specific function of the DA pathway (Fig. 2E). Moreover, the expression of this transgene shows a dosage-dependent progression of phenotypic outcomes. That is, mild expression rescues ectopic migration, while over-expression tips the balance to blocked migration.

The product of GTPCH enzymatic activity, BH4, is also a stabilizing cofactor of nitric oxide synthase (NOS), which is needed for hypoxia-induced outgrowth of terminal tracheal branches (Jarecki et al. 1999; Wingrove and O'Farrell 1999). However, the Pu mutant phenotypes reported here are most likely unrelated to this developmental process because of the following reasons: (1) Terminal branching occurs near the end of embryogenesis and during larval development (Jarecki et al. 1999; Wingrove and O'Farrell 1999) whereas the defects in primary and secondary branch migration occur during midembryogenesis. (2) Mutations in ple, which has no role in NOS function, also results in ectopic migration phenotypes (Fig. 2H). (3) DA and the DA pathway inhibitor can phenocopy the genetic mutants (Fig. 3). (4) DA treatment can rescue Pu/GTPCH mutant phenotypes (Fig. 7B), implicating a direct role of DA in primary and secondary branch migration. Interestingly, DA is enriched in the trachea (Fig. 3).

An inhibitory role for DA in the regulation of cell migration has been reported for a number of cell types over recent years. Yasunari et al. (1997; 2003) have demonstrated that DA is capable of blocking the migration of vascular smooth muscle cells, a factor in the formation of atherosclerotic lesions, in a process mediated through the D1 class of DA receptors. DA also was reported to interfere with activated neutrophil transendothelial migration (Sookhai et al. 2000). Similarly, DA, acting through D1 receptors, is reported to reduce the migration of regulatory T cells in damaged neural regions, thereby providing protection against T cell-mediated neurodegeneration (Kipnis et al. 2004). Finally, DA acting through the D2 class of DA receptors, can inhibit tumor angiogenesis in highly vascularized gastric and ovarian tumors (Basu et al. 2001, 2004; Chakroborty et al. 2004). It would be interesting to determine whether the mammalian GTPCH-TH-DA enzymatic pathways also have a non-neurological function in modulating tubulogenesis during development.

The modulation of surface FGFR by DA is dynamin-dependent

We found that DA pathway mutants show tracheal abnormalities that are strikingly similar to the perturbations of tracheal cell migration in embryos with abnormalities in FGF signaling. Diminution of DA expression in Drosophila embryos, either by mutations (Pu and ple) or by pharmacological depletion (3-IT treatment), has dramatic effects on the stereotyped migratory behavior of tracheal cells and patterning of the resulting branched structure. We observed that entire branches are misdirected and individual cells move away from the tracheal branches, a phenotype we term “run-away cells”. Under conditions of excess DA, via mutations (Catsup) or pharmacological modification (DA treatment), the converse phenotype, one of blocked migration, is evident. This phenotype is often associated with clustering of tracheal cells near the stunted ends of tracheal tubes. These opposing phenotypes are correlated with the increased or decreased levels of MAPK activation, the mediator of FGFR signaling (Fig. 4).

Our data further demonstrate that DA regulates FGF receptor turnover. We show that Btl::GFP fusion protein is down-regulated in the presence of DA and up-regulated in the presence of 3-IT, either in trachea or in cultured S2 cells (Figs. 5 and 6). These results are consistent with the model that DA promotes internalization of Btl/FGFR, leading to its degradation through the endocytic pathway. The role of DA in internalization of FGFR is further suggested by the genetic interaction between the DA synthesis and endocytic pathway genes. Mutations in the human tumor suppressor nucleoside diphosphate kinase (NDK) gene (nm23) are strongly associated with tumor metastatic activity (Steeg et al. 1988; Freije et al. 1997). Its functional homolog in Drosophila, abnormal wing discs (awd) (Biggs et al. 1990), has been shown to genetically interact with a temperature-sensitive allele of the shibire gene (shits), which encodes dynamin (Krishnan et al. 2001), a large GTPase required for the formation of clathrin-coated endocytic vesicles (Kosaka and Ikeda 1983; van der Bliek and Meyerowitz, 1991). Dammai et al. (2003) first demonstrated a function for awd/nm23 in the migratory phase of tracheal development and functional interactions with shi/dynamin that are required for the modulation of FGFR/Btl levels in tracheal cells. In this report, we show that Pu and ple phenotypes are exacerbated by awd and rescued by btl, while the Catsup phenotypes are rescued by awd but exacerbated by btl (Table 1). These results indicate that Pu/GTPCH and Ple/TH, and by extension, DA, are positive regulators of endocytosis.

We have extended this analysis by implicating direct involvement of DA in regulating the Shi/dynamin protein itself. Mutations in Pu/GTPCH, which regulates DA pools (Krishnakumar et al. 2000), result in reduction of the Shi/dynamin levels (Fig. 7) and, in consequence, the subsequent accumulation of FGFR/Btl in the plasma membranes of tracheal cells, thus accounting for ectopic migratory behavior of these mutant cells. Importantly, down-regulation of Shi/dynamin level in the Pu mutants can be rescued by treatment with DA. A direct correlation between DA and dynamin levels is also demonstrated in cultured S2 cells (Fig. 6). Thus, the genetic and pharmacological evidence in this report supports the hypothesis that the diminished DA pools that accompany loss-of-function mutations in the Pu/GTPCH and ple/TH genes result in deficits of DA-mediated signaling necessary for Shi/dynamin accumulation. The function of the DA synthesis pathway component in tracheal cell migration is illustrated in Figure 8.

Fig. 8.

Fig. 8

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A model for the action of DA synthesis pathway genes. In wild-type cells (middle), the surface level of Btl/FGFR is modulated by endocytosis via the action of Shi/dynamin, while the stability of Shi/dynamin is regulated by DA, presumably through a DA-receptor mediated signaling event. In Pu or ple mutants (right), or when DA synthesis is blocked by 3-IT, Shi/dynamin level is reduced, resulting in over-accumulation of Btl/FGFR and ectopic migration. In Catsup mutants, or in the presence of exogenously added DA, the level of Shi/dynamin is increased, resulting in down-regulation of surface Btl/FGFR and reduced migration.

Evidence of a role of DA in receptor endocytosis has emerged recently. For instance, DA can promote VEGFR endocytosis in cultured human endothelial cells (Basu et al. 2001). DA D3 receptor-mediated modulation of GABAA receptor (Chen et al. 2006) and DA-regulated endocytosis of the renal cell Na+, K+-ATPase (Efendiev et al. 2002) are similarly dynamin-mediated events. Why is a neurohormone also a mediator of endocytosis in other cell types? It is interesting to consider the possibility that the endocytic activity of DA may in fact be its ancestral function, which was adopted by the neurons and tubular cells later. Indeed, primitive, nerve-less multicellular organisms such as sponge can produce dopamine (Liu et al. 2004). The precise mechanism(s) by which DA regulates dynamin assembly is not yet clear. However, Efendiev et al (2002) reported that DA signaling promotes dynamin stabilization and assembly at the plasma membrane in cultured human kidney cells – and thus endocytosis – by activating protein phosphatase 2A which dephosphoylates dynamin-2. It is possible that a similar mechanism of action occurs in the tracheal cells and future experiments will help to address this possibility.

Supplementary Material

S1
S2

Acknowledgments

We thank A. Brand, T. Kornberg, D. Montell, and the Drosophila Stock Center for providing fly strains. This work was supported by Abney Scholarship to A.H., National Cancer Institute grant to J.O. and T.H. (R21CA099021) and National Cancer Institute grant to T.H. (RO1CA109860).

Footnotes

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S1
NIHMS28375-supplement-01.pdf (523.4KB, pdf)
S2
NIHMS28375-supplement-S2.pdf (100.3KB, pdf)

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