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Genetics. Oct 2008; 180(2): 885–893.
PMCID: PMC2567388

Bursicon Signaling Mutations Separate the Epithelial–Mesenchymal Transition From Programmed Cell Death During Drosophila melanogaster Wing Maturation

Abstract

Following eclosion from the pupal case, wings of the immature adult fly unfold and expand to present a flat wing blade. During expansion the epithelia, which earlier produced the wing cuticle, delaminate from the cuticle, and the epithelial cells undergo an epithelial–mesenchymal transition (EMT). The resulting fibroblast-like cells then initiate a programmed cell death, produce an extracellular matrix that bonds dorsal and ventral wing cuticles, and exit the wing. Mutants that block wing expansion cause persistence of intact epithelia within the unexpanded wing. However, the normal progression of chromatin condensation and fragmentation accompanying programmed cell death in these cells proceeds with an approximately normal time course. These observations establish that the Bursicon/Rickets signaling pathway is necessary for both wing expansion and initiation of the EMT that leads to removal of the epithelial cells from the wing. They demonstrate that a different signal can be used to activate programmed cell death and show that two distinct genetic programs are in progress in these cells during wing maturation.

GENETIC analyses of the morphogenetic mechanisms that convert the Drosophila embryo into the larva and subsequently into the adult fly have yielded valuable insights into conserved regulatory pathways and cellular strategies that are used reiteratively throughout development in many organisms. It is particularly useful to focus investigations on a tissue in which perturbations of development do not adversely affect the survival of the organism and provide easily observed phenotypes that aid in analyses of the underlying molecular defects. Drosophila wing development is an example of such a system, where defects in both wing patterning (Crozatier et al. 2004; O'Connor et al. 2006; Baker 2007) and the cellular mechanisms that regulate pupal morphogenesis of the wing structure (Dominguez-Giménez et al. 2007; Srivastava et al. 2007; O'Keefe et al. 2007) have provided insight into underlying conserved molecular mechanisms. Recent studies on the final stages of wing maturation following eclosion (Kiger et al. 2001, 2007; Kimura et al. 2004; Link et al. 2007) have added regulation of the epithelial–mesenchymal transition (EMT) and programmed cell death to the developmental mechanisms that can be addressed through study of the wing.

The sequence of events following eclosion of the adult fly from the pupal case has evolved to allow rapid maturation of the folded, flexible pupal wing into a functional flight organ. At eclosion, the wing is a compact structure, composed of tightly folded dorsal and ventral epithelia covered with a flexible cuticle. Shortly after eclosion, pressurization of the hemolymph initiates expansion of the folded wing blade. During this process, the dorsal and ventral epithelial cell layers delaminate from the overlying cuticle, undergo an EMT, and exit the wing blade, accompanied by initiation of a cell death program (Kiger et al. 2001, 2007). The dorsal and ventral wing cuticles become tightly bonded through deposition of an extracellular matrix by the departing epithelial cells and, possibly, by dispersed hemocytes also found in the wing at this time (Kiger et al. 2007). Subsequently, tanning of the cuticle completes the formation of a strong supple wing blade.

Our previous work found Armadillo/β-catenin to be an important regulator of events associated with wing maturation (Kiger et al. 2001, 2007). Gal4-driven ectopic expression in the wing epithelia of a number of molecules that interfere with Armadillo/β-catenin nuclear function (Pygopus, Shaggy/GSK3, stabilized Armadillo) block epithelial delamination and EMT. The wings expand, but wing epithelia are maintained within the wing blade and the wing surfaces fail to bond. Armadillo/β-catenin signaling is central to EMT in many systems (see Kiger et al. 2007). We also found that the batone mutation blocks both wing expansion and delamination of epithelial cells from the wing cuticle. Mosaic analysis of batone function in gynanders showed that the gene acts nonautonomously with a mutant focus proximal to or in the brain (Kiger et al. 2001). This focus is consistent with a role in production/release of a neurotransmitter or hormone. A number of other mutants exhibit failed wing expansion like batone. These mutants define a signaling pathway important for post-eclosion events, including wing expansion and cuticle tanning, consisting of the Rickets receptor protein and its ligand, Bursicon (McNabb et al. 1997; Baker et al. 1999; Baker and Truman 2002; Dewey et al. 2004).

The hormone Bursicon is released from specific neurons following ecdysis (Kim et al. 2006) and at the time of eclosion (Luan et al. 2006). This hormone belongs to the cystine knot family, which includes vertebrate glycoprotein hormones and transforming growth factor β (TGF-β) (Vitt et al. 2001). Bursicon is a heterodimer whose two subunits are encoded by the bursicon (burs) gene (Dewey et al. 2004) and the predicted gene CG15284, also called bursicon-β (Mendive et al. 2005) or partner-of-bursicon (Luo et al. 2005). Strong mutant alleles of bursicon exhibit failure of wing expansion following eclosion and cause a delay in sclerotization and melanization of the cuticle (Dewey et al. 2004). A mutant with a similar phenotype, pupal (pu), has been associated with the CG15284 locus (Lindsley and Zimm 1992; http://flybase.org/reports/FBrf0138570.html). Mutations in the rickets (rk) gene, which encodes the G-protein-coupled glycoprotein hormone receptor DLGR2, produce a similar phenotype, and Rickets has been shown to be the receptor for Bursicon (Baker and Truman 2002; Luo et al. 2005). Although the batone gene has not yet been characterized at the molecular level, the fact that it acts nonautonomously to produce a wing expansion defect similar to that produced by rickets suggests that it acts upstream of receptor activation. We therefore sought to investigate whether mutations in this group of genes affect the Armadillo-dependent EMT during wing maturation.

MATERIALS AND METHODS

Fly strains:

E. J. Koundakjian generously supplied many strains from the Zuker Collection (Koundakjian et al. 2004) that had been prescreened for wing defects. We carried out complementation tests between many of these strains and identified five allelic mutations that were independently identified by Dewey et al. (2004) to be alleles of the bursicon gene. The strain w1118; rickets4 was a gift of S. L. McNabb. The strain w1118; rk1 cn bw was obtained originally from the Bloomington Stock Center in 2004 and again in 2008. Homozygous phenotypes of rickets, bursicon, and pupal (pu) alleles were confirmed by analysis of hemizygotes using corresponding deficiency stocks from the Bloomington Stock Center [rk: w1118; Df(2L)BSC253/CyO, burs: w1118; Df(3R)Exel6187/TM6B,Tb1, pu: w1118; Df(2L)Exel6035/CyO)]. All other strains are described in Kiger et al. (2007) or were obtained from the Bloomington Stock Center.

Microscopy:

Adult wings were dissected and fixed as described in Kiger et al. (2007). Confocal microscopy was carried out with an Olympus FV-1000 system. Wings were routinely scanned by Z-section to identify the cellular location of all Arm-GFP and DAPI fluorescence present to assure that the images recorded were representative and to detect any changes in subcellular localization. Standard epifluorescence microscopy was performed on a Zeiss Axioplan equipped with a Kodak digital camera.

RESULTS

Genetic dissection of the EMT and cell death programs during wing maturation:

We previously analyzed the cellular events accompanying wing maturation by expressing green fluorescent protein (GFP) in the wing epithelial cells with a variety of transgenes (Kiger et al. 2007). This enabled us to follow progressive changes in cell behavior as the wing epithelia initially delaminated from the cuticle during wing expansion; cells then lost contact with each other and became round, extended processes and then became spindle shaped, elongating in the proximal-distal direction prior to exiting the wing. The course of the EMT was monitored using a GFP-tagged Armadillo/β-catenin (Arm-GFP) that allowed us to image adherens junctions between epithelial cells and to note that the loss of cellular contacts was accompanied by a redistribution of Arm-GFP from cellular membranes to the cytoplasm (Kiger et al. 2007; Figure 1). We also noted that the homozygous batone (bae) mutant, which blocks wing expansion, also blocks these epithelial changes at the delamination step.

Figure 1.
The batone mutation separates EMT and programmed cell death. In wings with wild-type batone function (A–D), confocal images show that redistribution of membrane-associated Armadillo/β-catenin (Arm-GFP fusion protein, green) accompanies ...

Here we investigate the effect of batone on intracellular distribution of Arm-GFP. The location of Arm-GFP in bae/+ heterozygotes is identical to that in wild-type wings analyzed previously (Kiger et al. 2007). Arm-GFP is clearly localized in subapical adherens junctions, just below the cellular apex where wing hairs are evident, at 5 min after eclosion (Figure 1A). DAPI-stained nuclei are clearly visible in each cell at the same level as the adherens junctions. Within ~60–75 min following eclosion, Arm-GFP redistributes from a primarily peripheral location in the junctions to a diffuse cytoplasmic location accompanied by rounding of the cells and loss of contact with their neighbors (Figure 1, B and C). Changes in Arm-GFP localization are accompanied by changes in appearance of the nuclei as programmed cell death is initiated, marked by condensation of the DAPI staining as nuclei become pycnotic. There is clearly variation in the timing of this event from fly to fly during this time period, wings of some flies exhibiting virtually normal cell contacts (Figure 1B) while wings of others having almost completed the transition to round cells (Figure 1C). By 18 hr post-eclosion in the bae/+ wings, virtually all of the cells in the wing blade have exited and the only cells visible are those of the wing veins, which persist in the expanded wing (Figure 1D).

The bae/Y male siblings of bae/+ females fail to expand their wings and the epithelium remains intact, as judged by persistence of membrane-associated Arm-GFP in adherens junctions. At 5 min post-eclosion the epithelium looks indistinguishable from heterozygous siblings (Figure 1E). By 65–75 min the bae/Y wings have not undergone redistribution of Arm-GFP (Figure 1F) and the adherens junctions remain intact at least through 18–22 hr (not shown). Nevertheless, changes in DAPI-stained nuclei appear to proceed normally. In Figure 1F, it is clear that in the majority of cells nuclei are no longer present, and in the remaining cells the chromatin is condensed and nuclei are pycnotic. Wild-type batone function is evidently required both to expand the wing and to initiate EMT within the wing epithelia, but it is not required to initiate programmed cell death.

The rickets gene is necessary for EMT but not for programmed cell death:

The wings of rickets mutants fail to fully expand when hemolymph is pressurized and then generally collapse, when hemolymph flows back into the thorax, to produce wings that resemble those of the batone mutant. We have studied the behavior of wing epithelial cells in homozygous-null mutant rk4/rk4 flies. This mutation introduces a stop codon in the transmembrane protein domain that should block production of a functional receptor (Baker and Truman 2002); the rk4/rk4 phenotype mimics that of a homozygous deficiency (Baker and Truman 2002). At 0–20 min post-eclosion, the epithelial adherens junctions imaged with Arm-GFP and the DAPI-stained nuclei appear to be normal (Figure 2A). Views of DAPI-stained wing blades at lower magnifications illustrate the uniform regular array of nuclei present throughout the wing blade at this time (Figure 2, B and C). However, at 175–185 min post-eclosion (Figure 2, E and F), few DAPI-stained nuclei are visible in the wing. Most of those that persist are in the wing vein cells or in scattered hemocytes (Kiger et al. 2007). At 18 hr post-eclosion, Arm-GFP persists at adherens junctions and the epithelial structure appears intact in rk4/rk4 wings (Figure 2D); only a few cells retain evidence of highly condensed DAPI-stained material.

Figure 2.
Mutations in the rickets gene (rk4/rk4) block Arm-GFP redistribution and dispersal of the adult wing epithelium without affecting chromatin loss. (A) 0- to 20-min wings with confocal view imaging Arm-GFP (green) within the subapical adherens junctions ...

To confirm that this phenotype is due to the homozygous rk4/rk4 mutation and not to a second mutation on the rk4 chromosome, we analyzed wings from a second rk homozygous null allele (rk1, Baker and Truman 2002), from the heteroallelic combination rk1/rk4, and from hemizygotes made using deletion chromosomes (Table 1; Figure 3). In all of these genotypes, only occasional fragmented or pycnotic nuclei remained in the intervein area of the wing blade at 16–24 hr post-eclosion (Table 1). Confocal microscopy of stocks carrying Arm-GFP in the mutant background confirmed that the adherens junctions are still present at this time (Figure 3, G–I). In some rk mutant wings (as well as in other mutants discussed below), particularly those that retain a balloon-like morphology rather than collapsing back to a folded wing structure, clouds of fragmented and pycnotic DAPI-stained nuclei are trapped in hemolymph within the lumen of the wing in between the two wing blades (Figure 3F). These clouds of DAPI-stained chromatin fragments are probably derived by blebbing of cellular contents from the apoptotic epithelial cells (Taylor et al. 2008), leaving behind the adherens junction framework and possibly other portions of the cell within the remaining epithelial layer.

Figure 3.
Analysis of heteroallelic mutant combinations and hemizygotes confirms that phenotypes are attributable to mutations in the bursicon-signaling pathway. In homozygotes of a rk1cn bw/rk1cn bw stock analyzed several years ago, normal DAPI-stained nuclei ...
TABLE 1
Fraction of intervein cells in the wing that contain normal or fragmented/pycnotic nuclei at 16–24 hr post-eclosion

We also note that in experiments carried out several years ago with homozygous rk1 flies, occasional wings that contained a uniform epithelial layer with nonapoptotic nuclei were observed at 25 hr post-eclosion (Figure 3A, Table 1). This was not observed in a homozygous stock of rk1 recently obtained from the Bloomington Stock Center. This was also not apparent in wings of hemizygotes or heteroallelic combinations of the rk1 chromosome (Figure 3, B and C; Table 1). We can conclude only that a second mutation, floating in the original rk1 stock, affects apoptosis.

Mutations in bursicon and in pupal block EMT without preventing cell death:

We screened a collection of stocks provided by the Zuker laboratory (Koundakjian et al. 2004) carrying mutagenized autosomes and exhibiting a variety of wing phenotypes, for phenotypes resembling batone or rickets. We found five stocks carrying mutant alleles of a complementation group on the right arm of chromosome III that exhibited collapsed wing phenotypes of varying severity. Subsequently, these mutations were shown by others to be alleles of the gene CG13419 (bursicon, encoding one of the Bursicon heterodimer subunits) and to be defective in Bursicon hormone activity (Dewey et al. 2004).

If Bursicon is the hormone that initiates EMT in the wing epithelia through Rickets, mutations in bursicon and its likely heterodimer partner pupal would be expected to mimic rickets. To test this hypothesis, the strongest bursicon alleles, bursZ4410 and bursZ5569, were chosen. The bursZ4410 mutation is a G-to-A conversion at the splice acceptor site for the second exon and is likely to lead to premature chain termination and an inactive polypeptide; the bursZ5569 mutation causes conversion of glycine 115 to cysteine (Dewey et al. 2004). In both mutant backgrounds at 30–90 min post-eclosion, confocal images show that some nuclear condensation can be observed but most nuclei are still present in the subapical region defined by Arm-GFP localization (Figure 4, A and C). At 20–26 hr post-eclosion, at a time when cells would be completely absent in a wild-type wing, subapical Arm-GFP is still present in a uniform wing epithelium and most nuclei are absent or pycnotic (Figure 4, B and D). Confocal images of wings of pupal (pu1) homozygous flies at 0–10 min (Figure 4E) and 20–22 hr (Figure 4F) post-eclosion are strikingly similar to those of the bursicon mutants, supporting the identity of pupal with CG15284. (While there is no doubt that pu1 is located in or very close to CG15284, we have not tried to identify a lesion in the CG15284 gene of the pu1 chromosome.) Heteroallelic combinations of the two bursicon alleles as well as hemizygotes of bursZ5569 and pu1 confirm that these phenotypes are associated with the burs and pu mutations (Table 1, Figure 3, E, F, H, and I).

Figure 4.
Phenotypes of mutations in burs and its proposed heterodimer partner pu are similar to those of rickets mutations. arm-GFP (II); bursZ4410 (A) 30–90 min post-eclosion and (B) 21–26 hr post-eclosion. arm-GFP (II); bursZ5569 (C) 30–90 ...

DISCUSSION

Completion of wing morphogenesis in Drosophila is an active process that is part of the suite of events initiated following eclosion of the fly from the pupal case. These events are known to be controlled by release of Bursicon from specific neurons in the abdominal ganglion (Baker and Truman 2002; Luo et al. 2005; Kim et al. 2006). We have investigated the possibility that the Bursicon/Rickets signaling pathway also plays a role in the events that remove the cellular epidermis from within the expanding wing. Our approach differs from that of other labs that have studied programmed cell death in the wing (Kimura et al. 2004; Link et al. 2007) in that we simultaneously follow a marker for nuclear breakdown associated with programmed cell death (DAPI chromatin staining) and a marker for the behavior and integrity of adherens junctions between cells (Arm-GFP). We observe that mutations in this signaling pathway separate the dissociation of the epithelial cells from initiation of programmed cell death. In wild-type wings, within about an hour after eclosion, redistribution of Arm-GFP from its location in the subapical adherens junctions to the cytoplasm is accompanied by changes in cell shape (Kiger et al. 2007; see Figure 1). Within the same time frame, we also observed chromatin condensation, indicating nuclear changes accompanying cell death, beginning in some nuclei and spreading to others. Loss-of-function mutations in several different components in the signaling pathway (rickets, bursicon, pupal) and a potential upstream component affecting the secretion of Bursicon (batone) all produce a similar phenotype marked by aberrant persistence of the epithelia with intact adherens junctions, extending at least to 24 hr post-eclosion. In contrast, in all of these mutant backgrounds, normal initiation of programmed cell death in the epithelial cells is observed with a time course similar to wild type. These results provide evidence for rk-dependent regulation of the EMT and existence of a cell-death-signaling pathway that is independent of signaling through the rk receptor. Our data might also be consistent with a model in which rk activation is capable of initiating both the EMT and cell death, but cell death induction can also occur via an independent and redundant pathway in the absence of wild-type rk function.

Contrary to our results, Kimura et al. (2004) reported the absence of wing epithelial cell death in homozygous rk1/rk1 mutants. We believe that it is likely that the rk1 stock used by Kimura contained a second site mutation, perhaps the same mutation discussed in our results above, and that this mutation rather than rk was responsible for the effect on apoptosis in the rk1/rk1 wings that they observed.

Elimination of the epithelial cells from the wing involves coordinated changes in the epithelia that involve delamination from the cuticle and loss of contacts between cells (Kiger et al. 2007), accompanied by rapid and widespread initiation of programmed cell death (Kimura et al. 2004; Link et al. 2007). Completion of the EMT is followed by removal of the cells from the wing. When the caspase inhibitor p35 is expressed in the epithelial cells, the cells complete the EMT and assume a rounded shape. A significant proportion of the disorganized cell population does not leave the wing (Kiger et al. 2007). A recent study by Link et al. (2007) also demonstrated that mutations that block programmed cell death in the wing epithelium produce persistence of nuclear staining long past the time when cells are normally cleared from the wing. While Link et al. (2007) did not employ methods that allowed visualization of the shape of whole cells or of adherens junctions, it is likely that persistence of the nuclei marked persistence of intact epithelial cells. These observations imply that completion of one or more steps in the cell death program are required in addition to the EMT for exit of epithelial cells from the wing. In this view, both activation of programmed cell death and the EMT are necessary for removal of the wing epithelium, but neither is sufficient. Link et al. (2007) have proposed that the cells disintegrate within the wing and their debris is flushed from the wing by flowing hemolymph. The results that we present here and elsewhere (Kiger et al. 2007) clearly show that EMT must accompany cell death for dispersal of the wing epithelium to occur. Programmed cell death without EMT does not cause loss of the intact wing epithelium.

On the basis of the spindle shape assumed by individual epithelial cells and the appearance of cell streaming in wings dissected from the body, we have suggested that active migration could facilitate removal of the cells from the wing, with the caveat that frequent grooming of the wings by the hind legs of the fly may be an important part of the process (Kiger et al. 2007). We suggest that loss of adhesion and acquisition of motility, grooming, and hemolymph flow may all contribute in vivo to rapid removal of cells. It seems likely that cells in which p35 is expressed, or in which other functions necessary for programmed cell death have been impaired, may make or retain attachments to the cuticle or extracellular matrix that prevent their removal from the wing. The ability to modulate such attachments, which may be necessary for clearing cells from the wing, could depend on appropriate regulation of caspase activity. The Drosophila caspase Dronc regulates border cell migration in the ovary independently of its role in apoptosis (Geisbrecht and Montell 2004). In the mouse, Caspase-8 is required for activation of calpain proteases, Rac, and lamellipodial assembly (Helfer et al. 2006). A similar requirement for caspase activity could link progress of a cell death program to the release of wing epithelial cells from their substrate attachments following EMT.

Our evidence for independent activation of epithelial cell death and the EMT raises the question of how these events are coordinated at eclosion. A complete answer to this question will have to await identification of other signal(s) that initiate programmed cell death or modulate the EMT. It is likely that overall synchronization is controlled via activation of the peptidergic networks within the central nervous system and coordinated release of signals that regulate specific aspects of ecdysis behavior (Truman 2005; Žitňan et al. 2007). For example, the same neurons that express Bursicon at eclosion also express other active signaling neuropeptides such as the crustacean cardioactive peptide (CCAP) and the myoinhibitory peptide (MIP) with potentially independent direct or indirect molecular targets (Dewey et al. 2004; Luo et al. 2005; Mendive et al. 2005; Kim et al. 2006; Luan et al. 2006). Sequential release of different regulators can also modulate a process, e.g., control of cuticle tanning via pre-eclosion CCAP stimulation of tyrosine hydroxylase (TH) protein accumulation followed by Bursicon-dependent activation of TH via phosphorylation after eclosion (Davis et al. 2007).

Coordination of the post-eclosion behavior of cells within the wing epithelium also potentially involves signals to the wing epithelial cells, prior to their death and dispersal, directing additional functions such as construction of the matrix that bonds the two cuticular surfaces of the mature wing. For example, the Timp gene (tissue inhibitor of metalloproteinases) is expressed in wings of newly eclosed flies and is required for normal wing bonding. Homozygous Timp/Timp flies expand their wings and eliminate the epithelial cells normally; however, the cuticular surfaces of the wing do not bond. Small Timp/Timp clones in the epithelia, produced by mitotic recombination, exhibit no bonding defects, consistent with secretion and diffusion of Timp from neighboring Timp+/Timpcells (Kiger et al. 2007). In contrast, large Timp/Timp clones, produced by mitotic recombination using the Minute technique, generate improperly bonded wings similar to those of homozygous Timp/Timp flies (J. Kiger, unpublished results). Thus, the genotype of the epithelial cells themselves affects bonding of the cuticular surfaces, establishing that the wing cells play a critical role in metabolizing and remodeling the wing extracellular matrix. This is in agreement with previous observations that premature death of the epithelial cells prevents bonding of the cuticular surfaces (Kiger et al. 2001). The timing and initiation of these cellular behaviors that affect wing bonding may also be coordinated via signals generated by the ecdysis cascade.

For survival of the fly following eclosion in the natural environment, it is important that the entire wing epidermis be removed quickly to ensure timely completion of wing blade bonding and formation of functional flight organs. It would be reasonable to postulate that imposition of an EMT along with initiation of programmed cell death would ensure a more rapid and uniform dispersal of the epidermis from the appendage. Indeed, it would appear that the genes for producing EMT form a cassette that can be deployed as needed during the evolution of developmental pathways: first during gastrulation, then during peripheral nervous system formation, and later in winged insects during wing maturation.

Acknowledgments

We thank Michael Paddy for his continuing assistance with confocal microscopy. We thank Steven Swanson for assistance with analysis of wing phenotypes of rk, burs, and pu hemizygous genotypes. We appreciate the comments of anonymous reviewers, whose suggestions improved this manuscript. This work was supported by funds of the Agricultural Experiment Station at the University of California at Davis to J.E.N. and J.A.K.

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