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Copper cells and stomach acid secretion in the Drosophila midgut
The International Journal of Biochemistry & Cell Biology 36 (2004) 745–752
Cells in Focus
Copper cells and stomach acid secretion in the Drosophila midgut Ronald R. Dubreuil∗ Department of Biological Sciences, University of Illinois Chicago, Chicago, IL 60607, USA Accepted 22 July 2003
Abstract Copper cells in the Drosophila larval midgut were originally named for their ability to accumulate dietary copper. Recent studies have uncovered a number of intriguing similarities between copper cells and the acid-producing gastric parietal cells of the mammalian stomach. In addition to their shared roles in stomach acidification, they share a peculiar invaginated morphology in which the apical cell surface is buried deep within the cytoplasm. These shared properties of morphology and function portend the identification of shared molecular mechanisms that account for their specialized roles in digestive physiology. © 2003 Elsevier Ltd. All rights reserved. Keywords: Copper cells; Acid-secreting cells; Gastric parietal cells; Labial gene
Cell facts • • • • •
Approximately 100 copper cells in larval middle midgut. Orange fluorescence after copper feeding. Specification during development is controlled by the labial gene. Apical surface is arranged as a flask-shaped invagination. Responsible for stomach acidification.
1. Introduction The gastric parietal cell in mammals is responsible for secretion of stomach acid through the activity of a proton pump that resides in the apical plasma membrane. In the resting cell, microvilli containing the proton-pumping ATPase are sequestered within tubulovesicles or canaliculi that are found throughout the cytoplasmic space. When activated to secrete stomach acid, parietal cells undergo a dramatic ∗
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transformation in which the tubulovesicles fuse with the apical surface of the cell and become dilated (reviewed by Yao & Forte, 2003). As a result, the apical surface of the acid-secreting cell acquires a deeply invaginated appearance (Fig. 1A; Duman, Pathak, Ladinsky, McDonald, & Forte, 2001). Much of the remaining space within the cytoplasm is occupied by a reticular network of mitochondria that presumably are required to drive the intense metabolic activity that accompanies acid secretion. Studies of the digestive tract in the fruit fly, Drosophila, have uncovered an equally peculiar cell type in the stomach of larvae and adults. These
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Fig. 1. Ultrastructure of acid secreting cells. (A) High magnification view of rabbit gastric parietal cells after stimulation with histamine for 28 min. The apical surface of the cell is deeply invaginated and densely covered with microvilli that contain the H,K ATPase proton pump. Many darkly stained mitochondria occupy the cytoplasmic spaces surrounding the invaginations. Bar = 2 M. Photo courtesy of Dr. J.G. Forte. (B) High magnification view of the copper cell region from an adult Drosophila midgut. The copper cell (C) like the gastric parietal cell, has a deeply invaginated apical domain (V) that is densely covered with microvilli (mv). Much of the cytoplasmic space surrounding the invagination is occupied by mitonchondria. Two interstitial cells (I) are shown separating the copper cell at the center from neighboring copper cells. Septate junctions (sj) appear as electron-dense plasma membrane regions where the shoulders of the copper cells make contact with the overarching apical region of interstitial cells. A thin sheath of muscle cells (mu) surrounds the gut epithelium opposite from the gut lumen. Bar = 2 M. Photo courtesy of Dr. O. Baumann. (C) A schematic cutaway view of the middle migut. Copper cells (orange) alternate in a checkerboard pattern with spindle-shaped interstitial cells (blue). The apical invagination of the copper cells is connected to the gut lumen through a narrow channel formed by the interstitial cells which overarch the apical region of the copper cells. Septate junctions form a collar (black) over the shoulders of the copper cells to form a permeability barrier between the gut lumen and the body cavity.
cells were first described by Strasburger (1932) and later by Poulson and coworkers who named them “cuprophilic” cells (Poulson & Bowen, 1952; Filshie, Poulson, & Waterhouse, 1971) because of their striking orange fluorescence in copper-fed larvae and their ability to accumulate radioactively-labeled copper. We now know that, if anything, copper cells should be considered “cuprophobic” since their normal physiological activity is inhibited by copper (described below). Hence, we refer to these cells simply as copper cells. Copper cells bear several striking similarities to mammalian gastric parietal cells. EM studies re-
vealed that copper cells have a deeply invaginated apical membrane domain that is densely covered with microvilli (Fig. 1B; Filshie et al., 1971; Dubreuil, Grushko, & Baumann, 2001). There is also a high density of mitochondria present throughout the cytoplasm. Dye feeding experiments have established that the food contents of the gut are strongly acidic in the copper cell region and in the stomach-like region immediately downstream (pH < 2.3; Strasburger, 1932; Dubreuil et al., 1998). Genetic experiments have shown that in the absence of copper cell differentiation there is no detectable stomach acid secretion (Dubreuil et al., 1998). Thus, copper cells from
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Drosophila share a number of unique features with the gastric parietal cells of mammals.
2. Copper cell environs The digestive tract of dipteran larvae is divided into conspicuous domains of morphology and function. Here, I will focus on the midgut of Drosophila melanogaster, which has received the most attention from cell and developmental biologists. Most of the length of the gut is termed the midgut. The remainder consists of the ectodermally derived foregut and hindgut which are relatively short compared to the midgut (Skaer, 1993). The midgut is probably the site where most digestion of food occurs. It is not widely appreciated that food passes through the digestive tract of many insects wrapped in a peritrophic membrane (Terra, 2001). This membrane, which resembles a microscopic sausage casing, is synthesized within the proventriculus (Pr, Fig. 2A) at the anterior end of the midgut. Four gastric caeca project from the base of the proventriculus (reaching upward alongside the
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proventriculus in the figure) where it feeds into the midgut proper. The anterior midgut (a) is a relatively short region in which the diameter of the epithelial tube gradually tapers between the proventriculus and the middle midgut (m). The slender middle midgut expands into a larger diameter stomach region which is typically filled with food (*). Another constriction of the gut below the stomach marks the site at which the pH of the food is neutralized (Dubreuil et al., 1998). The posterior midgut downstream of this contriction is the longest region of the midgut. It appears relatively homogeneous and is likely to be the principal site of food absorption. The copper cells can be distinguished from their neighbors in the light microscope by a number of criteria. In rhodamine–phalloidin labeled whole-mount preparations, the copper cells appear as bright crescents corresponding to the actin-rich microvilli of the apical domain (Lee, Coyne, Dubreuil, Goldstein, & Branton, 1993). The copper cells can also be detected by their conspicuous orange fluorescence in copper-fed larvae (Poulson & Bowen, 1952; McNulty, Puljung, Jefford, & Dubreuil, 2001).
Fig. 2. Anatomy of the Drosophila midgut. (A) Brightfield image of a dissected larval midgut. The proventriculus (Pr) marks the beginning of the anterior midgut (a), followed by the middle midgut (m) and the posterior midgut (p) which extends out of the field of view. The middle midgut is quite narrow in diameter at the beginning and becomes much broader in the stomach region where there is usually food visible in the gut lumen (*). (B) Fluorescent image of the field in A showing the domain of expression of a labial-GFP reporter gene that is exclusively expressed in copper cells of the midgut. A kink in the copper cell region is marked with an arrow for reference. (C) Higher magnification view of the GFP reporter pattern showing bright nuclear GFP fluorescence, weaker cytoplasmic fluorescence, and banana shaped gaps in the fluorescence pattern corresponding to the apical invagination of copper cells.
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Bienz and coworkers performed a series of elegant experiments addressing the mechanism of copper cell specification (Hoppler & Bienz, 1994). In their studies they described a -galactosidase reporter gene that is specifically expressed in copper cells. We produced a similar reporter construct that expresses green fluorescent protein (GFP) under control of the 5 -regulatory DNA that governs labial expression in the larval midgut (Chouinard & Kaufman, 1991). This reporter transgene labels the copper cells of the midgut in living embryos and larvae, and in the dissected midgut (Fig. 2B). Higher magnification views of the GFP labeled cells reveal the spherical shape of each copper cell as well as the invaginated apical domain which excludes the cytoplasmic GFP label (Fig. 2C, arrow). About 100 copper cells are detected in the middle midgut epithelium from its narrowest region through the proximal portion of the stomach. A more detailed account of the design and properties of the labial-GFP transgene will be published elsewhere.
3. Fine structure of cuprophilic cells Electron microscopy reveals that there are multiple distinct cell types in the middle midgut. The flattened, spherical copper cells (C) alternate with spindle-shaped interstitial cells (I; Fig. 1B) which surround and envelope each copper cell (shown schematically in Fig. 1C). The interstitial cells reach over the apical surface of the copper cells, leaving a narrow channel that connects the copper cell invagination to the gut lumen. The cytoplasm of the copper cells appears less dense than the cytoplasm of the interstitial cells, making it easy to see the boundary between them. The interstitial cells also have extensive infoldings of the basal plasma membrane that often extend close to the apical plasma membrane (Filshie et al., 1971; Dubreuil et al., 2001). The apical surface of the interstitial cells, facing the midgut lumen, is decorated with numerous short microvilli. Apart from their peculiar invaginated structure, copper cells have the same specialized plasma membrane domains found in other more conventional epithelial cells. The apical invagination is densely covered with microvilli that nearly fill the extracellular space within the invagination, thus accounting for the crescent shapes detected by rhodamine phalloidin staining
(Lee et al., 1993). The permeability barrier of the gut is provided by a specialized cell–cell contact known as the septate junction (sj). Septate junctions are functionally and morphologically similar to the tight junctions of mammalian cells (Tepass, Tanentzapf, Ward, & Fehon, 2001). They have an electron-dense character compared to the remainder of the basolateral plasma membrane. The molecular composition of the smooth septate junctions is not yet known. Antibodies against the cytoskeletal proteins spectrin and ankyrin appear to specifically label the electron dense region (Dubreuil et al., 2001, described below). There is close apposition between the copper cells and the interstitial cells in the basolateral domain below the level of the septate junction, but this plasma membrane region does not appear electron dense. The basal surfaces of copper cells and interstitial cells are flattened where they contact the thin muscle sheath that surrounds the gut epithelium. Specialized cell–cell contacts in this region have not yet been described. Several other general features are apparent in electron micrographs of the middle midgut. The nucleus of the copper cells (n) is located in the basal cytoplasm, whereas the nucleus of the interstitial cells (lower right in the figure) is found in the apical cytoplasm. The pore formed by the interstitial cells at the apex of the copper cells is often quite narrow, and in the section of an adult midgut shown here it appears to be nearly closed. However, the sum of evidence from EM analysis and fluorescent antibody staining suggests that the apical domain of copper cells is probably always open to the midgut lumen, unlike the situation in mammalian parietal cells where acid secretion is regulated by pinching off of tubulovesicles. High magnification views revealed that the apical microvilli of the copper cells are densely studded with lollipop-shaped particles that presumably represent a plasma membrane V-ATPase (Dubreuil et al., 2001). Moreover, an antibody against the Culex V-ATPase B subunit (Filippova, Ross, & Gill, 1998) specifically stains the apical membrane domain of copper cells (unpublished observation).
4. Specification and differentiation of cuprophilic cells Copper cells appear relatively early in embryonic development of the midgut. Bienz and coworkers
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established that expression of the homeotic gene labial is required for specification of copper cells and for maintenance of their differentiated state (Hoppler & Bienz, 1994). There is a gradient of labial expression within the copper cell domain with increasing labial expression from anterior to posterior (Bienz, 1996) which coincides with a more elaborate apical invagination in the most posterior cells (Dubreuil et al., 1998). There is a parallel gradient in the pattern of the septate junction (Dubreuil et al., 1998, 2001). In the most anterior cells the opening to the invagination is relatively large and is surrounded by a relatively thin septate junction collar. Toward the posterior the opening is severely constricted and the septate junction collar is concomitantly smaller in diameter, but also thicker. The zone of labial expression is determined by input from the morphogens decapentaplegic and wingless, whose expression in the visceral mesoderm is regulated by the homeotic gene Ubx (reviewed in Bienz, 1997). Interestingly, the boundaries of the copper cell domain can be shifted in response to changes in the dose of wingless (Hoppler & Bienz, 1995). Very high wingless levels can repress labial expression and copper cell development altogether whereas decreased wingless levels can lead to an expansion of the copper cell domain to include cells that would normally acquire a distinct fate. Most of the developmental studies to date have focused on the complex regulatory interactions of genes upstream of labial. An important future goal will be to elucidate the pathway downstream of labial that generates the peculiar shape and polarity of the copper cell. Differentiation of copper cells appears to depend on labial in both adults and larvae. A conditional labial mutation (labk3 ) blocks labial expression and interferes with copper cell differentiation in larvae but not in adults (Dubreuil et al., 2001). It appears that the molecular defect in this mutant affects expression of labial in the larva but not in the adult. Interestingly, since the mutant larvae which altogether lack copper cells (and therefore also lack stomach acid secretion) are nonetheless capable of normal development, it appears that stomach acid secretion is not essential at least during larval development. This unexpected observation fits well with the observation that stomach acid secretion can be inactivated pharmacologically in human patients with gastric disease without any dire consequences (Freston, Rose, Heller, Haber, &
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Jennings, 1999). Thus, the metabolically expensive process of stomach acid secretion appears to be dispensible, at least under controlled laboratory and clinical conditions.
5. Cell biology of copper cells One way to elucidate the molecular basis for the striking morphological properties of the copper cell will be to localize structural and regulatory proteins that are responsible for their unusual shape and polarity. So far only a handful of proteins have been localized. As mentioned above, staining with rhodamine phalloidin primarily detects the actin filaments of the apical microvilli and the muscle sheath (mu in Fig. 1B) that surrounds the gut epithelium (Lee et al., 1993). The two plasma membrane proteins that have been localized so far are the Na,K ATPase, which resides in the basolateral domain (Dubreuil, Wang, Dahl, Lee, & Goldstein, 2000), and the V-ATPase which resides in the apical microvilli (unpublished observation). Two isoforms of the cytoskeletal protein spectrin are expressed in copper cells. Spectrin associates with the cytoplasmic surface of the plasma membrane where it contributes to cell shape, membrane stability, and the organization of polarized plasma membrane domains (Bennett & Baines, 2001). The ␣ subunit of spectrin, which is shared by both Drosophila isoforms, is detected at all regions of the plasma membrane (Lee et al., 1993; Dubreuil et al., 2000). The  subunit is associated with the basolateral domain, including the septate junction and the H subunit is found exclusively in the apical plasma membrane domain (Dubreuil et al., 1998, 2001). Mutations in the  spectrin gene cause a gross mislocalization of the Na,K ATPase, indicating that spectrin is required for its normal expression in the basolateral domain (Dubreuil et al., 2000). Spectrin interacts with the Na,K ATPase via the linker protein ankyrin. While initial localization studies suggested that ankyrin was restricted to the septate junction (Dubreuil et al., 1998, 2001), we now know that other antibodies detect ankyrin codistributing with the Na,K ATPase throughout the basolateral membrane (unpublished observation). The original antibody may have recognized a unique subpopulation of ankyrin molecules, or may have cross-reacted with some other
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component of the septate junction. Thus the genetic requirement of  spectrin for the normal distribution of the Na,K ATPase is likely to be mediated via interactions between  spectrin, ankyrin and the Na,K ATPase. Many interesting cell biological issues are ripe for analysis in copper cells. The molecules that form the smooth septate junctions of the midgut are unknown, as is the mechanism that determines their size and shape. Interestingly, mutations in spectrin and labial both affect the diameter and thickness of the septate junction ring that surrounds the neck of the copper cell invagination (Lee et al., 1993; Dubreuil et al., 2000, 2001). These observations suggest that spectrin may be an important component of the pathway between labial expression and copper cell differentiation. An important future step will be to identify the adhesion molecules that attach copper cells to their neighbors, both in the septate junction and in the rest of the basolateral domain. Cell adhesion molecules are likely candidates to mediate the polarized assembly of spectrin and ankyrin within specific plasma membrane domains. Neuroglian, the only known adhesion molecule in Drosophila that interacts with the spectrin cytoskeleton (Dubreuil et al., 1996), is not expressed in the middle midgut. Another interesting area for further study is the identification of structural proteins that generate the invaginated morphology of the apical domain. The depth of the invagination is suggestive of a role for molecular motors such as members of the myosin or kinesin protein families. Neither has been studied in copper cells. Copper cells may also be a valuable system in which to study the mechanisms that underlie the development of plasma membrane polarity, since the polarized domains of these cells are especially well-defined. It will be interesting to ask if proteins such as Crumbs, Bazooka, Stardust, Scribble and Dlg, which have been studied in the development of ectodermally derived epithelial cells (Bilder, 2001; Knust & Bossinger, 2002), also have a role in the polarization of copper cells.
6. Implication of cuprophilic cells in stomach acid secretion The copper cells of the Drosophila midgut appear to be functionally related to the gastric parietal cells
of mammals. Beyond their mophological similarities, copper cells are present at precisely the site at which midgut acid secretion occurs, the magnitude of acid secretion is comparable between Drosophila and mammals, and genetic defects that perturb copper cells lead to a loss of stomach acid secretion in Drosophila. There are also some important differences between gastric parietal cells and copper cells. For one, copper cells do not appear to undergo the dramatic morphological transformation that accompanies activation of acid secretion in parietal cells. However, it remains possible that the apical invagination of copper cells can open and close to regulate acid secretion. If the copper cell invagination is not there to provide a regulatory mechanism, then it is not apparent what benefit this unusual morphology might offer. There is no a priori link between an invaginated morphology and the ability to secrete acid, although it is noteworthy that this shared peculiarity is rare in evolution. Perhaps there is some benefit in shielding the acid-secreting microvillar surface from the extracellular environment. Alternatively, it may be beneficial to shield neighboring cells from the site of proton translocation where the pH of the extracellular fluid should be lowest. A second difference is that the apical proton pump in copper cells appears to be a V-ATPase family member, while acid secretion in parietal cells is mediated by a H,K ATPase (which is not known to be expressed in Drosophila). The discovery that copper cells are responsible for acid secretion grew out of the initial characterization of Drosophila ␣ spectrin mutants. The shape and pattern of copper cells is grossly perturbed in these mutants (Lee et al., 1993). Since mammalian spectrin was closely linked to the H,K ATPase during activation of gastric parietal cells (Mercier, Teggio, Deviliers, Bataille, & Mangeat, 1989), we were interested to know if spectrin was also required for acid secretion in Drosophila. As it turns out, acid secretion was blocked in ␣ spectrin mutants (Dubreuil et al., 1998). However, it has not been a simple matter to determine the molecular basis for the defect. The contribution of spectrin to acid secretion is not yet known, in parietal cells or in copper cells. One hypothesis is that the apical population of ␣H spectrin in copper cells is somehow responsible for the correct targeting and/or function of the apical V-ATPase (Dubreuil et al., 1998). Spectrin could have a similar stabilizing effect on the H,K ATPase in mammals.
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Another hypothesis is that the basolateral population of ␣ spectrin regulates a basolateral activity that is indirectly required for acid secretion. One candidate is the anion exchanger which interacts with mammalian spectrins and has been implicated in the acid secretion process (Stuart-Tilley et al., 1994; Jons & Drenckhahn, 1998). Protons translocated by the proton pump are derived from splitting of water (Guyton & Hall, 1996). The resulting hydroxyl ions are then acted upon by carbonic anhydrase and CO2 to form HCO3 − which is then exchanged for Cl− at the basolateral surface of the cell. The Cl− ions pass through apical Cl channels, yielding a net stomach secretion of HCl. Failure to clear HCO3 − from the cell may exert feedback inhibition on the proton pumping process, as is the case with pharmacological inhibition of carbonic anhydrase (Janowitz, Colcher, & Hollander, 1952). Thus, the inhibition of acid secretion in spectrin mutants could be the result of an effect on the anion exchanger, analogous to the effect of  spectrin mutations on the Na,K ATPase (Dubreuil et al., 2000). It will be possible to test this hypothesis by examining the fate of a recently identified anion exchanger in the middle midgut of Drosophila ␣ spectrin mutants. 7. What makes a copper cell a copper cell? The mechanism behind the response of copper cells to dietary copper remains a mystery. Recent work suggests that the orange fluorescence observed in copper cells after copper feeding is due to formation of a complex between copper ions and a member of the metallothionein family of proteins (McNulty et al., 2001). However, it will be necessary to directly test this proposal once suitable genetic tools such as metallothionein mutants are in hand. The suggestion of a role for copper cells in metal detoxification seems less likely now that a role in the secretion of stomach acid has been established. Perhaps a more definitive test would be to examine the susceptibility of Drosophila larvae to copper toxicity under conditions in which development of copper cells has been blocked (i.e. in labial mutants). Interestingly, metallothionein is known to be expressed in the acid secreting cells of the dog stomach (Shimada, Yanagida, & Umemura, 1997). However, it was not possible to detect copper-dependent fluores-
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cence in response to copper feeding in the mouse stomach (our unpublished observation). It is clear that copper feeding in Drosophila inhibits stomach acid secretion (McNulty et al., 2001), which is consistent with binding of copper to an essential component of the acid secretion apparatus. Metallothionein may have an unanticipated role in stomach acid secretion that could explain why copper cells behave as a sink for the accumulation of dietary copper.
8. Future prospects Copper cells are a valuable experimental system in which to ask fundamental questions about epithelial cell biology as well as the mechanism of stomach acid secretion. The middle midgut of larvae is easily amenable to microscopic studies. It is a simple matter to dissect large number of larval midguts for staining experiments or physiological measurements. The true value of the Drosophila system is the ability to apply genetic strategies to problems of molecular function. For example, it will be possible to carry out genetic screens for mutations that affect the acid secretion pathway, which has been impractical in mammalian systems. This approach is expected to uncover novel gene products that contribute to the acid secretion process in Drosophila. Given the similarities between copper cells and gastric parietal cells it would not be surprising if new insights into the process of stomach acid secretion in mammals emerged as well.
Acknowledgements Special thanks to Drs. John Forte and Otto Baumann for providing electron micrographs, to Dr. Shaila Srinivasan, Christine Base and Pei San Ng for production and initial characterization of the labial-GFP reporter line and for comments on the manuscript, and to Susan M. Lundy, M.A.M.S. for artwork. This work was supported by NIH GM49301. References Bennett, V., & Baines, A. J. (2001). Spectrin and ankyrin-based pathways: Metazoan inventions for integrating cells into tissues. Physiological Reviews, 81, 1353–1388.
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Bienz, M. (1996). Induction of the endoderm in Drosophila. Seminars in Cell Developmental Biology, 7, 113–119. Bienz, M. (1997). Endoderm induction in Drosophila, the nuclear targets of the inducing signals. Current Opinion in Genetics & Development, 7, 683–688. Bilder, D. (2001). PDZ proteins and polarity: Functions from the fly. TIG, 17, 511–519. Chouinard, S., & Kaufman, T. C. (1991). Control of expression of the homeotic labial (lab) locus of Drosophila melanogaster: Evidence for both positive and negative autogenous regulation. Development, 113, 1267–1280. Dubreuil, R. R., MacVicar, G. R., Dissanayake, S., Liu, C., Homer, D., & Hortsch, M. (1996). Neuroglian-mediated adhesion induces assembly of the membrane skeleton at cell contact sites. Journal of Cell Biology, 133, 647–655. Dubreuil, R. R., Frankel, J., Wang, P., Howrylak, J., Kappil, M., & Grushko, T. (1998). Mutations of ␣ spectrin and labial block cuprophilic cell differentiation and acid secretion in the middle midgut of Drosophila larvae. Developmental Biology, 194, 1– 11. Dubreuil, R. R., Wang, P., Dahl, S. C., Lee, J. K., & Goldstein, L. S. B. (2000). Drosophila  spectrin functions independently of ␣ spectrin to polarize the Na,K ATPase in epithelial cells. Journal of Cell Biology, 149, 647–656. Dubreuil, R. R., Grushko, T., & Baumann, O. (2001). Differential effects of a labial mutation on the development, structure, and function of stomach acid secreting cells in Drosophila larvae and adults. Cell and Tissue Research, 306, 167–178. Duman, J. G., Pathak, N. J., Ladinsky, M. S., McDonald, K. L., & Forte, J. G. (2001). Three-dimensional reconstruction of cytoplasmic membrane networks in parietal cells. Journal of Cell Science, 115, 1251–1258. Filippova, M., Ross, L. S., & Gill, S. S. (1998). Cloning of the V-ATPase B subunit cDNA from Culex quinquefasciatus and expression of the B and C subunits in mosquitoes. Insect and Molecular Biology, 7, 223–232. Filshie, B. K., Poulson, D. F., & Waterhouse, D. F. (1971). Ultrastructure of the copper-accumulating region of the Drosophila larval midgut. Tissue and Cell, 3, 77–102. Freston, J. W., Rose, P. A., Heller, C. A., Haber, M., & Jennings, D. (1999). Safety profile of lansoprazole: The US clinical trial experience. Drug Safety, 20, 195–205. Guyton, A. C., & Hall, J. E. (1996). Medical physiology. Philadelphia, PA: Saunders. Hoppler, S., & Bienz, M. (1994). Specification of a single cell type by a Drosophila homeotic gene. Cell, 76, 689–702. Hoppler, S., & Bienz, M. (1995). Two different thresholds of wingless signalling with distinct developmental consequences in the Drosophila midgut. EMBO Journal, 14, 5016–5026.
Janowitz, H. D., Colcher, H., & Hollander, F. (1952). Inhibition of gastric secretion of acid in dogs by carbonic anhydrase inhibitor 2-acetylamino-1,3,4-thiadiaaole-5-sulphonamide. American Journal of Physiology, 171, 325–330. Jons, T., & Drenckhahn, D. (1998). Anion exchanger 2 (AE2) binds to erythrocyte ankyrin and is colocalized with ankyrin along the basolateral plasma membrane of human gastric parietal cells. European Journal of Cell Biology, 75, 232–236. Knust, E., & Bossinger, O. (2002). Composition and formation of intercellular junctions in epithelial cells. Science, 298, 1955– 1959. Lee, J., Coyne, R., Dubreuil, R. R., Goldstein, L. S. B., & Branton, D. (1993). Cell shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster. Journal of Cell Biology, 123, 1797–1809. McNulty, M., Puljung, M., Jefford, G., & Dubreuil, R. R. (2001). Evidence that a copper-metallothionein complex is responsible for fluorescence in acid secreting cells of the Drosophila midgut. Cell and Tissue Research, 304, 383–389. Mercier, F., Teggio, H., Deviliers, G., Bataille, D., & Mangeat, P. (1989). Membrane-cytoskeleton dynamics in rat parietal cells: Mobilization of actin and spectrin upon stimulation of gastric acid secretion. Journal of Cell Biology, 108, 441–453. Poulson, D. F., & Bowen, V. T. (1952). Organization and function of the inorganic constituents of nuclei. Experimental Cell Research, 2(Suppl.), 161–180. Shimada, A., Yanagida, M., & Umemura, T. (1997). An immunohistochemical study on the tissue-specific localization of metallothionein in dogs. Journal of Computer Pathology, 116, 1–11. Skaer, H. (1993). The alimentary canal. In M. Bate & A. M. Arias (Eds.), The development of Drosophila melanogaster (Vol. II, pp. 941–1012). Plainview, NY: Cold Spring Harbor Press. Strasburger, M. (1932). Bau, Funktion und variabilitat des darmtractus von Drosophila melanogaster. Zeitschrift fur Wissenchaft. Zoologie, 140, 539–649. Stuart-Tilley, A., Sardet, C., Pouyssegur, J., Schwartz, M. A., Brown, D., & Alper, S. L. (1994). Immunolocalization of anion exchanger AE2 and cation exchanger NHE-1 in distinct adjacent cells of the gastric mucosa. American Journal of Physiology, 266, C559–C568. Tepass, U., Tanentzapf, G., Ward, R., & Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annual Reviews in Genetics, 35, 747–784. Terra, W. R. (2001). The origin and functions of the insect peritrophic membrane and peritrophic gel. Archives of Insects in Biochemistry and Physics, 47, 47–61. Yao, X., & Forte, J. G. (2003). Cell biology of acid secretion by the parietal cell. Annual Reviews in Physiology, 65, 103–131.
Cells in Focus
Copper cells and stomach acid secretion in the Drosophila midgut Ronald R. Dubreuil∗ Department of Biological Sciences, University of Illinois Chicago, Chicago, IL 60607, USA Accepted 22 July 2003
Abstract Copper cells in the Drosophila larval midgut were originally named for their ability to accumulate dietary copper. Recent studies have uncovered a number of intriguing similarities between copper cells and the acid-producing gastric parietal cells of the mammalian stomach. In addition to their shared roles in stomach acidification, they share a peculiar invaginated morphology in which the apical cell surface is buried deep within the cytoplasm. These shared properties of morphology and function portend the identification of shared molecular mechanisms that account for their specialized roles in digestive physiology. © 2003 Elsevier Ltd. All rights reserved. Keywords: Copper cells; Acid-secreting cells; Gastric parietal cells; Labial gene
Cell facts • • • • •
Approximately 100 copper cells in larval middle midgut. Orange fluorescence after copper feeding. Specification during development is controlled by the labial gene. Apical surface is arranged as a flask-shaped invagination. Responsible for stomach acidification.
1. Introduction The gastric parietal cell in mammals is responsible for secretion of stomach acid through the activity of a proton pump that resides in the apical plasma membrane. In the resting cell, microvilli containing the proton-pumping ATPase are sequestered within tubulovesicles or canaliculi that are found throughout the cytoplasmic space. When activated to secrete stomach acid, parietal cells undergo a dramatic ∗
Tel.: +1-312-413-7831; fax: +1-312-996-2805. E-mail address: [email protected] (R.R. Dubreuil).
transformation in which the tubulovesicles fuse with the apical surface of the cell and become dilated (reviewed by Yao & Forte, 2003). As a result, the apical surface of the acid-secreting cell acquires a deeply invaginated appearance (Fig. 1A; Duman, Pathak, Ladinsky, McDonald, & Forte, 2001). Much of the remaining space within the cytoplasm is occupied by a reticular network of mitochondria that presumably are required to drive the intense metabolic activity that accompanies acid secretion. Studies of the digestive tract in the fruit fly, Drosophila, have uncovered an equally peculiar cell type in the stomach of larvae and adults. These
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Fig. 1. Ultrastructure of acid secreting cells. (A) High magnification view of rabbit gastric parietal cells after stimulation with histamine for 28 min. The apical surface of the cell is deeply invaginated and densely covered with microvilli that contain the H,K ATPase proton pump. Many darkly stained mitochondria occupy the cytoplasmic spaces surrounding the invaginations. Bar = 2 M. Photo courtesy of Dr. J.G. Forte. (B) High magnification view of the copper cell region from an adult Drosophila midgut. The copper cell (C) like the gastric parietal cell, has a deeply invaginated apical domain (V) that is densely covered with microvilli (mv). Much of the cytoplasmic space surrounding the invagination is occupied by mitonchondria. Two interstitial cells (I) are shown separating the copper cell at the center from neighboring copper cells. Septate junctions (sj) appear as electron-dense plasma membrane regions where the shoulders of the copper cells make contact with the overarching apical region of interstitial cells. A thin sheath of muscle cells (mu) surrounds the gut epithelium opposite from the gut lumen. Bar = 2 M. Photo courtesy of Dr. O. Baumann. (C) A schematic cutaway view of the middle migut. Copper cells (orange) alternate in a checkerboard pattern with spindle-shaped interstitial cells (blue). The apical invagination of the copper cells is connected to the gut lumen through a narrow channel formed by the interstitial cells which overarch the apical region of the copper cells. Septate junctions form a collar (black) over the shoulders of the copper cells to form a permeability barrier between the gut lumen and the body cavity.
cells were first described by Strasburger (1932) and later by Poulson and coworkers who named them “cuprophilic” cells (Poulson & Bowen, 1952; Filshie, Poulson, & Waterhouse, 1971) because of their striking orange fluorescence in copper-fed larvae and their ability to accumulate radioactively-labeled copper. We now know that, if anything, copper cells should be considered “cuprophobic” since their normal physiological activity is inhibited by copper (described below). Hence, we refer to these cells simply as copper cells. Copper cells bear several striking similarities to mammalian gastric parietal cells. EM studies re-
vealed that copper cells have a deeply invaginated apical membrane domain that is densely covered with microvilli (Fig. 1B; Filshie et al., 1971; Dubreuil, Grushko, & Baumann, 2001). There is also a high density of mitochondria present throughout the cytoplasm. Dye feeding experiments have established that the food contents of the gut are strongly acidic in the copper cell region and in the stomach-like region immediately downstream (pH < 2.3; Strasburger, 1932; Dubreuil et al., 1998). Genetic experiments have shown that in the absence of copper cell differentiation there is no detectable stomach acid secretion (Dubreuil et al., 1998). Thus, copper cells from
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Drosophila share a number of unique features with the gastric parietal cells of mammals.
2. Copper cell environs The digestive tract of dipteran larvae is divided into conspicuous domains of morphology and function. Here, I will focus on the midgut of Drosophila melanogaster, which has received the most attention from cell and developmental biologists. Most of the length of the gut is termed the midgut. The remainder consists of the ectodermally derived foregut and hindgut which are relatively short compared to the midgut (Skaer, 1993). The midgut is probably the site where most digestion of food occurs. It is not widely appreciated that food passes through the digestive tract of many insects wrapped in a peritrophic membrane (Terra, 2001). This membrane, which resembles a microscopic sausage casing, is synthesized within the proventriculus (Pr, Fig. 2A) at the anterior end of the midgut. Four gastric caeca project from the base of the proventriculus (reaching upward alongside the
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proventriculus in the figure) where it feeds into the midgut proper. The anterior midgut (a) is a relatively short region in which the diameter of the epithelial tube gradually tapers between the proventriculus and the middle midgut (m). The slender middle midgut expands into a larger diameter stomach region which is typically filled with food (*). Another constriction of the gut below the stomach marks the site at which the pH of the food is neutralized (Dubreuil et al., 1998). The posterior midgut downstream of this contriction is the longest region of the midgut. It appears relatively homogeneous and is likely to be the principal site of food absorption. The copper cells can be distinguished from their neighbors in the light microscope by a number of criteria. In rhodamine–phalloidin labeled whole-mount preparations, the copper cells appear as bright crescents corresponding to the actin-rich microvilli of the apical domain (Lee, Coyne, Dubreuil, Goldstein, & Branton, 1993). The copper cells can also be detected by their conspicuous orange fluorescence in copper-fed larvae (Poulson & Bowen, 1952; McNulty, Puljung, Jefford, & Dubreuil, 2001).
Fig. 2. Anatomy of the Drosophila midgut. (A) Brightfield image of a dissected larval midgut. The proventriculus (Pr) marks the beginning of the anterior midgut (a), followed by the middle midgut (m) and the posterior midgut (p) which extends out of the field of view. The middle midgut is quite narrow in diameter at the beginning and becomes much broader in the stomach region where there is usually food visible in the gut lumen (*). (B) Fluorescent image of the field in A showing the domain of expression of a labial-GFP reporter gene that is exclusively expressed in copper cells of the midgut. A kink in the copper cell region is marked with an arrow for reference. (C) Higher magnification view of the GFP reporter pattern showing bright nuclear GFP fluorescence, weaker cytoplasmic fluorescence, and banana shaped gaps in the fluorescence pattern corresponding to the apical invagination of copper cells.
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Bienz and coworkers performed a series of elegant experiments addressing the mechanism of copper cell specification (Hoppler & Bienz, 1994). In their studies they described a -galactosidase reporter gene that is specifically expressed in copper cells. We produced a similar reporter construct that expresses green fluorescent protein (GFP) under control of the 5 -regulatory DNA that governs labial expression in the larval midgut (Chouinard & Kaufman, 1991). This reporter transgene labels the copper cells of the midgut in living embryos and larvae, and in the dissected midgut (Fig. 2B). Higher magnification views of the GFP labeled cells reveal the spherical shape of each copper cell as well as the invaginated apical domain which excludes the cytoplasmic GFP label (Fig. 2C, arrow). About 100 copper cells are detected in the middle midgut epithelium from its narrowest region through the proximal portion of the stomach. A more detailed account of the design and properties of the labial-GFP transgene will be published elsewhere.
3. Fine structure of cuprophilic cells Electron microscopy reveals that there are multiple distinct cell types in the middle midgut. The flattened, spherical copper cells (C) alternate with spindle-shaped interstitial cells (I; Fig. 1B) which surround and envelope each copper cell (shown schematically in Fig. 1C). The interstitial cells reach over the apical surface of the copper cells, leaving a narrow channel that connects the copper cell invagination to the gut lumen. The cytoplasm of the copper cells appears less dense than the cytoplasm of the interstitial cells, making it easy to see the boundary between them. The interstitial cells also have extensive infoldings of the basal plasma membrane that often extend close to the apical plasma membrane (Filshie et al., 1971; Dubreuil et al., 2001). The apical surface of the interstitial cells, facing the midgut lumen, is decorated with numerous short microvilli. Apart from their peculiar invaginated structure, copper cells have the same specialized plasma membrane domains found in other more conventional epithelial cells. The apical invagination is densely covered with microvilli that nearly fill the extracellular space within the invagination, thus accounting for the crescent shapes detected by rhodamine phalloidin staining
(Lee et al., 1993). The permeability barrier of the gut is provided by a specialized cell–cell contact known as the septate junction (sj). Septate junctions are functionally and morphologically similar to the tight junctions of mammalian cells (Tepass, Tanentzapf, Ward, & Fehon, 2001). They have an electron-dense character compared to the remainder of the basolateral plasma membrane. The molecular composition of the smooth septate junctions is not yet known. Antibodies against the cytoskeletal proteins spectrin and ankyrin appear to specifically label the electron dense region (Dubreuil et al., 2001, described below). There is close apposition between the copper cells and the interstitial cells in the basolateral domain below the level of the septate junction, but this plasma membrane region does not appear electron dense. The basal surfaces of copper cells and interstitial cells are flattened where they contact the thin muscle sheath that surrounds the gut epithelium. Specialized cell–cell contacts in this region have not yet been described. Several other general features are apparent in electron micrographs of the middle midgut. The nucleus of the copper cells (n) is located in the basal cytoplasm, whereas the nucleus of the interstitial cells (lower right in the figure) is found in the apical cytoplasm. The pore formed by the interstitial cells at the apex of the copper cells is often quite narrow, and in the section of an adult midgut shown here it appears to be nearly closed. However, the sum of evidence from EM analysis and fluorescent antibody staining suggests that the apical domain of copper cells is probably always open to the midgut lumen, unlike the situation in mammalian parietal cells where acid secretion is regulated by pinching off of tubulovesicles. High magnification views revealed that the apical microvilli of the copper cells are densely studded with lollipop-shaped particles that presumably represent a plasma membrane V-ATPase (Dubreuil et al., 2001). Moreover, an antibody against the Culex V-ATPase B subunit (Filippova, Ross, & Gill, 1998) specifically stains the apical membrane domain of copper cells (unpublished observation).
4. Specification and differentiation of cuprophilic cells Copper cells appear relatively early in embryonic development of the midgut. Bienz and coworkers
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established that expression of the homeotic gene labial is required for specification of copper cells and for maintenance of their differentiated state (Hoppler & Bienz, 1994). There is a gradient of labial expression within the copper cell domain with increasing labial expression from anterior to posterior (Bienz, 1996) which coincides with a more elaborate apical invagination in the most posterior cells (Dubreuil et al., 1998). There is a parallel gradient in the pattern of the septate junction (Dubreuil et al., 1998, 2001). In the most anterior cells the opening to the invagination is relatively large and is surrounded by a relatively thin septate junction collar. Toward the posterior the opening is severely constricted and the septate junction collar is concomitantly smaller in diameter, but also thicker. The zone of labial expression is determined by input from the morphogens decapentaplegic and wingless, whose expression in the visceral mesoderm is regulated by the homeotic gene Ubx (reviewed in Bienz, 1997). Interestingly, the boundaries of the copper cell domain can be shifted in response to changes in the dose of wingless (Hoppler & Bienz, 1995). Very high wingless levels can repress labial expression and copper cell development altogether whereas decreased wingless levels can lead to an expansion of the copper cell domain to include cells that would normally acquire a distinct fate. Most of the developmental studies to date have focused on the complex regulatory interactions of genes upstream of labial. An important future goal will be to elucidate the pathway downstream of labial that generates the peculiar shape and polarity of the copper cell. Differentiation of copper cells appears to depend on labial in both adults and larvae. A conditional labial mutation (labk3 ) blocks labial expression and interferes with copper cell differentiation in larvae but not in adults (Dubreuil et al., 2001). It appears that the molecular defect in this mutant affects expression of labial in the larva but not in the adult. Interestingly, since the mutant larvae which altogether lack copper cells (and therefore also lack stomach acid secretion) are nonetheless capable of normal development, it appears that stomach acid secretion is not essential at least during larval development. This unexpected observation fits well with the observation that stomach acid secretion can be inactivated pharmacologically in human patients with gastric disease without any dire consequences (Freston, Rose, Heller, Haber, &
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Jennings, 1999). Thus, the metabolically expensive process of stomach acid secretion appears to be dispensible, at least under controlled laboratory and clinical conditions.
5. Cell biology of copper cells One way to elucidate the molecular basis for the striking morphological properties of the copper cell will be to localize structural and regulatory proteins that are responsible for their unusual shape and polarity. So far only a handful of proteins have been localized. As mentioned above, staining with rhodamine phalloidin primarily detects the actin filaments of the apical microvilli and the muscle sheath (mu in Fig. 1B) that surrounds the gut epithelium (Lee et al., 1993). The two plasma membrane proteins that have been localized so far are the Na,K ATPase, which resides in the basolateral domain (Dubreuil, Wang, Dahl, Lee, & Goldstein, 2000), and the V-ATPase which resides in the apical microvilli (unpublished observation). Two isoforms of the cytoskeletal protein spectrin are expressed in copper cells. Spectrin associates with the cytoplasmic surface of the plasma membrane where it contributes to cell shape, membrane stability, and the organization of polarized plasma membrane domains (Bennett & Baines, 2001). The ␣ subunit of spectrin, which is shared by both Drosophila isoforms, is detected at all regions of the plasma membrane (Lee et al., 1993; Dubreuil et al., 2000). The  subunit is associated with the basolateral domain, including the septate junction and the H subunit is found exclusively in the apical plasma membrane domain (Dubreuil et al., 1998, 2001). Mutations in the  spectrin gene cause a gross mislocalization of the Na,K ATPase, indicating that spectrin is required for its normal expression in the basolateral domain (Dubreuil et al., 2000). Spectrin interacts with the Na,K ATPase via the linker protein ankyrin. While initial localization studies suggested that ankyrin was restricted to the septate junction (Dubreuil et al., 1998, 2001), we now know that other antibodies detect ankyrin codistributing with the Na,K ATPase throughout the basolateral membrane (unpublished observation). The original antibody may have recognized a unique subpopulation of ankyrin molecules, or may have cross-reacted with some other
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component of the septate junction. Thus the genetic requirement of  spectrin for the normal distribution of the Na,K ATPase is likely to be mediated via interactions between  spectrin, ankyrin and the Na,K ATPase. Many interesting cell biological issues are ripe for analysis in copper cells. The molecules that form the smooth septate junctions of the midgut are unknown, as is the mechanism that determines their size and shape. Interestingly, mutations in spectrin and labial both affect the diameter and thickness of the septate junction ring that surrounds the neck of the copper cell invagination (Lee et al., 1993; Dubreuil et al., 2000, 2001). These observations suggest that spectrin may be an important component of the pathway between labial expression and copper cell differentiation. An important future step will be to identify the adhesion molecules that attach copper cells to their neighbors, both in the septate junction and in the rest of the basolateral domain. Cell adhesion molecules are likely candidates to mediate the polarized assembly of spectrin and ankyrin within specific plasma membrane domains. Neuroglian, the only known adhesion molecule in Drosophila that interacts with the spectrin cytoskeleton (Dubreuil et al., 1996), is not expressed in the middle midgut. Another interesting area for further study is the identification of structural proteins that generate the invaginated morphology of the apical domain. The depth of the invagination is suggestive of a role for molecular motors such as members of the myosin or kinesin protein families. Neither has been studied in copper cells. Copper cells may also be a valuable system in which to study the mechanisms that underlie the development of plasma membrane polarity, since the polarized domains of these cells are especially well-defined. It will be interesting to ask if proteins such as Crumbs, Bazooka, Stardust, Scribble and Dlg, which have been studied in the development of ectodermally derived epithelial cells (Bilder, 2001; Knust & Bossinger, 2002), also have a role in the polarization of copper cells.
6. Implication of cuprophilic cells in stomach acid secretion The copper cells of the Drosophila midgut appear to be functionally related to the gastric parietal cells
of mammals. Beyond their mophological similarities, copper cells are present at precisely the site at which midgut acid secretion occurs, the magnitude of acid secretion is comparable between Drosophila and mammals, and genetic defects that perturb copper cells lead to a loss of stomach acid secretion in Drosophila. There are also some important differences between gastric parietal cells and copper cells. For one, copper cells do not appear to undergo the dramatic morphological transformation that accompanies activation of acid secretion in parietal cells. However, it remains possible that the apical invagination of copper cells can open and close to regulate acid secretion. If the copper cell invagination is not there to provide a regulatory mechanism, then it is not apparent what benefit this unusual morphology might offer. There is no a priori link between an invaginated morphology and the ability to secrete acid, although it is noteworthy that this shared peculiarity is rare in evolution. Perhaps there is some benefit in shielding the acid-secreting microvillar surface from the extracellular environment. Alternatively, it may be beneficial to shield neighboring cells from the site of proton translocation where the pH of the extracellular fluid should be lowest. A second difference is that the apical proton pump in copper cells appears to be a V-ATPase family member, while acid secretion in parietal cells is mediated by a H,K ATPase (which is not known to be expressed in Drosophila). The discovery that copper cells are responsible for acid secretion grew out of the initial characterization of Drosophila ␣ spectrin mutants. The shape and pattern of copper cells is grossly perturbed in these mutants (Lee et al., 1993). Since mammalian spectrin was closely linked to the H,K ATPase during activation of gastric parietal cells (Mercier, Teggio, Deviliers, Bataille, & Mangeat, 1989), we were interested to know if spectrin was also required for acid secretion in Drosophila. As it turns out, acid secretion was blocked in ␣ spectrin mutants (Dubreuil et al., 1998). However, it has not been a simple matter to determine the molecular basis for the defect. The contribution of spectrin to acid secretion is not yet known, in parietal cells or in copper cells. One hypothesis is that the apical population of ␣H spectrin in copper cells is somehow responsible for the correct targeting and/or function of the apical V-ATPase (Dubreuil et al., 1998). Spectrin could have a similar stabilizing effect on the H,K ATPase in mammals.
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Another hypothesis is that the basolateral population of ␣ spectrin regulates a basolateral activity that is indirectly required for acid secretion. One candidate is the anion exchanger which interacts with mammalian spectrins and has been implicated in the acid secretion process (Stuart-Tilley et al., 1994; Jons & Drenckhahn, 1998). Protons translocated by the proton pump are derived from splitting of water (Guyton & Hall, 1996). The resulting hydroxyl ions are then acted upon by carbonic anhydrase and CO2 to form HCO3 − which is then exchanged for Cl− at the basolateral surface of the cell. The Cl− ions pass through apical Cl channels, yielding a net stomach secretion of HCl. Failure to clear HCO3 − from the cell may exert feedback inhibition on the proton pumping process, as is the case with pharmacological inhibition of carbonic anhydrase (Janowitz, Colcher, & Hollander, 1952). Thus, the inhibition of acid secretion in spectrin mutants could be the result of an effect on the anion exchanger, analogous to the effect of  spectrin mutations on the Na,K ATPase (Dubreuil et al., 2000). It will be possible to test this hypothesis by examining the fate of a recently identified anion exchanger in the middle midgut of Drosophila ␣ spectrin mutants. 7. What makes a copper cell a copper cell? The mechanism behind the response of copper cells to dietary copper remains a mystery. Recent work suggests that the orange fluorescence observed in copper cells after copper feeding is due to formation of a complex between copper ions and a member of the metallothionein family of proteins (McNulty et al., 2001). However, it will be necessary to directly test this proposal once suitable genetic tools such as metallothionein mutants are in hand. The suggestion of a role for copper cells in metal detoxification seems less likely now that a role in the secretion of stomach acid has been established. Perhaps a more definitive test would be to examine the susceptibility of Drosophila larvae to copper toxicity under conditions in which development of copper cells has been blocked (i.e. in labial mutants). Interestingly, metallothionein is known to be expressed in the acid secreting cells of the dog stomach (Shimada, Yanagida, & Umemura, 1997). However, it was not possible to detect copper-dependent fluores-
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cence in response to copper feeding in the mouse stomach (our unpublished observation). It is clear that copper feeding in Drosophila inhibits stomach acid secretion (McNulty et al., 2001), which is consistent with binding of copper to an essential component of the acid secretion apparatus. Metallothionein may have an unanticipated role in stomach acid secretion that could explain why copper cells behave as a sink for the accumulation of dietary copper.
8. Future prospects Copper cells are a valuable experimental system in which to ask fundamental questions about epithelial cell biology as well as the mechanism of stomach acid secretion. The middle midgut of larvae is easily amenable to microscopic studies. It is a simple matter to dissect large number of larval midguts for staining experiments or physiological measurements. The true value of the Drosophila system is the ability to apply genetic strategies to problems of molecular function. For example, it will be possible to carry out genetic screens for mutations that affect the acid secretion pathway, which has been impractical in mammalian systems. This approach is expected to uncover novel gene products that contribute to the acid secretion process in Drosophila. Given the similarities between copper cells and gastric parietal cells it would not be surprising if new insights into the process of stomach acid secretion in mammals emerged as well.
Acknowledgements Special thanks to Drs. John Forte and Otto Baumann for providing electron micrographs, to Dr. Shaila Srinivasan, Christine Base and Pei San Ng for production and initial characterization of the labial-GFP reporter line and for comments on the manuscript, and to Susan M. Lundy, M.A.M.S. for artwork. This work was supported by NIH GM49301. References Bennett, V., & Baines, A. J. (2001). Spectrin and ankyrin-based pathways: Metazoan inventions for integrating cells into tissues. Physiological Reviews, 81, 1353–1388.
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