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First published online May 15, 2009
Journal of Experimental Biology 212, 1731-1744 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.029306

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Epithelial ultrastructure and cellular mechanisms of acid and base transport in the Drosophila midgut

Shubha Shanbhag and Subrata Tripathi^*

Tata Institute of Fundamental Research, Colaba, Mumbai 400 005, India

View larger version (67K): [in this window] [in a new window]   Fig. 1. (A) Drawing of larval midgut of Drosophila melanogaster showing various segments and the intraluminal pH zones. The luminal content of the anterior segment and the anterior part of the posterior segment is between neutral to mild alkalinity (pH>7 and <8; green), the middle segment is highly acidic (pH<3.0; magenta dots) and the posterior part of the posterior segment is highly alkaline (pH>10; blue dots). GC, gastric caeca; HG, hindgut; MT, Malpighian tubules. (B) Schematic representation of a cross-section of the posterior region of the larval posterior midgut showing the arrangement of peritrophic membrane (magenta), epithelial cells (black), basement membrane (green), muscle fibres (red), regenerative cells or stem cells (light blue, marked S) and serosal barrier (brown). (C) General organization of two adjacent epithelial cells, lateral dimensions compressed. Colour coding is the same as in B. The arrow denotes very small lateral intercellular spaces. BEL, basal extracellular labyrinth; BM, basement membrane; CM, circular muscle; JC, junctional complex; LIS, lateral intracellular septum; LM, longitudinal muscle; M, mitochondria; MV, microvilli; N, nucleus; PM, peritrophic membrane; SB, serosal barrier; T, tracheole.

View larger version (137K): [in this window] [in a new window]   Fig. 2. Unperfused larval posterior midgut. (A) Section through two adjacent enterocytes of unperfused posterior midgut of larva depicting the intercellular septum from the junctional complex (JC) to the basal extracellular labyrinth (BEL). The lateral intracellular space is absent except for a few small dilations (arrows). The apical region of these cells contains microvilli (MV), and the basal region rests on the basement membrane (BM). An inner layer of circular muscles (CM) is also seen; magnification x13,200; scale bar, 1 µm. (B) Magnified view of an apical region with intercellular septal JC and dilations (arrows); magnification x37,300; scale bar, 0.5 µm. (C) Apical region of epithelial cell showing MV separated from the gut lumen by a single peritrophic membrane (PM); magnification x21,000; scale bar, 0.5 µm. (D) Cross-section of MV showing outer surfaces with fine fuzzy material and cytoplasmic surfaces containing portasome-like particles (arrowheads); magnification x84,000; scale bar, 0.5 µm. (E) Section through a basal region of an epithelial cell showing an extensive BEL closely associated with mitochondria (M). The basement membrane (BM) is separated from the haemolymph by inner CM, outer longitudinal muscle (LM) and the outermost thin serosal barrier (SB); magnification x16,700; scale bar, 0.5 µm. The BEL is lined by particles of <20 nm diameter (small arrows, in inset). The inset is a magnified view of the boxed region in E; magnification x54,000; inset scale bar, 0.25 µm.

View larger version (197K): [in this window] [in a new window]   Fig. 3. Perfused larval posterior midgut. (A–C) Sections of larval posterior midgut epithelial cell perfused with bilateral Hepes Ringer solution. (A) Microvillar brush border (MV) and extensive basal extracellular labyrinth (BEL); magnification x11,700. (B) Removal of the peritrophic membrane ensures direct access of intact microvilli (MV) to the luminal (L) perfusion solution. magnification x16,000. (C) Basal region of the cell shows basement membrane (BM), enclosing a significant BEL; magnification x17,400. (D–F) Sections of larval posterior midgut epithelial cell perfused with Hepes Ringer in the bath and Na+-free Hepes Ringer in the lumen. (D) The MV are intact, but BEL volume is reduced and several large intracellular vacuoles (V) are found in the cytoplasm; magnification x12,000. (E) MV have many vesicles budding off from them (arrows); magnification x16,700. (F) Magnified view of the basal region of the cell with reduced BEL volume; magnification x18,700. (G–I) Perfusion with bilateral Na+-free Hepes Ringer. (G) The cell is filled with a large number of intracellular vacuoles (V); magnification x11,700. (H) MV height is reduced, and there are several dilated vesicles (arrows) budding off from MV tips; magnification x16,700. (I) Basal region of the cell is devoid of basal extracellular labyrinth (asterisk); magnification x14,000. CM, circular muscle; SB, serosal barrier; T, tracheole. Scale bars (A–I), 1.0 µm.

View larger version (106K): [in this window] [in a new window]   Fig. 4. (A) Line diagram of adult gut showing the intraluminal pH in various midgut regions. The luminal contents of anterior and posterior midgut segments are mildly alkaline (pH 7–9; green dots), while the middle midgut segment is acidic (pH<4.0; magenta dots). C, cardia; H, head; HG, hindgut; MT, Malpighian tubules. (B–E) General organization of adjacent epithelial cells of various segments of adult midgut showing the arrangement of peritrophic membrane (magenta), epithelial cells (black), basement membrane (green), muscle fibres (red), regenerative cells or stem cells (light blue, marked *) and serosal barrier (brown). (B,C) Anterior and posterior region of the anterior midgut, respectively. (D) Acidic middle midgut. The apical region of the secretory cell (SC) extrudes a large number of electron-lucent vesicles (V) and vesicles containing electron-dense granules (G). (E) Posterior midgut. AC, absorptive cell; BEL, basal extracellular labyrinth; BM, basement membrane; CM, circular muscle; EcS, ectoperitrophic space; EnS, endoperitrophic space; G, granules; JC, junctional complex; LIS, lateral intracellular septum; LM, longitudinal muscle; M, mitochondria; MV, microvilli; N, nucleus; PM1 and PM2, peritrophic membranes; SB, serosal barrier; T, tracheole.

View larger version (104K): [in this window] [in a new window]   Fig. 5. Unperfused adult anterior midgut. (A,B) Light micrographs of cross-sections of anterior and posterior regions, respectively, of an adult anterior midgut. Epithelial cells show large spherical or oval nuclei at the centre. (A) Enterocytes of the anterior region are taller, with a central cytoplasmic dome into the lumen (arrowheads); magnification x400. (B) Posterior cells are large, cuboidal cells; magnification x400. Scale bars (A,B), 25 µm. (C) A single epithelial cell sectioned through the anterior region of the anterior adult midgut has a large oval-shaped nucleus (N) and a central cytoplasmic dome. The apical region of the cell contains a well-developed microvillar (MV) brush-border. Arrowheads denote the region from where the intracellular junctional complexes begin; magnification x4400; scale bar, 2 µm. (D) Section through double-layered peritrophic membranes (PM), an inner thick one (PM1) that surrounds the food, and an outer thin one (PM2) that bounds the ectoperitrophic space (asterisk) with the MV; star, ectoperitrophic space; magnification x18,700; scale bar, 0.5 µm. (E) Cross-sections of MV showing small portasome-like particles on their cytoplasmic side (arrows); magnification x100,000; scale bar, 0.1 µm. (F) An elongated regenerative or stem cell (S) located between the two enterocytes; magnification x8100; scale bar, 2.0 µm. (G) Longitudinal section through a portion of brush-border with MV; the apical region of the cell has several large mitochondria (M); magnification x20,100; scale bar, 0.5 µm. (H) Section through a basal region of an enterocyte showing infoldings of the basal extracellular labyrinth (BEL). The basement membrane (BM) is separated from the haemolymph by inner circular muscle (CM) and outer longitudinal muscle (LM). T, tracheole; magnification x8800; scale bar, 0.5 µm.

View larger version (215K): [in this window] [in a new window]   Fig. 6. Perfused adult anterior midgut. (A) Epithelium showing centrally located nucleus (N) and many apically located mitochondria (M). The basal extracellular labyrinth (BEL) extends up to almost half the cell height. L, lumen; MV, microvilli; JC, junctional complex; magnification x2700; scale bar, 5.0 µm. (B) Section through two adjacent epithelial cells of a perfused adult anterior midgut shows a basal regenerative cell (S) between them; magnification x3780; scale bar, 5.0 µm. (C) Apical region of the epithelial cell of perfused adult anterior midgut cells showing MV that are in direct access to the luminal solution; magnification x12,000; scale bar, 1.0 µm. (D) Basal region has many mitochondria (M). BM, basement membrane; CM, circular muscle; LM, longitudinal muscle; SB, serosal barrier; T, tracheole; x10,000; scale bar, 1.0 µm.

View larger version (187K): [in this window] [in a new window]   Fig. 7. Unperfused adult posterior midgut. (A) Single layer of cuboidal cells, inner circular muscle (CM) and outer longitudinal muscle (LM); magnification x680; scale bar, 25 µm. (B) The basal region of the cell shows highly dilated basal extracellular labyrinth (BEL); magnification x5700; scale bar, 2.0 µm. (C) Densely packed microvilli (MV); asterisk, ectoperitrophic space; magnification x28,500; scale bar, 0.5 µm. (D) Basal aspect of an adult posterior midgut epithelial cell with highly dilated BEL. BM, basement membrane; CM, circular muscle; SB, serosal barrier; T, tracheole; magnification x15,000; scale bar, 0.5 µm.

View larger version (113K): [in this window] [in a new window]   Fig. 8. Unperfused adult middle (or acidic) midgut. (A) Epithelial layer with alternating absorptive cells (AC) and cup-shaped secretory cells (SC). This region also contains several regenerative or stem cells (S). CM, circular muscle; L, lumen; PM, peritrophic membrane; magnification x560; scale bar, 25 µm. (B) Section through an apical region of an absorptive cell enclosed by double-layered peritrophic membranes PM1 and PM2 enclosing the ectoperitrophic space (asterisk) between the microvillar brush-border (MV) and PM2; magnification x27,000; scale bar, 0.5 µm. (C) Section through a cup-shaped secretory cell with basally located nucleus (N). Apical region of the cell has a well-developed brush-border (see E) and the basal region shows few membrane infoldings (see G). The cavity is usually filled with vesicles and secretory granules (G), and opens into the ectoperitrophic space (asterisk); magnification x4100; scale bar, 2.0 µm. (D) Section through an absorptive cell depicting the extensive array of deep parallel basal extracellular labyrinth extending to two-thirds of the cell height and perpendicular to the basement membrane (see H). CM, circular muscle; SB, serosal barrier; T, tracheole; magnification x3900; scale bar, 2.0 µm. (E) Apical region of a secretory cell contains long and densely packed MV. Several large mitochondria (M) are present in the cytoplasm but not in the microvilli; magnification x26,400; scale bar, 0.5 µm. (F) Section through an apical region of two adjacent absorptive cells showing small loosely packed MV. These cells also contain many large M. The long intracellular junctional complex (JC) has several dilations (arrows) but no lateral intracellular spaces; magnification x15,000; scale bar, 1.0 µm. (G) Section through the basal region of a secretory cell shows very few evaginations of basal extracellular labyrinth (BEL). BM, basement membrane; CM, circular muscle; magnification x16,000; scale bar, 1.0 µm. (H) Magnified view of the basal region of the absorptive cell (cf. D) with extensive array of BEL (open arrow) enclosed by BM. Several mitochondria (open arrowhead) lie parallel with their membranes closely opposed to the plasma membrane of the basal infoldings. The thin serosal barrier (SB) isolates the compartment between the BM and the haemolymph. T, tracheole. Magnification x16,700; scale bar, 1.0 µm.

View larger version (83K): [in this window] [in a new window]   Fig. 9. Unperfused adult middle midgut. (A,C) Low-magnification electron micrographs of a secretory cell (SC) located between two absorptive cells (AC) in closed and open configurations (open arrow). The secretory cell cavity in the open state contains electron-dense granules and vesicles that are liberated into the ectoperitrophic space (asterisk) of the midgut; magnification x2600; scale bar, 5.0 µm. (B,D) Diagrammatic representations of a secretory cell (SC) located between two absorptive cells (AC) in open and closed configurations (also see Fig. 10A,E). The luminal surface of SC has long, densely populated microvilli (MV), and the basal surface displays few infoldings (BEL). Absorptive cells (AC) have very short and sparsely distributed MV but long and extensive parallel arrays of BEL. BM, basement membrane; CC, closed cavity; CM, circular muscle; JC, intercellular junctional complex; LM, longitudinal muscle; N, nucleus; OC, open cavity; PM1 and PM2, peritrophic membranes; SB, serosal barrier; T, tracheole.

View larger version (97K): [in this window] [in a new window]   Fig. 10. Perfused adult middle midgut. (A). Section through a secretory cell (SC) with apical cavity open to the lumen (L). The peritrophic membrane is washed off, and intact microvilli (MV) are directly in contact with the luminal solution. The cell contains a large number of secretory granules (G), vesicles (V) and mitochondria (M); CM, circular muscle; LM, longitudinal muscle; SB, serosal barrier; T, tracheole; magnification x5000; scale bar, 5.0 µm. (B) Magnified view of apical region of a secretory cell shows vesicles with single or many electron-dense granules (G) arising mainly from the base of the adjacent microvilli and many electron-lucent vesicles (V) that bud off usually from the tip of the MV; magnification x18,700; scale bar, 1.0 µm. (C) Section through a portion of the cavity (asterisk) of the secretory cell, with many secretory granules and vesicles. M, mitochondria; MV, microvilli; N, nucleus; magnification x11,300; scale bar, 1.0 µm. (D) Intercellular junctional complex (JC) of an absorptive cell(s) seen closing the apical aperture of the secretory cavity; magnification x9300; scale bar, 1.0 µm. (E). Section through a secretory cell (SC) in the same plane shows the apical cell cavity closed by the intercellular JC of an absorptive cell (AC). CC, closed cavity; CM, circular muscle; LM, longitudinal muscle; M, mitochondria; SB, serosal barrier; magnification x5000; scale bar, 5.0 µm.

View larger version (145K): [in this window] [in a new window]   Fig. 11. Midguts of 3rd instar Drosophila larvae. (A,B) Larvae fed with food containing dyes alone or together with acetazolamide (C) to detect luminal pH. GC, gastric caeca; MT, Malpighian tubules. (A) Acidification was detected in the middle midgut (acid zone), where Bromophenol Blue (BPB) dye changed from blue to yellow (pH<3.0). (B) Very strong alkalinization was detected in the posterior half of the posterior midgut (alkaline zone), where Phenol Red (PR) dye changed from red to dark pink (pH>10.0). (C) Acetazolamide (100 µmol l–1) in food containing BPB led to dissipation of pH gradients in the larval midgut. (D) Luminal content pH detected by pH paper shows a distinct acid-secreting zone (acid zone, ochre, pH<3) and posterior alkaline zone (dark green). (E) Acetazolamide (100 µmol l–1) in food dissipated the pH gradients in the acid zone. Numbered panels below denote colours generated on universal pH paper by pH standards of 1–11. Scale bar, 1 mm.

View larger version (115K): [in this window] [in a new window]   Fig. 12. Carbonic anhydrase in the adult midgut. (A) Acidification was detected in the middle midgut (arrows, acid zone) where the colour of Bromophenol Blue (BPB) dye changed from blue to greenish brown (acid zone, pH<4.0); C, cardia; CR, crop; H, head; HG, hindgut; MT, Malpighian tubules. (B) Acetazolamide (100 µmol l–1) in food dissipated this gradient. (C) Luminal content pH detected by pH paper showed a distinct acid-secreting zone (arrows, pH<5) in the adult middle midgut. (D) Acetazolamide (100 µmol l–1) in food abolishes this acidification (arrows). Numbered panels below denote colours generated on universal pH paper by pH standards of 1–11. Scale bar, 1 mm.

View larger version (39K): [in this window] [in a new window]   Fig. 13. Carbonic anhydrase and H+ V-ATPase in the larval posterior midgut. (A) Intracellular pH measured in control solution (Sch-R) containing equal amounts of Schneider insect medium and insect Ringer perfusing the lumen and in the bath. Va, apical membrane potential; Vb, basal membrane potential; Vc, transepithelial potential at collection end; Vp, transepithelial potential at perfusion end; VpHin, potential of ion-selective microelectrode; VpH–Vb, intracellular pH corrected for membrane potential, referenced to ordinate at right. Acetazolamide (100 µmol l–1) applied to the bath reversibly alkalinizes the cell. (B) Extracellular scan of basal unstirred layer pH with an ion-selective microelectrode whose potential VpHout is shown as the brown trace, referenced to the ordinate on the right. In Sch-R, the extracellular pH of the bulk solution of the bath is 7.2. The pH microelectrode was advanced towards the gut wall by a piezo-stepper and positioned close to the serosal barrier and basal extracellular labyrinth (BEL; arrow); the increasingly positive VpHout potential reflects the pH gradient at the basal surface. The pH electrode was then retracted away to about 20 µm from the gut wall (arrowhead). Acetazolamide (100 µmol l–1) applied to the bath alkalinized the extracellular pH. Bafilomycin-A1 (1.5 µmol l–1) applied to the bath in addition to acetazolamide in the bath further alkalinized the extracellular pH. Washout with control Ringer restored H+ extrusion by the midgut, as seen by the positive change in VpHout at the end of the trace.

View larger version (28K): [in this window] [in a new window]   Fig. 14. (A–C) Adult midgut transepithelial potentials recorded from the anterior, middle and posterior segments, respectively, of Drosophila by the perfusion (Vp), collection (Vc) micropipettes and the basal cell membrane potential (Vb), after bilateral HCO3– Ringer (control Ringer) substitution. Current pulses (100 nA) cause displacements of Va, Vb, Vc and Vp. Va, apical membrane potential.

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