Peristalsis in the junction region of the Drosophila larval midgut is modulated by DH31 expressing enteroendocrine cells

  • Dennis R LaJeunesse1Email author,

    Affiliated with

    • Brooke Johnson1,

      Affiliated with

      • Jason S Presnell1,

        Affiliated with

        • Kathleen Kay Catignas1 and

          Affiliated with

          • Grzegorz Zapotoczny1

            Affiliated with

            BMC Physiology201010:14

            DOI: 10.1186/1472-6793-10-14

            Received: 24 June 2010

            Accepted: 10 August 2010

            Published: 10 August 2010

            Abstract

            Background

            The underlying cellular and molecular mechanisms that coordinate the physiological processes in digestion are complex, cryptic, and involve the integration of multiple cellular and organ systems. In all intestines, peristaltic action of the gut moves food through the various stages of digestion from the anterior end towards the posterior, with the rate of flow dependent on signals, both intrinsic and extrinsic to the gut itself.

            Results

            We have identified an enteroendocrine cell type that regulates gut motility in the Drosophila melanogaster larval midgut. These cells are located at the junction of the anterior and the acidic portions of the midgut and are a group of enteroendocrine cells that express the peptide hormone Diuretic Hormone 31 in this region of the gut. Using cell ablation and ectopic activation via expression of the Chlamydomonas reinhardtii blue light-activated channelopsin, we demonstrate that these enteroendocrine cells are both necessary and sufficient for the peristalsis in the junction region of the midgut and require the Diuretic Hormone 31 to affect normal peristalsis in this region. Within the same junction region of the midgut, we have also identified morphological features suggesting that this region acts as a valve that regulates the transit of food from the anterior midgut into the acidic portion of the gut.

            Conclusions

            We have characterized and described a set of enteroendocrine cells called the Midgut Junction DH31 expressing cells that are required for peristaltic movement in the junction region between the anterior portion and acidic region of the larval midgut of Drosophila melanogaster. We have shown that the Midgut Junction DH31 expressing cells are necessary and sufficient for motility and that the peptide hormone DH31 is required for peristalsis in the junction region of the midgut. The Drosophila model system will allow for a further dissection of the digestion process and provide a better understanding of the mechanisms that regulate digestion in all organisms.

            Background

            All higher metazoans have evolved digestive systems that extract sustenance from the environment for growth and survival [13]. Although digestive systems are adapted to diverse feeding behaviours, they share an overall similarity in their organization, and all coordinate nervous system and endocrine input to govern the movement and the processing of food within the alimentary canal [49]. Central to these concepts is the digestion of food within the gut. In all intestines, peristaltic action of the gut moves food through the various stages of digestion from the anterior end towards the posterior, with the rate of flow dependent on signals, both intrinsic and extrinsic to the gut itself [2, 3, 1013]. The exact cellular and molecular mechanisms of these controls, however, often remain enigmatic, and failure of these mechanisms results in inefficient digestion and improper movement of food.

            We are interested in understanding the mechanisms that regulate peristalsis of the larval midgut in Drosophila melanogaster. The Drosophila larval midgut is an endothelial tube composed of two cell types: enterocytes and enteroendocrine cells which arise from stem cell crypts located within the gut and differentiate into either cell type in a Notch signalling pathway-dependent fashion [1, 1417]. Depending on position cues within the gut tube, the enterocytes develop a wide variety of morphologies and functions and represent the majority of the cells within the midgut. While many of the enterocytes are involved in the absorption of nutrients at various stages of digestion, others, such as the acid-producing copper cells within the acid region of the midgut, are highly specialized [1, 1822]. Comprising a smaller portion of the cell population of the gut are the enteroendocrine cells, which have been shown to play several roles including the secretion of a number of peptide hormones and innate immune responses [2330]. Although many of these neuropeptide hormones are also expressed in the central nervous system where they have been shown to influence behaviour and circadian rhythm [4, 9, 31], the significance of neuropeptide hormone secretion by gut enteroendocrine cells remains unclear.

            The visceral muscles of the Drosophila midgut are organized into an inner group of circular muscles that wrap the circumference of the gut and an outer group of longitudinal muscles that traverse the length of the midgut [3235]. Unlike vertebrate intestines, the visceral musculature of the Drosophila midgut is a striated muscle type, most similar in structure to cardiac striated muscle [36] and originate from the same group of dorsal mesodermal cells that give rise to the dorsal vessel, the Drosophila heart [3741].

            The foregut, proventriculus, the anterior end of the midgut, and the hindgut are innervated by neurons emanating from the central nervous system [42, 43]. Feeding and ingestion of food into the midgut are mediated through interactions of the foregut with the nervous system via the stomatogastric nervous system [3, 44]. In contrast to the detailed understanding of the morphology and physiology of the stomatogastric nervous system (SNS) and the foregut, the neural/muscular physiology of the midgut has been less well characterized, despite it being the largest segment of the alimentary canal. Outside of its anterior-most end, the midgut appears to have little connection to the nervous system, and little is understood of the mechanisms that are responsible for maintaining and propagating gut motility and peristalsis through the midgut. In this report, we characterize an enteroendocrine cell type in the Drosophila larval midgut that regulates gut motility in the junction region of the larval midgut. These enteroendocrine cells express the peptide hormone DH31, express receptors to acetylcholine, and have stimulatory properties of excitable cell types. Using cell ablation and an ectopic blue-light activation technique, we demonstrate that these Midgut Junction DH31 expressing cells are essential for proper peristalsis within the junction region of the anterior midgut. A mutant analysis of DH31 demonstrates that this peristalsis utilizes a novel mechanism involving the secretion of a peptide hormone. We also show that the organization of the midgut junction region is more complex than previously thought. In addition, the Midgut Junction DH31 expressing cells involve the possible coordination of multiple components including a valve, cholinergic AllostatinB/MIP secreting cells, and enigmatic muscle tethers that link this portion of the midgut with the more anterior gastric caeca.

            Results

            Screen of midgut expressing Gal4 enhancer trap lines

            To better understand the different cell types within the larval midgut, we performed a screen of previously identified Gal4 enhancer trap lines and transgenic Gal4 constructs, reported to express in the gut, but which have not been characterized in detail [4547]. The goal of these experiments was to establish cellular markers to identify different cell types within the midgut, as well as to identify the genes associated with these various cell types, which will provide us with specific molecular tools to manipulate the midgut and better understand how the cells within the midgut interact to regulate peristalsis and the movement of food. Apart from the acid-producing cells and the visceral musculature, the larval midgut consists of several cell types including enteroendocrine cells, enterocytes, and tracheal cells [1, 20, 24]. Out of the thirty seven enhancer traps screened, nineteen of these Gal4 enhancer traps expressed in various patterns throughout the larval midgut (Table 1; Fig 1).
            Table 1

            Gal4 lines that express in larval midgut

            Gal4 Line

            Cytological location

            Gene/product

            Expression Pattern in Midgut

            Cha-Gal4

            3L,91C5

            Choline acetyltransferase, encodes an choline O-acetyltransferase and is involved in acetyl choline biosynthesis.

            MJDH31, VTG

            Ddc-Gal4

            2R, 37C1

            Dopa decarboxylase encodes an aromatic-L-amino-acid decarboxylase involved in the biosynthesis of serotonin and dopamine.

            MJDH31, VTG

            DJ752

            3L, 96F

            Enhancer of Split/HLHm7

            MJDH31

            bab-Gal4

            3L, 61E2-61F1

            bric-a-brac 1 encodes a transcription factor

            MJDH31, LM, some CM, some En

            MJ12

            2L, 22B1-22B2

            CG17646, encodes an ABC type 2 transporter

            MJDH31

            DJ761

            3R, 85D16-85D17

            Passila encodes a nuclear mRNA splicing factor

            anterior En

            C805

            2R, 48E6-48E7

            Developmental embryonic B/CG16972, RNA splicing factor

            MJDH31, trach, pattern in Pro

            CB20

            2L, 28B1

            Rapgap1 encodes a Ras GTPase activator activity;

            MJDH31, pattern in Pro

            5053A

            3L, 76C1

            teyrha-meyrah encodes a protein of unknown function

            LM

            Drm-Gal4

            2l, 24C1

            Drumstick encodes a small C2H2 zinc finger protein

            MJDH31, expression pattern in Pro

            DJ691

            3R, 85D22-85D24

            Mura encodes a zinc finger transcription factor

            En and CM

            DJ717

            3rd chromosome

             

            trach, MJDH31, pattern in Pro, Gob, some CM

            CB30

            2R, 56C8-56C9

            Tab2 encodes a zinc finger transcription factor

            CM, MJDH31, En,

            c564

            2nd chromosome

            Insert into repetitive DNA on 2nd

            En

            EDTP/DJ694

            2R, 54B7-54B15

            Encodes an egg-derived tyrosine phosphatase

            En

            MJ33a

            3rd chromosome

             

            Expression pattern in Pro, MJDH31, FG

            C135

            3rd chromosome

             

            MJDH31, ent, Gob

            T80

            2L, 23A3-23A3

            CG9894, protein of unknown function

            MJDH31, pattern in Pro, weak EN

            T155

            3rd chromosome

             

            pattern in Pro, weak EN

            Pro - proventriculus; MJDH31- Midgut Junction DH31 expressing cells; LM - Longitudinal muscles; CM - Circular Muscles; En - endothelial lining/enterocytes; GC - Gastric caeca; Gob -- Goblet cells; VTG - ventricular ganglion, ent - enteroendocrine cells, trach - tracheal cells and precursors.

            http://static-content.springer.com/image/art%3A10.1186%2F1472-6793-10-14/MediaObjects/12899_2010_Article_161_Fig1_HTML.jpg
            Figure 1

            Gal4 enhancer trap expression patterns in larval midgut. In all images, the anterior is toward the top and the posterior points towards the bottom. In B-F, the images are of the midgut junction region with the anterior midgut towards the top and acidic region towards the bottom. All size bars are 50 um except for the insert in C which is 10 um. A) The proventriculus is a large bulbous, multilayered portion of the foregut located at the junction of the foregut and midgut. bric-a-brac Gal4 expresses in the posterior within the inner layer of the proventriculus (thin arrow) and to a lesser extent within posterior cells of the outer endothelial layer (thick arrow). B) C135-Gal4 expresses within the Midgut Junction DH31 expressing cells (thick arrow) and the copper cells of the acidic region of the larval midgut (thin arrow). Expression can also be seen in tracheal precursor cells anterior to the Acidic region (arrow head). C) DJ717-Gal4 expresses in tracheal precursor cells (thick arrow and insert) as well as sporadically in the acid producing copper cells (thin arrow). D) CB30-Gal4 expresses in Midgut Junction DH31 expressing cells (thick arrow), tracheal precursor cells (thin arrow) and in circular muscles (arrow head). E) DJ761-Gal4 expresses in crypt cells (thick arrow) and in anterior midgut endothelial cells (thin arrow). F) c564-Gal4 expresses in Midgut Junction DH31 expressing cells (thick arrow) and throughout all cells in the endothelial lining of the larval midgut.

            We found five enhancer traps that express in the visceral musculature. One enhancer trap, bab-Gal4, an insertion into the bric-a-brac gene, expressed in the proventriculus (Fig 1A), circular and longitudinal muscles (Fig 1C, D), the endothelial lining, and in some enteroendocrine cells. Bric-a-brac encodes a transcription factor required for a variety of developmentally important patterning events, including tarsal formation in the leg and terminal filament formation in the ovary [48]. Interestingly, bab-Gal4 expressed in the circular muscle, in a pattern flanking the acidic region of the midgut, just upstream and downstream of this region, suggesting that there may be patterning within the circular visceral muscle, perhaps in relation to specific functional regions in the gut. Two other enhancer traps, DJ717-Gal4 and DJ691-Gal4, are also expressed in a pattern throughout the midgut. One enhancer trap, CB30, an insertion into the Tab2 transcription factor gene involved in the Drosophila innate immune response [23, 29], expressed throughout all circular muscles, as well as in the endoderm and a group of enteroendocrine cells (Fig 1D). The 5053A enhancer trap expressed exclusively in the longitudinal muscles. In this enhancer trap, the P[GawB] element has inserted into the teyrha-meyrha gene, which encodes a novel protein that has no homologues outside of Diptera.

            Four enhancer traps expressed in the enterocytes, two exclusively. One of these, c564-Gal, is an insert into repetitive DNA on the third chromosome, and the other, EDTP/DJ694, is an insert into an egg-derived tyrosine phosphatase [45] (Fig 1F). We also identified enhancer traps that expressed in a pattern within the endothelial lining. DJ761-Gal4 expressed only in the endothelium of the anterior midgut, stopping short of the acidic region (Fig 1E). DJ761-Gal4 is a P{GawB} into the pasilla gene which encodes an RNA splicing factor required for normal apical secretion from the salivary glands [49]. Another enhancer trap, C135-Gal4, labelled the copper cells of the acidic region, a variety of enteroendocrine cells within the midgut (Fig 1B), and larval fat body. We also identified an enhancer trap, DJ717Gal4 that expresses within the nascent tertiary tracheal cells located on the surface of the gut (Fig 1C). These cells are located on the surface of the gut, on top of visceral musculature, and vary greatly in their morphology as they develop into tracheal cells, often appearing round, spindle-shaped, or with multiple projections (inset, Fig 1C).

            Identification of the DH31 Expressing Cells of the Midgut Junction Region

            In twelve enhancer traps/Gal4 reporter transgenes, we observed expression of the GFP reporter gene in a distinct group of enteroendocrine cells located at the junction region of the anterior midgut and the acid-secreting portion of the larval midgut (Fig 2A). This junction region occurs at a conspicuous and characteristic U-shaped bend in the gut, which can be easily identified through the larval cuticle. In four lines, ChaGal4, DdcGal4, DJ752Gal4, and MJ12Gal4, the CD8GFP reporter expression was observed exclusively within the midgut in these cells (Table 1, Fig 2). The enteroendocrine cells are bottle-shaped and project an apical projection into the lumen of the gut (Fig 2G, I: Additional File 1). We observed an average of 7 ± 2 of these enteroendocrine cells per midgut (n = 37 midguts). Each cell extends through the endothelial epithelium and is associated with the overlying longitudinal muscle.
            http://static-content.springer.com/image/art%3A10.1186%2F1472-6793-10-14/MediaObjects/12899_2010_Article_161_Fig2_HTML.jpg
            Figure 2

            MIP and DH31 expression in Midgut Junction DH31 expressing cells and junction region. In A-F, midgut junction regions are oriented with the anterior in the upper right hand corner and the acidic region in the lower left hand side. G-I, the luminal/apical portion of the enteroendocrine cell is on the left and the basal portion of the cell is on the right. The size bars in A-F are 50 um the size bar for G-I is 20 um. A, D, G are DJ752 Gal4/UAS GFP-CD8 reporter gene expression. There are 4 Midgut Junction DH31 expressing cells in (A) and (D). (B) is expression of Allostatin B/MIP in the junction region, where there are eight cells expressing Allostatin B/MIP. C) is the merged image of A and B, note the distinct red and green cells that demonstrate two distinct groups of cells. (E) is the expression of DH31 peptide. (F) is the merged image of (D) and (E). Note that the DH31 expressing cells co-express GFP-CD8 and appear yellow. (G) through (I) are a close-up of the Midgut Junction DH31 expressing cells expressing the DH31 peptide hormone. In (G) the Midgut Junction DH31 expressing cells express the membrane bound GFP-CD8 outlining the cells basal body and luminal apical projection (arrow). (H) All of the DH31 peptide is localized to the basal portion of the cell but not in the apical projection. This is more apparent in (I), with the apical projection shown in green without co-localization of DH31. However, in the basal portion of the cell GFP and DH31 co-localize (yellow).

            Two peptide hormones are expressed in two different endocrine cell types within the midgut junction region. One group of midgut junction region enteroendocrine cells exclusively express Allatostatin B/MIP in the midgut [24]; and another group of enteroendocrine cells that are found in this region of the midgut express the peptide hormone Diuretic Hormone 31 (DH31). Other cells in the larvae anterior midgut and hindgut, however, also express the DH31 peptide hormone [24]. The cells were identified in our Gal4 screen expressing DH31 (Fig 2E, F) and do not express the AllostatinB peptide (Fig. 2B, C). These cells will be called Midgut Junction DH31 expressing cells; however not all of the DH31 expressing cells in the junction region express the markers used for the characterization of these cells (Fig 2F). Within the Midgut Junction DH31 expressing cells, the DH31 peptide is located within the cell body, but is excluded from the apical, luminal projection (Fig. 2H, I), suggesting that the secretory portion of these cells is located basally.

            Organization of the Midgut Junction Region

            In addition to the Midgut Junction DH31 expressing cells and the enteroendocrine cells expressing Allostatin B/MIP between the anterior midgut and the acid-expressing cells, we have also found that this junction region contains a pair of muscular tethers that link this region with the gastric caeca (Fig 3A, B, thin arrows; Additional File 2) and a valve (Fig 3A, thick arrows), which manifests as a reduction of the diameter of the gut without a change in the thickness of the endothelial lining. The gastric caeca emerge from the anterior midgut just posterior to the proventriculus as four blind-ended tubes [43, 50]. The tips of the ventral gastric caeca are linked to the anterior junction region via these muscular tethers (Additional File 3). The ratio of the diameter of the gut and the lumen at midgut junction region is the smallest within the entire midgut (Fig 3A) and is found at a conspicuous and characteristic bend in the gut. In addition to a bend at this region, the gut also appears to be twisted (LaJeunesse, personal observation). The muscular tethers are extremely labile and typically destroyed during most dissections; however the remnants of which are almost always observed on both the gastric caeca terminus and within the anterior junction region of the midgut (Additional File 4). These muscle tethers express higher levels of Disc Large protein, relative to other visceral muscles, and a preponderance of the Disc Large protein is found in internal plaques along the length of the muscle (Fig 3C). Disc Large protein is a component of the septate junctions in Drosophila epithelial cells, a part of the neuromuscular junction bouton, and the internalized plaque structures appear to be distinct from these previously identified subcellular localizations [51, 52]. The muscular tethers retain a striated sarcomeric organization of actin and myosin at the attachment to the midgut and at the origin on the gastric caeca. This striated organization, however, becomes less organized within the belly of the tether (data not shown).
            http://static-content.springer.com/image/art%3A10.1186%2F1472-6793-10-14/MediaObjects/12899_2010_Article_161_Fig3_HTML.jpg
            Figure 3

            Organization of the midgut junction region. A) The anterior midgut is at the top and the acidic region at the bottom. In green, individual Midgut Junction DH31 expressing cells labelled with ChaGal4 CD8GFP; in red, actin labelled with Phalloidin Alex 546. The Thin arrows denote the insertion of the muscular tethers within the junction region of the anterior midgut. The arrows heads show the constraint of the lumen within the junction region of the midgut outlined with a dotted line. Also note the yeast auto fluorescence in the lumen as well. Size bar is 50 um. B) Labelling with anti-Discs Large (in red) shows a remnant of the tether (arrow) inserting into the musculature of the anterior midgut junction region. In green (UAS CD8GFP/DJ752 Gal40) Midgut Junction DH31 expressing cells are shown. The size bar is 20 um. C) Within the modified longitudinal musculature tether are mono-nucleated (thin arrow) and contain plaques of the Discs Large Protein arranged lengthwise (arrows). The size bar is 20 um.

            Midgut Junction DH31 expressing cells express AChR and AllostatinB/MIP cells express CHAT

            In addition to DH31, we found that the Midgut Junction DH31 expressing cells express two Gal4 reporters for the genes involved in neurotransmitter biosynthesis, specifically Choline acetyltransferase (Cha) and Dopa decarboxylase (Ddc) [5355]. Since no ventricular ganglion axons project down the anterior midgut into this region, the Midgut Junction DH31 expressing cells are distinct from the nervous system. To determine whether these cells were expressing the enzyme Choline acetyltransferase (CHAT) that is encoded by the Cha gene, we examined the localization of CHAT protein in the midgut using a monoclonal antibody [56]. Curiously, although the Midgut Junction DH31 expressing cells express the ChaGal4 reporter (Fig 4B), they do not express CHAT protein (Fig 4A, C). This result demonstrates that the ChaGal4 transgene does not contain all of the regulatory elements for proper Cha expression in the midgut, and its expression in the Midgut Junction DH31 expressing cells appears to be an artefact. However, we observed CHAT expression (Fig 4D) in another group of cells within the midgut junction region, specifically the AllostatinB/MIP expressing cells (Fig 4E, F). Since the MIP/AllostatinB cells appear to be cholinergic, we looked for cells in the junctional region of the midgut that express acetylcholine receptors (AChR) using Alexa594 labelled α-bungarotoxin (BTX-594) which specifically bind nicotinic Acetyl Choline receptors [47, 57]. In live Midgut Junction DH31 expressing cells (as shown by ChaGal4 expression of CD8GFP; Fig 4G) we observe binding of the BTX-594 probe (Fig 4H, I); moreover these are the only cells within the midgut which bind this probe. While we haven't been able to find a physical connection or synapse between the Midgut Junction DH31 expressing cells and the AllostatinB/MIP expressing cells, the close proximity of cholinergic cells and cells expressing AChR suggests a functional connection between these two groups of cells.
            http://static-content.springer.com/image/art%3A10.1186%2F1472-6793-10-14/MediaObjects/12899_2010_Article_161_Fig4_HTML.jpg
            Figure 4

            Expression of CHAT and AChR in the junction region of the larval midgut. (A-C) Image of a larval midgut at the junction region labeled with ChaGal UASCD8GFP and anti-CHAT showing that CHAT is not expressed in the same cells as ChaGal4; there is no overlap of the red and green cells in (C). (A) The cells labeled in green are the ChaGal4 UASCD8GFP expressing Midgut Junction DH31 expressing cells. (B) The cells in red are cells expressing the CHAT protein. (C) Merged images showing distinct localization of CHAT and ChaGal4 in the junction region of the midgut. Error bar = 100 um. (D-F) Extended View confocal images of the co-localization of CHAT expression with AllostatinB/MIP. (D) Anti-CHAT expression in shown in green.(E) Allostatin B/MIP expression in relabeled with anti-AllostatinB/MIP in red. (F) Merged image shows that the co-localization of CHAT and AllostatinB/MIP. Bar = 10 um. (G-I) Images of living midgut at the midgut junction region. These images are a single plane taken near the basal portion of the cell. (G)The Midgut Junction DH31 expressing cells are labeled with ChaGal4 UAS-CD8GFP in green. (H) These same cells are labeled with alpha-bungarotoxin Alexa594 congugate. (I) the merged images show that the labels colocalize. Bar = 10 um.

            Ablation of Midgut Junction DH31 expressing cells/loss of DH31 abolishes normal peristalsis in the junction region of anterior midgut

            To determine the role of the Midgut Junction DH31 expressing cells in the midgut, we ablated the Midgut Junction DH31 expressing cells from the larval midgut using a system based on the ectopic expression of either the proapoptotic gene, UAS reaper, or the castor bean toxin, UAS ricin [58, 59]. We directed the expression of these ablation genes using the Gal80 ts conditional expression system and two Gal4 Drivers specific to the Midgut Junction DH31 expressing cells in the larval midgut, ChaGal4 and DJ752 [60]. Larval midguts with ablated Midgut Junction DH31 expressing cells were assessed in four separate assays: (1) a feeding assay to examine the movement of food through the alimentary canal; (2) a morphological assay in which we examined the structure of the midgut; (3) a functional assay in which we directly observed peristalsis within the anterior midgut junction region; and (4) an assay in which the pH of the food in acidic portion of the midgut was assessed using Bromophenol Blue dye.

            Although ablation of the Midgut Junction DH31 expressing cells did not change the passage of food through the gut (see Additional File 5) or generate any morphological changes to the anterior midgut junction region (see Additional File 6), we observed significant changes in peristalsis in the anterior midgut junction. We observed a significant decrease in the number of contractions versus the controls (Table 2; compare Additional File 7 with Additional File 8) demonstrating that these cells are central to peristalsis specifically in this region. However, peristalsis while not normal, is not completely lost and gut motility in other regions of the midgut appears to be unaffected. To determine whether the expression of the peptide hormone DH31 play any role in this peristalsis, we examined junction region peristalsis in larvae homozygous for a Dh31 KG09001 , a strong hypomorphic mutant allele that contains a P{SUPor-P} insertion into the second intron of the Dh31 gene [61, 62]. Larvae homozygous for Dh31 KG09001 have no detectable expression of DH31 peptide as determined by immunohistochemistry (Dennis LaJeunesse personal observation). Again, like the larval midguts from the ablation experiments, larval midgut from larvae homozygous for Dh31 KG09001 show greatly reduced peristalsis in the junction region of the larval midgut (Table 2; Additional File 9).
            Table 2

            Ablation of the Midgut Junction DH31 expressing cells from anterior midgut junction region alters peristalsis

            Genotype

            Condition/Temp treatment

            n

            Average # of contractions per minute ± SD

            w 1118

            29°C o/n; RT 3 hours

            33

            10.7 ± 3.1

            UAS ricin/+; DJ752Gal4/Gal80 ts

            29°C o/n; RT 3 hours

            30

            2.6 ± 2.8* A

            UAS reaper/+; DJ752Gal4/Gal80 ts

            29°C o/n; RT 3 hours

            20

            2.0 ± 2.7** A

            Dh31 KG09001 /Dh31 KG09001

            RT

            23

            1.6 ± 1.6***

            UAS ricin/+; Gal80 ts /+ (control)

            29°C o/n; RT 3 hours

            20

            10.2 ± 2.9

            UAS reaper/+; Gal80 ts /+ (control)

            29°C o/n; RT 3 hours

            15

            10.5 ± 2.1

            DJ752Gal4/+ (control)

            29°C o/n; RT 3 hours

            15

            9.6 ± 4.1

            w 1118 (wild type)

            RT

            43

            10.1 ± 3.9

            UAS ricin/+; DJ752Gal4/Gal80 ts

            RT

            20

            14.5 ± 5.9

            UAS reaper/+; DJ752Gal4/Gal80 ts

            RT

            20

            8.54 ± 4.5

            * significance P = 2.5E-16 when compare to w1118 (wild type) 29°C o/n; RT 3 hours control

            ** significance P = 4.56E-14 when compare to w1118 (wild type) 29°C o/n; RT 3 hours control

            ***significance P = 1.51E-18 when compare to w1118 (wild type) RT control

            A - "contractions" in these classes were uncoordinated twitches within the visceral musculature.

            o/n - overnight

            To further examine the role of the Midgut Junction DH31 expressing cells in peristalsis, we ectopically activated Midgut Junction DH31 expressing cells by expressing the Chlamydomonas reinhardtii Channelrhodopsin-2 (ChR2) in the Midgut Junction DH31 expressing cells and examining its effect on peristalsis. The ChR2 protein is a light-activated cation-selective ion channel that, when expressed in an excitable cell type and exposed to blue light (λ~488 nm), will initiate an action potential [63, 64]. Using this technique, we showed that the Midgut Junction DH31 expressing cells are not only necessary, but also sufficient for increasing the rate of peristalsis the midgut junction region. We observed a significant increase in the number of contractions in midguts with CHR2-expressing Midgut Junction DH31 expressing cells when exposed to blue light versus white light (+43% for DJ752Gal4 and +26% for ChaGal4; Table 3).
            Table 3

            Activation of the Midgut Junction DH31 expressing cells is sufficient to induce ectopic contractions in the anterior midgut junction region.

            Genotype

            n

            White light contractions/min

            Blue light contractions/min

            % change

            w 1118

            20

            12.7 ± 5.5

            12.8 ± 2.7

            +0.8%

            DJ752 Gal4; UASChR2X2(with retinal)

            20

            11.5 ± 4.2

            16.4 ± 4.0

            +43% *

            Cha Gal; UASChR2X2 (with retinal)

            20

            9.7 ± 4.5

            12.2 ± 4.0

            +26% **

            DJ752 Gal4; UASChR2X2 (no retinal)

            12

            11.3 ± 3.6

            9.2 ± 3.5

            -19%

            Cha Gal4; UASChR2X2 (no retinal)

            14

            11.1 ± 4.0

            10.3 ± 5.0

            -7%

            DJ752 Gal4 (control)

            16

            9.1 ± 5.0

            9.1 ± 4.5

            0%

            Cha Gal4 (control)

            12

            9.7 ± 4.3

            8.3 ± 3.4

            -14%

            UASChR2X2 (control)

            9

            11.5 ± 3.6

            11.5 ± 3.6

            0%

            * Significant increase in number of contractions during exposure to Blue Light when compared to the number of contractions during exposure to white light, P = 1.7E-4

            **Significant increase in number of contractions during exposure to Blue Light when compared to the number of contractions during exposure to white light, P = 6.4E-4

            Ablation of Midgut Junction DH31 expressing cells and loss of DH31 alters the pH within the acidic portion of the midgut

            Since the Midgut Junction DH31 expressing cells are found at valve-like structure poised at the entry of the acidic portion of the gut, we evaluated the pH within this region in midguts from larvae missing the Midgut Junction DH31 expressing cells and in larvae mutant for Dh31, with the idea of connecting the defects in peristalsis to some tangible consequence in the processing of food. Typically, larvae-fed food containing 2% Bromophenol Blue will have an anterior midgut with deep blue food and an acidic region marked by bright yellow food with a pH <3.0 [20]. In midguts from Midgut Junction DH31 expressing cells -ablated larvae or mutant for Dh31 we observed a significant mixing of acidified and non-acidified food in the acidic compartment of midgut. This result manifests as an increase in the number of larvae with green food instead of yellow food in the acidic region (Table 4). This suggests that Midgut Junction DH31 expressing cells regulate the passage of food into the acidic compartment of the larval midgut.
            Table 4

            Loss of Midgut Junction DH31 expressing cells results in Green food in the acidic region of the larval midgut.

            Genotype

            Condition/Temp treatment

            n

            % yellow food in acidic region

            % green food in acidic region

            w 1118

            29°C o/n; RT 3 hrs

            16

            94%

            6%

            UAS ricin/+; DJ752Gal4/Gal80 ts

            29°C o/n; RT 3 hrs

            30

            27%

            73%

            UAS reaper/+; DJ752Gal4/Gal80 ts

            29°C o/n; RT 3 hrs

            21

            38%

            62%

            DH31 KG09001 /DH31 KG09001

            RT

            28

            36%

            64%

            UAS ricin/+; Gal80 ts /+ (control)

            29°C o/n; RT 3 hrs

            11

            91%

            9%

            UAS reaper/+; Gal80 ts /+ (control)

            29°C o/n; RT 3 hrs

            11

            82%

            18%

            DJ752Gal4/+ (control)

            29°C o/n; RT 3 hrs

            30

            97%

            3%

            UAS ricin/+; DJ752Gal4/Gal80 ts

            RT

            17

            100%

            0%

            UAS reaper/+; DJ752Gal4/Gal80 ts

            RT

            14

            100%

            0%

            DJ752Gal4/+

            RT

            12

            100%

            0%

            Discussion

            Although intestinal enteroendocrine cells are a diverse group of cells and play a variety of roles including roles in sensing nutrient levels, immune response to pathogens, and secretion of hormones and neuropeptides [12, 24, 25, 28, 30, 6567], little work has been done on show the role of enteroendocrine cells in digestion and gut motility in insects [24, 25]. In this manuscript, we have identified a distinct type of enteroendocrine cell within the midgut junction region that we called the Midgut Junction DH31 expressing cells. Ablation of the Midgut Junction DH31 expressing cells from the larval midgut results in abnormal peristalsis in the junction region of the midgut and subsequent inappropriate mixing of acidified and non-acidified food in the acidic region of the midgut. The Midgut Junction DH31 expressing cells express and require the peptide hormone gene Dh31 for this peristalsis. While previous work in Drosophila has shown that DH31 can stimulate urine secretion from the Malpighian tubules though the activation of a cAMP pathway [4, 68], topical application of DH31 has been shown to stimulate contractions in the hindgut and dorsal vessel of the blood sucking insect Rhodnius prolixus [69]. Although this data suggests that DH31 stimulates visceral muscle motility whether this peptide hormone directly activates this contraction remains to be tested.

            The junction region of the midgut contains other features that suggest the Midgut Junction DH31 expressing cells controls a valve that regulates the rate of food input into acidic portion and functions (Fig 5A). The junction region is the narrowest portion of the midgut with a distinctive bend in a twisted gut - a geometry, which perhaps allows for greater control of the flow food through the gut at this point, like a bent/twisted garden hose. In addition to the Midgut Junction DH31 expressing cells, another group of enteroendocrine cells express AllostatinB/MIP. Despite the lack of any apparent physical connection between the Midgut Junction DH31 expressing cells and AllostatinB/MIP enteroendocrine cells, the fact that the AllostatinB/MIP cells are cholinergic and the Midgut Junction DH31 expressing cells express acetylcholine receptors suggests that these two groups of cells may communicate via an atypical, non-neural acetylcholine pathway. There are numerous cell types including skin epithelial cells, immune cells, within the smooth muscle of blood vessels that use acetylcholine signalling to mediate cell shape change and physiological response to various signals [7072]. This type of signalling is independent of the formation of synapses between cells and has been linked to paracrine, autocrine or even endocrine-like signalling [72, 73]. The regulation of peristalsis in the Drosophila larval midgut junction region may involve the secretion of ACh from the junction AllostatinB/MIP enteroendocrine cells that modulates the secretion of DH31 from the Midgut Junction DH31 expressing cells (Fig 5B). Conversely, one might imagine that the Midgut Junction DH31 expressing cells may secrete a different signal to regulate secretion of AllostatinB/MIP, which had myoinhibitory activity [74] from neighbouring enteroendocrine cells in the junction (Fig 5B). Questions remain on the potential nature of the luminal signals that regulate this potential cross talk. Both enteroendocrine cells have apical faces that contact the lumen and therefore could respond to specific signals in the lumen to modulate peristalsis. In the human duodenum, enteroendocrine cells called L-Cells regulate the peristaltic movement of food through the gut via the secretion of glucagon-like peptide-1 in response to the presence of glucose [67]. In the case of midgut junction peristalsis, the signal may be a digestive cue in the anterior midgut (i.e. the concentration of some metabolite) that triggers entry of food into the acidic region, or perhaps the signal is a reflux/retrograde signal from the acidic region (i.e. a change in pH) that keeps the valve closed.
            http://static-content.springer.com/image/art%3A10.1186%2F1472-6793-10-14/MediaObjects/12899_2010_Article_161_Fig5_HTML.jpg
            Figure 5

            Organization of the Midgut Junction Region. A) Schematic of the organization of the midgut junction region. Extensions of the longitudinal muscles (red stripes) of the gastric caeca attached to the midgut junction region which contain two groups of enteroendocrine cells, the Midgut Junction DH31 expressing cells (in green) and cholinergic Allostatin B/MIP expressing cells(mauve outlined/red). The lumen of this region of the gut is thinner when compare to the remainder of the midgut and acts like a valve (in orange). This region of the midgut is a conspicuous bent and twisted before the entry into the acidic region. B) Model of the regulation of peristalsis in the midgut. Midgut Junction DH31 expressing cells secrete DH31 basally to stimulate contractions. This secretion may be stimulated through the atypical ACh paracrine-like signalling from neighbouring Allostatin B/MIP secreting enteroendocrine cells. An unidentified reciprocal signal may emanate from the Midgut Junction DH31 expressing cells to regulate the expression of Allostatin B/MIP or ACh.

            Although little is known about the mechanisms that govern midgut motility in invertebrates, there is evidence of coordination between different portions of the gut. Elegant electrophysiological and experimental manipulation performed within the foregut of the blowfly Calliphora larvae, which has a similar organization and structure to the Drosophila foregut and SNS, has demonstrated that a complex network of interactions among all components of the SNS contribute to characteristic feeding and swallowing behaviours [3, 43, 44]. Of the SNS, only neurons emanating from the proventricular ganglion innervate the proventriculus and the anterior midgut and the gastric caeca. Interestingly, severing these neuronal connections to the midgut alters contractile activity in the foregut, suggesting that other mechanisms, perhaps of a myogenic nature, are modulating and coordinating the contractions within the foregut with digestive processes further along the alimentary canal[3, 43, 44]. We have observed coordinated movement within the junction region with feeding behaviour (LaJeunesse unpublished observations), and the muscular linkage between the junction region and the gastric caeca may serve as a physical connection between these two regions of the gut (Fig 5A). These atypical muscular tethers are continuous with the innervated gastric caecal longitudinal muscles and the longitudinal muscles of the gastric caeca are innervated by neurons from the proventricular ganglion[42, 43]. This linkage may provide a mechanism for communication between the SNS and the junction region of the midgut. The potential signal may be either mechanical, via a muscle contraction, or electrical, via an action potential. There is a precedent for the latter possibility; in vertebrate hearts, non-contractile muscular Purkinje fibres disperse electrical stimuli from the conduction system to the ventricular cardiac muscles[75, 76]. Given the ontological and structural similarities between the vertebrate cardiac system and the Drosophila larvae visceral muscle[36, 38], this remains an intriguing possibility. Moreover, such a linkage suggests that the entry of food into anterior midgut from the proventriculus and the exit of food into the acidic region are coordinated.

            Conclusions

            We have characterized and described a set of enteroendocrine cells called the Midgut Junction DH31 expressing cells that are required for peristaltic movement in the junction region between the anterior portion and acidic region of the larval midgut of Drosophila melanogaster. We have shown that the Midgut Junction DH31 expressing cells are necessary and sufficient for motility and that the peptide hormone DH31 is required for peristalsis in the junction region of the midgut. In addition to the Midgut Junction DH31 expressing cells, we have also described several features of this junction region that suggest a new complexity in the regulation of peristalsis in this region including cholinergic Allostatin B/MIP expressing cells, a valve, and a cryptic group of visceral muscle tethers that link the junction region to the more anterior gastric caeca. The movement of food from the anterior midgut into the acidic region of the larval gut represents a single step within digestion, and the ramifications of failure or disruption to these steps remain unclear. Our finding sheds light on the complexity of digestion, its regulation, and suggests a more complex mechanism for larval midgut motility that previously thought. Given its structural similarity to more complex digestive tracts, the Drosophila model system will allow for a further dissection of the digestion process and provide a better understanding of the mechanisms that regulate digestion in all organisms.

            Methods

            Drosophila Strains

            The stocks used in this study are as follows: w 1118 (wild-type control); P{GawB}DJ752 (Bloomington stock #8182); P{Cha-GAL4.7.4}19B (Bloomington stock #6793); P{UAS-mCD8::GFP.L}LL5 (Bloomington stock #5137); P{UAS-rpr.C}14 (Bloomington stock #5824); P{tubP-GAL80[ts]}2 (Bloomington stock #7017); P{GawB}bab1[Agal4-5] Bloomington stock #6802); y1; P{SUPor-P}Dh31KG09001 (Bloomington stock #16474); UAS-ricin (courtesy of K.G. Moffat, University of Warwick); UAS-ChR2 X2 (courtesy of Andre Fiala, Department of Genetics and Neurobiology, Theodor-Boveri-Institut, Julius-Maximilians-Universität Würzburg).

            Immunohistochemistry

            5-day-old larvae were dissected in 1× PBS containing freshly made 4% paraformaldehyde; larvae were fixed for 3 hours, washed, post0fixed in methanol for 1 hour. Larvae were washed 3× in 1 × PBS, incubated in PBT (1 × PBS/1%BSA/0.1%TritonX-100) for 30 minutes and then incubated overnight in primary antibody diluted in PBT. We used the anti-Disc Large antibody, monoclonal 4F3 (Developmental Studies Hybridoma Bank; 1:1000). The process was then repeated for the secondary antibody using Goat anti-Mouse Cy3 (Jackson Immunological, 115-165-062). For actin visualization, we performed the same procedure without the methanol post-fix and used Alex564 phalloidin (Molecular Probes; 1:2000). For CHAT localization (and any double labelling with anti-CHAT and another antigen) the process was performed as described above except for the following differences: we fixed overnight in 4% paraformaldehyde fixation and we used the ChAT4B1 antibody (Developmental Studies Hybridoma Bank; 1:100[56]). For DH31 and Allostatin B/MIP immunostaining, 5-day-old larvae were dissected in 10× PBS and immediately transferred to a (4% paraformaldehyde + 10× PBS) fix solution. Larval guts were fixed for 2.5 hours, washed six times (30 minutes per wash) in washing solution (0.1% Triton-X, 1% BSA, 10× PBS), then incubated for 1 hour in normal goat serum (10% goat serum in the washing solution) prior to overnight incubation in primary anti-DH31[24](1:500) and anti-AllostatinB/MIP[24] (1:1000). Same washing/goat serum incubation protocol was followed before one hour incubation in secondary antisera. Larval guts were mounted in Dako Fluorescent Mounting Medium. The double labelling procedures followed the protocol used for anti-Chat experiments except the secondary antibodies used where donkey anti-mouse FITC for visualizing CHAT localization (Jackson Immunological,715-095-150) and donkey anti-rabbit CY3 for visualizing AllostatinB/MIP localization (Jackson Immunological, 711-165-152). All micrographs were imaged using an Olympus IX81 inverted FV500 confocal microscope.

            α-bungarotoxin labelling

            4-5 day old larvae expressing ChaGal4 UASCD8GFP were collected, washed, and dissected in S2 insect cell culture media. Larval midguts were incubated for 30 minutes in a solution of 10 mM solution of α-bungarotoxin Alexa 594 (BTX-594; Molecular Probes, B-13423) in S2 insect cell culture media. To remove excess probe, the larval midguts were washed three times in fresh S2 media for 5 minute each time. Larval midgut were mounted on a clean glass slide and cover slip and viewed using an Olympus FV500 confocal microscope. Images were collected sequentially.

            Ablation/ectopic-activation of Midgut Junction DH31 expressing cells experiments

            Flies of the genotype UAS-ricin (or UAS-rpr) /CyOAct5cGFP; P{tubP-GAL80[ts]}2 and UAS-mCD8::GFP.L; DJ752Gal4 were crossed to each other. In third in start larvae, DJ752 is exclusively expressed in the Midgut Junction DH31 expressing cells of the intestine. Non-Act5cGFP larvae were collected using a Leica MZFLIII Fluorescent Stereomicroscope. Larvae were placed at 30°C for 3 hours to inactivate the temperature sensitive Gal80 inhibitory protein and activate/release the Gal4 expression of CD8GFP and either the cytotoxin gene ricin or the proapototic gene reaper. Larvae were then removed from the 30°C and reared at room temperature for several hours. Ablation of the Midgut Junction DH31 expressing cells was determined by the early (1 hour) GFP expression and subsequent later loss of expression of the same GFP reporter gene. We never saw the appearance of new Midgut Junction DH31 expressing cells as the numbers of Midgut Junction DH31 expressing cells at the one hour stage were always within the normal number found in wild type midguts. During imaging for the movies, the presence or absence of Midgut Junction DH31 expressing cells was determine by observing the junction region under GFP fluorescence illumination - the lack of any GFP expressing cells in this region signified a loss of the Midgut Junction DH31 expressing cells. To image the peristalsis in the junction region, larval midguts were dissected in S2 cell media and mounted on a glass slide with a coverslip with clay feet to prevent compression. A series of 120 images (2 images per second) were captured with UPlanFl20× dry objective using a CoolSnap CCD camera mounted on an Olympus BX51 upright compound microscope. Data was compiled with ImagePro software. Statistical analysis was performed using a two tailed T-Test assuming equal variances in Microsoft Excel. For the gain of function experiments, larvae of the appropriate genotype were cultured overnight on food containing 100 mM all-trans retinal (Sigma, R2500) and imaged the next day as above with one exception: the larvae were imaged under white light for thirty seconds, and then under an oscillating blue light (488 nm; 2 Hz) for another thirty seconds. One set of controls for these experiments was performed as described without the retinal feeding to allow for genotype control.

            Green Food/Yellow Food assay

            5-day-old larvae were cultured overnight in food containing 2% Bromophenol Blue, which is blue in basic solution and yellow in solutions with a pH lower than 3.0. Larvae were then dissected and the colour of the acidic portion of their midgut noted.

            Declarations

            Acknowledgements

            We would like to thank Dr. Amy Adamson and Dr. John Tomkiel for comments concerning the manuscript, Dr. Matina Kalcounis-Rüppell for help with creating the movie files. Dr. K.G. Moffat (University of Warwick) for the UAS ricin stocks, Bloomington Stock Center, Bloomington, Indiana for stocks, the Developmental Hybridoma Stock Center at the University of Iowa for antibodies, Dr. Jan Veenstra (University of Bordeaux) for antibodies, and Dr. Andre Fiala, (Department of Genetics and Neurobiology, Theodor-Boveri-Institut, Julius-Maximilians-Universität Würzburg) for the UASChR2 stocks. Correspondence and requests for materials should be addressed to Dennis LaJeunesse, drlajeun@uncg.edu.

            Authors’ Affiliations

            (1)
            Department of Biology, 312 Eberhart Bldg, University of North Carolina Greensboro

            References

            1. Nakagoshi H: Functional specification in the Drosophila endoderm. Dev Growth Differ. 2005, 47 (6): 383-92. 10.1111/j.1440-169X.2005.00811.x.View ArticlePubMed
            2. Benoit SC, Tracy AL: Behavioral controls of food intake. Peptides. 2008, 29 (1): 139-47. 10.1016/j.peptides.2007.10.019.PubMed CentralView ArticlePubMed
            3. Schoofs A, Niederegger S, Spiess R: From behavior to fictive feeding: anatomy, innervation and activation pattern of pharyngeal muscles of Calliphora vicina 3rd instar larvae. J Insect Physiol. 2009, 55 (3): 218-30. 10.1016/j.jinsphys.2008.11.011.View ArticlePubMed
            4. Coast GM, et al: The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol. 2001, 204 (Pt 10): 1795-804.PubMed
            5. Mirth C, Truman JW, Riddiford LM: The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster. Curr Biol. 2005, 15 (20): 1796-807. 10.1016/j.cub.2005.09.017.View ArticlePubMed
            6. Wu Q, et al: Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila. Proc Natl Acad Sci USA. 2005, 102 (37): 13289-94. 10.1073/pnas.0501914102.PubMed CentralView ArticlePubMed
            7. Zinke I, et al: Suppression of food intake and growth by amino acids in Drosophila: the role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system. Development. 1999, 126 (23): 5275-5284.PubMed
            8. Mirth CK, Riddiford LM: Size assessment and growth control: how adult size is determined in insects. Bioessays. 2007, 29 (4): 344-55. 10.1002/bies.20552.View ArticlePubMed
            9. Wu Q, Zhao Z, Shen P: Regulation of aversion to noxious food by Drosophila neuropeptide Y- and insulin-like systems. Nat Neurosci. 2005, 8 (10): 1350-5. 10.1038/nn1540.View ArticlePubMed
            10. Wood JD: Enteric nervous system: reflexes, pattern generators and motility. Curr Opin Gastroenterol. 2008, 24 (2): 149-58. 10.1097/MOG.0b013e3282f56125.View ArticlePubMed
            11. Melcher C, Pankratz MJ: Candidate Gustatory Interneurons Modulating Feeding Behavior in the <italic>Drosophila</italic> Brain. PLoS Biol. 2005, 3 (9): e305-10.1371/journal.pbio.0030305.PubMed CentralView ArticlePubMed
            12. Pfannkuche H, Gabel G: Glucose, epithelium, and enteric nervous system: dialogue in the dark. J Anim Physiol Anim Nutr (Berl). 2009, 93 (3): 277-86. 10.1111/j.1439-0396.2008.00847.x.View Article
            13. Peller CR, et al: Defective gut function in drop-dead mutant Drosophila. J Insect Physiol. 2009, 55 (9): 834-9. 10.1016/j.jinsphys.2009.05.011.PubMed CentralView ArticlePubMed
            14. Micchelli CA, Perrimon N: Evidence that stem cells reside in the adult Drosophila midgut epithelium. Nature. 2006, 439 (7075): 475-9. 10.1038/nature04371.View ArticlePubMed
            15. Ohlstein B, Spradling A: The adult Drosophila posterior midgut is maintained by pluripotent stem cells. Nature. 2006, 439 (7075): 470-4. 10.1038/nature04333.View ArticlePubMed
            16. Bardin AJ: Transcriptional control of stem cell maintenance in the Drosophila intestine. Development. 137 (5): 705-14. 10.1242/dev.039404.View Article
            17. Liu W, Singh SR, Hou SX: JAK-STAT is restrained by Notch to control cell proliferation of the Drosophila intestinal stem cells. J Cell Biochem. 109 (5): 992-9.
            18. Dubreuil RR, Grushko T, Baumann O: Differential effects of a labial mutation on the development, structure, and function of stomach acid-secreting cells in Drosophila melanogaster larvae and adults. Cell and Tissue Research. 2001, 306 (1): 167-178. 10.1007/s004410100422.View ArticlePubMed
            19. Dubreuil RR, et al: Drosophila beta spectrin functions independently of alpha spectrin to polarize the Na, K ATPase in epithelial cells. Journal of Cell Biology. 2000, 149 (3): 647-656. 10.1083/jcb.149.3.647.PubMed CentralView ArticlePubMed
            20. Dubreuil RR: Copper cells and stomach acid secretion in the Drosophila midgut. Int J Biochem Cell Biol. 2004, 36 (5): 745-52. 10.1016/j.biocel.2003.07.004.View ArticlePubMed
            21. Voght SP, et al: Drosophila NPC1b promotes an early step in sterol absorption from the midgut epithelium. Cell Metab. 2007, 5 (3): 195-205. 10.1016/j.cmet.2007.01.011.View ArticlePubMed
            22. Gutierrez E, et al: Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature. 2007, 445 (7125): 275-80. 10.1038/nature05382.View ArticlePubMed
            23. Gesellchen V, et al: An RNA interference screen identifies Inhibitor of Apoptosis Protein 2 as a regulator of innate immune signalling in Drosophila. EMBO Reports. 2005, 6 (10): 979-984. 10.1038/sj.embor.7400530.PubMed CentralView ArticlePubMed
            24. Veenstra JA: Peptidergic paracrine and endocrine cells in the midgut of the fruit fly maggot. Cell Tissue Res. 2009, 336 (2): 309-23. 10.1007/s00441-009-0769-y.View ArticlePubMed
            25. Veenstra JA, Agricola HJ, Sellami A: Regulatory peptides in fruit fly midgut. Cell Tissue Res. 2008, 334 (3): 499-516. 10.1007/s00441-008-0708-3.View ArticlePubMed
            26. Wu L, Silverman N: Fighting infection fly-style. Fly. 2007, 1 (2): 106-109.View ArticlePubMed
            27. Senger K, Harris K, Levine M: GATA factors participate in tissue-specific immune responses in Drosophila larvae. Proc Natl Acad Sci USA. 2006, 103 (43): 15957-62. 10.1073/pnas.0607608103.PubMed CentralView ArticlePubMed
            28. Haselton AT, Yin CM, Stoffolano JG: FMRFamide-like immunoreactivity in the central nervous system and alimentary tract of the non-hematophagous blow fly, Phormia regina, and the hematophagous horse fly, Tabanus nigrovittatus. J Insect Sci. 2008, 8: 1-17. 10.1673/031.008.6501.View ArticlePubMed
            29. Geuking P, Narasimamurthy R, Basler K: A genetic screen targeting the tumor necrosis factor/Eiger signaling pathway: identification of Drosophila TAB2 as a functionally conserved component. Genetics. 2005, 171 (4): 1683-1694. 10.1534/genetics.105.045534.PubMed CentralView ArticlePubMed
            30. Hansen MB, Witte A-B: The role of serotonin in intestinal luminal sensing and secretion. Acta Physiologica. 2008, 193 (4): 311-323. 10.1111/j.1748-1716.2008.01870.x.View ArticlePubMed
            31. Audsley N, Weaver RJ: Neuropeptides associated with the regulation of feeding in insects. Gen Comp Endocrinol. 2009, 162 (1): 93-104. 10.1016/j.ygcen.2008.08.003.View ArticlePubMed
            32. Klapper R, et al: The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc. Mechanisms of Development. 2002, 110 (1-2): 85-96. 10.1016/S0925-4773(01)00567-6.View ArticlePubMed
            33. Klapper R: The longitudinal visceral musculature of Drosophila melanogaster persists through metamorphosis. Mech Dev. 2000, 95 (1-2): 47-54. 10.1016/S0925-4773(00)00328-2.View ArticlePubMed
            34. Klapper R, et al: A new approach reveals syncytia within the visceral musculature of Drosophila melanogaster. Development. 2001, 128 (13): 2517-24.PubMed
            35. Wolfstetter G, et al: Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm. Mech Dev. 2009, 126 (8-9): 721-36. 10.1016/j.mod.2009.05.001.View ArticlePubMed
            36. Goldstein MA, Burdette WJ: Striated visceral muscle of drosophila melanogaster. J Morphol. 1971, 134 (3): 315-34. 10.1002/jmor.1051340305.View ArticlePubMed
            37. Furlong EE: Integrating transcriptional and signalling networks during muscle development. Curr Opin Genet Dev. 2004, 14 (4): 343-50. 10.1016/j.gde.2004.06.011.View ArticlePubMed
            38. Ranganayakulu G, et al: Divergent roles for NK-2 class homeobox genes in cardiogenesis in flies and mice. Development. 1998, 125 (16): 3037-48.PubMed
            39. Baylies MK, Bate M, Ruiz Gomez M: Myogenesis: a view from Drosophila. Cell. 1998, 93 (6): 921-7. 10.1016/S0092-8674(00)81198-8.View ArticlePubMed
            40. Broihier HT, et al: zfh-1 is required for germ cell migration and gonadal mesoderm development in Drosophila. Development. 1998, 125 (4): 655-66.PubMed
            41. Artero R, et al: The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2. Dev Biol. 1998, 195 (2): 131-43. 10.1006/dbio.1997.8833.View ArticlePubMed
            42. Budnik V, Wu CF, White K: Altered branching of serotonin-containing neurons in Drosophila mutants unable to synthesize serotonin and dopamine. J Neurosci. 1989, 9 (8): 2866-77.PubMed
            43. Spiess R, Schoofs A, Heinzel HG: Anatomy of the stomatogastric nervous system associated with the foregut in Drosophila melanogaster and Calliphora vicina third instar larvae. J Morphol. 2008, 269 (3): 272-82. 10.1002/jmor.10581.View ArticlePubMed
            44. Schoofs A, Spiess R: Anatomical and functional characterisation of the stomatogastric nervous system of blowfly (Calliphora vicina) larvae. J Insect Physiol. 2007, 53 (4): 349-60. 10.1016/j.jinsphys.2006.12.009.View ArticlePubMed
            45. Seroude L, et al: Spatio-temporal analysis of gene expression during aging in Drosophila melanogaster. Aging Cell. 2002, 1 (1): 47-56. 10.1046/j.1474-9728.2002.00007.x.View ArticlePubMed
            46. Hrdlicka L, et al: Analysis of twenty-four Gal4 lines in Drosophila melanogaster. Genesis. 2002, 34 (1-2): 51-57. 10.1002/gene.10125.View ArticlePubMed
            47. Ward EJ, et al: GAL4 enhancer trap patterns during Drosophila development. Genesis. 2002, 34 (1-2): 46-50. 10.1002/gene.10138.View ArticlePubMed
            48. Godt D, et al: Pattern formation in the limbs of Drosophila: bric a brac is expressed in both a gradient and a wave-like pattern and is required for specification and proper segmentation of the tarsus. Development. 1993, 119 (3): 799-812.PubMed
            49. Seshaiah P, et al: pasilla, the Drosophila homologue of the human Nova-1 and Nova-2 proteins, is required for normal secretion in the salivary gland. Developmental Biology. 2001, 239 (2): 309-322. 10.1006/dbio.2001.0429.View ArticlePubMed
            50. Tepass U, Hartenstein V: Epithelium formation in the Drosophila midgut depends on the interaction of endoderm and mesoderm. Development. 1994, 120 (3): 579-90.PubMed
            51. Mathew D, et al: Recruitment of Scribble to the Synaptic Scaffolding Complex Requires GUK-holder, a Novel DLG Binding Protein. Current Biology. 2002, 12 (7): 531-539. 10.1016/S0960-9822(02)00758-3.View ArticlePubMed
            52. Mendoza C, et al: Novel isoforms of Dlg are fundamental for neuronal development in Drosophila. Journal of Neuroscience. 2003, 23 (6): 2093-2101.PubMed
            53. Johard HA, et al: Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: relations to extrinsic neurons expressing different neurotransmitters. J Comp Neurol. 2008, 507 (4): 1479-96. 10.1002/cne.21636.View ArticlePubMed
            54. Haywood AF, Staveley BE: parkin counteracts symptoms in a Drosophila model of Parkinson's disease. BMC Neuroscience. 2004, 5 (14): 14-10.1186/1471-2202-5-14.PubMed CentralView ArticlePubMed
            55. Johard HA, et al: Peptidergic clock neurons in Drosophila: ion transport peptide and short neuropeptide F in subsets of dorsal and ventral lateral neurons. Journal of Comparative Neurology. 2009, 516 (1): 59-73. 10.1002/cne.22099.View ArticlePubMed
            56. Salvaterra PM, Kitamoto T: Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP. Brain Res Gene Expr Patterns. 2001, 1 (1): 73-82. 10.1016/S1567-133X(01)00011-4.View ArticlePubMed
            57. Lansdell SJ, Millar NS: Molecular characterization of Dalpha;6 and Dalpha7 nicotinic acetylcholine receptor subunits from Drosophila: formation of a high-affinity alpha-bungarotoxin binding site revealed by expression of subunit chimeras. Journal of Neurochemistry. 2004, 90 (2): 479-489. 10.1111/j.1471-4159.2004.02499.x.View ArticlePubMed
            58. Hidalgo A, Urban J, Brand AH: Targeted ablation of glia disrupts axon tract formation in the Drosophila CNS. Development. 1995, 121 (11): 3703-12.PubMed
            59. Rulifson EJ, Kim SK, Nusse R: Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science. 2002, 296 (5570): 1118-20. 10.1126/science.1070058.View ArticlePubMed
            60. Suster ML, et al: Refining GAL4-driven transgene expression in Drosophila with a GAL80 enhancer-trap. Genesis. 2004, 39 (4): 240-5. 10.1002/gene.20051.View ArticlePubMed
            61. Tweedie S: FlyBase: enhancing Drosophila Gene Ontology annotations. Nucleic Acids Res. 2009, D555-9. 10.1093/nar/gkn788. 37 Database
            62. Bellen HJ, et al: The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics. 2004, 167 (2): 761-81. 10.1534/genetics.104.026427.PubMed CentralView ArticlePubMed
            63. Hornstein NJ, Pulver SR, Griffith LC: Channelrhodopsin2 mediated stimulation of synaptic potentials at Drosophila neuromuscular junctions. J Vis Exp. 2009, 25
            64. Schroll C, et al: Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae. Curr Biol. 2006, 16 (17): 1741-7. 10.1016/j.cub.2006.07.023.View ArticlePubMed
            65. Coast GM: Diuresis in the housefly (Musca domestica) and its control by neuropeptides. Peptides. 2001, 22 (2): 153-60. 10.1016/S0196-9781(00)00372-7.View ArticlePubMed
            66. Wu P, et al: Correlation between the expressions of gastrin, somatostatin and cyclin and cyclin-depend kinase in colorectal cancer. World J Gastroenterol. 2005, 11 (45): 7211-7.View ArticlePubMed
            67. Jang HJ, et al: Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1. Proc Natl Acad Sci USA. 2007, 104 (38): 15069-74. 10.1073/pnas.0706890104.PubMed CentralView ArticlePubMed
            68. Johnson EC, et al: A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. Journal of Experimental Biology. 2005, 208 (Pt 7): 1239-1246. 10.1242/jeb.01529.View ArticlePubMed
            69. Brugge VA, Schooley DA, Orchard I: Amino acid sequence and biological activity of a calcitonin-like diuretic hormone (DH31) from Rhodnius prolixus. J Exp Biol. 2008, 211 (Pt 3): 382-90. 10.1242/jeb.013771.View ArticlePubMed
            70. Arredondo J, et al: Receptor-mediated tobacco toxicity: acceleration of sequential expression of alpha5 and alpha7 nicotinic receptor subunits in oral keratinocytes exposed to cigarette smoke. FASEB J. 2008, 22 (5): 1356-68. 10.1096/fj.07-9965.com.View ArticlePubMed
            71. Chernyavsky AI: Auto/paracrine control of inflammatory cytokines by acetylcholine in macrophage-like U937 cells through nicotinic receptors. Int Immunopharmacol. 10 (3): 308-15. 10.1016/j.intimp.2009.12.001.PubMed CentralView ArticlePubMed
            72. Wessler I, Kirkpatrick CJ: Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans. Br J Pharmacol. 2008, 154 (8): 1558-71. 10.1038/bjp.2008.185.PubMed CentralView ArticlePubMed
            73. Grando SA, et al: Recent progress in understanding the non-neuronal cholinergic system in humans. Life Sci. 2007, 80 (24-25): 2181-5. 10.1016/j.lfs.2007.03.015.View ArticlePubMed
            74. Blackburn MB, et al: The identification of two myoinhibitory peptides, with sequence similarities to the galanins, isolated from the ventral nerve cord of Manduca sexta. Regul Pept. 1995, 57 (3): 213-9. 10.1016/0167-0115(95)00034-9.View ArticlePubMed
            75. Dun W, Boyden PA: The Purkinje cell; 2008 style. J Mol Cell Cardiol. 2008, 45 (5): 617-24. 10.1016/j.yjmcc.2008.08.001.PubMed CentralView ArticlePubMed
            76. Hirose M, et al: Function of Ca(2+) release channels in Purkinje cells that survive in the infarcted canine heart: a mechanism for triggered Purkinje ectopy. Circ Arrhythm Electrophysiol. 2008, 1 (5): 387-95. 10.1161/CIRCEP.107.758110.PubMed CentralView ArticlePubMed

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            This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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