**2.** *Drosophila* **as an emerging model system for studying gut: comparison of human and fly gut**

Easy genetic manipulation and effortless genetic tools make flies an insect of choice to study inter organ neuronal signaling including gut-brain axis neuronal connectivity. To understand human metabolic diseases and how a GI play a key role there is a recent focus of research. Some progressive studies also draw a link between neurodegenerative disorders and gut microbiota of humans and other insects. *Drosophila's* assistance for studying these and metabolic diseases with respect to its gut will be covered in this chapter.

*Gut Feeding the Brain:* Drosophila *Gut an Animal Model for Medicine to Understand… DOI: http://dx.doi.org/10.5772/intechopen.96503*

Fruit flies have a simple and similar to humans- gut system (**Figure 1**). In mammals including humans, the esophagus (*Drosophila* foregut) passes the consumed food to the stomach (crop in flies), where food stores and digestion proceeds. Nutrient absorption takes place in the small intestine (anterior midgut in flies). Later nutrient, water and electrolyte absorption commences in the large intestine (fly hindgut). Finally, it reaches the rectum and anus for excretion (**Figure 1**) [1, 2]. In flies, after food passes through the middle midgut (a region of low pH, contains the iron and copper cells), it transits through the posterior midgut for further absorption and through the hindgut and rectum to exchange water and electrolytes and finally reaches the anus for excretion. Malpighian tubules (renal-like structures) are tubular excretory organs in flies connected to the midgut-hindgut junction and they absorb solutes, water and waste from the surrounding hemolymph, and release them in the gut in the form of solid nitrogenous compounds (**Figure 1**) [3]. Though the malpighian tubules are drastically different from human kidneys, similarities have been seen in function and development.

The adult *Drosophila* gut is like a tube structure lined by an epithelial monolayer comprising of four cell types: intestinal stem cells (ISCs), absorptive enterocytes (ECs), secretory enteroendocrine (EE) cells, and enteroblasts (EBs) (**Figures 1** and **2**). Ectodermally derived foregut consists of esophagus, crop, and cardia (**Figures 1** and **2**). The crop is a diverticulated structure unique to Diptera. A complex array of valves and sphincters ensure passage of intestinal matter in and

#### **Figure 1.**

*Comparison between human and Drosophila gut. Organs with similar functions are coded with same colors. Drosophila contains many tissues/organs that functionally resemble to most essential human gastrointestinal system: Esophagus (foregut), midgut (small intestine) and large intestine (hindgut), stomach (crop), kidneys (malpighian tubules).*

#### **Figure 2.**

*Fly gut anatomy. (A) the Drosophila gut-brain axis consists of central nervous system (brain and ventral nerve cord-VNC; shown in white), gastrointestinal system (foregut, midgut and hindgut), crop and malpighian tubules. (B) the whole fly gut is divided into foregut (esophagus, crop and proventriculus), midgut (R1-R5), and hindgut (gray). pH divisions are also observed in midgut. (C) In Drosophila gut epithelia, the epithelium is protected by the peritrophic matrix and thin mucus layer apically and is covered in a basal lamina and visceral muscle cells. The fly midgut is composed of absorptive enterocytes (ECs) and secretory enteroendocrine cells (EE) that stand up from differentiation of the basally embedded intestinal stem cells (ISCs). Enteroblasts (EBs) are transient progenitors destined to differentiate into ECs.*

out of the crop into the main alimentary canal. Crop function is poorly determined, its function in processes like early digestion, detoxification, microbial control, and food storage in flies has been speculated from other insects [4]. The cardia (or proventriculus) is a complex bulb-shaped structure composed of three epithelial layers. It makes the peritrophic matrix (site of antimicrobial peptide production) [5, 6] which may act as a valve, regulating the entry of ingested food into the midgut (**Figures 1, 2B** and **C**). Posterior to the cardia is endodermally derived midgut (with average length of 6 mm in adult flies), the main digestive/absorptive portion [7, 8] (**Figures 1, 2A** and **B**). It has been found that fly midgut epithelial cells have an opposite arrangement of junctions, with occluding junctions above *adherens* junctions, as in mammals [9]. The visceral muscle surrounds the epithelium. It is protected toward the lumen by secreted mucus and, posterior to the foregut, by a chitinous layer (peritrophic matrix) [10] (**Figure 2C**).

The fly midgut has been segmented into the anterior, middle and the posterior midgut (**Figures 1, 2A** and **B**). It has been further subdivided morphologically and molecularly into 10–14 regions (**Figure 2B**) [11–13]. Midgut regionalization has been

#### *Gut Feeding the Brain:* Drosophila *Gut an Animal Model for Medicine to Understand… DOI: http://dx.doi.org/10.5772/intechopen.96503*

seen in the muscles, trachea and neurons that surround it [12–15]. Physical properties (*e.g.*, luminal pH), histological and cellular features (villi size, lumen width), stem cell proliferation rates, and gene expression profiles [11–13, 16, 17] have been used to characterized all midgut regions. The middle midgut (R3) contains a copper cell region in R3ab, which produces gastric acid, followed by a large flat cell region (R3c) with uncertain role (**Figure 2B**). Two boundaries flanking this region are inflection points where the midgut folds stereotypically inside the body cavity.

The Malpighian tubules release at the junction between the midgut and hindgut (**Figures 1,2A** and **B**). The water/ion exchange occurs in the hindgut which consist of pylorus (a second valve-like structure), ileum, and rectum [7, 8]. The muscles that surround the epithelium in flies are striated, as opposed to the smooth muscles found in mammalian intestines [18]. An outer layer of longitudinal muscles found surrounding the midgut. Circular muscles are found to be present throughout the fly tract. Physiology of the intestine is maintained and regulated by autonomic innervation and by hormones. The tracheal system forms a branched structure surrounding the gut during development [15] and may influence epithelial regeneration in the adult. Owning to similarity of flies and human gut, we will be discussing further how gut of the flies is handled and controlled to understand about neural circuitry drawing it closer to the brain and diseases related to intestinal illnesses.

#### **2.1 Intestinal anatomy of** *Drosophila* **and human**

Both humans and fly intestines share similar tissue, anatomy and physiological function [19, 20]. Their gut are of endothelial origin in nature [21, 22] and comprise of an epithelial monolayer of columnar or cuboidal ECs. A series of sequential depressions called the crypts of Lieberkühn, along the small and large intestine, and protruding villi along the internal surface of the small intestine in mammalian intestinal epithelium maximize its surface area [23]. Extensive folding has not been reported in the *Drosophila* intestine. Cytoplasmic extensions (microvilli) of the apical side of ECs and ISCs [24] do increase the cellular surface area facing the gut lumen in both flies and mammals. Microvilli spread parallel to each other toward the lumen to form the brush border [24–26]. A layer of mucus present above the brush border protects the host from intestinal microbes. The peritrophic matrix in *Drosophila* gut helps to sequester microbes from coming in contact with the midgut and hindgut [27, 28] (**Figure 2C**).

In both flies and mammals, the epithelial monolayer is associated on its basal side on an extracellular collagenous matrix (known as basement membrane) [29]. A checkerboard of innervated and trachea-oxygenated longitudinal and circular muscles tissue underneath the basement membrane in flies drive the peristaltic movements [30] (**Figure 2C**). Mammalian intestine has a similar organization of intestinal external musculature in the outer layers where musculature is also innervated and oxygenated by a plexus of vasculature [31, 32]. Layers including, the submucosa, (a dense layer of connective tissue containing nerves and lymphatic and blood vessels); muscularis mucosae (an additional muscle layer); and the lamina propria underling the intestinal epithelium and contains connective tissue, lymph nodes (Peyer's patches), immune cells (leukocytes, and dendritic and mast cells), vessels and myofibroblasts [33], fill the space between the outer musculature and the basement membrane in mammals.

## **2.2 ISCs, ECs, and EE cells of fly gut**

About 65% of human-disease causing genes are shared as a functional homolog in fruit flies. This shows conservation of genes and function at an evolutionary

level. Fundamental processes such as digestion is also conserved from flies to humans. *Drosophila* intestine is composed of many cell types of heterogeneous developmental origin. Adult multipotent ISCs are present in both fly and mammalian guts [34–37]. ISCs differentiate throughout to self-renew and form new specialized cells namely absorptive type ECs and secretory type EE cells (**Figure 2C**) [38]. ECs and EE cells are found in both mammals and flies. ECs help in absorbing nutrients. EE cells release hormones for gut mobility and function. They also have antimicrobial purposes which are fulfilled by analogous cells in humans such as goblet and Paneth cells [39, 40]. *Drosophila* intestine produce both mucus and AMPs, but secretory cells (mucus-producing goblet cells) and the AMP-producing Paneth cells of mammalian gut have not been found in *Drosophila* midgut [28, 41]. Mammalian ECs and secretory cells are located at the bottom of the crypts and specifically express Lgr5 and/or Bmi1 (stem cell markers). Both of these cell types can give rise to all lineages of intestinal cells, including the transient amplifying (TA) cells that lie immediately above ISCs. TA gradually move upwards while maturing to eventually reach complete maturation close to the opening of the crypts. There is a continuous turnover of TA cells, which are either shed or become apoptotic upon maturation [23, 42].

The lineage of fly posterior midgut with only one type of mature absorptive cell and one main type of secretory cell is very simple. Although asymmetric ISC divisions in the fly midgut produce transient cells (EBs), these cells do not undergo further cell division and remain close to the ISCs before maturation. Fly midgut ISCs are situated basally and are broadly dispersed in the intestinal epithelium. The cellular composition and regeneration in *Drosophila* hindgut are interestingly similar to mammals. As in mammals, the ISCs of the hindgut are specified anteriorly and move posteriorly, as TA cells do, before their further differentiation in the posterior hindgut [37]. Nonetheless fly hindgut has not been examined as extensively as the midgut which has served as the prototype *Drosophila* tissue for the study of intestinal pathology [43–45].

#### **2.3 The fly intestine functions**

Like the regional specialization of digestive functions, the expression of digestive enzymes has also been found to confined to specific segments of the digestive tract in flies [12, 46, 47]. In addition to its roles in nutrient extraction and utilization, the digestive tract responds to the food and bacteria in its lumen. Digestion takes place in fly midgut [48] which can be further modulated by various factors like temperature, redox potential, pH, and intestinal transit [8, 48]. It has been shown that the expression and activity of digestive enzymes are tightly regulated in many insects like enzymes involved in the breakdown of sugars in flies are enriched in anterior (R1/R3) portions of the adult midgut and Peptidase genes express more posteriorly [47].

The enzymatic activity of the intestine is a key factor determining availability of certain nutrients. A substantial reduction of intestinal digestive enzyme activities (trypsin, chymotrypsin, aminopeptidase, and acetate esterase) has been reported in flies lacking EE cells [49]. Though not extensively investigated in *Drosophila,* modulation by nutrient quality and quantity, neuronal activity, and endocrine signals has been described in many insects [8, 50, 51]. Models suggesting role of ECs in integrating information about sugar uptake (sensed intrinsically in the intestine by Mondo-Bigmax) and the carbohydrate status of the fat body (relayed by TGF-β/Activin signaling) to modulate expression of the carbohydrate digestive enzymes have been proposed. Repression mechanism involving the TGF-β/Activin ligand Dawdle (Daw) which, upon refeeding with nutritious sugars *Gut Feeding the Brain:* Drosophila *Gut an Animal Model for Medicine to Understand… DOI: http://dx.doi.org/10.5772/intechopen.96503*

(but not non-nutritious sugars) after a period of starvation, reduces the expression of carbohydrate digestive enzymes in the adult ECs [52]. Activation of the intracellular sugar sensor complex Mondo-Bigmax promotes the expression of both Daw and the transcription factor *sugarbabe (sug)* [53]. Sug further repress the expression of amylases. Low cholesterol in the diet upregulate expression of the Hr96 nuclear receptor (homologous to the vertebrate LXR receptor involved in regulated cholesterol homeostasis) [54]. Hr96 binds cholesterol and promotes the expression of genes involved in cholesterol homeostasis and lipid breakdown including Magro (Mag) [54–56]. Mag plays a dual role in breaking down intestinal cholesterol esters to maintain cholesterol homeostasis. It also enables triacylglyceride (TAG) breakdown, required for intestinal lipid absorption and peripheral fat accumulation [56, 57]. It has been suggested that intestinal mag expression can also repressed by a sugar-rich diet in a foxo-dependent manner [58]. Such a mechanism becomes chronically active in the aging intestine due to disrupting lipid homeostasis and activation of JNK pathway affecting the metabolic homeostasis [58].

## *2.3.1 Role in nutrients absorption*

**Carbohydrates:** A diverse array of transporters internalizes simple sugars into the ECs for further digestion and absorption [59] in insects like Glucose transporters, the GLUT/Slc2 family of facilitative glucose transporters and the SGLT/Slc5 family of Na<sup>+</sup> -glucose symporters [60–64]. GLUT-like gene has been described in flies [65]. *Drosophila* genome harbors homologs of other glucose transporters including a homolog of SWEET family of sugar transporters [66, 67] a disaccharide transporter Slc45–1 [68]; trehalose transporters (Tret1–1 and Tret1–2) [69] and Slc45–1 (can transport sucrose) [68, 70]. The possible intestinal activity of many of these transporters deserves further investigation.

**Proteins:** A mixture of amino acids, di- and tri-peptides are products of protein break down. This chemical diversity is handled by a broad range of apical and basolateral transport systems (many are homologous to known mammalian transporter systems) [59, 71]. *Drosophila* homologs of cationic amino acid transporters [72], ion-dependent and independent amino acid transporters for neutral amino acids [73–76] and oligopeptide transporters [77, 78] are some examples. Intestinal expression of amino acid transporters Pathetic [74] has also been reported. Minidiscs [73], NAT1 and other Slc6 family members [75, 79], and the oligopeptide transporters Yin and CG2930, with enriched expression in proventriculus/hindgut and midgut [77, 78] has been shown. The nature, physiological modulation, and significance of many of these amino acid/oligopeptide transporters remains to be investigated.

**Lipids and sterols:** Intestinal lipid transport in *Drosophila* is still undetermined. Intestinal cells absorb free fatty acids, glycerol, mono- and diacylglycerols, and phospholipid derivatives (products of lipid digestion) along with dietary sterols. Diffusion and emulsification have been proposed for absorption [80]. Vertebrates emulsify by covering lipids with bile salts, but in insects emulsification is achieved by forming fatty acid-amino acid and glycolipid complexes, as well as fatty acids and lysophospholipid micelles [80]. In ECs, the products of lipid breakdown are used to resynthesize diacylglycerols and TAG. They get packaged together with cholesterol and fat body-derived carrier proteins to form lipoprotein particles and trafficked throughout the body [81] ensuring that the products of lipid breakdown are kept at low concentrations inside the ECs, which may facilitate diffusion. Mutants in which lipoprotein secretion from the fat body is compromised has revealed both anterior and posterior midgut regions as sites of lipid efflux [81]. The absorption of sterols is crucial to insects as they cannot synthesize sterols and require a dietary source of sterol for the synthesis of the steroid molting hormone ecdysone. In *Drosophila*

Niemann-Pick Chomologs-Npc1a and Npc2a are broadly required for intracellular sterol trafficking [82], whereas Npc1b is expressed in the midgut and is required for intestinal sterol absorption [83].

Changes in the expression of p38 kinase or the Atf3 and Foxo transcription factors cause accumulation of neutral lipid in ECs [58, 84]. It has been shown that neutral lipid increase following depletion of the EE hormone Tk [85], or in sterile female flies after mating [86]. It has been suggested that activation of intestinal lipogenesis is key to survival in diet-restricted flies. Indeed, nutrient scarcity induces expression of the sugar sensor transcription factor *sug* in the intestine which, in turn, promotes intestinal lipogenesis. Internal nutritional challenges may be equally dependent on deployment of these intestinal adaptations [86].

#### *2.3.2 Intestinal pH*

Many animals generate localized regions of low pH inside the intestinal lumen to facilitate protein breakdown, absorption of minerals and metals, and limit the survival of ingested microbes. While mammalian digestion takes place in acidic conditions, insect digestion occurs at neutral or basic pH including *Drosophila* (neutral or mildly alkaline). Luminal pH does, however, display consistent transitions along the length of the intestine and becomes strongly acidic (pH 2–4) in the copper cell region of both larvae and adults [24, 87, 88]. Posterior to this region, the midgut lumen becomes mildly alkaline again (pH 7–9), but is again acidified in the hindgut (pH 5), partly as a result of discharges from the malpighian tubules. Diet affects the acidity of rectal ampulla where final pH adjustments may take place [14]. Copper cells are specialized ECs with a highly invaginated apical membrane, similar to the mammalian gastric parietal cells [89]. During aging in adult flies, genetic interference with copper cell identity or their progressive loss are associated with loss of gut acidity [90].

The contribution of five ion transporters enriched in the acidic region have been studied [88]. These include: the potassium/chloride symporter Kazachoc (Kcc), a member the Slc12 family of electroneutral cation-chloride transporters (express in intestine) [91, 92]; the Slowpoke pore-forming subunit of a calcium-activated K<sup>+</sup> channel (express in neurons, muscles, tracheal cells, and two types of midgut ECs in the copper and iron cell regions) [93]; the ligand-gated chloride channel pHCL-2 which, in addition to regulating fluid secretion in malpighian tubules (express in the copper cell, iron, and large flat cell regions of the midgut) [94, 95]; the carbonic anhydrase CAH1; and the bicarbonate/chloride exchanger CG8177, belonging to the Slc4a1–3 subfamily of anion exchangers (express in a specific midgut pattern similar to that of pHCl-2) [96]. Collectively, these findings suggest that the transport of H+ , Cl− , K+ , and HCO3<sup>−</sup> contributes to acid generation in the *Drosophila* midgut.

#### *2.3.3 Water and osmolytes*

Flies extract water from their diet to maintain hydration and ionic balance. This compensates for substantial water loss resulting from metabolic and physiological processes. Although malpighian tubules are important for this process, but intestine also contributes. Water absorption from the food occurs in the insect midgut and in rectal pads of rectum [8]. The rectal pads are also the crucial site for reabsorption of ions. Ions and water can cross the intestinal epithelium through or between cells and their transport play an important role in the maintenance of ion gradients that sustain active transport in the intestinal epithelium. The scanning ion-selective electrode technique (SIET) provides a way to probe intestinal gradients for ions such as K<sup>+</sup> , Na+ , H+ , or Cl− [24, 97]. K+ and Na+ absorption occur largely in the large flat cell and posterior regions of the midgut and, also in the anterior hindgut in the

#### *Gut Feeding the Brain:* Drosophila *Gut an Animal Model for Medicine to Understand… DOI: http://dx.doi.org/10.5772/intechopen.96503*

case of Na<sup>+</sup> [97]. The two *Drosophila* Nha members express in intestine epithelia and their ubiquitous knockdown decrease survival, especially under Na+ stress [12, 98–100]. Including kcc, four different genes encoding homologs of the cation-Cl<sup>−</sup> Slc12 cotransporters express in osmoregulatory organs (gut, anal pads, and Malpighian tubules) [91, 92].
