Degenerative and regenerative processes involved in midgut pseudotumor formation in the stick insect (Carausius morosus)

Spontaneous and experimentally induced pseudotumor formation in Carausius morosus impairs the midgut tissue homeostasis. Spontaneous pseudotumor formation begins by the break down of a single or a small group of columnar cells (CCs) and is followed by the degeneration of neighboring CCs. There are not only marked similarities but also decisive differences between normal dying CCs in healthy specimens and the degeneration of CCs leading to pseudotumors: in both cases, the apical cell parts with the nucleus are extruded into the midgut lumen, but only during of pseudotumor formation an “amorphous substance” originates from the basal parts of the CCs. Hemocytes are attracted to this substance and form a nodule‐like aggregation, which is responsible for the phenotype of pseudotumors. Pseudotumor infestation has also an impact on the midgut nidi, which consist of an intestinal stem cell and several CC progenitor cells. In healthy specimens only one progenitor cell per nidus differentiates at a time, but, several to all progenitor cells differentiate simultaneously in pseudotumor‐infested specimens. Extirpation of the ingluvial ganglion in healthy specimens results in an immediate onset of pseudotumor formation and a dramatic acceleration of pseudotumor growth. Importantly, the ultrastructural characteristics of spontaneous and experimentally induced pseudotumors are identical. This supports the idea that the stomatogastric nervous system plays an integral role in the maintenance of midgut tissue homeostasis. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Heparan sulfate maintains adult midgut homeostasis in Drosophila

Tissue homeostasis is controlled by the differentiated progeny of residential progenitors (stem cells). Adult stem cells constantly adjust their proliferation/differentiation rates to respond to tissue damage and stresses. However, how differentiated cells maintain tissue homeostasis remains unclear. Here, we find that heparan sulfate (HS), a class of glycosaminoglycan (GAG) chains, protects differentiated cells from loss to maintain intestinal homeostasis. HS depletion in enterocytes (ECs) leads to intestinal homeostasis disruption, with accumulation of intestinal stem cell (ISC)‐like cells and mis‐differentiated progeny. HS‐deficient ECs are prone to cell death/stress and induced cytokine and epidermal growth factor (EGF) expression, which, in turn, promote ISC proliferation and differentiation. Interestingly, HS depletion in ECs results in the inactivation of decapentaplegic (Dpp) signaling. Moreover, ectopic Dpp signaling completely rescued the defects caused by HS depletion. Together, our data demonstrate that HS is required for Dpp signal activation in ECs, thereby protecting ECs from ablation to maintain midgut homeostasis. Our data shed light into the regulatory mechanisms of how differentiated cells contribute to tissue homeostasis maintenance.     

Identification, physiological actions, and distribution of TPSGFLGMRamide: a novel tachykinin-related peptide from the midgut and stomatogastric nervous system of Cancer crabs

In most invertebrates, multiple species-specific isoforms of tachykinin-related peptide (TRP) are common. In contrast, only a single conserved TRP isoform, APSGFLGMRamide, has been documented in decapod crustaceans, leading to the hypothesis that it is the sole TRP present in this arthropod order. Previous studies of crustacean TRPs have focused on neuronal tissue, but the recent demonstration of TRPs in midgut epithelial cells in Cancer species led us to question whether other TRPs are present in the gut, as is the case in insects. Using direct tissue matrix assisted laser desorption/ionization Fourier transform mass spectrometry, in combination with sustained off-resonance irradiation collision-induced dissociation, we found that at least one additional TRP is present in Cancer irroratus, Cancer borealis, Cancer magister, and Cancer productus. The novel TRP isoform, TPSGFLGMRamide, was present not only in the midgut, but also in the stomatogastric nervous system (STNS). In addition, we identified an unprocessed TRP precursor APSGFLGMRG, which was detected in midgut tissues only. TRP immunohistochemistry, in combination with preadsorption studies, suggests that APSGFLGMRamide and TPSGFLGMRamide are co-localized in the stomatogastric ganglion (STG), which is contained within the STNS. Exogenous application of TPSGFLGMRamide to the STG elicited a pyloric motor pattern that was identical to that elicited by APSGFLGMRamide, whereas APSGFLGMRG did not alter the pyloric motor pattern.     

Effect of sublethal Bacillus thuringiensis crystal endotoxin treatment on the larval midgut of a moth, Manduca: SEM study

The effect of a single, sublethal dose of B. thuringiensis crystal endotoxin on the midgut of the moth Manduca sexta larvae was monitored during acute and recovery stages. Initially both goblet and columnar cells swelled. Many columnar cells produced membrane extrusions. In some cases the affected cells ruptured, extruding cellular debris into the midgut lumen. Following the acute stage, the midgut tissue recovered, the damaged cells being extruded into the midgut lumen apparently as newly regenerated cells rose to take their place. The insects appeared to recover completely and continue normal development.     

Primary and continuous midgut cell cultures from Pseudaletia unipuncta (Lepidoptera: Noctuidae)

Midgut epithelial cells were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several cells lines were obtained from these P. unipuncta primary cultures, and they have been subcultured and maintained for over 24 mo. The three major midgut cell types were present in the cultures, including stem (regenerative), columnar, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem cells were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells every 7-8 wk. After approximately six passages, the cell density in T-flasks appeared to be somewhat constant, reaching 10(3)-10(4) cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these cells with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.     

Tachykinin-related peptides in invertebrates: a review

Peptides with sequence similarities to members of the tachykinin family have been identified in a number of invertebrates belonging to the mollusca, echiuridea, insecta and crustacea. These peptides have been designated tachykinin-related peptides (TRPs) and are characterized by the preserved C-terminal pentapeptide FX1GX2Ramide (X1 and X2 are variable residues). All invertebrate TRPs are myostimulatory on insect hindgut muscle, but also have a variety of additional actions: they can induce contractions in cockroach foregut and oviduct and in moth heart muscle, trigger a motor rhythm in the crab stomatogastric ganglion, depolarize or hyperpolarize identified interneurons of locust and the snail Helix and induce release of adipokinetic hormone from the locust corpora cardiaca. Two putative TRP receptors have been cloned from Drosophila; both are G-protein coupled and expressed in the nervous system. The invertebrate TRPs are distributed in interneurons of the CNS of Limulus, crustaceans and insects. In the latter two groups TRPs are also present in the stomatogastric nervous system and in insects endocrine cells of the midgut display TRP-immunoreactivity. In arthropods the distribution of TRPs in neuronal processes of the brain displays similar patterns. Also in coelenterates, flatworms and molluscs TRPs have been demonstrated in neurons. The activity of different TRPs has been explored in several assays and it appears that an amidated C-terminal hexapeptide (or longer) is required for bioactivity. In many invertebrate assays the first generation substance P antagonist spantide I is a potent antagonist of invertebrate TRPs and substance P. Locustatachykinins stimulate adenylate cyclase in locust interneurons and glandular cells of the corpora cardiaca, but in other tissues the putative second messenger systems have not yet been identified. The heterologously expressed Drosophila TRP receptors coupled to the phospholipase C pathway and could induce elevations of inositol triphosphate. The structures, distributions and actions of TRPs in various invertebrates are compared and it is concluded that the TRPs are multifunctional peptides with targets both in the central and peripheral nervous system and other tissues, similar to vertebrate tachykinins. Invertebrate TRPs may also be involved in developmental processes.     

Embryonic development of the alimentary canal and ectodermal derivatives in the primitive moth,Neomicropteryx nipponensis issiki (Lepidoptera,Micropterygidae)

The formation of the alimentary canal, nervous system, and of other ectodermal derivatives in the embryo of the primitive moth, Neomicropteryx nipponensis Issiki, is described. The stomodaeum is formed from an invagination in the medioposterior portion of the protocephalon. The proctodaeum arises as an extension of the amnioproctodaeal cavity. The midgut epithelium orginates from anterior and posterior rudiments in blind ends of the stomodaeum and proctodaeum. The decondary dorsal organ is formed in developing midgut. The development of the brain is typical of insects. The ventral nerve cord originates in large part from neuroblasts arising in 3 gnathal, 3 thoracic, and 11 abdominal segments. Intrasegmental median cord cells probably differentiate into both ganglion cells and glial elements of the ventral nerve cord; intersegmental cells appear not to participate in the formation of the nervous system. The stomatogastric nervous system develops from three evaginations in the dorsal wall of the stomodaeum, and consists of the frontal, hypocerebral, and ventricular ganglia, the recurrent nerve, and corpora cardiaca. Five stemmata arise from the epidermis on each side of the head. Five pairs of ectodermal invaginations are formed in the cephalognathal region to produce the tentorium, mandibular apodemes, corpora allata, and silk glands. Prothoracic glands orginate in the prothorax. Mesothoracic spiracles shift anteriorly to the prothorax during development. Oenocytes arise in the first seven abdominal segments. Invaginated pleuropodia are formed in the first abdominal segment.     

Intestinal stem cell proliferation and epithelial homeostasis in the adult Drosophila midgut

Adult tissue homeostasis requires a tight balance between the removal of old or damaged cells and the production of new ones. Such processes are usually driven by dedicated stem cells that reside within specific tissue locations or niches.The intestinal epithelium has a remarkable regenerative capacity, which has made it a prime paradigm for the study of stem cell-driven tissue self-renewal. The discovery of the presence of stem cells in the adult midgut of the fruit fly Drosophila melanogaster has significantly impacted our understanding of the role of stem cells in intestinal homeostasis. Here we will review the current knowledge of the main mechanisms involved in the regulation of tissue homeostasis in the adult Drosophila midgut, with a focus on the role of stem cells in this process. We will also discuss processes involving acute or chronic disruption of normal intestinal homeostasis such as damage-induced regeneration and ageing.     

EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila

Many tissues in higher animals undergo dynamic homeostatic growth, wherein damaged or aged cells are replaced by the progeny of resident stem cells. To maintain homeostasis, stem cells must respond to tissue needs. Here we show that in response to damage or stress in the intestinal (midgut) epithelium of adult Drosophila, multiple EGFR ligands and rhomboids (intramembrane proteases that activate some EGFR ligands) are induced, leading to the activation of EGFR signaling in intestinal stem cells (ISCs). Activation of EGFR signaling promotes ISC division and midgut epithelium regeneration, thereby maintaining tissue homeostasis. ISCs defective in EGFR signaling cannot grow or divide, are poorly maintained, and cannot support midgut epithelium regeneration after enteric infection by the bacterium Pseudomonas entomophila. Furthermore, ISC proliferation induced by Jak/Stat signaling is dependent upon EGFR signaling. Thus the EGFR/Ras/MAPK signaling pathway plays central, essential roles in ISC maintenance and the feedback system that mediates intestinal homeostasis.     

Histochemical characterization of cell death in honeybee larvae midgut after treatment with Paenibacillus larvae, Amitraz and Oxytetracycline

A number of techniques were employed to assess cell death induced in honeybee larvae midgut after per os inoculation of bacterium Paenibacillus larvae var. larvae, the causative agent of American foulbrood disease, and separately with acaricide Amitraz and antibiotic Oxytetracycline. In honeybee larvae exposed to Amitraz, which demonstrates both necrosis and apoptosis, cell death was found in 82% of midgut columnar and in 50% of regenerative epithelial cells, 24 h after treatment. Cell death reduced to 36% in the epithelial cells, 48 h after treatment. In Oxytetracycline-treated larvae, cell death was identified in 40% of midgut epithelial cells, 24 h after inoculation and increased to 55% over the next 24 h. In Paenibacillus -infected larvae, all midgut epithelial cells died. Using ApopTag (Oncor) to label the multiple DNA ends generated by DNA fragmentation showed programmed cell death in 49% of columnar midgut cells 24 h after Amitraz application. Cell death was reduced to 9% over the next 24 h. Our data indicate that cell death could be identified and quantified in situ, using TUNEL techniques. This study also shows that the acaricide Amitraz is a trigger for programmed cell death in the midgut epithelial cells of honeybee larvae, unlike Paenibacillus which induces necrosis only. The data show that immunohistochemical methods are useful for studying in situ tissue pathology, and indicate possibilities for monitoring the effects of infective and chemical environmental stressors on cell death in honeybee larvae tissue.     

Localization of allatostatin-immunoreactive material in the central nervous system,stomatogastric nervous system,and gut of the cockroach Blattella germanica

Immunoreactivity against peptides of the allatostatin family having a typical YXFGL-NH₂ C-terminus has been localized in different areas of the central nervous system, stomatogastric nervous system and gut of the cockroach Blattella germanica. In the protocerebrum, the most characteristic immunoreactive perikarya are situated in the lateral and median neurosecretory cell groups. Immunoreactive median neurosecretory cells send their axons around the circumesophageal connectives to form arborizations in the anterior neuropil of the tritocerebrum. A group of cells in the lateral aspect of the tritocerebrum project to the antennal lobes in the deutocerebrum, where immunoreactive arborizations can be seen in the periphery of individual glomeruli. Nerve terminals were shown in the corpora allata. These terminals come from perikarya situated in the lateral neurosecretory cells in the pars lateralis and in the subesophageal ganglion. Immunoreactive axons from median neurosecretory cells and from cells positioned in the anteriormost part of the tritocerebrum enter together in the stomatogastric nervous system and innervate foregut and midgut, especially the crop and the valve between the crop and the midgut. The hindgut is innervated by neurons whose perikarya are located in the last abdominal ganglion. Besides immunoreactivity in neurons, allatostatin-immunoreactive material is present in endocrine cells distributed within the whole midgut epithelium. Possible functions for these peptides according to their localization are discussed. Arch. Insect Biochem. Physiol. 37:269–282, 1998. © 1998 Wiley-Liss, Inc.     

Gradients in the in vivo intestinal stem cell compartment and their in vitro recapitulation in mimetic platforms

Intestinal tissue, and specifically its mucosal layer, is a complex and gradient-rich environment. Gradients of soluble factor (BMP, Noggin, Notch, Hedgehog, and Wnt), insoluble extracellular matrix proteins (laminins, collagens, fibronectin, and their cognate receptors), stromal stiffness, oxygenation, and sheer stress induced by luminal fluid flow at the crypt-villus axis controls and supports healthy intestinal tissue homeostasis. However, due to current technological challenges, very few of these features have so far been included in in vitro intestinal tissue mimetic platforms. In this review, the tightly defined and dynamic microenvironment of the intestinal tissue is presented in detail. Additionally, the authors introduce the current state-of-the-art intestinal tissue mimetic platforms, as well as the design drawbacks and challenges they face while attempting to capture the complexity of the intestinal tissue’s physiology. Finally, the compositions of an “idealized” mimetic system is presented to guide future developmental efforts.     

Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR‐8–Escargot switch

The intestinal epithelium is remarkably robust despite perturbations and demand uncertainty. Here, we investigate the basis of such robustness using novel tracing methods that allow simultaneously capturing the dynamics of stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. We found that intestinal stem cells (ISCs) divide “ahead” of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. They extend long membrane protrusions that make cell–cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial–mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR‐8/miR‐200 in “pausing” enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse MET by antagonizing escargot. Our findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand.     

Identification of adult midgut precursors in Drosophila

The adult Drosophila midgut is thought to arise from an endodermal rudiment specified during embryogenesis. Previous studies have reported the presence of individual cells termed adult midgut precursors (AMPs) as well as “midgut islands” or “islets” in embryonic and larval midgut tissue. Yet the precise relationship between progenitor cell populations and the cells of the adult midgut has not been characterized. Using a combination of molecular markers and directed cell lineage tracing, we provide evidence that the adult midgut arises from a molecularly distinct population of single cells present by the embryonic/larval transition. AMPs reside in a distinct basal position in the larval midgut where they remain through all subsequent larval and pupal stages and into adulthood. At least five phases of AMP activity are associated with the stepwise process of midgut formation. Our data shows that during larval stages AMPs give rise to the presumptive adult epithelium; during pupal stages AMPs contribute to the final size, cell number and form. Finally, a genetic screen has led to the identification of the Ecdysone receptor as a regulator of AMP expansion.     

Ultrastructural changes in the midgut epithelium of the first larva of Allacma fusca (Insecta, Collembola, Symphypleona)

Abstract. In the newly hatched larva in Allacma fusca , the midgut epithelium was fully developed and formed by flattened epithelial cells surrounding the yolk mass in the midgut lumen. Immediately after hatching, the first larva began to feed; the migut lumen was filled with the yolk mass and food (mainly algae). Regenerative cells typical of the developing midgut epithelium of many insects were not observed. Initially, midgut cells of the larva were cuboidal but became columnar in shape with distinct regionalization in the distribution of cell organelles. Furthermore, urospherites appeared in the midgut cell cytoplasm, i.e., structures characteristic for the midgut epithelium of insects having no Malpighian tubules. As a result, cells with the capacity for digestion, absorption, and excretion were observed to be completely formed in the first larval stage.     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.     

Ultrastructure of the posterior half of the intestine of the channel catfish,Ictalurus punctatus

The posterior half of the channel catfish intestine has a similar histological organization to that of other teleost fishes. This region is organized into a muscosa, a submucosa, a double layered muscularis and a serosa. A “stratum compactum” of dense connective tissue was confirmed for the submucosa. In its histology and cytology, the midgut resembles the hindgut, except that in the hindgut the muscularis is thicker, the microvilli are shorter, there are fewer absorptive inclusions in the columnar cells and there are more goblet cells. With the exception of the serosa, the tissue layers of the intestine of the 6 cm juvenile catfish are fully developed. The most notable difference between the intestines of the juvenile and adult catfish occurs in the columnar epithelial cells. The mucosal cells of the juvenile catfish contain an abundance of large clear vacuoles while the mucosal cells of the mature catfish contain smaller dense granules. With few exceptions, the ultrastructural details of the cells in the catfish intestine are identical to those of the same cell types of the mammalian intestine.     

The stomatogastric nervous system of the house cricket Acheta domesticus L. II. Iontophoretic study of neuron anatomy

The anatomy of neurons of the stomatogastric nervous system of Ascheta domesticus was studied using heavy metal iontophoresis through cut nerve ends followed by silver intensification. Nineteen categories of neuron are described and compared with neurons known from the stomatogastric nervous system of other insects. Possible functions for the neurons are suggested. Motor neuron candidates are suggested for all parts of the gut served by the stomatogastric nervous system, and axons of sensory neurons of the anterior pharynx are located. There are four neuron types that cannot readily be assigned motor, sensory, or interneuron functions: large dorsal cells of the frontal ganglion; the two neurons of the nervus connectivus, and two categories of neurons in the median neurosecretory cell group of the pars intercerebralis, the axons of which are contained in the stomatogastric nerves.     

Cell renewal in adjoining intestinal and tracheal epithelia of Manduca

Cell renewal continuously replaces dead or dying cells in organs such as human and insect intestinal (midgut) epithelia; in insects, control of self-renewal determines insects’ responses to any of the myriad pathogens and parasites of medical and agricultural importance that enter and cross their midgut epithelia. Regenerative cells occur in the midgut epithelia of many, if not all, insects and are probably derived from a distinctive population of stem cells. The control of proliferation and differentiation of these midgut regenerative cells is assumed to be regulated by an environment of adjacent cells that is referred to as a regenerative cell niche. An antibody to fasciclin II marks cell surfaces of tracheal regenerative cells associated with rapidly growing midgut epithelia. Tracheal regenerative cells and their neighboring midgut regenerative cells proliferate and differentiate in concert during the coordinated growth of the midgut and its associated muscles, nerves and tracheal cells.     

Degeneration and cell regeneration in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae) during post-embryonic development

Cell death, proliferation, and differentiation in some developmental stages of insects have been studied in the midgut of ametabolous, which undergo only continuous growth, and holometabolous, which undergo complete metamorphosis. However, in hemimetabolous insects, evolutionarily intermediate between ametabolous and holometabolous, midgut reorganization during the post-embryonic development has been poorly studied. The present study evaluates the post-embryonic development of the midgut of a hemimetabolous insect, Podisus nigrispinus, to test the hypothesis that these insects have programmed cell death and proliferation followed by differentiation of regenerative cells during midgut growth from nymphs to adult. The morphometrical data showed a 6-fold increase in midgut length from the first instar nymph to the adult, which did not result from an increase in the size of the midgut cells, suggesting that the growth of the midgut occurs by an increase in cell number. Cell death was rarely found in the midgut, whereas proliferation of regenerative cells occurred quite frequently. The growth of the midgut of P. nigrispinus appears to result from the proliferation of regenerative cells present in the epithelium; unlike ametabolous and holometabolous insects, the midgut of P. nigrispinus does not undergo extensive remodeling, as shown by the low frequency of digestive cell death.