Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

Midgut tissue undergoes remodeling during metamorphosis in insects belonging to orders Lepidoptera and Diptera. We investigated the developmental and hormonal regulation of these remodeling events in lepidopteran insect, Heliothis virescens. In H. virescens, programmed cell death (PCD) of larval midgut cells as well as proliferation and differentiation of imaginal cells began at 108 h after ecdysis to the final larval instar (AEFL) and proceeded through the pupal stages. Expression patterns of pro- cell death factors (caspase-1 and ICE) and anti-cell death factor, Inhibitor of Apoptosis (IAP) were studied in midguts during last larval and pupal stages. IAP, Caspase-1 and ICE mRNAs showed peaks at 48 h AEFL, 96 h AEFL and in newly formed pupae, respectively. Immunohistochemical analysis substantiated high caspase-3 activity in midgut at 108 h AEFL. Application of methoprene, a juvenile hormone analog (JHA) blocked PCD by maintaining high levels of IAP, downregulating the expression of caspase-1, ICE and inhibiting an increase in caspase-3 protein levels in midgut tissue. Also, the differentiation of imaginal cells was impaired by methoprene treatment. These studies demonstrate that presence of JHA during final instar larvae affects both midgut remodeling and larval-pupal metamorphosis leading to larval/pupal deformities in lepidopteran insects, a mechanism that is different from that in mosquito, Ae. aegypti where JHA uncouples midgut remodeling from metamorphosis.     

Stem cells from midguts of Lepidopteran larvae: clues to the regulation of stem cell fate

Previously, we showed that isolated stem cells from midguts of Heliothis virescens can be induced to multiply in response to a multiplication protein (MP) isolated from pupal fat body, or to differentiate to larval types of mature midgut cells in response to either of 4 differentiation factors (MDFs) isolated from larval midgut cell-conditioned medium or pupal hemolymph. In this work, we show that the responses to MDF-2 and MP in H. virescens stem cells decayed at different time intervals, implying that the receptors or response cascades for stem cell differentiation and multiplication may be different. However, the processes appeared to be linked, since conditioned medium and MDF-2 prevented the action of MP on stem cells; MP by itself appeared to repress stem cell differentiation. Epidermal growth factor, retinoic acid, and platelet-derived growth factor induced isolated midgut stem cells of H. virescens and Lymantria dispar to multiply and to differentiate to mature midgut cells characteristic of prepupal, pupal, and adult lepidopteran midgut epithelium, and to squamous-like cells and scales not characteristic of midgut tissue instead of the larval types of mature midgut epithelium induced by the MDFs. Midgut stem cells appear to be multipotent and their various differentiated fates can be influenced by several growth factors.     

Differential effects of a<Emphasis Type="Italic"> labial</Emphasis> mutation on the development,structure, and function of stomach acid-secreting cells in<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> larvae and adults

The differentiation of copper cells, which secrete stomach acid in Drosophila larvae, has been shown previously to be sensitive to the labialk3 mutation. Here we found that stomach acid secretion in adults was insensitive to labk3. The basis for this stage-specific effect was elucidated by characterizing the development, structure, and function of the adult midgut. First, we demonstrated by copper-dependent fluorescence and morphology that copper cells were present in the adult stomach. Fine-structure analysis of adult copper cells led to the identification of a previously unrecognized plasma membrane domain: apicolateral contacts between copper cells and their neighbors consisted of smooth septate junctions that were enriched in alphabeta-spectrin and ankyrin. Second, we demonstrated that adult copper cells were present in labk3/labvd1 (conditional/null) adults. The labial protein was expressed in adult labk3/labvd1 copper cells, but not in larvae. Thus the labk3 mutation had a stage-specific effect on midgut labial expression, but did not appear to affect protein function. Surprisingly, stomach acidification was dispensable during larval development, since labk3/labvd1 mutant larvae that lacked midgut acidification developed into fertile adults.     

Lysozyme in the midgut of Manduca sexta during metamorphosis.

Low levels of lysozyme were found in the midgut epithelium of the tobacco hornworm, Manduca sexta, during the early part of the fifth larval stadium. This was observed in control insects as well as in bacterially challenged insects. No lysozyme was detected in the gut contents of either group of insects which were actively eating or in the early stages of metamorphosis. However, high levels of lysozyme activity were detected in homogenates of midgut tissue collected from insects later in the stadium. Immunocytochemical studies demonstrated that lysozyme accumulates in large apical vacuoles in regenerative cells of the midgut during the larval-pupal molt. These cells, initially scattered basally throughout the larval midgut epithelium, multiply and form a continuous cell layer underneath the larval midgut cells. At the larval/pupal ecdysis the larval midgut epithelium is sloughed off and the regenerative cells, now forming the single cell layer of the midgut, release the contents of their vacuoles into the midgut lumen. This release results in high lysozyme activity in the lumen of the pupal midgut and is thought to confer protection from bacterial infection. This is the first indication that the lysozyme gene may be developmentally regulated in a specific tissue in the absence of a bacterial infection.     

Midgut epithelium formation in Thermobia domestica (Packard) (Insecta, Zygentoma)

The origin of midgut epithelium may begin either from yolk cells (energids), tips of stomo- and proctodaeum (ectoderm), inner layer (endoderm) or from both kinds of the above mentioned cells. The origin of the midgut epithelium in wingless insects (Apterygota) has still not been determined. In Thermobia domestica the formation of midgut is much delayed, and it completes in the post-embryonic stage, while the stomo- and the proctodaeum are well-developed in the embryonic period. The energids, which remain inside the yolk, start to migrate to its periphery, where they arrange singly close to cell membrane. The yolk mass with the energids at the 14th day of embryogenesis are referred to as the primary midgut. During the first instar larval stage more and more energids migrate to the yolk periphery and the cell membrane starts to form numerous foldings surrounding the groups of energids, which in turn lead to formation of isolated regenerative cell groups. Eventually the cell membrane invaginations reach the center of the yolk mass. Large cells of the primary epithelium, surrounding the newly formed midgut lumen are formed. The cells of the primary epithelium are filled with yolk and are equipped with microvilli pointing to the midgut lumen. As the yolk is being digested, the process of the primary epithelium cells degeneration begins. The cells are getting shorter and start to degenerate. The definitive midgut epithelium is formed from proliferating regenerative cells. It consists of regularly spaced regenerative cell groups as well as the epithelial cells. The ultrastructure of both these cell groups has been described.     

The role of stem cells in midgut growth and regeneration

Summary TheManduca sexta (L.) [Lepidoptera: Sphingidae] andHeliothis virescens (F.) [Lepidoptera: Noctuidae] midguts consist of a pseudostratified epithelium surrounded by striated muscle and tracheae. This epithelium contains goblet, columnar, and basal stem cells. The stem cells are critically important in that they are capable of massive proliferation and differentiation. This growth results in a fourfold enlargement of the midgut at each larval molt. The stem cells are also responsible for limited cell replacement during repair. While the characteristics of the stem cell population vary over the course of an instar, stem cells collected early in an instar and those collected late can start in vitro cultures. Cultures of larval stem, goblet, and columnar cells survive in vitro for several mo through proliferation and differentiation of the stem cells. One of the two polypeptide differentiation factors which have been identified and characterized from the culture medium has now been shown to be present in midgut in vivo. Thus the ability to examine lepidopteran midgut stem cell growth in vitro and in vivo is proving to be effective in determining the basic features of stem cell action and regulation. Mention of any product in this publication does not imply endorsement by the USDA.     

Distribution of the wingless gene product in Drosophila embryos: a protein involved in cell-cell communication

wingless, a segment polarity gene required in every segment for the normal development of the Drosophila embryo, encodes a cysteine-rich protein with a signal peptide. A polyclonal antiserum localizes the wingless protein in approximately the same region of the embryo as the wingless mRNA. The pattern of antigen localization changes rapidly during development. In the extended germband stage, stripes of wingless staining are present in the trunk region just anterior to the parasegment boundary; wingless-expressing cells abut engrailed-expressing cells across that boundary. wingless antigen is seen both inside and outside the cell by electron microscopy: inside the cell, in small membrane-bound vesicles and in multivesicular bodies; outside the cell, close to or on the plasma membrane and associated with material in the intercellular space. The multivesicular bodies containing the wingless protein are occasionally found in engrailed-positive cells, suggesting that the wingless protein behaves as a paracrine signal.     

A novel tissue in an established model system: the <Emphasis Type="Italic">Drosophila</Emphasis> pupal midgut

The Drosophila larval and adult midguts are derived from two populations of endodermal progenitors that separate from each other in the early embryo. As larval midgut cells differentiate into an epithelial layer, adult midgut progenitors (AMPs) remain as small clusters of proliferating, undifferentiated cells attached to the basal surface of the larval gut epithelium. During the first few hours of metamorphosis, AMPs merge into a continuous epithelial tube that overgrows the larval layer and differentiates into the adult midgut; at the same time, the larval midgut degenerates. As shown in this paper, there is a second, transient pupal midgut that develops from the AMPs at the beginning of metamorphosis and that intercalates between the adult and larval midgut epithelia. Cells of the transient pupal midgut form a multilayered tube that exhibits signs of differentiation, in the form of septate junctions and rudimentary apical microvilli. Some cells of the pupal midgut develop as endocrine cells. The pupal midgut remains closely attached to the degenerating larval midgut cells. Along with these cells, pupal midgut cells are sequestered into the lumen where they form the compact “yellow body.” The formation of a pupal midgut has been reported from several other species and may represent a general feature of intestinal metamorphosis in insects.     

Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta

While the larval midgut of Manduca sexta has been intensively studied as a model for ion transport, the developmental origins of this organ are poorly understood. In our study we have used light and electron microscopy to investigate the process of midgut epithelial cell differentiation in the embryo. Our studies were confined to the period between 56 and 95 hr of embryonic development (hatching is at 101 hr at 25 degrees C), since preliminary studies indicated that all morphologically visible differentiation of the midgut epithelium occurs during this time. At 56 hr the midgut epithelium is organized into a ragged pseudostratified epithelium. Over the next 10 hr, the embryo molts and the midgut epithelium takes on a distinctive character in which the future goblet and columnar cells can be identified. With further differentiation, closed vesicles in the goblet cells expand and subsequently communicate to the outside by way of a valve. The columnar cells form numerous microvilli on their apical surfaces that extend over the goblet cells. Both cell types form basal folds from a series of plasmalemmal invaginations. Differentiation occurs concurrent with a six-fold elongation of these cells.     

The BEAF insulator regulates genes involved in cell polarity and neoplastic growth

Boundary Element Associated Factor-32 (BEAF-32) is an insulator protein predominantly found near gene promoters and thought to play a role in gene expression. We find that mutations in BEAF-32 are lethal, show loss of epithelial morphology in imaginal discs and cause neoplastic growth defects. To investigate the molecular mechanisms underlying this phenotype, we carried out a genome-wide analysis of BEAF-32 localization in wing imaginal disc cells. Mutation of BEAF-32 results in miss-regulation of 3850 genes by at least 1.5-fold, 794 of which are bound by this protein in wing imaginal cells. Up-regulated genes encode proteins involved in cell polarity, cell proliferation and cell differentiation. Among the down-regulated genes are those encoding components of the wingless pathway, which is required for cell differentiation. Miss-regulation of these genes explains the unregulated cell growth and neoplastic phenotypes observed in imaginal tissues of BEAF-32 mutants.     

Secretion and movement of wingless protein in the epidermis of the Drosophila embryo.

The segment polarity gene wingless encodes a cysteine rich protein which is essential for pattern formation in Drosophila. Using polyclonal antibodies against the product of the wingless gene, we demonstrate that this protein is secreted in the embryo and that it is taken up by neighbouring cells. The protein can be found two or three cell diameters away from the cells in which it is synthesized. We discuss the possible mechanisms which are responsible for this spatial distribution and its regulation during embryogenesis.     

Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action

In holometabolous insects such as mosquito, Aedes aegypti, midgut undergoes remodeling during metamorphosis. Insect metamorphosis is regulated by several hormones including juvenile hormone (JH) and 20-hydroxyecdysone (20E). The cellular and molecular events that occur during midgut remodeling were investigated by studying nuclear stained whole mounts and cross-sections of midguts and by monitoring the mRNA levels of genes involved in 20E action in methoprene-treated and untreated Ae. aegypti. We used JH analog, methoprene, to mimic JH action. In Ae. aegypti larvae, the programmed cell death (PCD) of larval midgut cells and the proliferation and differentiation of imaginal cells were initiated at about 36h after ecdysis to the 4th instar larval stage (AEFL) and were completed by 12h after ecdysis to the pupal stage (AEPS). In methoprene-treated larvae, the proliferation and differentiation of imaginal cells was initiated at 36h AEFL, but the PCD was initiated only after ecdysis to the pupal stage. However, the terminal events that occur for completion of PCD during pupal stage were blocked. As a result, the pupae developed from methoprene-treated larvae contained two midgut epithelial layers until they died during the pupal stage. Quantitative PCR analyses showed that methoprene affected midgut remodeling by modulating the expression of ecdysone receptor B, ultraspiracle A, broad complex, E93, ftz-f1, dronc and drice, the genes that are shown to play key roles in 20E action and PCD. Thus, JH analog, methoprene acts on Ae. aegypti by interfering with the expression of genes involved in 20E action resulting in a block in midgut remodeling and death during pupal stage.     

The larval alimentary canal of the Antarctic insect, Belgica antarctica

On the Antarctica continent the wingless midge, Belgica antarctica (Diptera, Chironomidae) occurs further south than any other insect. The digestive tract of the larval stage of Belgica that inhabits this extreme environment and feeds in detritus of penguin rookeries has been described for the first time. Ingested food passes through a foregut lumen and into a stomodeal valve representing an intussusception of the foregut into the midgut. A sharp discontinuity in microvillar length occurs at an interface separating relatively long microvilli of the stomodeal midgut region, the site where peritrophic membrane originates, from the midgut epithelium lying posterior to this stomodeal region. Although shapes of cells along the length of this non-stomodeal midgut epithelium are similar, the lengths of their microvilli increase over two orders of magnitude from anterior midgut to posterior midgut. Infoldings of the basal membranes also account for a greatly expanded interface between midgut cells and the hemocoel. The epithelial cells of the hindgut seem to be specialized for exchange of water with their environment, with the anterior two-thirds of the hindgut showing highly convoluted luminal membranes and the posterior third having a highly convoluted basal surface. The lumen of the middle third of the hindgut has a dense population of resident bacteria. Regenerative cells are scattered throughout the larval midgut epithelium. These presumably represent stem cells for the adult midgut, while a ring of cells, marked by a discontinuity in nuclear size at the midgut-hindgut interface, presumably represents stem cells for the adult hindgut.