Raziel S. Hakim

Regulation of Midgut Growth, Development, and Metamorphosis

The insect midgut is an important site of entry for pathogens and insect control agents. This review focuses on recent information related to midgut epithelial growth, metamorphosis, and repair as a defense against pathogens. The roles of stem cell mitogens and differentiation factors are described. Included is a discussion of apoptosis and autophagy in the yellow body. Sloughing, also described, protects the midgut from virus infections and bacterial toxins through death and replacement of affected cells. The mechanisms by which the repair process reduces the effectiveness of pest control strategies are discussed. Primary tissue culture methods also are described, and their value in understanding the mechanisms by which biologically based insecticides work is discussed.

Regeneration of cultured midgut cells after exposure to sublethal doses of toxin from two strains of Bacillus thuringiensis.

Toxin from two strains of Bacillus thuringiensis (Bt), AA 1-9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae. HD-73 toxin was more effective although, at the doses used, not all cells were killed. After 2 days of exposure to 0.8 pg/µl AA 1-9 or 0.06 pg/µl HD-73, columnar and goblet cell numbers declined to ca 20% of controls. In contrast, stem and differentiating cells increased to 140-200% of controls. The dynamic of depletion and replacement depended on toxin type and concentration. Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid population corrections in vitro. Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells was induced by the cultured midgut cells themselves. Controls and cells treated with toxin from Bt strain AA 1-9 were stained using a polyclonal antibody to Lepidopteran midgut differentiation factor 1 (MDF1). With Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis of this differentiation factor during increased stem cell differentiation. The response of cultured midgut cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.

Insect midgut epithelium in vitro: an insect stem cell system

Abstract Mixed cell cultures and stem cell cultures were prepared from midguts of Manduca sexta pharate fourth instar and mid-wandering fifth instar larvae. An extract prepared from the fat body was able to promote stem cell proliferation and affect differentiation in a dose-dependent manner. DNA synthesis activity was confirmed by use of [3H]thymidine. Immunohistological localization of bromodeoxyuridine (BrdU), a thymidine analog, indicated that dividing stem cells incorporated the label. In many cases, one of the daughter cells incorporated the label while the other did not; often this daughter appeared morphologically different from its sister cell. These results implied that one of the sister stem cells remained as a proliferating stem cell while the other sister was committed to differentiate. Studies strongly suggest that these midgut cell cultures comprise a true stem cell system. Cell-free conditioned medium from cultures of differentiating pharate fourth instar midgut cells induced development of larval columnar cells from mid-wandering fifth instar midgut stem cells. Conversely, conditioned medium from differentiating cultures of mid-wandering fifth instar midgut induced development of mid-wandering fifth instar low columnar cells from midgut stem cells isolated from pharate fourth instar larvae. Therefore, it appears that differentiating cells produce soluble cytokines which direct specific modes of differentiation by M. sexta stem cells.

The role of stem cells in midgut growth and regeneration

SummaryTheManduca 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.

Proliferation and differentiation of midgut epithelial cells from Manduca sexta, in vitro

Summary We have developed an insect midgut primary cell culture from pharate fourth instar larvae of Manduca sexta. An enriched Graces medium supplemented with pupal fat body from Lymantria dispar and 20-hydroxyecdysone (20HE) supported stem cell proliferation and differentiation and maintained larval columnar and goblet cell morphology. Cell kinetics indicate that stem cells differentiate to columnar and goblet cells in culture.

Two polypeptide factors that promote differentiation of insect midgut stem cells in vitro.

Isolated stem cells from the midguts of Manduca sexta and Heliothis virescens can be induced to differentiate in vitro by either of two polypeptide factors. One of the peptides was isolated from culture medium conditioned by differentiating mixed midgut cells; we used high performance liquid chromatographic separation and Edman degradation of the most prominent active peak. It is a polypeptide with 30 amino acid residues (3,244 Da), with the sequence HVGKTPIVGQPSIPGGPVRLCPGRIRYFKI, and is identical to the C-terminal peptide of bovine fetuin. A portion of this molecule (HVGKTPIVGQPSIPGGPVRLCPGRIR) was synthesized and was found to be very active in inducing differentiation of H. virescens midgut stem cells. It was designated Midgut Differentiation Factor 1 (MDF1). Proteolysis of bovine fetuin with chymotrypsin allowed isolation of a pentamer, Midgut Differentiation Factor 2 (MDF2) with the sequence HRAHY corresponding to a portion of the fetuin molecule near MDF1. Synthetic MDF2 was also biologically active in midgut stem cell bioassays. Dose response curves indicate activity in physiological ranges from 10(-14) to 10(-9) M for MDF1 and 10(-15) to 10(-5) M for MDF2.

Apoptosis in cultured midgut cells from Heliothis virescens larvae exposed to various conditions.

We exposed midgut cells from primary cultures of Heliothis virescens larvae to cell-free previously used medium, the Vaughn X and HyQ SFtrade mark media used for serum-free culture of insect cell lines which do not support H. virescens midgut cells, and to toxin from Bacillus thuringiensis. A statistically significant increase in the percent of dying cells was counted in cell populations in Vaughn X medium. Use of the TUNEL method to detect apoptosis indicated a low rate (7.2%) of apoptosis in control cultures grown in Heliothis medium, an increase to approximately 20% in previously used and HyQ SFtrade mark media, and to approximately 45% of cells remaining after exposure to and initial destruction by B. thuringiensis toxin. Apoptotic nuclei were predominant (approximately 6%) in mature columnar cells in control cultures. Approximately 1% of goblet, stem, and differentiating cells were apoptotic. However, apoptosis rose to 12% in stem and differentiating cells exposed to used and unsuitable medium. B. thuringiensis exposure to toxin for 2-3 days resulted in visible membrane damage and necrosis, causing the death of 84% of the cells as measured by both the TUNEL and Annexin methods. Some of the columnar cells and stem and differentiating cells that remained also contained apoptotic nuclei. Stem and differentiating cells normally replace dying mature cells in the midgut. Thus, exposure of cultures of H. virescens midgut cells to adverse environments such as unsuitable or poisonous media appeared to induce down-regulation of the cell populations by apoptosis.

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.

Cultured midgut cells of Heliothis virescens (Lepidoptera): fibronectin and integrin β1, immunoreactivity during differentiation in vitro

Summary The midgut epithelium of larval Lepidoptera consists of a monolayer of mature columnar and goblet cells in vivo with loosely bound stem cells at its base. Cultures of Heliothis virescens midgut contained quiescent and dividing stem cells, differentiating and mature cells as well as small rows of attached cells, randomly distributed in the culture vessel. Fixed cultures were immunostained with polyclonal antibodies to fibronectin and integrin β1. Many stem cells stained darkly; lighter staining stem cells imply that some stem cells can become less adhesive. Developing cells were pale, lacking integrin and fibronectin epitopes on their surfaces, and were probably poorly adhesive. During a molt in vivo, differentiating stem cells insert between existing epithelial cells, increasing gut size; lack of stickiness would enable them to do so. As pre-columnar cells differentiated in vitro, stainability reappeared. Mature columnar cells were decorated with a pattern of intense surface immunostaining materia...

Change of form of septate and gap junctions during development of the insect midgut

In insects, smooth septate junctions join cells derived from the embryonic midgut, and pleated septate junctions are found in all other tissues. Relatively little is known about either type of septate junction or the relationship between them, but they have been treated as two different junctions in the literature. The gap junctions which are associated with these septate junctions also differ. Crystalline gap junctions are found in the midgut, associated with smooth septate junctions, and irregular gap junctions are found in tissues where pleated septate junctions are located. We have examined the development of smooth septate junctions and crystalline gap junctions and the relationship between them, by studying the embryogenesis of the midgut in Manduca sexta (tobacco hornworm). At 56 hr of development (hatching is at 104 hr) pleated septate junctions and irregular gap junctions joined the midgut epithelial cells. At 65 hr, the septate junctions had disappeared, but gap junctions persisted. At 70 hr, smooth septate junctions had replaced the earlier pleated septate junctions and gap junctions associated with these smooth septate junctions were often of the crystalline form. In later embryos, the smooth septate junctions matured and enlarged, while all gap junctions became crystalline in form.