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.     

Regenerative cells and the architecture of beetle midgut epithelia

The architectural ground plan of beetle and other insect midguts is represented by a monolayer of epithelial cells arranged in a cylindrical configuration. Proliferation and differentiation of regenerative cells maintain the integrity of this monolayer in the face of continual losses of individual cells through cytoplasmic budding and/or expulsion of entire epithelial cells. Peritrophic membranes have conventionally been considered universal features of insect midguts that function to protect vulnerable microvillar surfaces of the midgut epithelium from abrasion by ingested food; however, peritrophic membranes were found in only a small fraction of the adult beetle species examined in this study. In adult beetles, midgut epithelial cells are continually replaced by cells recruited from populations of mitotic regenerative cells that are interspersed among the differentiated epithelial monolayer. To remain contiguous with the other cells in the midgut monolayer, some of these proliferating populations have adopted evaginated configurations of cells that extend for varying distances from the basal surface of the monolayer. These configurations are referred to as regenerative crypts or pouches and consist of progenitor cells and stem cells. The presence, the relative densities, and the relative lengths of these regenerative pouches vary considerably among families of beetles. Placement of longitudinal muscles of the midgut relative to the proximodistal axes of these regenerative pouches also varies among species of beetles. The presence, the size, and the density of regenerative cell populations are related to 1) feeding habits of adult beetles, 2) presence of peritrophic membranes, and 3) expulsion of entire midgut epithelial cells or fragments of these epithelial cells into midgut lumens. © 2012 Wiley Periodicals, Inc.     

Differentiation of regenerative cells in the midgut epithelium of Epilachna cf nylanderi (Mulsant 1850) (Insecta, Coleoptera, Coccinellidae)

Differentiation of regenerative cells in the midgut epithelium of Epilachna cf nylanderi (Mulsant 1850) (Insecta, Coleoptera, Coccinellidae), a consumer of the Ni-hyperaccumulator Berkheya coddii (Asteracae) from South Africa, has been monitored and described. Adult specimens in various developmental phases were studied with the use of light microscopy and transmission electron microscopy. All degenerated epithelial cells are replaced by newly differentiated cells. They originate from regenerative cells which act as stem cells in the midgut epithelium. Just after pupal-adult transformation, the midgut epithelium of E. nylanderi is composed of columnar epithelial cells and isolated regenerative cells distributed among them. The regenerative cells proliferate intensively and form regenerative cell groups. In each regenerative cell group the majority of cells differentiate into new epithelial cells, while some of them still act as stem cells and persist as a reservoir of cells capable for proliferation and differentiation. Because this species is an obligate monophage of plants which accumulate nickel, proliferation and differentiation of midgut stem cells follow degeneration intensively and in a typical manner.     

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.     

The role of actin cables in directing the morphogenesis of the pharyngeal pouches

The pharyngeal arches are separated by endodermal outpocketings, the pharyngeal pouches. These are structures of considerable importance; they are required to segregate the mesenchymal populations of each arch and to induce the formation of arch components, and they generate specific derivatives, including the parathyroid and the thymus. The pharyngeal pouches are first evident as localised sites at which the endoderm contacts the ectoderm, and they then expand along the proximodistal axis to generate the narrow, tight morphology of the mature pouch. We currently have no knowledge of the morphogenetic mechanisms that direct formation of the pharyngeal pouches. Here, in chick, we show that cells within the pharyngeal pouch endoderm have an abundance of apically located actin fibres that are networked within the endodermal sheet, via their insertion into N-cadherin adherens junctions, to form a web of supra-cellular actin cables. Cytochalasin D disruption of these actin structures results in the formation of aberrant pouches that fail to generate their normal slit-like morphology. This suggests that the process of pharyngeal pouch morphogenesis involves the constraining influence of these actin cables that direct expansion, within the pouch, along the proximodistal axis. These results, importantly, provide us with vital insights into how the pharyngeal pouches form their normal morphology. They also give evidence, for the first time, of actin cables functioning as constraints during complex vertebrate morphogenetic episodes.     

Fine structure of the midgut epithelium in two Archaeognatha, Lepismachilis notata and Machilis hrabei (Insecta), in relation to its degeneration and regeneration

In two archaeognathans, Lepismachilis notata and Machilis hrabei, the midgut epithelium and processes of its regeneration and degeneration have been described at the ultrastructural level. In both analysed species, the midgut epithelium is composed of epithelial and regenerative cells (regenerative nests). The epithelial cells show distinct regionalization in organelles distribution with the basal, perinuclear, and apical regions being distinguished. Degeneration of epithelial cells proceeds in a necrotic way (continuous degeneration) during the entire life of adult specimens, but just before each moult degeneration intensifies. Apoptosis has been observed. Regenerative cells fulfil the role of midgut stem cells. Some of them proliferate, while the others differentiate into epithelial cells. We compared the organisation of the midgut epithelium of M. hrabei and L. notata with zygentoman species, which have just been described.     

An amuse-bouche of stem cell regulation: Underlying principles and mechanisms from adult Drosophila intestinal stem cells

Stem cells have essential functions in the development and maintenance of our organs. Improper regulation of adult stem cells and tissue homeostasis can result in cancers and age-dependent decline. Therefore, understanding how tissue-specific stem cells can accurately renew tissues is an important aim of regenerative medicine. The Drosophila midgut harbors multipotent adult stem cells that are essential to renew the gut in homeostatic conditions and upon stress-induced regeneration. It is now a widely used model system to decipher regulatory mechanisms of stem cell biology. Here, we review recent findings on how adult intestinal stem cells differentiate, interact with their environment, and change during aging.     

Fine structure of the midgut epithelium of Nicoletia phytophila Gervais, 1844 (Zygentoma: Nicoletiidae: Nicoletiinae) with special emphasis on its degeneration

The midgut epithelium of Nicoletia phytophila is composed of columnar digestive cells and regenerative cells that form regenerative nests. The cytoplasm of midgut epithelial cells shows typical regionalization in organelle distribution. Two types of regenerative cells have been distinguished: cells which are able to divide intensively and cells which differentiate. Spot desmosomes have been observed between neighboring regenerative cells. The occurrence of intercellular junctions is discussed. The midgut epithelium degenerates both in an apoptotic and necrotic way. Necrosis proceeds during each molting period (cyclic manner), while apoptosis occurs between each molting, when the midgut epithelium is responsible for e.g. digestion. These processes of epithelium degeneration are described at the ultrastructural level. Our studies not only add new information about fine structure of the midgut epithelium of N. phytophila, but contribute to resolving the relationships within the Zygentoma. There are no doubts about the very close sister position of Nicoletiidae and Ateluridae. The midgut epithelium characters confirm their close relationship. However we do not recommend classifying the atelurid genera only within Nicoletiidae: Nicoletiinae.     

Ultrastructural studies of midgut epithelium formation in Lepisma saccharina L. (Insecta, Zygentoma)

At the end of embryogenesis of Lepisma saccharina L. (Insecta, Zygentoma), when the stomodaeum and proctodaeum are completely formed, the midgut epithelium is replaced by the primary midgut, a yolk mass is surrounded by a cell membrane. Midgut epithelium formation begins in the 1st larval stage. Energids migrate toward the yolk periphery and aggregate just beneath the cell membrane. They are gradually enclosed by cell membrane folds of the primary midgut. Single cells are formed. Succeeding energids join just formed cells. Thus, groups of cells, regenerative cell groups, are formed. Their number gradually increases. The external cells of the regenerative cell groups transform into epithelial cells and their basal regions spread toward the next regenerative cell groups. Epithelial cells of neighboring regenerative cell groups join each other to form the epithelium. At the end of the 2nd larval stage, just before molting, degeneration of newly the formed epithelium begins. Remains of organelles and basal membrane occur between the regenerative cell groups. The new epithelium is formed from the regenerative cell groups, which are now termed stem cells of the midgut epithelium.     

An essential role for Fgfs in endodermal pouch formation influences later craniofacial skeletal patterning

Fibroblast growth factor (Fgf) proteins are important regulators of pharyngeal arch development. Analyses of Fgf8 function in chick and mouse and Fgf3 function in zebrafish have demonstrated a role for Fgfs in the differentiation and survival of postmigratory neural crest cells (NCC) that give rise to the pharyngeal skeleton. Here we describe, in zebrafish, an earlier, essential function for Fgf8 and Fgf3 in regulating the segmentation of the pharyngeal endoderm into pouches. Using time-lapse microscopy, we show that pharyngeal pouches form by the directed lateral migration of discrete clusters of endodermal cells. In animals doubly reduced for Fgf8 and Fgf3, the migration of pharyngeal endodermal cells is disorganized and pouches fail to form. Transplantation and pharmacological experiments show that Fgf8 and Fgf3 are required in the neural keel and cranial mesoderm during early somite stages to promote first pouch formation. In addition, we show that animals doubly reduced for Fgf8 and Fgf3 have severe reductions in hyoid cartilages and the more posterior branchial cartilages. By examining early pouch and later cartilage phenotypes in individual animals hypomorphic for Fgf function, we find that alterations in pouch structure correlate with later cartilage defects. We present a model in which Fgf signaling in the mesoderm and segmented hindbrain organizes the segmentation of the pharyngeal endoderm into pouches. Moreover, we argue that the Fgf-dependent morphogenesis of the pharyngeal endoderm into pouches is critical for the later patterning of pharyngeal cartilages.     

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.     

Aedes aegypti midgut remodeling during metamorphosis

The Aedes aegypti midgut is restructured during metamorphosis; its epithelium is renewed by replacing the digestive and endocrine cells through stem or regenerative cell differentiation. Shortly after pupation (white pupae) begins, the larval digestive cells are histolized and show signs of degeneration, such as autophagic vacuoles and disintegrating microvilli. Simultaneously, differentiating cells derived from larval stem cells form an electron-dense layer that is visible 24 h after pupation begins. Forty-eight hours after pupation onset, the differentiating cells yield an electron-lucent cytoplasm rich in microvilli and organelles. Dividing stem cells were observed in the fourth instar larvae and during the first 24 h of pupation, which suggests that stem cells proliferate at the end of the larval period and during pupation. This study discusses various aspects of the changes during midgut remodeling for pupating A. aegypti.     

The fine structure of the midgut epithelium in a centipede,Scolopendra cingulata (Chilopoda,Scolopendridae), with the special emphasis on epithelial regeneration

Scolopendra cingulata has a tube-shaped digestive system that is divided into three distinct regions: fore-, mid- and hindgut. The midgut is lined with a pseudostratified columnar epithelium which is composed of digestive, secretory and regenerative cells. Hemocytes also appear between the digestive cells of the midgut epithelium. The ultrastructure of three types of epithelial cells and hemocytes of the midgut has been described with the special emphasis on the role of regenerative cells in the protection of midgut epithelium. The process of midgut epithelium regeneration proceeds due to the ability of regenerative cells to proliferate and differentiate according to a circadian rhythm. The regenerative cells serve as unipotent stem cells that divide in an asymmetric manner.Additionally, two types of hemocytes have been distinguished among midgut epithelial cells. They enter the midgut epithelium from the body cavity. Because of the fact that numerous microorganisms occur in the cytoplasm of midgut epithelial cells, we discuss the role of hemocytes in elimination of pathogens from the midgut epithelium. The studies were conducted with the use of transmission electron microscope and immunofluorescent methods.     

The differential use of cheek pouches in a troop ofPapio ursinus

Cheek pouches, one of the distinguishing characters of the Cercopithecinae, are structures used for the temporary storage of food. Their size and frequency of use within a given species are related primarily to the amount of conspecific competition for food. In relation to total body size, members of the genusPapio are considered to have relatively small cheek pouches which are said to be used only occasionally to maximize food harvest when local clusters of food are encountered. This investigation represents 165 hours of observation on a troop ofPapio ursinus at Mkuzi Game Reserve in Natal, South Africa. At the time of observation the choice of foods in the home range was restricted and usually found in abundance only in small clusters of trees. Given that conspecific competition would have been pronounced under these conditions, it was thought that any differences in the frequency of cheek pouch use related to age, sex, rank, or the reproductive state of an animal would be readily recognizable. The results indicated that cheek pouches were used differentially throughout the day, with virtually all ages of each sex displaying the same general pattern of maximal cheek pouch use during the middle of the day. In each sex there was an age graded diminution of the frequency of cheek pouch use from juveniles to adults. This trend was more pronounced in males resulting in a substantial sex difference in the use of cheek pouches between adult males and females. In addition, there was some indication that differences in the frequency of cheek pouch use between adult males were correlated with rank. Although a pattern of cheek pouch use and rank was not evident amongst adult females, there was an association between reproductive state and the frequency of cheek pouch use. Overall, body size, dominance, and energetic demands appeared to be the most significant factors underlying the differences in cheek pouch use in this troop.     

Monitoring stem cell proliferation and differentiation in primary midgut cell cultures from Heliothis virescens larvae using flow cytometry

In the midgut of Heliothis virescens larvae, proliferation and differentiation of stem cell populations allow for midgut growth and regeneration. Basic epithelial regenerative function can be assessed in vitro by purifying these two cell type populations, yet efficient high throughput methods to monitor midgut stem cell proliferation and differentiation are not available. We describe a flow cytometry method to differentiate stem from mature midgut cells and use it to monitor proliferation, differentiation and death in primary midgut stem cell cultures from H. virescens larvae. Our method is based on differential light scattering and vital stain fluorescence properties to distinguish between stem and mature midgut cells. Using this method, we monitored proliferation and differentiation of H. virescens midgut cells cultured in the presence of fetal bovine serum (FBS) or AlbuMAX II. Supplementation with FBS resulted in increased stem cell differentiation after 5 days of culture, while AlbuMAX II-supplemented medium promoted stem cell proliferation. These data demonstrate utility of our flow cytometry method for studying stem cell-based epithelial regeneration, and indicate that AlbuMAX II-supplemented medium may be used to maintain pluripotency in primary midgut stem cell cultures.     

Cdx2 determines the fate of postnatal intestinal endoderm

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.     

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.