Differential localization and processing of apoptotic proteins in Malpighian tubules of Drosophila during metamorphosis

Drosophila development proceeds through three larval stages, before it pupates to reach adulthood. During pupation, larval tissues are destructed by programmed cell death and replaced by adult structures. Programmed cell death is a tightly regulated process accomplished by the induction of three closely linked pro-apoptotic genes reaper, hid and grim, ultimately leading to the activation of caspases, DRONC and DRICE and results in cell death. Unlike other larval tissues, Malpighian tubules are unique in not undergoing characteristic ecdysone-induced apoptosis and are carried to the adults. In this paper we show that apoptotic proteins, HID, GRIM, DRONC and DRICE are expressed in the Malpighian tubules, however they are sequestered in the nucleus. Significantly DRONC and DRICE are not enzymatically processed to active forms in the Malpighian tubules, however, ectopic expression of pro-apoptotic proteins leads to malformed Malpighian tubules and lethality. We also show that the Drosophila inhibitor of apoptotic protein 1, DIAP1, is localized and processed differently in Malpighian tubules. These results provide first evidence in favor of differential activity of apoptotic proteins in Malpighian tubules.     

Regulatory interactions and role in cell type specification of the Malpighian tubules by the cut, Krüppel, and caudal genes of Drosophila.

Krüppel and caudal genes are both required for normal segmentation of the embryo, and the developmental regulatory gene cut is necessary for the normal specification of external sensory organs. These three genes are also expressed in the Malpighian tubules before and during differentiation. Two of the genes, Krüppel and cut, are known to be required for development of the tubules. We report that the absence of maternal and zygotic caudal function reduces their normal growth and elongation. Normal Krüppel function, which is known to be required for caudal expression, is also required for cut expression, while cut and caudal are expressed independently of each other. Cell type transformations of Malpighian tubules were studied by examining the effects of mutations on the expression of markers specific to Malpighian tubules, hindgut, or midgut of normal embryos. Loss of Krüppel activity confers hindgut characteristics on those cells that normally form the Malpighian tubules with all markers tested. Loss of cut function alters the expression of some markers but not others. The pathway of tissue specific gene regulation, apparently, branches beyond Krüppel to form at least a cut and a caudal branch.     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interactions of Krüppel with other terminal genes. The gap genes tailless and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

crossveinless-c is a RhoGAP required for actin reorganisation during morphogenesis

Members of the Rho family of small GTPases are required for many of the morphogenetic processes required to shape the animal body. The activity of this family is regulated in part by a class of proteins known as RhoGTPase Activating Proteins (RhoGAPs) that catalyse the conversion of RhoGTPases to their inactive state. In our search for genes that regulate Drosophila morphogenesis, we have isolated several lethal alleles of crossveinless-c (cv-c). Molecular characterisation reveals that cv-c encodes the RhoGAP protein RhoGAP88C. During embryonic development, cv-c is expressed in tissues undergoing morphogenetic movements; phenotypic analysis of the mutants reveals defects in the morphogenesis of these tissues. Genetic interactions between cv-c and RhoGTPase mutants indicate that Rho1, Rac1 and Rac2 are substrates for Cv-c, and suggest that the substrate specificity might be regulated in a tissue-dependent manner. In the absence of cv-c activity, tubulogenesis in the renal or Malpighian tubules fails and they collapse into a cyst-like sack. Further analysis of the role of cv-c in the Malpighian tubules demonstrates that its activity is required to regulate the reorganisation of the actin cytoskeleton during the process of convergent extension. In addition, overexpression of cv-c in the developing tubules gives rise to actin-associated membrane extensions. Thus, Cv-c function is required in tissues actively undergoing morphogenesis, and we propose that its role is to regulate RhoGTPase activity to promote the coordinated organisation of the actin cytoskeleton, possibly by stabilising plasma membrane/actin cytoskeleton interactions.     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interaction of Krüppel with other terminal genes. The gap genes tailles and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

Tissue specific effects of ommochrome pathway mutations in Drosophila melanogaster

The tissue-specific effects of 17 mutations affecting the synthesis of brown eye pigment (xanthommatin) have been investigated by combining them with chocolate and red cells, two mutations causing ectopic pigmentation of the Malpighian tubules and larval fat body (which normally only synthesize pigment precursors). The majority of mutations block the pigmentation of four organs; the normally pigmented eyes and ocelli, and ectopically pigmented tubules and fat body. They represent genes that would appear to be required for the normal operation of the pathway per se and are likely to encode structural proteins. Mutations at 5 loci affect pigmentation of a subset of organs: cd and po affect only the eyes and ocelli; kar affects the eyes, ocelli and fat body; car causes excretion of pigment from tubules; and z affects pigmentation of the eyes alone. Of these loci, only z has been shown to encode a regulatory protein and the role of the remaining four gene products is not clear. Two mutations affecting the red eye pigments (drosopterins), bw and mal, do not substantially perturb brown pigment synthesis in any of the four organs.     

Genes controlling posterior gut development in theDrosophila embryo

Our present detailed understanding of the genetic mechanisms controlling segmentation has been made possible, in large part, by comprehensive screens of cuticular morphology that identified genes involved in epidermal patterning. To systematically identify genes involved in internal morphogenesis, specifically development of the gut, we have screened mutant embryos produced by a collection of 53 embryonic lethal mutations affecting embryonic pattern formation or differentiation, and a collection of 161 deficiencies covering, in aggregate, approximately 70% of the genome. Staining with the anti-crumbs antibody was used to characterize the Malpighian tubules and hindgut, as well as other internal organs. The geneshuckebein, tailless andwingless, and two previously undescribed loci at 24C/D and 68D/E, are required to establish the primordia for the posterior midgut and hindgut/Malpighian tubules. A locus in region 30A/C is required for extension of the midgut epithelium to surround the yolk, and region 36E/37F is required for outbudding of the Malpighian tubule primordia. Several deficiencies were identified that uncover loci with specific effects on the morphogenesis (elongation, lumen formation) of the hindgut and Malpighian tubules and on the formation of constrictions in the midgut.     

昆虫内向整流钾离子通道的结构与功能

内向整流钾离子通道(inwardly-rectifying potassium channels, Kir)在动物体内承担着重要的生理功能。有关昆虫Kir的研究虽然不多,但近5年来却取得许多重要进展,本文就昆虫Kir近年来的研究进展进行评述。目前对昆虫Kir的研究主要集中在双翅目与半翅目,对基因组的分析以及基因克隆研究表明,昆虫Kir基因数量较少,远低于哺乳动物。双翅目的冈比亚按蚊Anopheles gambiae与埃及伊蚊Aedes aegypti有5~6个Kir基因,黑腹果蝇Drosophila melanogaster仅3个Kir基因,半翅目的褐飞虱Nilaparvata lugens与热带臭虫Cimex lectularius也只有3个Kir基因,而大豆蚜Aphis glycines的Kir基因数则减少到2个,第3个Kir基因的丢失可能与其马氏管的退化有关。系统进化分析表明昆虫Kir具有3个亚家族,但与哺乳动物的7个Kir亚家族没有直系同源关系。尽管如此,昆虫Kir具有与哺乳动物Kir类似的基本结构特征:由4个亚基组成四聚体通道,每个亚基具有2个跨膜区(TM1与TM2),TM1与TM2之间具K^+选择过滤序列。昆虫的Kir基因主要在唾液腺与马氏管中高水平表达,Kir抑制剂可阻断唾液腺与马氏管的分泌活性,从而影响昆虫的取食与排泄活动并使昆虫致死,说明这2类组织器官的分泌活性与Kir有关,Kir介导的K^+跨膜转运驱动了这类组织中上皮细胞的分泌活动。更为重要的发现是氟啶虫酰胺对褐飞虱Kir具有很高的阻断活性,并影响其唾液分泌与排泄功能,证明了Kir就是该杀虫剂的分子靶标。最后本文还对昆虫Kir研究中存在的科学问题进行了分析,展望了开发靶向Kir的新型杀虫剂的研究前景。     

Post-embryonic development of the Malpighian tubules in <Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera) workers: morphology,remodeling, apoptosis,and cell proliferation

The honeybee Apis mellifera has ecological and economic importance; however, it experiences a population decline, perhaps due to exposure to toxic compounds, which are excreted by Malpighian tubules. During metamorphosis of A. mellifera, the Malpighian tubules degenerate and are formed de novo. The objective of this work was to verify the cellular events of the Malpighian tubule renewal in the metamorphosis, which are the gradual steps of cell remodeling, determining different cell types and their roles in the excretory activity in A. mellifera. Immunofluorescence and ultrastructural analyses showed that the cells of the larval Malpighian tubules degenerate by apoptosis and autophagy, and the new Malpighian tubules are formed by cell proliferation. The ultrastructure of the cells in the Malpighian tubules suggest that cellular remodeling only occurs from dark-brown-eyed pupae, indicating the onset of excretion activity in pupal Malpighian tubules. In adult forager workers, two cell types occur in the Malpighian tubules, one with ultrastructural features (abundance of mitochondria, vacuoles, microvilli, and narrow basal labyrinth) for primary urine production and another cell type with dilated basal labyrinth, long microvilli, and absence of spherocrystals, which suggest a role in primary urine re-absorpotion. This study suggests that during the metamorphosis, Malpighian tubules are non-functional until the light-brown-eyed pupae, indicating that A. mellifera may be more vulnerable to toxic compounds at early pupal stages. In addition, cell ultrastructure suggests that the Malpighian tubules may be functional from dark-brown-eyed pupae and acquire greater complexity in the forager worker bee.     

Developmental changes in Malpighian tubule cell structure.

Structural changes which occur in the Malpighian tubule yellow region primary cells during larval-pupal-adult development of the skipper butterfly Calpodes ethlius are described. The developmental changes in cell structure are correlated with functional changes in fluid transport (Ryerse, 1978a) in a way which supports osmotic gradient models of fluid secretion. Larval tubules are specialized for fluid secretion with deep basal infolds and elongate mitochondria-containing apical microvilli which provide channels in which osmotic gradients could be set up. The Malpighian tubule cells are extensively remodelled at pupation when fluid transport is switched off, but they persist intact through metamorphosis. At this time, the basement membrane doubles in thickness, the mitochondria are retracted from the microvilli and are isolated for degradation in autophagic vacuoles, and both apical and basal plasma membranes are internalized via coated vesicles for degradation in multivesicular bodies, which results in the shortening of the microville and the disappearance of the basal infolds. Mitochondria are re-inserted into the microvilli, and the basal infolds re-form in pharate adult stage Malpighian tubules when fluid secretion resumes. Adult tubules are similar in general structure to larval tubules and contain mitochondria in the microvilli and basal infolds. However, they differ from larval tubules in that they are capable of very rapid fluid transport, have a reduced tubule diameter and tubule wall thickness, a much thicker basement membrane and peripherally associated tracheoles. Mineral concretions of calcium phosphate accumulate in larval tubules, persist through metamorphosis and decline in number in adults, suggesting they serve some anabolic role.     

Histopathology of secondary infections of Malpighamoeba locustae (Protozoa,Amoebidae) in the desert locust, Schistocerca gregaria (Orthoptera,Acrididae)

Schistocerca gregaria, the desert locust, harbors the protozoan parasite Malpighamoeba locustae. Twelve to 16 days after infection, trophozoites begin to multiply rapidly inside the Malpighian tubules of the gut. Swelling and rupture of the Malpighian tubules leads to the release of large numbers of cysts and trophozoites into the hemocoel. The classic insect defense response results in these cysts and trophozoites becoming encapsulated by the hemocytes of the host. Hemocytes of the phagocytic type become attached to, and lodged between, a variety of tissues and organs of the locust and black hemocytic capsules are produced. The extent to which the different tissues are involved is graded. Some tissues are completely blackened and encapsulated by masses of hemocytes but others are so lightly affected that the small specks of blackened pigment they lay down are discernible only on close examination. The trophozoites themselves do not divide outside the Malpighian tubules. The graded response in the host tissues is related to (1) the presence of sheets and lobes of fat body and (2) the presence of phagocytic hemocytes.     

Non-apoptotic function of apoptotic proteins in the development of Malpighian tubules of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Drosophila metamorphosis is characterized by the histolysis of larval structures by programmed cell death, which paves the way for the establishment of adult-specific structures under the influence of the steroid hormone ecdysone. Malpighian tubules function as an excretory system and are one of the larval structures that are not destroyed during metamorphosis and are carried over to adulthood. The pupal Malpighian tubules evade destruction in spite of expressing apoptotic proteins, Reaper, Hid, Grim, Dronc and Drice. Here we show that in the Malpighian tubules expression of apoptotic proteins commences right from embryonic development and continues throughout the larval stages. Overexpression of these proteins in the Malpighian tubules causes larval lethality resulting in malformed tubules. The number and regular organization of principal and stellate cells of Malpighian tubules is disturbed, in turn disrupting the physiological functioning of the tubules as well. Strikingly, the localization of beta-tubulin, F-actin and Disclarge (Dlg) is also disrupted. These results suggest that the apoptotic proteins could be having non-apoptotic function in the development of Malpighian tubules.     

Genetic control of development of the Malpighian vessels in Drosophila melanogaster

Malpighian tubules of insects are a functional analog of mammalian kidneys and serve as a classical model for studying the structure and functions of transport epithelium. The review contains the data on structural organization, functioning, and formation of the Malpighian tubules during embryogenesis in Drosophila melanogaster. Various systems of genes are described that control the program of development of the renal (Malpighian) tubules in D. melanogaster. A special attention is paid to the ways of signal transduction and factors involved in cell differentiation, proliferation, and morphological transformation during development of the Malpighian tubules. Evolutionarily conservative genetic systems are considered that are involved in the control of development of both the renal epithelium of Drosophila and mammalian kidneys. A relationship was noted between the disturbed balance of genetic material and congenital defects of the human excretory system.     

Two New Species of Nosema (Microsporida: Nosematidae) from the Mexican Bean Beetle Epilachna varivestis (Coleoptera: Coccinellidae)1,2

Two new species of the genus Nosema (Microsporida: Nosematidae) are described from the Mexican bean beetle, Epilachna varivestis (Coleoptera: Coccinellidae) and their life cycle stages studied by light and electron microscopy. Both species are monomorphic and disporous: they develop in direct contact with the cytoplasm of host cells and the nuclei of all stages are diplokaryotic. The more virulent species produces systemic infections most extensively in the adipose tissue, muscles, and Malpighian tubules of larvae and also invades the reproductive tissues of adult beetles. During merogonic development, it forms chains of diplokaryotic meronts. The fine structure of the sporoblast nuclei shows clumped material in the pole of each nucleus opposite their common plane of apposition. Spores are straight to slightly curved and ovocylindrical in shape and they measure 5.3 ± 0.13 × 2.1 ± 0.03 μm. The less virulent species also invades most host tissues but does not develop in the midgut epithelium; the Malpighian tubules are the principal site of its development and it also invades the ovaries and testes of adult beetles. Merogony occurs exclusively as the result of binary fission of diplokaryotic meronts. The plasmalemma of the meronts is covered with a thin deposit of exospore material upon which are located closely packed tubules that encircle the body transversely. A thickened deposit of exospore material on the surface of the diplokaryotic sporonts later obscures these tubules. Other tubules occur free in the host cell cytoplasm or attached to the plasmalemma of meronts and sporonts. Secretory granules also occur free or in chains in the host cytoplasm and are probably produced from the surface of the sporoblasts. Sporoblasts also contain an unusual cup-shaped organelle associated with a dense body, which is apparently involved in the formation of the polar tube and its associated organelles in the anterior part of the spore. Spores are ellipsoidal to slightly pyriform and measure 4.7 ± 0.06 × 2.6 ± 0.03 μm.     

Changes in cytoskeletal actin patterns in the Malpighian tubules of the fleshfly,Sarcophaga bullata (Parker) (Diptera : Calliphoridae), during metamorphosis

Using the rhodamine-labelled phalloidin staining method in combination with detergent extraction, metamorphic changes in actin filament patterns were investigated in the Malpighian tubules of the fleshfly, Sarcophaga bullata (Parker) (Diptera : Calliphoridae). Metamorphosis in this organ implies a process of dedifferentiation, followed by a process of redifferentiation. During dedifferentiation, the large basal actin bundles of the primary cells disappear and the microvillar membrane surface of these cells decreases. Concomitantly, several vesicles are pinched off from infoldings of the brush border. In older pupae, the Malpighian tubules redifferentiate to give rise to adult tubules with actin patterns similar to those of larvae. During redifferentiation of the tubules, the secondary cells display a marked increase in the number of actin filaments in their protrusions. The primary cells in the distal part of the anterior Malpighian tubules of late pupae display a well-developed basal pattern of thick parallel actin bundles. In most cases, major changes in actin filament patterns are found simultaneously with major changes in cell shape, indicating a close relationship between these actin filaments and the process of cellular remodelling.     

Genetic Control of Development of Malpighian Tubules in Drosophila melanogaster

Malpighian tubules of insects are a functional analog of mammalian kidneys and serve as a classical model for studying the structure and functions of transport epithelium. The review contains the data on structural organization, functioning, and formation of the Malpighian tubules during embryogenesis in Drosophila melanogaster. Various systems of genes are described that control the program of development of the renal (Malpighian) tubules in D. melanogaster. A special attention is paid to the ways of signal transduction and factors involved in cell differentiation, proliferation, and morphological transformation during development of the Malpighian tubules. Evolutionarily conservative genetic systems are considered that are involved in the control of development of both the renal epithelium ofDrosophila and mammalian kidneys. A relationship was noted between the disturbed balance of genetic material and congenital defects of the human excretory system.     

Four Distinct Regulatory Regions of the Cut Locus and Their Effect on Cell Type Specification in Drosophila

The cut gene in Drosophila is necessary in at least one cell type, the external sensory organs, for proper cell type specification and morphogenesis. It is also expressed in a variety of other tissues, where its function is less well characterized. Previous work has demonstrated that mutations affecting all the tissues map in the transcribed and translated portion of the gene, while mutations that are tissue specific in their effects map in the 140 kb upstream of the most 5' exon known. Within that 140 kb, the mutations fall into four subregions, two of which contain mutations affecting unique sets of tissues and the other two of which contain mutations that affect a third set. Our examination of the defects of mutants, their complementation behavior, and their effect on the distribution of the cut protein in embryos, alters the picture in three important ways. First, some mutations convert the cells of the Malpighian tubules into what appear to be gut cells, suggesting that cut is necessary for cell type specification and morphogenesis in a variety of tissues. Second, mutations in each of the four subregions in the 140 kb of upstream DNA cause a different set of phenotypes, suggesting that the regulatory region contains at least four separate units with different tissue specific functions. And third, mutations have now been identified that map in the transcribed and translated portion of the gene but that have tissue specific effects.     

Dual origin of the renal tubules in Drosophila: mesodermal cells integrate and polarize to establish secretory function

Organs are made up of cells from separate origins, whose development and differentiation must be integrated to produce a physiologically coherent structure. For example, during the development of the kidney, a series of interactions between the epithelial mesonephric duct and the surrounding metanephric mesenchyme leads to the formation of renal tubules. Cells of the metanephric mesenchyme first induce branching of the mesonephric duct to form the ureteric buds, and they then respond to signals derived from them. As a result, mesenchymal cells are recruited to the buds, where they undergo a mesenchymal-to-epithelial transition as they condense to form nephrons. In contrast, the simple renal tubules of invertebrates, such as insect Malpighian tubules (MpTs), have always been thought to arise from single tissue primordia, epithelial buds that grow by cell division and enlargement and from which a range of specialized subtypes differentiate. Here, we reveal unexpected parallels between the development of Drosophila MpTs and vertebrate nephrogenesis by showing that the MpTs also derive from two cell populations: ectodermal epithelial buds and the surrounding mesenchymal mesoderm. The mesenchymal cells are recruited to the growing tubules, where they undergo a mesenchymal-to-epithelial transition as they integrate and subsequently differentiate as a physiologically distinctive subset of tubule cells, the stellate cells. Strikingly, the normal incorporation of stellate cells and the later physiological activity of the mature tubules depend on the activity of hibris, an ortholog of mammalian NEPHRIN.