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.     

Hid can induce, but is not required for autophagy in polyploid larval Drosophila tissues

The major cell death pathways are apoptosis and autophagy-type cell death in Drosophila. Overexpression of proapoptotic genes in developing imaginal tissues leads to the activation of caspases and apoptosis, but most of them show no effect on the polytenic cells of the fat body during the last larval stage. Surprisingly, overexpression of Hid induces caspase-independent autophagy in the fat body, as well as in most other larval tissues tested. Hid mutation results in inhibition of salivary gland cell death, but the disintegration of the larval midgut is not affected. Electron microscopy shows that autophagy is normally induced in fat body, midgut and salivary gland cells of homozygous mutant larvae, suggesting that Hid is not required for autophagy itself. Constitutive expression of the caspase inhibitor p35 produces identical phenotypes. Our results show that the large, post-mitotic larval cells do not react or activate autophagy in response to the same strong apoptotic stimuli that trigger apoptosis in small, mitotically active imaginal disc cells.     

Reprogramming in the absence of DNA synthesis in Galleria larval epidermis.

Larval epidermal cells from a day-1 penultimate instar Galleria larva on implantation into day-5 last instar larva metamorphose and deposit a pupal cuticle at the same time as the host pupates. DNA synthesis in the implanted larval cell was monitored with 3-H-thymidine. Various regimens of 3-H-thymidine application were used and under no conditions did the larval cells incorporate label during the period from implantation to deposition of pupal cuticle. This suggests that a wax moth larval ectoderm cell can reprogram its genome to secrete a pupal cuticle without a precedent cell division.     

The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropia. I. Generalized epidermal cells

The ultrastructure of wing epidermis of the giant silkmoth, Hyalophora cecropia, was studied during pupal diapause and the first half of development to the adult. In diapause, the generalized epidermal cells are characterized by many free ribosomes, some vesicles and small lamellae of rough endoplasmic reticulum, some scattered short mitochondria and a few small Golgi complexes. During the early states of post-diapause development, before and after the time of apolysis (separation of the epidermis from the overlying cuticle), there is a marked increase in structures often associated with synthetic functions, such as polyribosomes, lamellate rough endoplasmic reticulum and Golgi complexes. On day five of post-apolysis development, just after the appearance of scale-forming and socket-forming cells, the generalized epidermal cells lay down the cuticulin layer of the adult cuticle. At this stage and later, the polyribosomes and lamellate rough endoplasmic reticulum decrease in abundance. Cell nuclei show three phases of temporary transition from predominantly lobed to predominantly round profile, which correspond to periods of reported DNA synthesis. Throughout this developmental process, therefore, there is good correlation of fine structure with changes in macromolecular synthesis recorded elsewhere.     

Cellular features of an apoptotic form of programmed cell death during the development of the ascidian, Boltenia villosa

The phylogenetic position of ascidians near the base of the chordate tree makes them ideal organisms for evolutionary developmental studies of programmed cell death (PCD). In the present study, the following key features of an apoptotic form of PCD are described in Boltenia villosa: fragmentation of DNA, increases in plasma membrane permeability, decreases in mitochondrial activity, production of reactive oxygen species (ROS), and caspase activation. First, evidence is presented for apoptosis of cells within the ovary. Later in development, during the early phase of larval tail resorption at the beginning of metamorphosis, some notochord nuclei showed DNA fragmentation and their cell corpses were rapidly eliminated from the larval body. In striking contrast to the rapid demise of notochord cells, larval muscle cells persisted for more than a week within developing juveniles. Rhodamine 123 and MTT experiments suggest that mitochondria within some of the resorbed larval tail muscle cells were metabolically active for more than a week. Furthermore, resorbed tail muscle cells contained a muscle-specific intermediate filament, termed p58, despite relatively high levels of ROS activity and the ubiquitination of their plasma membranes at day two. Corpses of larval tail muscle cells containing aggregated pigment granules survived within juveniles for more than a month, in contrast to the rapid elimination of notochord cells. Evidence consistent with the formation of larval muscle cell apoptotic bodies is presented. The most surprising result of the present study was that caspase-8, usually associated with apoptotic signaling, was activated in larval endoderm cells that develop into adult structures. When the present results were compared to features of PCD previously reported in other ascidians, significant species differences in PCD were revealed.     

Conditional expression of anti-apoptotic protein p35 by Cre-mediated DNA recombination in cardiomyocytes from loxP-p35-transgenic mice

p35, a viral inhibitor of caspase, prevents cell death induced by various stimuli. We established an experimental system to study the involvement of caspases in cell death, using primary cultured cells from p35 transgenic mice in which the p35 open reading frame (ORF) had been disrupted by the insertion of a DNA segment flanked by loxP sites, the Cre recognition sites. In this system, p35 expression can be initiated by Cre recombinase. Cardiomyocytes, which are highly sensitive to hypoxic stress, were infected with an adenovirus carrying the cre gene (AxCANCre). Expression of p35 by infection with AxCANCre resulted in inhibition of caspase-3 activation and resistance to hypoxia-induced cell death. Hypoxia-induced cytochrome c release was also attenuated in p35-expressing cardiomyocytes. Our transgenic mice can be used as an experimental model for studying the involvement of caspases in various degenerative diseases as well as programmed cell death both in vitro and in vivo.     

Pattern regulation during the development of the dorsal abdomen in the flesh fly,Sarcophaga agryostoma

Summary In dipteran flies the adult abdominal epidermis is formed from small nests of diploid histoblast cells which spread out and replace the larval epidermis during metamorphosis. The pattern of nest outgrowth and fusion in Sarcophaga shows that the large dorsal hemitergite is normally formed by the two dorsal nests, the spiracle nest and part of the ventral nest (which also forms the hemisternite). By rotating the dorsal histoblast nests, we demonstrate that the adult segment border lies between the flexible intersegmental membrane (ISM) and the naked anterior strip of tergite, the acrotergite. Deletion of histoblast nests often results in a corresponding deletion of adult structures, accompanied by enlargement of adjacent structures within the segment and in neighbouring segments. Pattern formation is not strictly coupled to cell division (as in imaginal discs), since the nests remaining after an ablation, in spreading to fill vacant areas, generate more cells and larger structures than normal. Nest deletions can also result in regeneration, with remaining nests forming additional structures in the dorsal-ventral or anterior-posterior axis of the segment. The deletion of strips of anterior and intersegmental larval epidermis without histoblasts results in the formation of double-posterior duplications of the adult hemitergite. Although these operations damage adjacent histoblast nests, several features of the results suggest that the duplications arise from the interaction (after healing) of histoblasts with larval cells which they would not normally encounter, leading to the intercalation of histoblast cells bearing intervening anterior-posterior positional values. A similar process of intercalation may occur in normal development, as the histoblasts spread from their local origins across the larval epidermal sheet, replacing the larval cells to form the entire epidermis of the adult segment. Offprint requests to: V. French     

Tissue-specific effects of the juvenile hormone analogue ZR 515 during metamorphosis in Drosophila cell cultures

The juvenile hormone analogue ZR 515 has specific effects on ecdysone-induced metamorphic differentation of Drosophila cells cultured in vitro. The number of vesicles containing imaginal cuticular structures is reduced to 10% of control levels. Similarly, the differentiation of adult fat body is partly inhibited by ZR 515. The differentiation of adult tubular and fibrillar muscles, however, is not affected. ZR 515 does not inhibit cuticle secretion by tracheal cells and larval epidermal cells.     

Structure and cuticle formation of the reticulated carapace of the ostracode Bicornucythere bisanensis

Electron microscopic examination revealed fine structures of the adult carapace of Bicornucythere bisanensis , which consists mainly of epicutiele, procuticle, membranous layer, epidermal cells and subdermal cells. Piled membrane structure is recognized as an organic framework of the procuticle. Newly formed cuticle examined with specimens at various stages of the molt cycle clarified the process of cuticle formation. Analysis of the complicated process of cuticular ridge formation permits morphogenetic discussion of surface features of the ostracode carapace of many species. Cuticle formation in close relation to cell boundaries clearly indicates that each polygon of the carapace reticulation is formed by one epidermal cell. The cell-polygon correspondence suggests that ostracode reticulation is important for elucidation of the phylogenic and ontogenic development of multicellular organisms at cell level.
□ Ostracoda, carapace structure, biomineralization, cuticle formation.     

Segmental organisation of the tail region in the embryo of Drosophila melanogaster

Summary The segmental organisation of the tail region in the embryo of Drosophila melanogaster, which is defined here as the epidermal region posterior to the boundary between abdominal segments A7 and A8, has been investigated by means of ultraviolet (UV) laser fate-mapping and phenotypic analysis of embryonic mutants that alter the segmental pattern of the larval cuticle. Wild-type embryos were irradiated in the presumptive tail region with a UV- laser microbeam of 20 m diameter at the blastoderm stage. The ensuing defects were scored in the cuticle pattern of the tail region of the first-instar larva, which is described in detail in this paper. The spatial distribution of defect frequencies was used to construct a blastoderm fate-map of the cuticle structures of the larval tail region. The segmental origin of the larval tail structures was inferred from the phenotypic analysis of segmentation and homoeotic mutants, which revealed pattern repetition throughout the embryonic tail region corresponding to four segment anlagen, A8 to A11, and a non-segmental telson. These data enabled the transformation of the blastoderm fate-map of cuticle structures into a map of tail segment anlagen. The tail anlage occupies about 10% of the egg length (EL), bounded by segment A7 anteriorly at 20% EL and by the proctodaeum posteriorly at 10% EL, as measured from the posterior pole. The anlagen of segments A8 and A9 appear to be narrow dorso-ventral strips of blastoderm cells similar to the anlagen of the trunk segments, whereas the anlagen of A10 and A11 are smaller and produce fewer pattern elements. The telson is represented in the cuticle by the tuft which derives from a very dorsal posterior position. The antero-posterior axis of the entire tail anlage appears curved upward posteriorly. Differences in the mode of development between tail and trunk segments are discussed, as are similarities of larval and imaginal tail development in Drosophila. Comparison with tail development in other insects suggests that, during evolution, the transition from semi-long-germ to long-germ development modified the organisation of the tail region without affecting its primary subdivision into metameric units.     

Illuminating the role of caspases during Drosophila oogenesis

Cell death is a prominent feature of animal germline development. In Drosophila, the death of 15 nurse cells is linked to the development of each oocyte. In addition, females respond to poor environmental conditions by inducing egg chamber death prior to yolk uptake by the oocyte. To study these two forms of cell death, we analyzed caspase activity in the germline by expressing a transgene encoding a caspase cleavage site flanked by cyan fluorescent protein and yellow fluorescent protein. When expressed in ovaries undergoing starvation-induced apoptosis, this construct was an accurate reporter of caspase activity. However, dying nurse cells at the end of normal oogenesis showed no evidence of cytoplasmic caspase activity. Furthermore, although expression of the caspase inhibitors p35 or Drosophila inhibitor of apoptosis protein 1 blocked starvation-induced death, it did not affect normal nurse cell death or overall oogenesis in well-fed females. Our data suggest that caspases play no role in developmentally programmed nurse cell death.     

What are and what are not imaginal discs: reevaluation of some basic concepts (Insecta, Holometabola).

Some general aspects of the concept of imaginal discs in the Holometabola are reevaluated. Their monolayer character and continuity with the surrounding epidermis are confirmed. Studies on the imaginal discs of the silkworm (Bombyx mori) and data from the literature show that the discs and their peripodial cells produce cuticle during larval life, as well as at metamorphosis. In B. mori it is demonstrated that adult and larval antennae are produced by the same cells or their progeny. The results also suggest that segments of the typically three-segmented larval antenna of Holometabola are not scape, pedicel, and one-segmented flagellum; at least segments 2 and 3 are of flagellar origin. Based on these and some additional facts it is argued that: (1) No larval organs are "replaced" at metamorphosis, but strict "sequential homology" is always maintained. (2) Imaginal discs are not undifferentiated structures destined to form the adult after larval breakdown, cannot be unambiguously defined, and do not represent qualitatively different epidermal structures. Classical imaginal discs (invaginated and present also in pre-final larval instars) arose several times independently and were not present in the larvae of ancestral Holometabola. (3) Since the disc cells are not undifferentiated and "embryonic" (if these words have a defined meaning at all), it is unreasonable to expect that the processes taking place in discs at metamorphosis would differ fundamentally from those occurring in other diploid metamorphosing epidermal cells.     

Cuticulogenesis correlated with ultrastructural changes in oenocytes and epidermal cells in the late cockroach embryo

During late embryogenesis in a cockroach, the epidermal cells secrete two cuticles: the embryonic cuticle and the pharate first larval cuticle. Late embryogenesis begins with the deposition of the cuticulin layer of the embryonic cuticle. The embryonic cuticle is an atypical one. It remains relatively thin and a well lamellated endocuticle is usually lacking. After general apolysis of the embryonic cuticle the epidermis secretes the epicuticle of the first larval cuticle and, subsequently, a typical lamellate procuticle. During the penultimate phase of late embryogenesis (i.e. before general apolysis) the epidermis becomes larvally committed. Some epidermal cells start to differentiate into specialized structures of the dermal glands, whereas the differentiated oenocytes appear to have acquired some stability. Nevertheless, shortly before general apolysis some oenocytes display signs of an increased alteration of the SER. When general apolysis occurs, the oenocytes contain a well-developed SER. The whole of the oenocyte population is programmed to regress after epicuticle deposition of the first larval cuticle. The correlation of oenocyte regression with available data on cuticulogenesis, ecdysteroid titres and cuticular lipid synthesis is discussed.     

Ultrastructure of the Anterior End in Three Ontogenetic Stages of Nectonema munidae (Nematomorpha)

Abstract The structure of the anterior end of three ontogenetically successive stages of Nectonema munidae (Nematomorpha) is investigated by light and transmission electron microscopy. During development, an adult cuticle is formed under a larval cuticle, which is subsequently moulted. Only one moult can be documented for Nectonema. The brain has a main subpharyngeal portion and a weak suprapharyngeal commissure. It is circumpharyngeal only in early developmental stages, the dorsal commissure is reduced in the adult stage. Four giant cells and a cerebral cavity are adult features. Although the morphology of the giant cells is elucidated, their function remains unclear, but a sensory function is probable. A septum marks the posterior border of the anterior end and divides a cerebral cavity from a body cavity. A precursor of the septum is present in the first stage observed, but it lies next to the epidermis and does not separate a cerebral cavity. Cuticular structures in the pre-pharyngeal region of the early stages are interpreted as remnants of the larval boring organ. They are moulted together with the larval cuticle. The morphology of the pharynx and the anterior part of the intestine is shown.     

Development of the adult leg epidermis in <Emphasis Type="Italic">Manduca sexta</Emphasis>: contribution of different larval cell populations

During metamorphosis of the tobacco hornworm Manduca sexta, the simple thoracic legs of the larva are remodeled into the more complex adult legs. Most of the adult leg epidermis derives from the adult primordia, small sets of epidermal cells located in specific regions of the larval leg, which proliferate rapidly in the final larval instar. In contrast, the contribution of the epidermal cells outside the primordia is unknown. In this study we have determined their contribution to the adult leg by labeling them with 5-bromodeoxyuridine (BUdR) and following their fate. Although the labeled cells diminished drastically in number, small groups of these cells persisted into the midpupal stage suggesting that they do contribute to the adult leg epidermis. We also found that during the wandering stage the adult primordia went through active proliferation and very little cell death, while the cells outside the primordia went through extensive cell death accounting for the decrease in their number. Our results indicate that two distinct cell populations exist outside the adult primordia. Most cells belong to the first population, which is larval-specific and disappears through apoptosis early in metamorphosis. The second population consists of polymorphic cells that contribute to the larval, pupal and adult leg epidermis.Edited by D. Tautz     

Live imaging of epidermal morphogenesis during the development of the adult abdominal epidermis of Drosophila

During larval stages of Drosophila development, the abdominal epidermis is composed of histoblasts (adult precursors) and larval epidermal cells (LECs). During metamorphosis, histoblasts proliferate and colonize the territories occupied by the LECs, which die and become engulfed by macrophages. This morphogenetic process is an excellent model for in vivo analysis of epithelial migration, cell division, cell death, patterning and differentiation. Here, we describe a protocol for time-lapse recording of the developing epidermis during metamorphosis. The protocol describes the removal of the pupal case (which acts as an opaque barrier to effective imaging) and mounting and imaging of specimens of different stages so that normal developmental processes are preserved. This method enables high-resolution studies over long time periods using fluorescent markers and confocal microscopy. The protocol requires 1 h for pupal dissection and mounting and, depending on the stages and genotypes to be analyzed, several more hours for preprocessing and aging and developmental staging of flies and pupae.     

Isolation,characterization, and in vitro culture of larval and adult epidermal cells of the frogXenopus laevis

Summary Methods for the isolation and in vitro culture of larval and adultXenopus laevis epidermal cells have been developed. Epidermal cells of stage 52–54 tadpoles and adult epidermal cells were enzymatically dissociated and purified (98%) by Percoll-density centrifugation and unit-gravity sedimentation. Both cell types attached on fibronectin-coated dishes and proliferated for 1 wk when the proper medium was used. There were four significant differences between larval and adult cells: a) Adult cells had a greater buoyant density than larval cells. b) Keratin synthesis patterns were markedly different. c) A combination of medium F12 and Eagle's minimum essential medium was optimal for growth of larval cells whereas MCDB151 medium was optimal for adult cells. d) Adult cells needed fetal bovine serum (>5%) whereas larval cells grew without fetal bovine serum. In contrast to these differences, larval and adult cells had two similar properties: a) Insulin had a potent effect on the growth of both cells, and b) The optimal Ca⁺⁺ concentration for cell growth was quite low for both cell types; 0,1 mM for larval cells and below 0.05 mM for adult cells. These results suggest that low Ca⁺⁺ levels are essential for both cornifying (adult) and uncornifying (larval) amphibian keratinocytes. The culture techniques described herein for larval and adult epidermal cells provide a new in vitro model for analyzing development of the epidermis during amphibian metamorphosis. This study was supported by grant (HD 24438) from the National Institutes of Health, Bethesda, MD.     

Visualization of proprioceptors in Drosophila larvae and pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs). Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis. The development of larval ChOs during embryogenesis has been studied extensively. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system. Multiple ChOs have been described in the adult fly. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy. In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary. Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.     

orthodenticle activity is required for the development of medial structures in the larval and adult epidermis of Drosophila.

Lethal alleles of orthodenticle (= otd) cause abnormalities in the embryonic head that reflect an early role in anterior pattern formation. In addition, otd activity is required for the development of the larval and adult epidermis. Clonal analysis of both viable and lethal alleles shows that the adult requirement for otd is restricted to medial regions of certain discs. When otd activity is reduced or removed, some medial precursor cells produce bristles and cuticle characteristic of more lateral structures. Similar medial defects are observed in the larval epidermis of embryos homozygous for lethal otd alleles. Antibodies to otd recognize a nuclear protein found at high levels in the medial region of the eye antennal discs, the leg discs, the genital discs and along the ventral midline of the ventral epidermis of the embryo. These results suggest that the otd gene product is required to specify medial cell fates in both the larval and adult epidermis.