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.     

Metamorphosis of midgut epithelial cells in the silkworm (Bombyx Mori L.) with special regard to the calcium salt deposits in the cytoplasm. I. light microscopy

The morphological changes of the metamorphosing midgut cell in the silkworm were traced light-microscopically. The regenerative cells of the larval midgut proliferate rapidly during larval-pupal molt and finally replace the larval midgut, establishing new pupal midgut tissue composed of only one cell type. Pupal midgut cells contain numerous basophilic granules which are believed on histological grounds to be the deposits of calcium salts. Calcium seems to be transported from hemolymph to the pupal midgut cells and stored there temporarily as insoluble salts such as phosphate or carbonate, and then finally discharged into the lumen in a merocrine fashion. The midgut cells of the adult no longer contain calcium deposits.     

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.     

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.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions

In the central nervous system (CNS) of pupal Calliphora, dramatic alterations occur in the perineurial and glial gap junctions. Having formed macular plaques by late larval stages, in early pupae cell migration causes the EF intramembranous junctional particles to disaggregate and move apart into linear and then disorganised arrays as shown by freeze-fracture. After nerve and glial cell reorganisation into the adult pattern, the gap junctions begin to reform in the late pupae, again seemingly by particle migration into linear arrays and clusters. Ultimately the particles form numerous macular plaques between both perineurial and glial cells. Statistical analyses support the contention that these are performed EF particles which undergo translateral movement from macular larval junctions into the disaggregated particles of early pupae and that the same particles appear to undergo realignment and reclustering in late pupae to form the mature gap junctions of adults. This is the first report to indicate breakdown and reformation of gap junctions in vivo involving reutilisation of the same intramembranous particles. Perineurial “tight” junctions are not to be found in early pupal stages and their absence can be correlated with the free entry of ionic lanthanum into the CNS observed during that period. In late pupae, when the tight junctional moniliform ridges have apparently reformed, the entry of the tracer lanthanum becomes restricted to the level of the perineurium, penetrating no deeper. This is also the case in the adult, where the blood-brain barrier is maintained. PF particles in the form of short linear ridges and clustered particle arrays in nerve cell membranes are present throughout pupal and adult stages; their continued presence throughout the whole of development suggests some role in neuronal function, as yet unclear.     

Metamorphosis of midgut epithelial cells in the silkworm (Bombyx mori L.) with special regard to the calcium salt deposits in the cytoplasm. II. Electron microscopy

The mode of formation and fate of calcium salt deposits (calcospherites) appearing in the pupal midgut cells of the silkworm Bombyx mori was followed in the electron microscope. The larval midgut absorbs calcium ions from the haemolymph primarily via the goblet cells, while in the pupal midgut this occurs throughout the entire tissue. Part of the calcium absorbed by the pupal midgut accumulates in small vesicles, probably derived from the Golgi body and these develop into the concentrically laminated calcospherites. Late in the pupal period, these are discharged into the midgut lumen in a merocrine fashion. The midgut in the adult insect retains only a few poorly defined calcospherites in the cytoplasm.     

Hormonal and nutritional regulation of insect fat body development and function

The insect fat body is an organ analogue to vertebrate adipose tissue and liver and functions as a major organ for nutrient storage and energy metabolism. Similar to other larval organs, fat body undergoes a developmental “remodeling” process during the period of insect metamorphosis, with the massive destruction of obsolete larval tissues by programmed cell death and the simultaneous growth and differentiation of adult tissues from small clusters of progenitor cells. Genetic ablation of Drosophila fat body cells during larval‐pupal transition results in lethality at the late pupal stage and changes sizes of other larval organs indicating that fat body is the center for pupal development and adult formation. Fat body development and function are largely regulated by several hormonal (i.e. insulin and ecdysteroids) and nutritional signals, including oncogenes and tumor suppressors in these pathways. Combining silkworm physiology with fruitfly genetics might provide a valuable system to understand the mystery of hormonal regulation of insect fat body development and function. © 2009 Wiley Periodicals, Inc.     

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.     

Development of the Drosophila entero-endocrine lineage and its specification by the Notch signaling pathway

In this paper we have investigated the developmental–genetic steps that shape the entero-endocrine system of Drosophila melanogaster from the embryo to the adult. The process starts in the endoderm of the early embryo where precursors of endocrine cells and enterocytes of the larval midgut, as well as progenitors of the adult midgut, are specified by a Notch signaling-dependent mechanism. In a second step that occurs during the late larval period, enterocytes and endocrine cells of a transient pupal midgut are selected from within the clusters of adult midgut progenitors. As in the embryo, activation of the Notch pathway triggers enterocyte differentiation and inhibits cells from further proliferation or choosing the endocrine fate. The third step of entero-endocrine cell development takes place at a mid-pupal stage. Before this time point, the epithelial layer destined to become the adult midgut is devoid of endocrine cells. However, precursors of the intestinal midgut stem cells (pISCs) are already present. After an initial phase of symmetric divisions which causes an increase in their own population size, pISCs start to spin off cells that become postmitotic and express the endocrine fate marker, Prospero. Activation of Notch in pISCs forces these cells into an enterocyte fate. Loss of Notch function causes an increase in the proliferatory activity of pISCs, as well as a higher ratio of Prospero-positive cells.     

Stem cells of the beetle midgut epithelium

At the completion of metamorphosis, adult insect cells have traditionally been assumed to halt cell divisions and terminally differentiate. While this model of differentiation holds for adult ectodermal epithelia that secrete cuticular specializations of exoskeletons, adult endodermal epithelia are populated by discrete three-dimensional aggregates of stem cells that continue to divide and differentiate after adult emergence. Aggregates of these presumptive adult stem cells are scattered throughout larval and pupal midgut monolayers. At the beginning of adult development (pupal-adult apolysis), the number of cells within each aggregate begins to increase rapidly. Dividing cells form three-dimensional, coherent populations that project as regenerative pouches of stem cells into the hemocoel surrounding the midgut. Stem cell pouches are regularly spaced throughout endodermal monolayers, having adopted a spacing pattern suggesting that each incipient pouch inhibits the formation of a similar pouch within a certain radius of itself—a process referred to as lateral inhibition. At completion of adult development (pupal-adult ecdysis), a distinct basal-luminal polarity has been established within each regenerative pouch. Dividing stem cells occupying the basal region are arranged in three-dimensional aggregates. As these are displaced toward the lumen, they transform into two-dimensional monolayers of differentiated epithelial cells whose apical surfaces are covered by microvilli. This organization of stem cell pouches in insect midguts closely parallels that of regenerative crypts in mammalian intestines.     

Lepidopteran larval midgut during prepupal instar: digestion or self-digestion?

Programmed cell death (PCD) is crucial in body restructuring during metamorphosis of holometabolous insects (those that have a pupal stage between the final larval and adult stages). Besides apoptosis, an increasing body of evidence indicates that in several insect species programmed autophagy also plays a key role in these developmental processes. We have recently characterized the midgut replacement process in Heliothis virescens larva, during the prepupal phase, responsible for the formation of a new pupal midgut. We found that the elimination of the old larval midgut epithelium is obtained by a combination of apoptotic and autophagic events. In particular, autophagic PCD completely digests decaying tissues, and provides nutrients that are rapidly absorbed by the newly formed epithelium, which is apparently functional at this early stage. The presence of both apoptosis and autophagy in the replacement of midgut cells in Lepidoptera offers the opportunity to investigate the functional peculiarities of these PCD modalities and if they share any molecular mechanism, which may account for possible cross-talk between them.     

The development and function of the female germ line in Drosophila melanogaster: a cell lineage study.

X-ray-induced mitotic recombination was used to follow the development and function of the female germ line in Drosophila melanogaster. Clones marked by maternal effect mutations which alter the morphology of the egg [fs(1)K10] or the phenotype of the resulting progeny (maroonlike) were produced in trans-heterozygotes irradiated during embryonic, larval, or pupal development or as 5-day-old adults. Judging from the size of clones induced at the blastoderm stage, only five to ten of the pole cells observed on the surface of the embryo contribute to the germ line. Most of the K10 clones induced during embryonic and larval development were associated with mal twin spots, indicating that both daughters of the irradiated germ cell remained in the germ line and gave rise to eggs in the adult. During larval life the number of cells increases logarithmically and reaches a maximum of 110 at 24 hr after pupation. The same value was obtained for 5-day-old adults. In contrast to the mosaic females produced as embryos and larvae, mosaics obtained after pupal and adult irradiations were of two types, those laying only one K10 egg and those laying several K10 eggs distributed over the lifespan of the adult. This result indicates that the stem cell divisions characteristic of the adult period have begun shortly after pupation. About 9 to 11 days are required for an irradiated stem cell to produce its first clonal K10 egg, and two-thirds of this time is spent in the germarium. Each ovariole possesses on the average two to three functioning stem cells. This multiplicity of stem cells was confirmed by the recovery of mosaic ovarioles when mal heterozygotes irradiated as adults or late larvae were stained for aldehyde oxidase activity.     

The wing imaginal disc

The Drosophila wing imaginal disc is a tissue of undifferentiated cells that are precursors of the wing and most of the notum of the adult fly. The wing disc first forms during embryogenesis from a cluster of ∼30 cells located in the second thoracic segment, which invaginate to form a sac-like structure. They undergo extensive proliferation during larval stages to form a mature larval wing disc of ∼35,000 cells. During this time, distinct cell fates are assigned to different regions, and the wing disc develops a complex morphology. Finally, during pupal stages the wing disc undergoes morphogenetic processes and then differentiates to form the adult wing and notum. While the bulk of the wing disc comprises epithelial cells, it also includes neurons and glia, and is associated with tracheal cells and muscle precursor cells. The relative simplicity and accessibility of the wing disc, combined with the wealth of genetic tools available in Drosophila, have combined to make it a premier system for identifying genes and deciphering systems that play crucial roles in animal development. Studies in wing imaginal discs have made key contributions to many areas of biology, including tissue patterning, signal transduction, growth control, regeneration, planar cell polarity, morphogenesis, and tissue mechanics.