Signaling pathways directing the movement and fusion of epithelial sheets: lessons from dorsal closure in Drosophila

Wound healing in embryos and various developmental events in metazoans require the spreading and fusion of epithelial sheets. The complex signaling pathways regulating these processes are being pieced together through genetic, cell biological, and biochemical approaches. At present, dorsal closure of the Drosophila embryo is the best-characterized example of epithelial sheet movement. Dorsal closure involves migration of the lateral epidermal flanks to close a hole in the dorsal epidermis occupied by an epithelium called the amnioserosa. Detailed genetic studies have revealed a network of interacting signaling molecules regulating this process. At the center of this network is a Jun N-terminal kinase cascade acting at the leading edge of the migrating epidermis that triggers signaling by the TGF-beta superfamily member Decapentaplegic and which interacts with the Wingless pathway. These signaling modules regulate the cytoskeletal reorganization and cell shape change necessary to drive dorsal closure. Activation of this network requires signals from the amnioserosa and input from a variety of proteins at cell-cell junctions. The Rho family of small GTPases is also instrumental, both in activation of signaling and regulation of the cytoskeleton. Many of the proteins regulating dorsal closure have been implicated in epithelial movement in other organisms, and dorsal closure has emerged as an ideal model system for the study of the migration and fusion of epithelial sheets.     

果蝇背闭合行为中DJNK信号转导途径研究进展

Jun氨基末端激酶 (JunN terminalkinase ,JNK)是一种重要的细胞信号传递者。它参与了细胞生长、分化、程序性死亡等生理过程 ,而且在调节上皮细胞运动和形态发生等方面也起着重要作用。大量研究证实 ,在果蝇Drosophila的背闭合行为 (dorsalclosure,DC)中 ,DJNK(DrosophilaJNK)的调节是关键。文章就果蝇DC的发生过程以及DJNK信号途径的研究进展作一简要的综述。     

Unravelling the distinct contribution of cell shape changes and cell intercalation to tissue morphogenesis: the case of the Drosophila trachea

Intercalation allows cells to exchange positions in a spatially oriented manner in an array of diverse processes, spanning convergent extension in embryonic gastrulation to the formation of tubular organs. However, given the co-occurrence of cell intercalation and changes in cell shape, it is sometimes difficult to ascertain their respective contribution to morphogenesis. A well-established model to analyse intercalation, particularly in tubular organs, is the Drosophila tracheal system. There, fibroblast growth factor (FGF) signalling at the tip of the dorsal branches generates a ‘pulling’ force believed to promote cell elongation and cell intercalation, which account for the final branch extension. Here, we used a variety of experimental conditions to study the contribution of cell elongation and cell intercalation to morphogenesis and analysed their mutual requirements. We provide evidence that cell intercalation does not require cell elongation and vice versa. We also show that the two cell behaviours are controlled by independent but simultaneous mechanisms, and that cell elongation is sufficient to account for full extension of the dorsal branch, while cell intercalation has a specific role in setting the diameter of this structure. Thus, rather than viewing changes in cell shape and cell intercalation as just redundant events that add robustness to a given morphogenetic process, we find that they can also act by contributing to different features of tissue architecture.     

Developmental expression of Rab11, a small GTP‐binding protein in Drosophila epithelia

Intracellular membrane trafficking regulates a wide variety of developmental processes, including cell and tissue morphogenesis. Here we report developmental expression of Drosophila Rab11, a small GTP‐binding protein, required for both endocytic recycling and exocytosis. Rab11 is expressed in the epithelial cell types of diverse lineages at all developmental stages, beginning from the cellular blastoderm in early embryos to adult primordia and adult tissues, like the columnar epithelia lining male ejaculatory bulb. A robust expression of Rab11 is seen both in the amnioserosa and in the lateral epidermis during embryonic dorsal closure, a morphogenetic event that involves spreading and fusion of the contra‐lateral sides of epidermis. Rab11 mutant embryos fail to display the characteristic morphological changes in these two epithelial tissues during dorsal closure, providing a strong basis to dissect the role of Rab11 in coordinated epithelial sheet movements. genesis 47:32–39, 2009. © 2008 Wiley‐Liss, Inc.     

Terminal filament cell organization in the larval ovary of Drosophila melanogaster: ultrastructural observations and pattern of divisions

The adult ovary of Drosophila is composed of approximately twenty parallel repetitive structures called ovarioles. The ovarioles appear at the prepupal stage and their formation requires the presence of stacks of discshaped cells called the terminal filaments. Terminal filaments form in a progressive manner during the third larval instar. We have looked at the beginning of formation of both the terminal filaments and ovarioles at an ultrastructural level. Moreover, we studied the pattern of division of the terminal filament cell precursors using the base analog, BrdU. Two main waves of division are observed. The first wave consists of divisions of almost all the terminal filament cell precursors during a short period of time at the transition between the second and third larval instar. The second wave, in which the precursors carry out their final divisions before differentiating, occurs gradually, going from the medial to the lateral side of the ovary during the first half of the third larval instar.     

Pharmacological analysis of heartbeat in Drosophila

Analysis of the mechanisms underlying cardiac excitability can be faciliated greatly by mutations that disrupt ion channels and receptors involved in this excitability. With an extensive repertoire of such mutations, Drosophila provides the best available genetic model for these studies. However, the use of Drosophila for this purpose has been severely handicapped by lack of a suitable preparation of heart and a complete lack of knowledge about the ionic currents that underlie its excitability. We describe a simple preparation to measure heartbeat in Drosophila. This preparation was used to ask if heartbeat in Drosophila is myogenic in origin, and to determine the types of ion channels involved in influencing the heart rate. Tetrodotoxin, even at a high concentration of 40 μM, did not affect heart rate, indicating that heartbeat may be myogenic in origin and that it may not be determined by Na⁺ channels. Heart rate was affected by PN200–110, verapamil, and diltiazem, which block vertebrate L-type Ca²⁺ channels. Thus, L-type channels, which contribute to the prolonged plateau of action potentials in vertebrate heart, may play a role in Drosophila cardiac excitability. It also suggests that Drosophila heart is subject to a similar intervention by organic Ca²⁺ channel blockers as the vertebrate heart. A role for K⁺ currents in the function of Drosophila heart was suggested by an effect of tetraethylammonium, which blocks all the four identified K⁺ currents in the larval body wall muscles, and quinidine, which blocks the delayed rectifier K⁺ current in these muscles. The preparation described here also provides an extremely simple method for identifying mutations that affect heart rate. Such mutations and pharmacological agents will be very useful for analyzing molecular components of cardiac excitability in Drosophila. © 1995 John Wiley & Sons, Inc.     

Morphology of the pupal heart,adult heart,and associated tissues in the fruit fly,Drosophila melanogaster

The early pupal heart of the fruit fly Drosophila melanogaster has recently been the subject of intense physiological and molecular work, yet it has not been well described, nor has it been compared with the heart of the adult fly. In the work reported here, the hearts of adults and early pupae of D. melanogaster were studied by scanning and transmission electron microscopy and by light microscopy. The hearts of adults and early pupae both consist of a tube of circular striated muscle one cell in thickness. The alary muscles, which suspend the heart, are more delicate in the adult compared to the early pupa. The pericardial cells in both early pupae and adults are connected to the heart by connective tissue radiating from the alary muscles or dorsal diaphragm. We confirm that four major changes occur in the heart during metamorphosis: 1) a conical chamber is formed de novo in the first and second abdominal segments; 2) the adult heart curves to conform to the contour of the abdomen; 3) a layer of longitudinal striated muscle appears on the ventral surface of the heart; 4) a fourth pair of ostia is added to the three already present in the early pupa; and note additionally that 5) the ostia appear as simple openings in the heart of the early pupa but are valve‐like in the adult. J. Morphol. 240:225–235, 1999. © 1999 Wiley‐Liss, Inc.     

dPak is required for integrity of the leading edge cytoskeleton during Drosophila dorsal closure but does not signal through the JNK cascade

The Pak kinases are effectors for the small GTPases Rac and Cdc42 and are divided into two subfamilies. Group I Paks possess an autoinhibitory domain that can suppress their kinase activity in trans. In Drosophila, two Group I kinases have been identified, dPak and Pak3. Rac and Cdc42 participate in dorsal closure of the embryo, a process in which a hole in the dorsal epidermis is sealed through migration of the epidermal flanks over a tissue called the amnioserosa. Dorsal closure is driven in part by an actomyosin contractile apparatus at the leading edge of the epidermis, and is regulated by a Jun amino terminal kinase (JNK) cascade. Impairment of dPak function using either loss-of-function mutations or expression of a transgene encoding the autoinhibitory domain of dPak led to disruption of the leading edge cytoskeleton and defects in dorsal closure but did not affect the JNK cascade. Group I Pak kinase activity in the amnioserosa is required for correct morphogenesis of the epidermis, and may be a component of the signaling known to occur between these two tissues. We conclude that dorsal closure requires Group I Pak function in both the amnioserosa and the epidermis.     

Embryonic morphogenesis signaling pathway mediated by JNK targets the transcription factor JUN and the TGF-β homologue decapentaplegic

The dorsal surface of the Drosophila embryo is formed by the migration of the lateral epithelial cells to cover the amnioserosa. The Drosophila cJun-N-terminal kinase (DJNK) is essential for this process. Mutations in DJNK or the DJNK activator hemipterous (HEP) lead to incomplete dorsal closure, resulting in a hole in the dorsal cuticle. The molecules downstream of DJNK in this signaling pathway have not been established. Here we demonstrate that the basket1 (bsk1) mutation of DJNK causes decreased interaction with DJUN. Expression of decapentaplegic (DPP), a TGF-β homologue, in the leading edge of the dorsal epithelium, is identified as a genetic target of the JNK pathway. A constitutive allele of JUN is able to rescue the dorsal closure defect of bsk1 and restores DPP expression. Furthermore, ectopic DPP rescues the defects in dorsal closure caused by bsk1. These data indicate that the interaction of DJNK with DJUN contributes to the dorsal closure signaling pathway and targets DPP expression. J. Cell. Biochem. 67:1–12, 1997. © 1997 Wiley-Liss, Inc.     

Specific expression of the Drosophila midline-jumper retro-transposon in embryonic CNS midline cells

Here we describe of a novel Drosophila LTR-type retrotransposon that is expressed in the embryonic CNS midline glia and in the embryonic germ cells. The element is related to the gypsy and burdock retrotransposons and was termed midline-jumper. In addition to cDNA clones generated from internal retrotransposon sequences, we have identified one cDNA clone that appears to reflect a transposition event, indicating that the midline-jumper retrotransposon is not only transcribed but also able to transpose during Drosophila development.     

Case studies of ends‐out gene targeting in Drosophila

Ends‐in and ends‐out gene replacement approaches have been successfully used to disrupt Drosophila genes involved in a variety of biological processes. These methods combine double‐strand breaks and homologous recombination to replace a targeted chromosome region with a designed DNA sequence. Unfortunately, these methods require large numbers of single animal crosses, making them both time consuming and labor intensive. Here, we designed a single complete targeting vector for use in a mass crossing ends‐out gene targeting study. Importantly, our gene targeting method included a balancer chromosome to block endogenous homologous chromosome pairing and to promote pairing between the foreign targeting DNA fragment and the targeted chromosome. This technique provided successful and efficient gene replacement, greatly facilitating the gene knockout procedure. genesis 47:305–308, 2009. © 2009 Wiley‐Liss, Inc.     

RNAi-mediated inhibition of gene function in the follicle cell layer of the Drosophila ovary

RNA-mediated interference (RNAi) has been reported to be an effective reverse genetic approach for studying gene function in various organisms. To assess RNAi as a means of examining genes expressed in ovarian follicle cells for their involvement in embryonic dorsal-ventral patterning, we tested the ability of transgenically expressed double-stranded RNA (dsRNA) directed against the dorsal group gene windbeutel to generate phenotypic effects in the progeny of expressing females. We observed that expression in follicle cells under the control of Gal4 transcribed from the strong and widely expressed alphaTub84B or Actin5C promoters led to efficient dorsalization of progeny embryos. Surprisingly, a variety of strongly expressed follicle cell-specific Gal4 enhancer trap lines failed to elicit an RNAi phenotype in combination with the windbeutel-specific dsRNA. These results stress the importance of careful choice of expression system and of conditions for use in transgenic RNAi-mediated studies of gene function.     

Intestinal stem cells in the adult Drosophila midgut

Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury.     

Regulation of Drosophila matrix metalloprotease Mmp2 is essential for wing imaginal disc:trachea association and air sac tubulogenesis

The Drosophila Dorsal Air Sac Primordium (ASP) is a tracheal tube that grows toward Branchless FGF-expressing cells in the wing imaginal disc. We show that the ASP arises from a tracheal branch that invades the basal lamina of the disc to juxtapose directly with disc cells. We examined the role of matrix metalloproteases (Mmps), and found that reducing Mmp2 activity perturbed disc-trachea association, altered peritracheal distributions of collagen IV and Perlecan, misregulated ASP growth, and abrogated development of the dorsal air sacs. Whereas the function of the membrane-tethered Mmp2 in the ASP is non-cell autonomous we find that it may have distinct tissue-specific roles in the ASP and disc. These findings demonstrate a critical role for Mmp2 in tubulogenesis post-induction, and implicate Mmp2 in regulating dynamic and essential changes to the extracellular matrix.     

The pioneer gene, apontic, is required for morphogenesis and function of the Drosophila heart

In an effort to isolate genes required for heart development and to further our understanding of cardiac specification at the molecular level, we screened PlacZ enhancer trap lines for expression in the Drosophila heart. One of the lines generated in this screen, designated B2-2-15, was particularly interesting because of its early pattern of expression in cardiac precursor cells, which is dependent on the homeobox gene tinman, a key determinant of heart development in Drosophila. We isolated and characterized a gene in the vicinity of B2-2-15 that exhibits an identical expression pattern than the reporter gene of the enhancer trap. The product of his gene, apontic (apt; see also Gellon et al., 1997), does not appear to have any homology with known genes. apt mutant embryos show distinct abnormalities in heart morphology as early as mid-embryonic stages when the heat tube assembles, in that segments of heart cells (those of myocardial and pericardial identity) are often missing. Most strikingly, however, apt mutant embryos or larvae only develop a much reduced heart rate, perhaps because of defects in the assembly of an intact heart tube and/or because of defects in the function or physiological control of the myocardial cells, which normally mediate heart contractions. These cardiac defects may be the cause of death of these mutants during late embryonic or early larval stages.     

Stage-specific regulation of actin genes in Drosophila wing cells

Extreme and rapid changes in the synthesis of messenger RNAs and proteins accompany differentiation in wing tissues of Drosophila. Of the six actin genes, at least three are expressed in wing cells, some during the most extreme changes in cell shape. However, different messages of the set appear, decay, and reappear on a regulated temporal program. These results show that actin expression is stage-specific in a single cell type.     

Juvenile hormone and methyl farnesoate production in cockroach embryos in relation to dorsal closure and the reproductive modes of different species of cockroaches

Juvenile hormone (JH), produced by the corpora allata (CA), is first detectable after dorsal closure, a conspicuous event in embryogenesis. The present research found that the timing of dorsal closure was consistently at about 45% of the total embryonic development time across most of the oviparous and ovoviviparous cockroach species examined. These included the ovoviviparous cockroaches Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, Phoetalia pallida, Schultesia lampyridiformis, and Panchlora nivea, as well as the oviparous cockroaches Blatta orientalis, Periplaneta americana, Eurycotis floridana, and Supella longipalpa. However, the only known viviparous cockroach Diploptera punctata completed dorsal closure at 20.8% of embryo development time. Methyl farnesoate (MF), the immediate precursor of JH III, is considered a functional molecule in crustaceans; however, in insects its function is still unclear. To understand the role of JH and MF in cockroach embryos, I compared JH and MF biosynthesis and release in several cockroach species of known phylogenetic relationships. Using a radiochemical assay, the present research showed that cockroach embryos representing all three reproductive modes produced and released both JH and MF, as previously shown for B. germanica, N. cinerea, and D. punctata. Members of a pair of embryonic CA from B. discoidalis, B. fumigata, R. maderae, and D. punctata were incubated with and without farnesol. MF accumulated in large amounts only in CA of R. maderae in the presence of farnesol, which indicates that control of the last step of biosynthesis of JH, conversion of MF into JH by MF epoxidase, is probably a rate-limiting step in this species.     

Specialized extraembryonic cells connect embryonic and extraembryonic epidermis in response to Dpp during dorsal closure in Drosophila

Dorsal closure in Drosophila embryogenesis involves expansion of the dorsal epidermis, followed by closure of the opposite epidermal edges. This process is driven by contractile force generated by an extraembryonic epithelium covering the yolk syncytium known as the amnioserosa. The secreted signaling molecule Dpp is expressed in the leading edge of the dorsal epidermis and is essential for dorsal closure. We found that the outermost row of amnioserosa cells (termed pAS) maintains a tight basolateral cell-cell adhesion interface with the leading edge of dorsal epidermis throughout the dorsal closure process. pAS was subject to altered cell motility in response to Dpp emanating from the dorsal epidermis, and this response was essential for dorsal closure. alphaPS3 and betaPS integrin subunits accumulated in the interface between pAS and dorsal epidermis, and were both required for dorsal closure. Looking at alphaPS3, type I Dpp receptor, and JNK mutants, we found that pAS cell motility was altered and pAS and dorsal epidermis adhesion failed under the mechanical stress of dorsal closure, suggesting that a Dpp-mediated mechanism connects the squamous pAS to the columnar dorsal epidermis to form a single coherent epithelial layer.     

Ovarian defects in hybrids of the species pair Drosophila virilis and Drosophila texana

In interspecific matings between Drosophila virilis and Drosophila texana female sterility is observed in F₂ hybrid females. A previous study has shown that no vitellogenin synthesis occurs in the fat body of sterile hybrid females. The results presented in this paper show that hybrid ovaries of sterile females transplanted into the abdomens of females of the parental species are not able to develop upon maturity. With few exceptions, the hybrid ovaries remained alive in the host environment, but their oocytes failed to develop to vitellogenic stages. Thus, in hybrid females between Drosophila virilis and Drosophila texana sterility is the result of defects in both the two main developmental processes of egg maturation, the synthesis of vitellogenins in the fat body and the uptake of vitellogenins by the ovary. Dev Genet 20:47–52, 1997. © 1997 Wiley-Liss, Inc.