Egfr/Ras pathway mediates interactions between peripodial and disc proper cells in Drosophila wing discs

All imaginal discs in Drosophila are made up of a layer of columnar epithelium or the disc proper and a layer of squamous epithelium called the peripodial membrane. Although the developmental and molecular events in columnar epithelium or the disc proper are well understood, the peripodial membrane has gained attention only recently. Using the technique of lineage tracing, we show that peripodial and disc proper cells arise from a common set of precursors cells in the embryo, and that these cells diverge in the early larval stages. However, peripodial and disc proper cells maintain a spatial relationship even after the separation of their lineages. The peripodial membrane plays a significant role during the regional subdivision of the wing disc into presumptive wing, notum and hinge. The Egfr/Ras pathway mediates this function of the peripodial membrane. These results on signaling between squamous and columnar epithelia are particularly significant in the context of in vitro studies using human cell lines that suggest a role for the Egfr/Ras pathway in metastasis and tumour progression.     

Novel signaling from the peripodial membrane is essential for eye disc patterning in Drosophila

The Drosophila eye disc is a sac of single layer epithelium with two opposing sides, the peripodial membrane (PM) and the disc proper (DP). Retinal morphogenesis is organized by Notch signaling at the dorsoventral (DV) boundary in the DP. Functions of the PM in coordinating growth and patterning of the DP are unknown. We show that the secreted proteins, Hedgehog, Wingless, and Decapentaplegic, are expressed in the PM, yet they control DP expression of Notch ligands, Delta and Serrate. Peripodial clones expressing Hedgehog induce Serrate in the DP while loss of peripodial Hedgehog disrupts disc growth. Furthermore, PM cells extend cellular processes to the DP. Therefore, peripodial signaling is critical for eye pattern formation and may be mediated by peripodial processes.     

Decapentaplegic head capsule mutations disrupt novel peripodial expression controlling the morphogenesis of the Drosophila ventral head

Drosophila adult structures derive from imaginal discs, which are sacs with apposed epithelial sheets, the disc proper (DP) and the peripodial epithelium (PE). The Drosophila TGF-beta family member decapentaplegic (dpp) contributes to the development of adult structures through expression in all imaginal discs, driven by enhancers from the 3' cis-regulatory region of the gene. In the eye/antennal disc, there is 3' directed dpp expression in both the DP and PE associated with cell proliferation and eye formation. Here, we analyze a new class of dpp cis-regulatory mutations, which specifically disrupt a previously unknown region of dpp expression, controlled by enhancers in the 5' regulatory region of the gene and limited to the PE of eye/antennal discs. These are the first described Drosophila mutations that act by solely disrupting PE gene expression. The mutants display defects in the ventral adult head and alter peripodial but not DP expression of known dpp targets. However, apoptosis is observed in the underlying DP, suggesting that this peripodial dpp signaling source supports cell survival in the DP.     

Wg and Egfr signalling antagonise the development of the peripodial epithelium in Drosophila wing discs

Imaginal discs contain a population of cells, known as peripodial epithelium, that differ morphologically and genetically from the rest of imaginal cells. The peripodial epithelium has a small contribution to the adult epidermis, though it is essential for the eversion of the discs during metamorphosis. The genetic mechanisms that control the identity and cellular morphology of the peripodial epithelia are poorly understood. In this report, we investigate the mechanisms that pattern the peripodial side of the wing imaginal disc during early larval development. At this time, the activities of the Wingless (Wg) and Epidermal growth factor receptor (Egfr) signalling pathways specify the prospective wing and notum fields, respectively. We show that peripodial epithelium specification occurs in the absence of Wingless and Egfr signalling. The ectopic activity in the peripodial epithelium of any of these signalling pathways transforms the shape of peripodial cells from squamous to columnar and resets their gene expression profile. Furthermore, peripodial cells where Wingless signalling is ectopically active acquire hinge identity, while ectopic Egfr activation results in notum specification. These findings suggest that suppression of Wg and Egfr activities is an early step in the development of the peripodial epithelium of the wing discs.     

Peripodial cells regulate proliferation and patterning of Drosophila imaginal discs

Cells employ a diverse array of signaling mechanisms to establish spatial patterns during development. Nowhere is this better understood than in Drosophila, where the limbs and eyes arise from discrete epithelial sacs called imaginal discs. Molecular-genetic analyses of pattern formation have generally treated discs as single epithelial sheets. Anatomically, however, discs comprise a columnar cell monolayer covered by a squamous epithelium known as the peripodial membrane. Here we demonstrate that during development, peripodial cells signal to disc columnar cells via microtubule-based apical extensions. Ablation and targeted gene misexpression experiments demonstrate that peripodial cell signaling contributes to growth control and pattern formation in the eye and wing primordia. These findings challenge the traditional view of discs as monolayers and provide foundational evidence for peripodial cell function in Drosophila appendage development.     

Postembryonic development of the wing imaginal discs in the female wingless bagworm moth Eumeta variegata (Lepidoptera,Psychidae)

The process of wing disc development and degeneration in the bagworm moth Eumeta variegata was investigated histologically. Morphological differences between two sexes first appear in the penultimate (eighth) larval instar. In the male, wing discs proliferate rapidly in the penultimate larval instar and continue proliferating; a conspicuous peripodial epithelium forms in the last (ninth) larval instar. The hemopoietic organs break down in this stage and disappear completely by the prepupal stage. In the female, in contrast, the wing discs remain as in the previous (seventh) instar, without proliferation of cells inside. No peripodial epithelium forms in the penultimate instar or later. Hemopoietic organs are still attached to the wing discs in the last larval instar and the entire wing discs transform into a plain, thick epidermis in the prepupal period. It is suggested that the hemopoietic organs may prevent the wing discs from developing in E. variegata.     

Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). Here, we uncover a new morphogenetic/cellular mechanism for disc eversion. We show that imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. We found that the JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells.     

The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis.

In Drosophila, the Jun-N-terminal Kinase-(JNK) signaling pathway is required for epithelial cell shape changes during dorsal closure of the embryo. In the absence of JNK pathway activity, as in the DJNKK/hemipterous (hep) mutant, the dorsolateral ectodermal cells fail both to elongate and move toward the dorsal midline, leading to dorsally open embryos. We show here that hep and the JNK pathway are required later in development, for correct morphogenesis of other epithelia, the imaginal discs. During metamorphosis, the imaginal discs undergo profound morphological changes, giving rise to the adult head and thoracic structures, including the cuticle and appendages. hep mutant pupae and pharate adults show severe defects in discs morphogenesis, especially in the fusion of the two lateral wing discs. We show that these defects are accompanied by a loss of expression of puckered (puc), a JNK phosphatase-encoding gene, in a subset of peripodial cells that ultimately delineates the margins of fusing discs. In further support of a role of puc in discs morphogenesis, pupal and adult hep phenotypes are suppressed by reducing puc function, indicative of a negative role of puc in disc morphogenesis. Furthermore, we show that the small GTPase Dcdc42, but not Drac1, is an activator of puc expression in a hep-dependent manner in imaginal discs. Altogether, these results demonstrate a new role for the JNK pathway in epithelial morphogenesis, and provide genetic evidence for a role of the peripodial membrane in disc morphogenesis. We discuss a general model whereby the JNK pathway regulates morphogenesis of epithelia with differentiated edges.     

Lumenal transmission of decapentaplegic in Drosophila imaginal discs

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.     

Hox proteins coordinate peripodial decapentaplegic expression to direct adult head morphogenesis in Drosophila

The Drosophila BMP, decapentaplegic (dpp), controls morphogenesis of the ventral adult head through expression limited to the lateral peripodial epithelium of the eye-antennal disc by a 3.5kb enhancer in the 5' end of the gene. We recovered a 15bp deletion mutation within this enhancer that identified a homeotic (Hox) response element that is a direct target of labial and the homeotic cofactors homothorax and extradenticle. Expression of labial and homothorax are required for dpp expression in the peripodial epithelium, while the Hox gene Deformed represses labial in this location, thus limiting its expression and indirectly that of dpp to the lateral side of the disc. The expression of these homeodomain genes is in turn regulated by the dpp pathway, as dpp signalling is required for labial expression but represses homothorax. This Hox-BMP regulatory network is limited to the peripodial epithelium of the eye-antennal disc, yet is crucial to the morphogenesis of the head, which fate maps suggest arises primarily from the disc proper, not the peripodial epithelium. Thus Hox/BMP interactions in the peripodial epithelium of the eye-antennal disc contribute inductively to the shape of the external form of the adult Drosophila head.     

Drosophila peripodial cells,more than meets the eye?

Drosophila imaginal discs (appendage primordia) have proved invaluable for deciphering cellular and molecular mechanisms of animal development. By combining the accessibility of the discs with the genetic tractability of the fruit fly, researchers have discovered key mechanisms of growth control, pattern formation and long-range signaling. One of the principal experimental attractions of discs is their anatomical simplicity - they have long been considered to be cellular monolayers. During larval stages, however, the growing discs are 2-sided sacs composed of a columnar epithelium on one side and a squamous 'peripodial' epithelium on the other. Recent studies suggest important roles for peripodial epithelia in processes previously assumed to be confined to columnar cell monolayers.     

Jun N-terminal kinase signaling makes a face

decapentaplegic (dpp), the Drosophila ortholog of BMP 2/4, directs ventral adult head morphogenesis through expression in the peripodial epithelium of the eye-antennal disc. This dpp expressing domain exerts effects both on the peripodial epithelium, and the underlying disc proper epithelium. We have uncovered a role for the Jun N-terminal kinase (JNK) pathway in dpp-mediated ventral head development. JNK activity is required for dpp's action on the disc proper, but in the absence of dpp expression, excessive JNK activity is produced, leading to specific loss of maxillary palps. In this review we outline our hypotheses on how dpp acts by both short range and longer range mechanisms to direct head morphogenesis and speculate on the dual role of JNK signaling in this process. Finally, we describe the regulatory control of dpp expression in the eye-antennal disc, and pose the problem of how the various expression domains of a secreted protein can be targeted to their specific functions.     

Cellular events within peripodial epithelia that accompany evagination of Manduca wing discs: Conversion of cuboidal epithelia to columnar epithelia

In the wing disc of Manduca, a sheet of peripodial epithelium completely covers the apical surface of another epithelium destined to form the wing blade. The cubodial cells of the peripodial epithelium not only are attached to a thick basal lamina but also their lateral and basal surfaces are highly convoluted and stain intensely with ruthenium red (RR). In contrast, the columnar cells of the wing epithelium lack both a basal lamina and RR-positive surfaces. During evagination, the RR-positive material disappears and the extent of lateral cell contact within the peripodial epithelium increases. Concurrently with this lateral “zippering”, the entire peripodial sheet contracts and slides over the wing blade epithelium, thereby exposing the wing to the external surface of the insect. Trypsin treatment of Manduca discs accelerates both evagination and the disappearance of RR-positive material from the surfaces of cells in the peripodial epithelium. Apparently contraction of the peripodial sheet and the increase in its lateral cell contacts is accompanied by the disappearance of acidic glycoproteins from its lateral and basal cell surfaces.     

The role of the peripodial membrane in the morphogenesis of the eye-antennal disc ofDrosophila melanogaster

Summary The early morphogenesis of the eye-antennal disc ofDrosophila in response to 20-hydroxy ecdysone involves the curling of the eye anlagen dorsally over the antenna. During this process, the area of the peripodial membrane is substantially reduced. The peripodial membrane is taut at this stage, and if it is cut the curling of the disc cannot continue, and the eye anlagen returns to its original position within one minute of the operation. In contrast, cutting the columnar epithelium between the eye and antennal anlagen does not disrupt curling, but actually facilitates it. During curling, the cells of the peripodial membrane appear healthy, and exhibit basal extensions. We suggest that the curling of the eye is mediated by the conversion of cuboidal peripodial membrane cells into pseudostratified columnar epithelium at the edges of the peripodial membrane. Subsequently, cells of the peripodial membrane secrete first a pupal cuticle, and then an imaginal cuticle.     

器官成形素调控果蝇翅芽细胞形貌的研究进展

果蝇翅芽是研究细胞形貌发生的模式系统。在果蝇翅芽的发育过程中,器官成形素由浓度高的区域(成形素表达细胞)向浓度低的区域(接收细胞)移动,形成动态的浓度梯度。器官成形素信号通路的激活调控翅芽细胞的形貌发生、存活、生长和分化。目前已鉴定的在翅芽细胞表达的器官成形素包括Hedgehog(Hh),Decapentaplegic(Dpp)和Wingless(Wg)。结合国际最新研究进展,本文综述了3种器官成形素在翅芽细胞形貌发生过程中的重要作用,讨论了细胞形貌发生的分子机制。     

Patterns of cell division in imaginal discs of Drosophila

A transmission electron microscopic study of cell division in serially sectioned imaginal discs of early third instar fruitfly larvae revealed that mitotic cells maintain a relationship with the basal surface of the disc through thin cytoplasmic extensions abutting on vesicular material. Two patterns of cell division were discerned. In one, cell divisions were isolated and usually found near the peripodial membrane-disc epithelium junction; in the other, cell divisions were clustered. Observations on cell death and cell division in the peripodial membrane are also reported.     

Intercellular adhesivity and pupal morphogenesis inDrosophila melanogaster

Summary Leg and wing imaginal discs of mature larvae ofDrosophila melanogaster when treated with 0.1% trypsin for 5–10 min underwent a change in shape that closely resembled normal pupal morphogenesis. Simultaneously, the cells of the disc epithelium changed in shape from tall columnar to cuboidal. Colcemid eliminated microtubules but was without effect on the shape of the imaginal discs or their cells. Tryptic digestion reduced non-junctional intercellular adhesivity but septate desmosomes and gap junctions remained intact.It is proposed that the structure of imaginal discs permits the packaging of the anlagen of the adult integument so that they can change shape and replace the larval structures in a brief period. Apparently most of the definitive form of the pupal leg is built into the disc and becomes visible within a few minutes as intercellular adhesivity is changed.     

Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps.     

Towards Long Term Cultivation of Drosophila Wing Imaginal Discs In Vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.     

Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism

Imaginal discs of Drosophila are simple epithelial tissues that undergo dramatic changes in shape during metamorphosis, including elongation to form adult appendages such as legs and wings. We have examined the cellular basis of leg disc morphogenesis by staining filamentous actin to outline cell boundaries in discs and observing cell shapes with scanning confocal laser microscopy (SCLM). Surprisingly, we found that prior to the onset of morphogenesis, cells in the dorsal-lateral regions of leg discs are compressed in the proximal-distal axis and greatly elongated circumferentially. These cells are also asymmetric in the apical-basal axis, being more elongated in the apical-most region of the cell than they are subapically, and frequently contacting different sets of neighbors apically and basally. Elongated cells were first observed in early third instar discs, and persisted through several rounds of cell division as the discs matured. During appendage elongation in vivo and trypsin-accelerated elongation in vitro, these highly asymmetric cells became isometric. As the apical cell profiles changed shape, apical and basal cell contacts came into register. Measurements of apical cell dimensions suggest that changes in cell shape account for most of the elongation in the basitarsal and tibial leg segments between 0 and 6 h after puparium formation (AP). The conversion of a stable population of anisometric cells to isometric dimensions constitutes a novel mechanism for altering the proportions of an epithelial sheet during development.