Imaginal Ring Research Articles

Polyploid mitosis and depolyploidization promote chromosomal instability and tumor progression in a Notch-induced tumor model.

Ploidy variation is a cancer hallmark and is frequently associated with poor prognosis in high-grade cancers. Using a Drosophila solid-tumor model where oncogenic Notch drives tumorigenesis in a transition-zone microenvironment in the salivary gland imaginal ring, we find that the tumor-initiating cells normally undergo endoreplication to become polyploid. Upregulation of Notch signaling, however, induces these polyploid transition-zone cells to re-enter mitosis and undergo tumorigenesis. Growth and progression of the transition-zone tumor are fueled by a combination of polyploid mitosis, endoreplication, and depolyploidization. Both polyploid mitosis and depolyploidization are error prone, resulting in chromosomal copy-number variation and polyaneuploidy. Comparative RNA-seq and epistasis analysis reveal that the DNA-damage response genes, also active during meiosis, are upregulated in these tumors and are required for the ploidy-reduction division. Together, these findings suggest that polyploidy and associated cell-cycle variants are critical for increased tumor-cell heterogeneity and genome instability during cancer progression.

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector.

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an autoinjector apparatus, trained operators can achieve more efficient and consistent transplantation results compared to those obtained using a manual injector. Here, we cover topics in a chronological fashion: from the crossing of Drosophila lines, to the induction and dissection of the primary tumor, transplantation of the primary tumor into a new adult host and continued generational transplantation of the tumor for extended studies. As a demonstration, here we use Notch intracellular domain (NICD) overexpression induced salivary gland imaginal ring tumors for generational transplantation. These tumors can first be reliably induced in a transition-zone microenvironment within larval salivary gland imaginal rings, then allografted and cultured in vivo to study continued tumor growth, evolution, and metastasis. This allotransplantation method can be useful in potential drug screening programs, as well as for studying tumor-host interactions.

Abstract 4636: Oncogenic Notch triggers neoplastic-tumorigenesis in a transition-zone like tissue microenvironment

Abstract The “seed and soil” hypothesis proposed by Stephen Paget (1889) highlights the importance of tissue microenvironment for secondary tumor formation. The microenvironment that promotes primary tumor origination, however, remains largely unclear. Transition zones, where two types of epithelial tissue meet, have been characterized as high-risk sites for tumorigenesis. Here we show that the Drosophila salivary gland (SG) imaginal ring (ImR) can be used as a model to study tumorigenesis in transition zones. We found that constitutive activation of Notch signaling during the third larval instar drives overproliferation and tumor formation in the SG ImR. Interestingly, tumorigenesis always occurs at the posterior end of the ImR, which is a transitional area between the polyploid giant cells and diploid ImR cells. These Notch-induced tumors display disrupted epithelial organization, grow continuously and can metastasize once transplanted into the abdomen of adult flies, suggesting that they have the characteristics of malignant neoplasms. Further analyses revealed that the SG ImR transition zone possesses endogenously high levels of Janus kinase/signal transducers and activators of transcription (JAK-STAT) and c-Jun amino-terminal kinases (JNK), both of which are necessary for tumor growth. In this region, JNK signaling induces the expression of matrix metalloproteinase 1 (MMP1), which is also required for tumor formation in this model system. Furthermore, we found that ectopic MMP1 expression can transform the anterior end of the SG ImR, which is normally refractory to oncogenic Notch-induced tumorigenesis, into a tumor hotspot. Together, these studies reveal that local endogenous activation of JNK and JAK-STAT signaling creates a niche-like tissue microenvironment that is susceptible to oncogene-induced neoplastic tumor formation. Citation Format: Wu-Min Deng, Sheng-An Yang. Oncogenic Notch triggers neoplastic-tumorigenesis in a transition-zone like tissue microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4636.

Oncogenic Notch Triggers Neoplastic Tumorigenesis in a Transition-Zone-like Tissue Microenvironment

During the initial stages of tumorigenesis, the tissue microenvironment where the pro-tumor cells reside plays a crucial role in determining the fate of these cells. Transition zones, where two types of epithelial cells meet, are high-risk sites for carcinogenesis, but the underlying mechanism remains largely unclear. Here, we show that persistent upregulation of Notch signaling induces neoplastic tumorigenesis in a transition zone between the salivary gland imaginal ring cells and the giant cells in Drosophila larvae. In this region, local endogenous JAK-STAT and JNK signaling creates a tissue microenvironment that is susceptible to oncogenic-Notch-induced tumorigenesis, whereas the rest of the salivary gland imaginal ring is refractory to Notch-induced tumor transformation. JNK signaling activates a matrix metalloprotease (MMP1) to promote Notch-induced tumorigenesis at the transition zone. These findings illustrate the significance of local endogenous inflammatory signaling in primary tumor formation.

Serrate/Notch Signaling Regulates the Size of the Progenitor Cell Pool in Drosophila Imaginal Rings.

Drosophila imaginal rings are larval tissues composed of progenitor cells that are essential for the formation of adult foreguts, hindguts, and salivary glands. Specified from subsets of ectoderm in the embryo, imaginal ring cells are kept quiescent until midsecond larval instar, and undergo rapid proliferation during the third instar to attain adequate numbers of cells that will replace apoptotic larval tissues for adult organ formation. Here, we show that Notch signaling is activated in all three imaginal rings from middle embryonic stage to early pupal stage, and that Notch signaling positively controls cell proliferation in all three imaginal rings during the third larval instar. Our mutant clonal analysis, knockdown, and gain-of-function studies indicate that canonical Notch pathway components are involved in regulating the proliferation of these progenitor cells. Both trans-activation and cis-inhibition between the ligand and receptor control Notch activation in the imaginal ring. Serrate (Ser) is the ligand provided from neighboring imaginal ring cells that trans-activates Notch signaling, whereas both Ser and Delta (Dl) could cis-inhibit Notch activity when the ligand and the receptor are in the same cell. In addition, we show that Notch signaling expressed in middle embryonic and first larval stages is required for the initial size of imaginal rings. Taken together, these findings indicate that imaginal rings are excellent in vivo models to decipher how progenitor cell number and proliferation are developmentally regulated, and that Notch signaling in these imaginal tissues is the primary growth-promoting signal that controls the size of the progenitor cell pool.

Specification of cell fates within the salivary gland primordium

The Drosophila salivary gland is a simple tubular organ derived from a contiguous epithelial primordium, which is established by the activities of the homeodomain-containing proteins Sex combs reduced (SCR), Extradenticle (EXD), and Homothorax (HTH). EGF signaling along the ventral midline specifies the salivary duct fate for cells in the center of the primordium, while cells farther away from the source of EGF signal adopt a secretory cell fate. EGF signaling works, at least in part, by repressing expression of secretory cell genes in the duct primordium, including fork head (fkh), which encodes a winged-helix transcription factor. FKH, in turn, represses trachealess (trh), a duct-specific gene initially expressed throughout the salivary gland primordium. trh encodes a basic helix–loop–helix PAS-domain containing transcription factor that has been proposed to specify the salivary duct fate. In conflict with this model, we find that three genes, dead ringer (dri), Serrate (Ser), and trh itself, are expressed in the duct independently of trh. Expression of all three duct genes is repressed in the secretory cells by FKH. We also show that SER in the duct cells signals to the adjacent secretory cells to specify a third cell type, the imaginal ring cells. Thus, localized EGF- and Notch-signaling transform a uniform epithelial sheet into three distinct cell types. In addition, Ser directs formation of actin rings in the salivary duct.

Ultrastructure of the hindgut of Drosophila larvae, with special reference to the domains identified by specific gene expression patterns.

The hindgut of Drosophila larvae consists of nine domains that have been distinguished by specific gene expression patterns. In the present study, we examined the ultrastructure of the hindgut of Drosophila larvae, with special reference to the domains, in order to determine whether or not the domains are morphologically distinct functional units. Each domain showed specific ultrastructural features that suggested specific corresponding functions. According to the morphological features, terms are proposed for each domain: the imaginal ring; the "pylorus," which has a thick cuticular layer and well-developed sphincter muscles; the "large intestine," which occupies a major middle portion of the hindgut and has a unique dorsal and ventral subdivision; "border cells," which delineate the anterior and posterior borders of the large intestine and the border between the dorsal and ventral domains of the large intestine; and the "rectum," which is situated at the posterior end of the hindgut and has a thick cuticular layer and sphincter muscles. The morphological features indicate that the large intestine has active absorptive activities. The domains, which have been distinguished by gene expressions, were demonstrated to be functional tissue units of the gut.

Expression of cell proliferation‐related PCNA and E2F genes inDrosophilagut and inhibitory effect of nitric oxide

To understand the late gut development and differentiation, identification and characterization of target genes of homeotic genes involved in gut development are required. We have previously reported that homeodomain proteins can regulate expression of the cell proliferation‐related genes. We investigated here the expression of the Drosophila proliferating cell nuclear antigen(PCNA) and E2F(dE2F) genes in larval and adult guts using trans‐genic flies bearing lacZ reporter genes. Both PCNA and dE2F genes were expressed strongly in whole regions of the larval and adult guts including the esophagus, proventriculus, midgut and hindgut, showing higher expression in foregut and hindgut imaginal rings of larva. Nitric Oxide(NO) has been known to be involved in cell proliferation and tumor growth and also to have an antiproliferative activity. Therefore, we also investigated effects of NO on the expression of PCNA and dE2F genes in gut through analyses of lacZ reporter expression level in the SNP (NO donor)‐treated larval guts. Expressions of both PCNA and dE2F were greatly declined by SNP. The inhibitory effect of NO was shown in whole regions of the gut, especially in hindgut, while the internal region of proventriculus, esophagus, foregut imaginal ring and hindgut imaginal ring was resistant. Our results suggest that this inhibitory effect may be related with the antiproliferative activity of NO.

The Drosophila Cyclin D-Cdk4 complex promotes cellular growth

Mammalian cyclin D-Cdk4 complexes have been characterized as growth factor-responsive cell cycle regulators. Their levels rise upon growth factor stimulation, and they can phosphorylate and thus neutralize Retinoblastoma (Rb) family proteins to promote an E2F-dependent transcriptional program and S-phase entry. Here we characterize the in vivo function of Drosophila Cyclin D (CycD). We find that Drosophila CycD-Cdk4 does not act as a direct G(1)/S-phase regulator, but instead promotes cellular growth (accumulation of mass). The cellular response to CycD-Cdk4-driven growth varied according to cell type. In undifferentiated proliferating wing imaginal cells, CycD-Cdk4 caused accelerated cell division (hyperplasia) without affecting cell cycle phasing or cell size. In endoreplicating salivary gland cells, CycD-Cdk4 caused excessive DNA replication and cell enlargement (hypertrophy). In differentiating eyes, CycD-Cdk4 caused cell enlargement (hypertrophy) in post-mitotic cells. Interaction tests with a Drosophila Rb homolog, RBF, indicate that CycD-Cdk4 can counteract the cell cycle suppressive effects of RBF, but that its growth promoting activity is mediated at least in part via other targets.

The Drosophila Pax gene eye gone is required for embryonic salivary duct development.

What are the developmental mechanisms required for conversion of an undifferentiated, two-dimensional field of cells into a patterned, tubular organ? In this report, we describe the contribution of the Drosophila Pax gene eye gone to the development of the embryonic salivary glands and ducts. eye gone expression in salivary tissues is controlled by several known regulators of salivary fate. After the initial establishment of the salivary primordium by Sex combs reduced, fork head excludes eye gone expression from the pregland cells so that its salivary expression is restricted to the posterior preduct cells. trachealess, in contrast, activates eye gone expression in the posterior preduct cells. We have previously described the process by which fork head and the EGF receptor pathway define the border between the gland and duct primordia. Here we show that eye gone is required for the subdivision of the duct primordium itself into the posterior individual duct and the anterior common duct domains. In the absence of eye gone, individual ducts as well as the precursor of the adult salivary glands, the imaginal ring, are absent. We took advantage of this ductless phenotype to show that Drosophila larvae do not have an obligate requirement for salivary glands and ducts. In addition to its role in the salivary duct, eye gone is required in the embryo for the development of the eye-antennal imaginal disc and the chemosensory antennal organ.

ArrowheadEncodes a LIM Homeodomain Protein That Distinguishes Subsets ofDrosophilaImaginal Cells

TheArrowheadgene encodes a LIM-homeodomain transcription factor required for establishment of a subset of imaginal tissues: the abdominal histoblasts and the salivary gland imaginal rings. Consistent with its role in development, during embryogenesisArrowheadis expressed in each abdominal segment and in the labial segment. Late in embryonic development, expression is refined to the abdominal histoblasts and salivary gland imaginal ring cells themselves. When ectopically expressed in imaginal disc cells,Arrowheadcauses programmed cell death and loss of corresponding adult structures. Therefore,Arrowheadexpression is required for development of one set of imaginal cells and is incompatible with development of another, emphasizing the specificity ofArrowheadand the sensitivity of different target cells to its expression. Loss-of-function mutations inArrowheadaffect conserved or invariant amino acids in the LIM- and homeo-domains demonstrating the importance of these residues in LIM homeodomain protein activity.

Establishment of Drosophila imaginal precursor cells is controlled by the Arrowhead gene.

Metamorphosis in Drosophila melanogaster requires synchronization of numerous developmental events that occur in isolated imaginal precursor tissues. The imaginal primordia are established during embryonic stages and are quiescent for much of larval life. The Arrowhead gene is necessary for establishment of proper numbers of cells within a subset of imaginal precursor tissues. Loss-of-function mutations in Arrowhead reduce the number of abdominal histoblasts and salivary gland imaginal ring cells before the proliferative stages of their development. The number of abdominal histoblasts in mutant animals is approximately half that of wild-type, as might result from failure of a single early division of these cells. A neomorphic Arrowhead allele results in the specific loss of the retinal precursors by the early third instar, before they have begun to differentiate. Since Arrowhead mutations affect only subsets of imaginal tissue, there must be distinctions in the developmental regulation of different imaginal precursors. Arrowhead may be part of a regulatory pathway responsible for establishing the proper number of abdominal histoblasts and salivary gland imaginal ring cells. The neomorphic Arrowhead allele, which may cause misexpression of the Arrowhead gene in the eye-antenna imaginal disc, interferes with the establishment or proliferation of retinal precursor cells.

Intrinsic growth control in the imaginal primordia of Drosophila, and the autonomous action of a lethal mutation causing overgrowth

Cell proliferation in Drosophila imaginal discs appears to be regulated by a disc-intrinsic mechanism involving local cell interactions that also control the formation of patterns of differentiation. This growth-control mechanism breaks down in animals homozygous for the mutation lethal (2) giant discs (l(2)gd) which remain as larvae for up to 9 days longer than normal. During this time cell proliferation continues in the imaginal discs as well as in the imaginal rings for the salivary glands, foregut, and hindgut, so that these tissues become greatly overgrown. When wild-type wing discs from mid-third instar larvae were removed and cultured for up to 28 days in wild-type female adult hosts, they grew and terminated growth at a cell number close to that which would be attained in situ by the time of pupariation. On the other hand, wing discs from l(2)gd homozygotes grew rapidly and continuously when cultivated in wild-type hosts, reached an enormous size, and acquired abnormal folding patterns. Overgrowth of mutant imaginal rings also continued during culture of these tissues in wild-type hosts. We conclude that overgrowth in this mutant is due to an autonomous defect in the imaginal primordia, which requires an extended larval period for its expression in situ.

The mode of action of “discless” mutations in Drosophila melanogaster

The development of five different “discless” lethal mutants of Drosophila melanogaster was studied by using whole mounts and histological procedures as well as by radiation-induced mitotic recombination. Rudimentary imaginal discs were found in both newly hatched first-instar and late-third-instar larvae homozygous for each of the mutations. Rather than interfering with the establishment of imaginal discs, the mutations were found to interfere with cell proliferation not only in the imaginal discs but also in the larval central nervous system, the imaginal rings of the salivary gland and foregut, and the gonads. Production of blood cells by the lymph gland was severely reduced in all of the mutants. Autonomous effects on the growth of homozygous clones in a heterozygous background were demonstrated for four of the five mutations. Although some of the mutations had, in general, a more extreme effect on cell proliferation than did others, the effects on different organ systems were not clearly correlated. The mutations also caused a slight reduction in the final cell size in the nonproliferating larval epidermis and histoblasts, but only the most extreme mutation studied caused a reduction in cell number in these tissues. It is concluded that these mutations are not imaginal disc specific, but that they interfere with functions required for cell proliferation in general. Several possible explanations for their lack of effects on embryonic cell proliferation are discussed.

Tissue distribution of isocitrate dehydrogenase in Drosophila melanogaster

Distribution of the soluble form of isocitrate dehydrogenase (ICDH) in imaginal discs, some larval and some adult Drosophila melanogaster structures was determined. ICDH distribution in imaginal discs provides an opportunity to study in morphologically similar cells the degree of biochemical differentiation, the ontogeny of ICDH pattern formation, tissue determination at the disc level and in some instances the position of developmental compartments. Differential activity for ICDH was observed in the eye-antennal disc, dorsal prothoracic disc, all leg discs, wing and haltere discs, genital disc, larval gut, plus the adult ovaries and the male genital structures. Uniform activity was found in the labial disc, larval salivary gland cells, nests of abdominal histoblasts and in larval fat bodies. No detectable ICDH activity was observed in the imaginal cells of the clypeus-labrum, the imaginal cells of the foregut, the imaginal ring of the hindgut, or in clusters of imaginal cells of ICDH negative regions of the midgut. It is concluded that ICDH, like aldehyde oxidase, is non-random in its tissue distribution and does provide an additional internal histochemical marker for developmental studies.

Histological analysis of the dynamics of growth of imaginal discs and histoblast nests during the larval development ofDrosophila melanogaster.

Histological analysis of the dynamics of growth of imaginal discs and histoblast nests during the larval development ofDrosophila melanogaster.

Development of foregut of six-spotted ladybird beetle, Chilomenes sexmaculata fabr. (Coleoptera: Coccinellidae)

A definite anterior imaginal ring is absent in the larva of the six-spotted ladybird beetle, Chilomenes sexmaculata. The oesophageal valve of the larva may be considered to function as an imaginal ring since it forms new cells. The larval cells of the oesophagus are passed into the adult after some cellular differentiation. Some of the cells in the posterior region of the oesophagus degenerate and form darkly staining globular bodies. In the region of the oesophageal valve new cells are formed by mitotic activity. These cells, together with the old larval cells, reconstitute the posterior region (proventriculus and oesophageal valve) of the adult foregut. The anterior region of the oesophagus passes into the adult without any apparent change.

Metamorphic changes in posterior region of foregut of Papilio aristolochiae F. (Lepidoptera: Papilionidae)

The cells of the anterior imaginal ring in Papilio aristolochiae divide mitotically during the early pupal period and increase in number. Some of the cells of the anterior imaginal ring evaginate on the ventral side and form the anlage of ventral oesophageal diverticulum. After the formation of the diverticulum the imaginal ring again becomes divided into anterior and posterior regions, which are the anlagen of the proventriculus and oesophageal valve respectively. The cells of the latter 2 regions remain columnar in the beginning but soon become squamous and folded. The folds of the oesophageal valve form an intussception with the anterior part of the midgut, prior to the imaginal emergence.

Development of ventral oesophageal diverticulum in Papilio aristolochiae F. (Lepidoptera: Papilionidae)

A single ventral oesophageal diverticulum is present in adult Papilio aristolochiae (Lepidoptera: Papilionidae), and a histologic development of this has been studied in detail. The larval oesophageal valve from which the ventral diverticulum arises, has been divided into 5 different regions. The fate of all these regions has been followed during pupation. Cells of the anterior imaginal ring in the pupa divide mitotically and furnish the cells of ventral evagination, which forms the ventral diverticulum. The ventral evagination grows as a result of increase in the number of cells and also cell enlargement. Regions 4 and 5 degenerate completely in the adult, and the degenerated cells are thrown into the body cavity.

Histology of the alimentary canal during the metamorphosis of Hyalophora cecropia (L.)

AbstractThe histology of the alimentary canal is described for the last instar larva, pupa, and adult Hyalophora cecropia (L.). Particular attention is given to the changes occurring in the midgut during the larval‐pupal transformation and in the hindgut during the pupal‐adult transformation. Descriptions are made as to the nature of the imaginal rings at each end of the midgut and a new structure is described in the larval ileum. The formation of adult rectal pads is followed in detail.