Ben Ewen-Campen

Single-cell RNA sequencing (scRNA-seq) is a powerful approach for characterizing gene expression at single-cell resolution and identifying distinct cell populations within complex tissues. However, tissue dissociation disrupts the spatial relationships among cells, making it difficult to determine their original anatomical locations and interpret gene expression patterns in the context of tissue organization. To address this limitation, we developed FlyAnatoView ( https://www.flyrnai.org/tools/rna_seq_base/web/svg_files/ ), a resource that projects scRNA-seq data onto anatomical diagrams of Drosophila melanogaster . By integrating gene expression data with anatomical representations, FlyAnatoView helps restore spatial context and enables users to explore expression patterns across tissues, organs, and cell types. This resource provides an intuitive way to connect transcriptomic profiles with anatomical structures, facilitating the interpretation and exploration of scRNA-seq datasets.

The Drosophila proventriculus is a bulb-shaped structure at the juncture of the foregut and the midgut, which plays important roles in ingestion, peritrophic membrane synthesis, and the immune response to oral pathogens. A previous study identified a population of cells in the proventriculus which incorporate bromodeoxyuridine (BrdU), a marker of DNA synthesis, and proposed that these cycling cells are multipotent stem cells that replace dying cells elsewhere in the tissue. Here, we re-investigate these cycling cells and find that they do not undergo mitosis, do not generate clonal lineages, and do not proliferate in response to tissue damage, and are therefore not stem cells. Instead, we find that these cells continually endocycle throughout the fly’s life, increasing their ploidy and size, while at the same time cells in this tissue are lost into the gut lumen as the fly ages. Functionally, these cells play a critical role in the synthesis of peritrophic membrane components, and we show that when their endocycling is experimentally increased or decreased, there is a concomitant change in ploidy, tissue size, and peritrophic membrane synthesis. Further, we show that inhibition of endocycling makes flies more susceptible to orally infectious bacteria. Altogether, we show that continual endocycling of these cells is critical for maintaining tissue size and function in the face of cell loss due to aging or tissue damage.

The Drosophila proventriculus is a bulb-shaped structure at the juncture of the foregut and the midgut, which plays important roles in ingestion, peritrophic membrane synthesis, and the immune response to oral pathogens. A previous study identified a population of cells in the proventriculus which incorporate bromodeoxyuridine (BrdU), a marker of DNA synthesis, and proposed that these cycling cells are multipotent stem cells that replace dying cells elsewhere in the tissue. Here, we re-investigate these cycling cells and find that they do not undergo mitosis, do not generate clonal lineages, and do not proliferate in response to tissue damage, and are therefore not stem cells. Instead, we find that these cells continually endocycle throughout the fly’s life, increasing their ploidy and size, while at the same time cells in this tissue are lost into the gut lumen as the fly ages. Functionally, these cells play a critical role in the synthesis of peritrophic membrane components, and we show that when endocycling in these cells is experimentally increased or decreased, there is a concomitant change in ploidy, tissue size, and peritrophic membrane synthesis. Further, we show that inhibition of endocycling makes flies more susceptible to orally infectious bacteria. Altogether, we show that continual endocycling of these cells is critical for maintaining tissue size and function in the face of cell loss due to aging or tissue damage.

During development, sexually reproducing animals must specify and maintain the germ line, the lineage of cells that gives rise to the next generation of animals. In the fruit fly Drosophila melanogaster , germ cell-less ( gcl ) is required for the formation of primordial germ cells in the form of cells that cellularize at the posterior pole of the embryo, called pole cells. Forming pole cells is a mechanism of germ cell formation unique to a subset of insects. Even though most animals do not form pole cells as primordial germ cells, gcl is conserved across Metazoa, raising the question of how this conserved gene acquired its central role in the evolutionarily derived process of pole cell formation. Here, we examine the functions of gcl in two other insects with different modes of germ cell specification: the milkweed bug Oncopeltus fasciatus and the cricket Gryllus bimaculatus . We found that gcl is involved in germ cell development, but not strictly required for germ cell specification, in O. fasciatus , although it appears to function through a mechanism different from that in D. melanogaster . In contrast, we could not detect any impact on the embryonic germ line upon gcl knockdown in G. bimaculatus . This work serves as a case study into how the roles of genes in the process of germ line development can change over evolutionary time across animals. Highlights: germ cell-less knockout reduces germ cell number in Oncopeltus fasciatus . germ cell-less knockdown does not affect germ cell number in Gryllus bimaculatus . The genetic mechanisms through which gcl affects germ cell number have diverged among insects. Graphical

Communication between cells in metazoan organisms is mediated by a remarkably small number of highly conserved signaling pathways. Given this small number of signaling pathways, the existence of multiple related ligands for many of these pathways represents a key evolutionary innovation for encoding complexity into cell-cell signaling. Relatedly, crosstalk between pathways is another critical feature which allows a modest number pathways to ultimately generate an enormously diverse range of outcomes. It would thus be useful to have genetic tools to identify and manipulate not only those cells which express a given signaling ligand, but also those cells that specifically co-express pairs of signaling ligands. We present a collection of split-Gal4 knock-in lines targeting many of the ligands for highly conserved signaling pathways in Drosophila (Notch, Hedgehog, FGF, EGF, TGFβ, JAK/STAT, JNK, and PVR). We demonstrate that these lines faithfully recapitulate the endogenous expression pattern of their targets, and that they can be used to identify cells and tissues that co-express pairs of ligands. As a proof of principle, we demonstrate that the 4th chromosome TGFβ ligands myoglianin and maverick are broadly co-expressed in muscles and other tissues of both larva and adults, and that the JAK/STAT ligands upd2 and upd3 are partially co-expressed from cells of the midgut following gut damage. Together with our previously collection of split-Gal4 lines targeting the seven Wnt ligands, this resource allows Drosophila researchers to identify and genetically manipulate cells that specifically express pairs of conserved ligands from nearly all the major intercellular signaling pathways.

Communication between cells in metazoan organisms is mediated by a remarkably small number of highly conserved signaling pathways. Given the relatively small number of signaling pathways, the existence of multiple related ligands for many of these pathways is thought to represent a key evolutionary innovation for encoding complexity into cell-cell signaling. Relatedly, crosstalk and other interactions between pathways is another critical feature which allows a modest number pathways to ultimately generate an enormously diverse range of outcomes. It would thus be useful to have genetic tools to identify and manipulate not only those cells which express a given signaling ligand, but also those cells that specifically co-express pairs of signaling ligands. Here, we present a collection of split-Gal4 knock-in lines targeting many of the ligands for highly conserved signaling pathways in Drosophila (Notch, Hedgehog, FGF, EGF, TGF β , JAK/STAT, JNK, and PVR). We demonstrate that these lines faithfully recapitulate the endogenous expression pattern of their targets, and that they can be used to specifically identify the cells and tissues that co-express pairs of signaling ligands. As a proof of principle, we demonstrate that the 4th chromosome TGF β ligands myoglianin and maverick are broadly co-expressed in muscles and other tissues of both larva and adults, and that the JAK/STAT ligands upd2 and upd3 are partially co-expressed from cells of the midgut following gut damage. Together with our previously collection of split-Gal4 lines targeting the seven Wnt ligands, this resource allows Drosophila researchers to identify and genetically manipulate cells that specifically express pairs of conserved ligands from nearly all the major intercellular signaling pathways.

Despite the deep conservation of the DNA damage response (DDR) pathway, cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo, and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila, activates epidermal growth factor receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which, in turn, modulates the DDR in a Chk2-, p53-, and E2F1-dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with prosurvival, antiapoptotic activities, thereby facilitating developmental robustness in the face of genomic damage.

Despite the deep conservation of the DNA damage response pathway (DDR), cells in different contexts vary widely in their susceptibility to DNA damage and their propensity to undergo apoptosis as a result of genomic lesions. One of the cell signaling pathways implicated in modulating the DDR is the highly conserved Wnt pathway, which is known to promote resistance to DNA damage caused by ionizing radiation in a variety of human cancers. However, the mechanisms linking Wnt signal transduction to the DDR remain unclear. Here, we use a genetically encoded system in Drosophila to reliably induce consistent levels of DNA damage in vivo , and demonstrate that canonical Wnt signaling in the wing imaginal disc buffers cells against apoptosis in the face of DNA double-strand breaks. We show that Wg, the primary Wnt ligand in Drosophila , activates Epidermal Growth Factor Receptor (EGFR) signaling via the ligand-processing protease Rhomboid, which in turn modulates the DDR in a Chk2, p53, and E2F1- dependent manner. These studies provide mechanistic insight into the modulation of the DDR by the Wnt and EGFR pathways in vivo in a highly proliferative tissue. Furthermore, they reveal how the growth and patterning functions of Wnt signaling are coupled with pro-survival, anti-apoptotic activities, thereby facilitating developmental robustness in the face of genomic damage. Author Summary: Ectopic activation of the highly conserved Wnt signaling pathway has been previously demonstrated to promote resistance to radiation and chemoradiation therapy in a variety of human cancers, yet the mechanisms by which Wnt modulates the DDR pathway are not clearly established. Furthermore, putative interactions between Wnt signaling and the DDR outside the context of pathological Wnt over-expressing tumors have not been clearly elucidated. Here, we show that, in Drosophila , loss of canonical Wnt signaling during development of the highly proliferative wing imaginal disc sensitizes cells to DNA damage, biasing them towards apoptosis and ultimately disrupting normal wing development. In contrast, ectopic Wnt signaling reduces the level of apoptosis for a given level of DNA damage. Mechanistically, we demonstrate that Wnt signaling acts via Epidermal Growth Factor Receptor (EGFR) signaling, a well characterized pro-survival pathway, by activating the ligand-processing protease Rhomboid, and that this effect requires core DDR components Chk2, p53, and E2F1. Altogether, we show that Wnt signaling can promote developmental robustness by opposing apoptosis in the face of DNA damage, and reveal a mechanism by which Wnt signaling modulates the DDR via EGFR signaling.

The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila . However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of “split-intein Gal4” in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type–specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm (“Two Against Background” or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.