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Controlled signaling activity is vital for normal tissue homeostasis and oncogenic signaling activation facilitates tumorigenesis. Here, we combine single-cell transcriptomics with in-depth genetic and imaging analysis to investigate the role of the EGFR-Ras and Hedgehog signaling pathways in homeostasis of the Drosophila follicle stem cell lineage. We find that Hedgehog signaling simultaneously promotes an undifferentiated state and induces differentiation via activation of the epithelial-mesenchymal-transition associated transcription factor Zfh1. Overactivation of Hedgehog signaling generates a mixed transcriptional state comparable to partial epithelial-mesenchymal-transition. EGFR-Ras overactivation induces cell cycle defects by activating the transcription factors Pointed and E2f1 and impedes differentiation. Overactivation of both pathways blocks differentiation and induces tumor-like growth where follicle cells exhibit a loss of tissue architecture, sustained proliferation and a reduced lifespan of the host. These findings provide new insight into how signaling pathways converge at the transcriptional level to prevent malignant cell behavior. Aberrant signaling pathway activation is a key driver of tumorigenesis. Using single-cell RNA sequencing, the authors unravel how EGFR-Ras and Hedgehog signaling promote distinct steps of tumor progression in a Drosophila ovarian tumor model.

Neuropeptides play crucial roles in regulating context-dependent behaviors, but the underlying mechanisms remain elusive. We investigate the role of the neuropeptide SIFa and its receptor SIFaR in regulating two distinct mating duration behaviors in male Drosophila : Longer-Mating-Duration (LMD) and Shorter-Mating-Duration (SMD). We found that SIFaR expression in specific neurons is required for both LMD and SMD behaviors. Social context and sexual experience lead to synaptic reorganization between SIFa and SIFaR neurons, altering internal states of brain. We revealed that the SIFa-SIFaR/Crz-CrzR neuropeptide relay pathway is essential for generating distinct interval timing behaviors, with Crz neurons being responsive to the activity of SIFa neurons. Additionally, CrzR expression in glial cell population is critical for regulating LMD behavior. Our study provides insights into how neuropeptides and their receptors modulate context-dependent behaviors through synaptic plasticity and calcium signaling, with implications for understanding the neural circuitry underlying interval timing and neuropeptidergic system modulation of behavioral adaptations.

During Drosophila epithelial development, dynamic signalling filopodia (cytonemes) establish direct contacts between distant cells to facilitate the formation of the Hedgehog signalling gradient. However, not much is known about how cytonemes are regulated. In this study, we show that cytoneme dynamics and Hedgehog signalling in the Drosophila epithelia depend on the Epidermal Growth Factor pathway and on its downstream effector Ras1. We describe that EGFR/Ras1 pathway is required to maintain in the wing disc epithelium the basal plasma membrane levels of Interference Hedgehog (Ihog), a critical Hh co-receptor and adhesion protein. In addition, our data demonstrate that filamin A or Cheerio in Drosophila , responds to both Ihog and EGFR pathway and recruited to the basal site of the plasma membrane. This recruitment contributes to Ihog’s role in stabilizing cytonemes. The Hedgehog signalling gradient is established by dynamic signalling filopodia (cytonemes). Here, the authors show that EGF signalling may regulate cytoneme dynamics by maintaining basal plasma membrane levels of the Hedgehog co-receptor Ihog in the wing disc epithelium.

JNK signaling is a critical regulator of inflammation and regeneration, but how it is controlled in specific tissue contexts remains unclear. Here we show that, in the Drosophila intestine, the TNF-type ligand, Eiger (Egr), is expressed exclusively by intestinal stem cells (ISCs) and enteroblasts (EBs), where it is induced by stress and during aging. Egr preferentially activates JNK signaling in a paracrine fashion in differentiated enterocytes (ECs) via its receptor, Grindelwald (Grnd). N -glycosylation genes ( Alg3 , Alg9 ) restrain this activation, and stress-induced downregulation of Alg3 and Alg9 correlates with JNK activation, suggesting a regulatory switch. JNK activity in ECs induces expression of the intermembrane protease Rhomboid (Rho), driving secretion of EGFR ligands Keren (Krn) and Spitz (Spi), which in turn activate EGFR signaling in progenitor cells (ISCs and EBs) to stimulate their growth and division, as well as to produce more Egr. This study uncovers an N -glycosylation-controlled, paracrine JNK-EGFR-JNK feedforward loop that sustains ISC proliferation during stress-induced gut regeneration. Zhang et al. illustrate a spatial activation pattern of JNK signaling within the intestinal stem cell lineage and uncover a paracrine JNK-EGFR-JNK feedforward loop that sustains ISC proliferation during stress-induced gut regeneration.

Proper gonad development is a pre-requisite for gametogenesis and reproduction. During female gonad formation in Drosophila , the EGF receptor (EGFR) signalling pathway ensures the correct number of primordial germ cells (PGCs) populate the larval gonad. We study the gene pointed (pnt) , which acts downstream of the EGFR receptor and belongs to the ETS transcription factor family, with a previously unknown function in gonadogenesis. We report that pnt is expressed in female larval gonads and later in the adult ovarian germline niche and that it is required to sustain proper gametogenesis. Loss of pnt function in female larval gonads, similar to the EGFR, induced PGC overproliferation. Conversely, we isolated a novel mutant allele gene, termed pnt aga , which resulted in agametic gonads and ovaries. While pnt aga embryos developed gonads containing a normal complement of PGCs, these are subsequently lost by apoptosis during late larval and pupal stages. Molecular characterization of pnt aga revealed reduced expression levels of the different pnt isoforms, unveiling a complex autoregulatory network involving the three Pnt proteins. We propose that germline survival in Drosophila gonads requires a precise tuning of EGFR signalling to ensure the appropriate transcriptional activation of its target pnt .

Signalling molecules often contribute to several downstream pathways that produce distinct transcriptional outputs and cellular phenotypes. One of the major unanswered questions in cell biology is how multiple activities of signalling molecules are coordinated in space and time in vivo. Here, we focus on the Signal Transducer and Activator of Transcription (STAT) protein as a paradigm of signalling molecules involved in several independent signalling pathways. Using Drosophila wing discs as an in vivo model, we demonstrate an interplay of at least three STAT activities in this tissue. In addition to the ‘canonical’ pathways, in which STAT is phosphorylated and activated by Janus Kinases, STAT is involved in two ‘non-canonical’ pathways. In one pathway, STAT is activated by the Epidermal Growth Factor Receptor (EGFR), promoting apoptosis. In the other, it binds the Heterochromatin Protein 1 (HP1) to enhance heterochromatin formation. We provide evidence that while the ‘canonical’ STAT signalling is dominant over ‘non-canonical’ pathways, EGFR:STAT and HP1:STAT pathways compete for the availability of unphosphorylated STAT. We also describe a central role for the cell-cell adhesion protein E-cadherin, with both EGFR and STAT colocalising with E-cadherin at cell-cell junctions and on intracellular vesicles. We show that elevated intracellular E-cadherin promotes EGFR:STAT pathway leading to apoptosis, which is prevented by inhibiting E-cad endocytosis. Taken together, we conclude that E-cadherin controls the balance between two non-canonical STAT activities. We hypothesise that this balance represents a tumour-suppressive mechanism, in which junctional disassembly in dysregulated epithelial-to-mesenchymal transitions would shift this balance towards the EGFR:STAT signalling to promote apoptosis.

Mitochondria are essential organelles that have recently emerged as hubs for several metabolic and signaling pathways in the cell. Mitochondrial morphology is regulated by constant fusion and fission events to maintain a functional mitochondrial network and to remodel the mitochondrial network in response to external stimuli. Although the role of mitochondria in later stages of spermatogenesis has been investigated in depth, the role of mitochondrial dynamics in regulating early germ cell behavior is relatively less-well understood. We previously demonstrated that mitochondrial fusion is required for germline stem cell (GSC) maintenance in the Drosophila testis. Here, we show that mitochondrial fission is also important for regulating the maintenance of early germ cells in larval testes. Inhibition of Drp1 in early germ cells resulted in the loss of GSCs and spermatogonia due to the accumulation of reactive oxygen species (ROS) and activation of the EGFR pathway in adjacent somatic cyst cells. EGFR activation contributed to premature germ cell differentiation. Our data provide insights into how mitochondrial dynamics can impact germ cell maintenance and differentiation via distinct mechanisms throughout development.

The Hippo pathway regulates proliferation and survival in Drosophila and mammals, although shared transcriptional targets of their effectors have not been identified. Mammalian YAP controls expression of the EGFR ligand amphiregulin to regulate epithelial-to-mesenchymal transition in mammary epithelial cells, and the EGFR pathway genetically interacts with Yorkie in Drosophila. The Hippo signalling pathway regulates cellular proliferation and survival, thus has profound effects on normal cell fate and tumorigenesis 1 , 2 , 3 . The pivotal effector of this pathway is YAP (yes-associated protein), a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation 4 , 5 , 6 , 7 , 8 , 9 , 10 . So far, studies of YAP target genes have focused on cell-autonomous mediators; here we show that YAP-expressing MCF10A breast epithelial cells enhance the proliferation of neighbouring untransfected cells, implicating a non-cell-autonomous mechanism. We identify the gene for the epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) as a transcriptional target of YAP, whose induction contributes to YAP-mediated cell proliferation and migration, but not EMT. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1 and 2 (large tumour suppressor 1 and 2) is sufficient to induce AREG expression, consistent with physiological regulation of AREG by the Hippo pathway. Genetic interaction between the Drosophila YAP orthologue Yorkie and Egfr signalling components supports the link between these two highly conserved signalling pathways. Thus, YAP-dependent secretion of AREG indicates that activation of EGFR signalling is an important non-cell-autonomous effector of the Hippo pathway, which has implications for the regulation of both physiological and malignant cell proliferation.

Epithelial tissues undergo cell turnover both during development and for homeostatic maintenance. Removal of cells is coordinated with the increase in number of newly dividing cells to maintain barrier function of the tissue. In Drosophila metamorphosis, larval epidermal cells (LECs) are replaced by adult precursor cells called histoblasts. Removal of LECs must counterbalance the exponentially increasing adult histoblasts. Previous work showed that the LEC removal accelerates as endocytic activity decreases throughout all LECs. Here, we show that the acceleration is accompanied by a mode switching from isolated single-cell apoptosis to clustered ones induced by the endocytic activity reduction. We identify the epidermal growth factor receptor (EGFR) pathway via extracellular-signal regulated kinase (ERK) activity as the main components downstream of endocytic activity in LECs. The reduced ERK activity, caused by the decrease in endocytic activity, is responsible for the apoptotic mode switching. Initially, ERK is transiently activated in normal LECs surrounding a single apoptotic LEC in a ligand-dependent manner, preventing clustered cell death. Following the reduction of endocytic activity, LEC apoptosis events do not provoke these transient ERK up-regulations, resulting in the acceleration of the cell elimination rate by frequent clustered apoptosis. These findings contrasted with the common perspective that clustered apoptosis is disadvantageous. Instead, switching to clustered apoptosis is required to accommodate the growth of neighboring tissues.

Neuronal birth and specification must be coordinated across the developing brain to generate the neurons that constitute neural circuits. We used the Drosophila visual system to investigate how development is coordinated to establish retinotopy, a feature of all visual systems. Photoreceptors achieve retinotopy by inducing their target field in the optic lobe, the lamina neurons, with a secreted differentiation cue, epidermal growth factor (EGF). We find that communication between photoreceptors and lamina cells requires a signaling relay through glia. In response to photoreceptor-EGF, glia produce insulin-like peptides, which induce lamina neuronal differentiation. Our study identifies a role for glia in coordinating neuronal development across distinct brain regions, thus reconciling the timing of column assembly with that of delayed differentiation, as well as the spatiotemporal pattern of lamina neuron differentiation.