Jean-Paul Vincent

Recovery from tissue stress requires the elimination of damaged cells and the commensurate production of healthy cells. How these two processes are coordinated to ensure tissue recovery remains poorly understood. Here we show that the c-Jun N-terminal kinase (JNK) pathway orchestrates apoptosis and proliferation, two seemingly opposite cellular behaviours, in response to epithelial stress. We took advantage of new optogenetic tools to trigger a pulse of JNK signalling activity in a defined domain of Drosophila wing primordia and mapped the response in time and space. Within 3 h after induction, the cells within the activation domain arrest proliferation, while a subset undergo apoptosis. As JNK signalling returns to background level, apoptosis subsides and proliferation resumes to allow completion of development with high fidelity. Using a sensitive reporter, we find that while JNK triggers apoptosis, the level of JNK activity does not correlate with apoptosis on a cell-by-cell basis. We show that the cell cycle phase modulates the pro-apoptotic activity of JNK signalling: G2 arrest is protective, while, in G1, mild JNK signalling activity suffices to trigger apoptosis. Transcriptomic analysis identified the genes encoding Dally and Dally-like protein (Dlp) among the most upregulated by excess JNK signalling. We show that these glypicans contribute to tissue repair by enhancing surface retention of the ligands Wingless (a Wnt) and Dpp (a BMP). We suggest that the resulting local increase in morphogen signalling provides subsequent protection from the pro-apoptotic effect of JNK signalling, allowing surviving cells to re-enter the cell cycle and resume development. Accordingly, recovery from ribosomal protein deficiency or irradiation-induced stress is impaired upon reduction of Dlp activity. Our work shows how a single stress pathway can selectively trigger apoptosis while protecting cells that can contribute to tissue recovery during resumption of normal development.

Summary: Cells and tissues have the ability to respond to different stresses and maintain their homeostasis. The b-Zip transcription factor Xrp1 was found to play a pivotal role in this context in Drosophila . Xrp1 is required for the response to irradiation, in genetic conditions of ribosomal protein (Rp) deficiency leading to cell competition, and more generally in mediating the Integrated Stress Response (ISR). However, how these diverse stresses converge to and activate Xrp1 is still unclear. Our results demonstrate the existence of three separate mechanisms of Xrp1 induction. In response to Rp stress, a short isoform of Xrp1 is produced and requires the function of the ribosomal protein RpS12. In response to irradiation, a specific mRNA isoform is transcriptionally induced, which encodes the Xrp1-short protein isoform, in a p53-dependent manner. In response to the ISR, the long isoform of Xrp1 is translated by the derepression of a uORF present on the Xrp1 5’UTR, a mechanism that requires eIF2α phosphorylation, RpS12, and a transcriptional induction by ATF4. Therefore, our work provides a comprehensive understanding of the role of Xrp1 in different situations of cellular stress, illustrating the complex level of integration of the mechanisms controlling this central gene.

Glycans impact every aspect of physiology. Despite the rapid increase in relevance and translational potential, there remains a lack of tools for studying glycosylation in vivo . Here, we present FlyMOE, a chemical-genetic platform for targeted glycoprotein profiling in Drosophila melanogaster . Transgenic flies are equipped with biosynthetic capabilities to install bioorthogonally-tagged monosaccharides into the cellular glycoproteome. The genetic tractability of Drosophila allows for versatile application based on developmental stages, genetic drivers and glycosyltransferase enzymes. We employ FlyMOE to create an overview of the glycoproteome of Drosophila tissues and embryos based on mass spectrometry glycoproteomics, and investigate the substrates of individual members of the PGANT glycosyltransferase family in vivo . FlyMOE will allow the detailed investigation of the glycoproteome in Drosophila and similarly genetically modifiable in vivo model systems.

The Wnt family ligands carry a lipid moiety that must be shielded from the aqueous environment. During secretion, this is achieved by the cargo receptor Wntless. The effect of acute endocytosis inhibition suggests that Wingless dissociates from Wntless at the apical plasma membrane but is prevented from being released extracellularly by rapid endocytosis. Super-resolution microscopy shows that Wingless then traffics, without Wntless, through specialized Rab7/Rab4-positive endosomes before basolateral release. In the absence of endocytosis, apical release is allowed, albeit in punctae. Similar punctae are formed upon abrogation of the ceramide synthase Schlank. We suggest that they represent insoluble aggregates since they are ‘solubilized’ by the lipid-binding protein Dally-like protein. Moreover, purified Wingless forms similar-looking structures that are solubilised by liposomes in vitro. These findings suggest that membrane lipid composition could modulate the stability of the Wingless–Wntless complex and/or the lipid bilayer’s ability to accommodate the Wingless lipid. Together with rapid endocytosis, this ensures orderly transfer of Wingless from Wntless to the membrane, preventing aggregation, and promoting basolateral secretion.

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Certain animals exhibit remarkable regenerative capacity after injury ¹ . Regeneration demonstrates that differentiated tissues can re-initiate organogenic program to rebuild lost structures ² . Whether this latent developmental plasticity can be broadly reawakened in vivo without injury remains unknown. Here we show that a single transcription factor, Dichaete, the Drosophila homolog of Sox2, is sufficient to induce entire wings and sporadically well-patterned eyes, antennae and leg structures from their corresponding host organs. In contrast to transdetermination, these newly formed organs consistently retain their original identity. The activation of early cell fate markers suggests that Dichaete triggers an organ-primordium competence program whose identity is specified by local positional cues. We isolated the reprogrammed progenitor cell populations and profiled the downstream factors. Genetic analysis identifies a minimal combination of three downstream mediators, Escargot, Maelstrom and Unpaired1, that largely recapitulates wing induction. Furthermore, heterologous expression of the mouse Sox2 is also sufficient to induce nascent wing discs, indicating evolutionary conservation of Dichaete/Sox2 in inducing organogenesis. Together, our results demonstrate that defined factors can unlock latent organogenic potential, inducing de novo formation of entire organs. This study opens a potential avenue for in vivo replacement of diseased or damaged organs.

Wnts are secreted signalling molecules that regulate development and adult homeostasis. Most Wnts carry a lipid moiety that must be shielded from the aqueous environment. In the secretory pathway, this is achieved by a hydrophobic tunnel in Wntless, a multipass transmembrane protein. However, the Wnt lipid moiety must be released from Wntless before Wnts can engage with Frizzled receptors on receiving cells. Here we address the cell biological basis of Wnt-Wntless dissociation, using as a model the secretion of Drosophila Wingless in wing primordia. Super-resolution microscopy shows that Wingless first reaches the apical surface before being re-internalized to reach, without Wntless, specialized Rab7/Rab4-positive endosomes. From there Wingless traffics to the basolateral membrane where it can engage with glypicans to form a basolateral gradient. Acute inhibition of endocytosis, either with a temperature-sensitive dynamin mutant or a novel optogenetic means of inhibiting clathrin, leads to apical Wingless release in abnormal punctae devoid of Wntless, suggesting that Wingless-Wntless dissociation commences at the apical surface, perhaps because of a distinct lipid composition there. Indeed, similar looking punctae are produced upon genetic abrogation of the ceramide synthase Schlank, specifically in Wingless-producing cells. These punctae resemble insoluble aggregates that form in vitro upon detergent removal. Accordingly, punctae formation can be prevented by shielding the Wingless lipid, in vivo with excess Dally-like protein (Dlp) or in vitro with liposomes. Our results show that membrane lipid composition modulates the orderly transfer of Wingless lipid from Wntless to the inner endosomal surface thus preventing aggregation and ensuring seamless secretion in the basolateral space.

Growth deceleration before growth termination is a universal feature of growth during development. Transcriptomics analysis reveals that during their two-day period of growth deceleration, wing imaginal discs of Drosophila undergo a progressive metabolic shift from oxidative phosphorylation towards glycolysis. Ultra-sensitive reporters of HIF-1α stability and activity show that imaginal discs become increasingly hypoxic during development in normoxic conditions, suggesting that limiting oxygen supply could underlie growth deceleration. We confirm the expectation that rising levels of HIF-1α dampen TOR signalling activity through transcriptional activation of REDD1. Conversely, excess TOR leads, in a tissue-size-dependent manner, to hypoxia, which boosts HIF-1α levels and activity. Thus, HIF-1α mediates a negative feedback loop whereby TOR signalling triggers hypoxia, which in turn reduces TOR signalling. Abrogation of this feedback by Sima/HIF-1α knockdown leads to cellular stress, which is alleviated by reduced TOR signalling or a modest increase in environmental oxygen. We conclude that Sima/HIF-1α prevents TOR-mediated growth from depleting local oxygen supplies during normal development.