RESUMO
Investigating organ biology often requires methodologies to induce genetically distinct clones within a living tissue. However, the 3D nature of clones makes sample image analysis challenging and slow, limiting the amount of information that can be extracted manually. Here we develop PECAn, a pipeline for image processing and statistical data analysis of complex multi-genotype 3D images. PECAn includes data handling, machine-learning-enabled segmentation, multivariant statistical analysis, and graph generation. This enables researchers to perform rigorous analyses rapidly and at scale, without requiring programming skills. We demonstrate the power of this pipeline by applying it to the study of Minute cell competition. We find an unappreciated sexual dimorphism in Minute cell growth in competing wing discs and identify, by statistical regression analysis, tissue parameters that model and correlate with competitive death. Furthermore, using PECAn, we identify several genes with a role in cell competition by conducting an RNAi-based screen.
Assuntos
Carya , Animais , Competição entre as Células , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional , Aprendizado de MáquinaRESUMO
Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, our understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, we have monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). We show that the scrib mutation impacts the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch-dedicated transcription factor. The Notch-dependent neoplasms require, however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1 and basic leucine zipper factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally, our work highlights some Notch/scrib specificities, in particular the role of the PAR domain-containing basic leucine zipper transcription factor and Notch direct target Pdp1 for neoplastic growth.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Receptores Notch/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Carcinogênese , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Larva/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Interferência de RNA , Transdução de Sinais , Asas de Animais/metabolismoRESUMO
Cell competition induces the elimination of less-fit "loser" cells by fitter "winner" cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. Here we provide evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. Our data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers.
Assuntos
Competição entre as Células , Proteínas de Ligação a DNA , Proteínas de Drosophila , Proteínas Ribossômicas , Animais , Apoptose/genética , Competição entre as Células/genética , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Estresse Oxidativo/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
During development, cell numbers are tightly regulated, ensuring that tissues and organs reach their correct size and shape. Recent evidence has highlighted the intricate connections between the cytoskeleton and the regulation of the key growth control Hippo pathway. Looking for apical scaffolds regulating tissue growth, we describe that Drosophila melanogaster big bang (Bbg), a poorly characterized multi-PDZ scaffold, controls epithelial tissue growth without affecting epithelial polarity and architecture. bbg-mutant tissues are smaller, with fewer cells that are less apically constricted than normal. We show that Bbg binds to and colocalizes tightly with the ß-heavy-Spectrin/Kst subunit at the apical cortex and promotes Yki activity, F-actin enrichment, and the phosphorylation of the myosin II regulatory light chain Spaghetti squash. We propose a model in which the spectrin cytoskeleton recruits Bbg to the cortex, where Bbg promotes actomyosin contractility to regulate epithelial tissue growth.
