RESUMO
Deciphering contributions of specific cell types to organ function is experimentally challenging. The Drosophila midgut is a dynamic organ with five morphologically and functionally distinct regions (R1-R5), each composed of multipotent intestinal stem cells (ISCs), progenitor enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs), and visceral muscle (VM). To characterize cellular specialization and regional function in this organ, we generated RNA-sequencing transcriptomes of all five cell types isolated by FACS from each of the five regions, R1-R5. In doing so, we identify transcriptional diversities among cell types and document regional differences within each cell type that define further specialization. We validate cell-specific and regional Gal4 drivers; demonstrate roles for transporter Smvt and transcription factors GATAe, Sna, and Ptx1 in global and regional ISC regulation, and study the transcriptional response of midgut cells upon infection. The resulting transcriptome database (http://flygutseq.buchonlab.com) will foster studies of regionalization, homeostasis, immunity, and cell-cell interactions.
Assuntos
Drosophila/metabolismo , Intestinos/citologia , Transcriptoma , Músculos Abdominais/citologia , Músculos Abdominais/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Drosophila/genética , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Células Enteroendócrinas/citologia , Células Enteroendócrinas/metabolismo , Fatores de Transcrição GATA/antagonistas & inibidores , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Mucosa Intestinal/metabolismo , Análise de Componente Principal , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição da Família Snail , Células-Tronco/citologia , Células-Tronco/metabolismo , Simportadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Drosophila melanogaster presents itself as a powerful model for studying the somatic stem cells of the gut and how bacteria affect intestinal homeostasis. The Gal4/UAS/Gal80 (ts) system allows for temporally controlled expression of fluorescent proteins, RNAi knock-down, and other genetic constructs targeted to specific cell populations in the midgut. Similarly, FLP/FRT-mediated somatic recombinations in intestinal stem cells (ISCs) are utilized to visualize and analyze the clonal lineages of individual or populations of stem cells. Live imaging microscopy and immunofluorescence allow both qualitative and quantitative characterization of stem cell shape, proliferation, and differentiation. Here, we detail the use of these tools and techniques for studying gut performance during and following a bacterial infection in the adult fruit fly.
