Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation, and loss
Abstract
Organ renewal is governed by the dynamics of cell division, differentiation, and loss. To study these dynamics in real time, we present a platform for extended live imaging of the adult Drosophila midgut, a premier genetic model for stem cell-based organs. A window cut into a living animal allows the midgut to be imaged while intact and physiologically functioning. This approach prolongs imaging sessions to 12-16 hours and yields movies that document cell and tissue dynamics at vivid spatiotemporal resolution. Applying a pipeline for movie processing and analysis, we uncover new, intriguing cell behaviors: that mitotic stem cells dynamically re-orient, that daughter cells use slow kinetics of Notch activation to reach a fate-specifying threshold, and that enterocytes extrude via ratcheted constriction of a junctional ring. By enabling real-time study of midgut phenomena that were previously inaccessible, our platform opens a new realm for dynamic understanding of adult organ renewal.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files for figures have also been uploaded to Dryad (https://dx.doi.org/10.5061/dryad.1v1g1b0).
-
Data from: Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation, and lossDryad Digital Repository, doi:10.5061/dryad.1v1g1b0.
Article and author information
Author details
Funding
National Institutes of Health (R01GM116000-01A1)
- Judy Lisette Martin
- Erin Nicole Sanders
- Paola Moreno-Roman
- Leslie Ann Jaramillo Koyama
- Shruthi Balachandra
- XinXin Du
- Lucy Erin O'Brien
National Institutes of Health (1F31GM123736-01)
- Leslie Ann Jaramillo Koyama
National Institutes of Health (Stanford Discovery Fund Innovation Program)
- Lucy Erin O'Brien
Stanford University (Center for Biomedical Imaging at Stanford Seed Grant)
- Judy Lisette Martin
- Lucy Erin O'Brien
National Science Foundation (GRFP DGE-1656518)
- Erin Nicole Sanders
National Institutes of Health (2T32GM00779038)
- Erin Nicole Sanders
- Leslie Ann Jaramillo Koyama
William K. Bowes, Jr. Foundation (Stanford Bio X Bowes Graduate Fellowship)
- Paola Moreno-Roman
Stanford University (Stanford DARE (Diversifying Academia Recruiting Excellence) Fellowship)
- Paola Moreno-Roman
National Institutes of Health (NRSA 1F32GM115065)
- XinXin Du
Stanford University (Stanford Dean's Postdoctoral Fellowship)
- XinXin Du
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Martin et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 8,547
- views
-
- 974
- downloads
-
- 56
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Stem Cells and Regenerative Medicine
Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.
-
- Stem Cells and Regenerative Medicine
Human induced pluripotent stem cells (hiPSCs) are promising resources for producing various types of tissues in regenerative medicine; however, the improvement in a scalable culture system that can precisely control the cellular status of hiPSCs is needed. Utilizing suspension culture without microcarriers or special materials allows for massive production, automation, cost-effectiveness, and safety assurance in industrialized regenerative medicine. Here, we found that hiPSCs cultured in suspension conditions with continuous agitation without microcarriers or extracellular matrix components were more prone to spontaneous differentiation than those cultured in conventional adherent conditions. Adding PKCβ and Wnt signaling pathway inhibitors in the suspension conditions suppressed the spontaneous differentiation of hiPSCs into ectoderm and mesendoderm, respectively. In these conditions, we successfully completed the culture processes of hiPSCs, including the generation of hiPSCs from peripheral blood mononuclear cells with the expansion of bulk population and single-cell sorted clones, long-term culture with robust self-renewal characteristics, single-cell cloning, direct cryopreservation from suspension culture and their successful recovery, and efficient mass production of a clinical-grade hiPSC line. Our results demonstrate that precise control of the cellular status in suspension culture conditions paves the way for their stable and automated clinical application.