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.
Reviewing Editor
- Allan C Spradling, Howard Hughes Medical Institute, Carnegie Institution for Science, United States
Version history
- Received: February 27, 2018
- Accepted: November 12, 2018
- Accepted Manuscript published: November 14, 2018 (version 1)
- Version of Record published: December 3, 2018 (version 2)
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
-
- 8,057
- views
-
- 938
- downloads
-
- 53
- 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
-
- Stem Cells and Regenerative Medicine
We developed a 96-well plate assay which allows fast, reproducible, and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (polyethylene glycol [PEG]) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural readouts.
-
- Stem Cells and Regenerative Medicine
Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.