Image3C, a multimodal image-based and label independent integrative method for single-cell analysis
Abstract
Image-based cell classification has become a common tool to identify phenotypic changes in cell populations. However, this methodology is limited to organisms possessing well characterized species-specific reagents (e.g., antibodies) that allow cell identification, clustering and convolutional neural network (CNN) training. In the absence of such reagents, the power of image-based classification has remained mostly off-limits to many research organisms. We have developed an image-based classification methodology we named Image3C (Image-Cytometry Cell Classification) that does not require species-specific reagents nor pre-existing knowledge about the sample. Image3C combines image-based flow cytometry with an unbiased, high-throughput cell cluster pipeline and CNN integration. Image3C exploits intrinsic cellular features and non-species-specific dyes to perform de novo cell composition analysis and to detect changes in cellular composition between different conditions. Therefore, Image3C expands the use of imaged-based analyses of cell population composition to research organisms in which detailed cellular phenotypes are unknown or for which species-specific reagents are not available.
Data availability
All original data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1390. Image3C code and description are freely available at the GitHub repository https://github.com/stowersinstitute/LIBPB-1390-Image3C.
Article and author information
Author details
Funding
Howard Hughes Medical Institute
- Alejandro Sánchez Alvarado
National Science Foundation (1923372)
- Nicolas Rohner
National Institutes of Health (GM127872,DP2DP2AG071466)
- Nicolas Rohner
Stowers Institute for Medical Research
- Andrew C Box
- Christopher Wood
- Alejandro Sánchez Alvarado
- Nicolas Rohner
Deutsche Forschungsgemeinschaft (PE 2807/1-1)
- Robert Peuß
American Association for Anatomy
- Alice Accorsi
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Research and animal care were approved by the Institutional Animal Care and Use Committee (IACUC) of the Stowers Institute for Medical Research. protocol (#2019-080)
Copyright
© 2021, Accorsi 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
-
- 3,090
- views
-
- 394
- downloads
-
- 6
- 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
-
- Developmental Biology
- Stem Cells and Regenerative Medicine
Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment in mice, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. By profiling the chromatin-bound proteome (chromatome) through genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos leads to developmental arrest, reduction of H3K9me3, and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.
-
- Developmental Biology
Notch signaling has been identified as a key regulatory pathway in patterning the endocardium through activation of endothelial-to-mesenchymal transition (EMT) in the atrioventricular canal (AVC) and proximal outflow tract (OFT) region. However, the precise mechanism underlying Notch activation remains elusive. By transiently blocking the heartbeat of E9.5 mouse embryos, we found that Notch activation in the arterial endothelium was dependent on its ligand Dll4, whereas the reduced expression of Dll4 in the endocardium led to a ligand-depleted field, enabling Notch to be specifically activated in AVC and OFT by regional increased shear stress. The strong shear stress altered the membrane lipid microdomain structure of endocardial cells, which activated mTORC2 and PKC and promoted Notch1 cleavage even in the absence of strong ligand stimulation. These findings highlight the role of mechanical forces as a primary cue for endocardial patterning and provide insights into the mechanisms underlying congenital heart diseases of endocardial origin.