Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for Drosophila research
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eLife assessment
This useful work describes the establishment and characterization of new cell lines derived from specific tissues of the fruit fly Drosophila. The evidence supporting the claims of the authors is solid, although the characterization of the cell lines is incomplete and the genomic findings are not presented in a user-friendly manner. These lines could be a useful resource that complements in vivo Drosophila genetics, improving biochemistry and facilitating high-throughput screening.
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Abstract
Expression of activated Ras, Ras V12 , provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted Ras V12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad Ras V12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center.
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eLife assessment
This useful work describes the establishment and characterization of new cell lines derived from specific tissues of the fruit fly Drosophila. The evidence supporting the claims of the authors is solid, although the characterization of the cell lines is incomplete and the genomic findings are not presented in a user-friendly manner. These lines could be a useful resource that complements in vivo Drosophila genetics, improving biochemistry and facilitating high-throughput screening.
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Reviewer #1 (Public Review):
In this manuscript, the authors were trying to achieve the generation of continuous cell lines after lineage-restricted mis-expression of RasV12 in vivo followed by primary cell culture. They used glia-, epithelia-, and muscle-specific Gal4s, to get glial, epithelial, and muscle cell lines, as well as the RU-inducible Gene Switch Gal4, to get neuronal and blood cell lines. They performed RNA-seq analysis of the cell lines and showed that they are most similar to each by principal component analysis. They compared their RNA-seq to the Fly Cell Atlas and showed that the cell lines were quite similar to their in vivo counterparts. They treated cell lines with the steroid hormone ecdysone and found that many of the cell lines differentiate. These cell lines also contain an attP site, allowing for CRISPR-based …
Reviewer #1 (Public Review):
In this manuscript, the authors were trying to achieve the generation of continuous cell lines after lineage-restricted mis-expression of RasV12 in vivo followed by primary cell culture. They used glia-, epithelia-, and muscle-specific Gal4s, to get glial, epithelial, and muscle cell lines, as well as the RU-inducible Gene Switch Gal4, to get neuronal and blood cell lines. They performed RNA-seq analysis of the cell lines and showed that they are most similar to each by principal component analysis. They compared their RNA-seq to the Fly Cell Atlas and showed that the cell lines were quite similar to their in vivo counterparts. They treated cell lines with the steroid hormone ecdysone and found that many of the cell lines differentiate. These cell lines also contain an attP site, allowing for CRISPR-based screens. These cell lines could be passaged for many generations, but robust effects were found in the early passages. These cell lines have been deposited at a public resource center (The Drosophila Genomics Resource Center, DGRC).
The major strengths of the paper include rigorous analysis of characteristics, gene expression, and differentiation potential of the cell lines. There were only a few minor weaknesses related to editorial changes in the manuscript.
The authors provide convincing results that support their conclusions and as such the authors achieved their aims.
This work is likely to have a positive impact on the Drosophila community. These cell lines will serve as a solid foundation for both low- and high-throughput screens.
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Reviewer #2 (Public Review):
The authors describe the derivation of new and stable fly cell lines through a strategy of tissue-specific RasV12 expression and in some cases single cell cloning. Lines with molecular and, in some cases, phenotypic characteristics of the targeted tissue are identified: muscle, neural, glial, epithelial, and macrophage-like. These are (for the most part) karyotypically normal and amenable to genetic manipulation including transient and attP-mediated insertion. This paper reports a publicly available resource that will be of great use to many. The cell lines are ready for the well-established tools available for high-throughput screening using CRISPR, RNAi, and small molecules, and allow scalable biochemistry which has been a limitation of using Drosophila for some research questions. Moreover, the …
Reviewer #2 (Public Review):
The authors describe the derivation of new and stable fly cell lines through a strategy of tissue-specific RasV12 expression and in some cases single cell cloning. Lines with molecular and, in some cases, phenotypic characteristics of the targeted tissue are identified: muscle, neural, glial, epithelial, and macrophage-like. These are (for the most part) karyotypically normal and amenable to genetic manipulation including transient and attP-mediated insertion. This paper reports a publicly available resource that will be of great use to many. The cell lines are ready for the well-established tools available for high-throughput screening using CRISPR, RNAi, and small molecules, and allow scalable biochemistry which has been a limitation of using Drosophila for some research questions. Moreover, the Ras-targeting approach is potentially a general way to make additional tissue-specific cells, and the authors describe several failures as well as successes in deriving tissue-specific lines. Overall it is a highly valuable piece of work. Ways that the paper reporting this work could be enhanced for the reader include 1) a more critical analysis of the limitations of these lines to represent their prospective in vivo tissues; 2) a more explicit comparison of these lines next to existing fly cell lines including but not limited to the workhorse S2, and 3) any information on the ease of use and behavior of these cells in the types of high-throughput/high-volume formats where they are likely to be most valuable.
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Reviewer #3 (Public Review):
This is a clearly written, straightforward, resource paper describing the creation of several new cell lines that may prove useful to the Drosophila community. They are to be distributed through the Drosophila Genomics Resource Center and might be put to use at the Drosophila RNAi screening center.
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