Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for Drosophila research

Decision letter after peer review:

Thank you for submitting your article "Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for Drosophila research" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Erika A Bach as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Claude Desplan as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: David Bilder (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. Increase the 'identity' of the cell lines

– Provide information and possibly new data to increase the utility of Table 2

– Provide information on what expected marker genes are NOT expressed in the new cell lines

– Compare the new lines to existing lines, especially the workhorse S2 cells.

– Provide information/commentary on growing in new cells line in large numbers or employing them in high-throughput screening.

2. Improve user access to genomics data. Downloading and accessing GEO files are not user-friendly. Genomic data should be accessible to the potential users of these cell lines.

– Provide tabular genome-wide data for each cell line.

– Consider generating a website with these data

3. Improve the images and figure legends

– Provide higher magnification views to support the conclusions.

– Figure 2: show Ecad staining on a non-epithelial cell line with comparable density.

– Figure 3D: show more clearly the multinucleate cells.

– Provide information on magnification for images.

4. Methods

– Mention how specific the GAL4 drivers are in embryos (possibly by providing real time expression, maybe using G-trace).

– Provide a detailed description of how the new cell lines were generated (rather than cite Simcox, 2013).

– Provide a detailed description of the culture conditions for the new cell lines.

– Provide reagent tables and RRIDs for all reagents

5. Improve karyotyping.

Reviewer #1 (Recommendations for the authors):

1. The authors should review their reference manager because not all of the references are cited in the same way. Most are last name comma first initial but A. Simcox is frequently cited.

2. Under the Methods section, the authors should provide a description of how they generated the cell lines (rather than cite Simcox, 2013).

3. Is it possible to put scale bars on the micrographs? If not, could the authors please state the magnification?

Reviewer #2 (Recommendations for the authors):

1) The critical assignment of the 'identity' of the cell lines would benefit from more detail and explanation. The process is a bit confusing. For instance, in Table 2, why is the dataset Whole body for some and a specific organ for others? In the latter case, why does Btl8 map to the oenocyte sample? On line 182, the authors say that the glial lines did not result in as clear a similarity as other lines, but that is not obvious from the values in Table 2. Is it possible/useful to compare the enrichment scores of Table 2 to those of well-characterized tissue-specific mammalian cell lines and their in vivo equivalents (say MDCK cells and renal tubule cells) for perspective?

2). Related, the paper would benefit from an explicit critical discussion of the cell lines. For instance, what expected marker genes are NOT expressed? E.g. the tracheal-derived cell lines L8 do not express trh, L7 downregulates trh. In what ways are these not simple immortalized cells of in vivo equivalents -that may be important for people thinking about using them? The authors' simple statements e.g. line 212 do a disservice to the nuances of identity.

3) The authors could make clear how these new lines compare to existing lines. For instance, S2 was generally thought to be hemocyte-like, how does it compare to Act GSI-3? What reason would researchers interested in hemocyte biology want to use one over the other? An example of this value is the comparison of transfection efficiency to S2 (line 237).

4) Any experience with growing in large numbers, or in high-throughput screening format? Since these are promoted as uses, it would be very helpful to have a perspective from the authors, who include those most experienced with the variety of existing cell lines and their use in these contexts.

Other points – not absolutely needed for accceptance

In general, the figures are rather sparse, there is room for more data such as those suggested above.

Any speculation as to why so many of the lines correlate with an adult, rather than embryonic, profiles when the tissue source was embryos?

Figure 2: would be nice to show Ecad staining on a non-epithelial cell line with comparable density.

Would be nice to mention how specific the GAL4 drivers are in embryos

The culture conditions for the cell lines, which is critical information, are buried in a section about 'RNA extraction'.

Figure 3D: would be nice to show more clearly the multinucleate cells.

For the lines where there was a failure to derive (e.g. Mef2; elav) -is it possible that the levels of active Ras were too high to be compatible with life and/or immortalization?

The new lines all group more closely with each other in Figure S1 than with any previous line, regardless of the supposed tissue represented. Presumably, this is due to an activated Ras expression signature. Or is their contribution from their more recent time of derivation, or their more similar genetic background? If you 'filter out' the generic Ras expression signature, does that help with the assignment of the cell type?

Discussion about the role of continuous Ras expression would be nice. Inspection of the figure legends 4C and 5C suggests that turning off GAL4 or Ras activity makes the cells quiescent, but explicit information about this, how long they can live in this state, whether there are obvious effects on differentiation, etc. would be useful.

Reviewer #3 (Recommendations for the authors):

The paper is clear and well-written, but I believe that the work needs to be extended to make it useful.

1) The RNAseq characterization is perhaps the most important and global aspect of the utility, because someone interested in a particular gene, could check to see if it was well expressed and in which of the novel (or existing) cell lines. I did not see tabular genome-wide data that would fit this bill. The expression data is basically not presented in the manuscript. The authors are used to presenting data to the community through the DGRC, DRSC, and FlyBase. They need to make these data easy to access. It is hard to judge the GEO submission as it is still listed as private.

2) The phase images are not sufficient to understand the cell biology nature of these cell lines. High magnification views that show higher content information on the cells are needed. It is nice to show the hormonally induced differentiation onset using one or a few markers, but for a resource, I would hope for more granularity. Again it is difficult to make choices of cell lines for experiments with this resolution.

3) Although I do not recall reading it explicitly, I assume that the chromosome spreads were scored based on chromosome size. This is not very satisfying in the era of chromosome paints and especially DNAseq (e.g. PMID: 25262759).

4) The figure legends should indicate what is being detected. "Expressed" is too vague.

5) Reagent tables are much easier to read than in-text methods with names and undescribed identifiers.