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Discovery of Progenitor Cell Signatures by Time-Series Synexpression Analysis During Drosophila Embryonic Cell Immortalization

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Discovery of Progenitor Cell Signatures by Time-Series Synexpression Analysis During Drosophila Embryonic Cell Immortalization Correction DEVELOPMENTAL BIOLOGY Correction for “Discovery of progenitor cell signatures by time- series synexpression analysis during Drosophila embryonic cell immortalization,” by Mary-Lee Dequéant, Delphine Fagegaltier, Yanhui Hu, Kerstin Spirohn, Amanda Simcox, Gregory J. Hannon, and Norbert Perrimon, which appeared in issue 42, October 20, 2015, of Proc Natl Acad Sci USA (112:12974–12979; first published October 5, 2015; 10.1073/pnas.1517729112). The authors note that Delphine Fagegaltier should be credited for designing research and performing research. The authors also note that Delphine Fagegaltier, Amanda Simcox, and Gregory J. Hannon should be credited for contributing to the writing of the paper. The corrected author contributions footnote appears below. Author contributions: M.-L.D., D.F., A.S., G.J.H., and N.P. designed research; M.-L.D., D.F., K.S., and A.S. performed research; M.-L.D., D.F., and Y.H. analyzed data; and M.-L.D. and N.P. wrote the paper with contributions from D.F., A.S., and G.J.H. www.pnas.org/cgi/doi/10.1073/pnas.1520482112 E6408 | PNAS | November 17, 2015 | vol. 112 | no. 46 www.pnas.org Downloaded by guest on September 25, 2021 Discovery of progenitor cell signatures by time-series synexpression analysis during Drosophila embryonic cell immortalization Mary-Lee Dequéanta,1, Delphine Fagegaltierb, Yanhui Hua, Kerstin Spirohna, Amanda Simcoxc, Gregory J. Hannond, and Norbert Perrimona,e,1 aDepartment of Genetics, Harvard Medical School, Boston, MA 02115, bCold Spring Harbor Laboratories, Cold Spring Harbor, NY 11724; cDepartment of Molecular Genetics, The Ohio State University, Columbus, OH 43210; dHoward Hughes Medical Institute, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 11724; and eHoward Hughes Medical Institute, Harvard Medical School, Boston, MA 02115 Contributed by Norbert Perrimon, September 10, 2015 (sent for review May 18, 2015; reviewed by Peter Cherbas, Gary Karpen, and Renato Paro) The use of time series profiling to identify groups of functionally population contributing to adult muscles (4–7). Consistently, treat- related genes (synexpression groups) is a powerful approach for the ment of the immortalized cells with ecdysone, a steroid hormone discovery of gene function. Here we apply this strategy during RasV12 triggering adult muscle differentiation (8), induced in vitro terminal immortalization of Drosophila embryonic cells, a phenomenon not myogenic differentiation. Our coexpression network analysis further well characterized. Using high-resolution transcriptional time-series predicted a number of candidate regulators of AMPs, allowing us to datasets, we generated a gene network based on temporal expres- validate the transcription factor CG9650, an ortholog of the mam- sion profile similarities. This analysis revealed that common immortal- malian genes Bcl11a/b, as a previously unidentified regulator of AMP ized cells are related to adult muscle precursors (AMPs), a stem cell- proliferation. In conclusion, we show the value of this newly char- like population contributing to adult muscles and sharing properties acterized Drosophila embryonic progenitor culture system for time- with vertebrate satellite cells. Remarkably, the immortalized cells re- series genomic approaches to identify stem/progenitor cell regulators. tained the capacity for myogenic differentiation when treated with Results the steroid hormone ecdysone. Further, we validated in vivo the tran- Generation of Immortalized Cell Lines. We established primary cul- scription factor CG9650, the ortholog of mammalian Bcl11a/b,asa V12 tures from Act5C > UAS-Ras , UAS-GFP embryos in which regulator of AMP proliferation predicted by our analysis. Our study V12 ubiquitously expressed Gal4 drives the expression of both Ras demonstrates the power of time series synexpression analysis to char- GFP acterize Drosophila embryonic progenitor lines and identify stem/pro- and (Fig.1)(3).Inearlypassages, the cultures showed het- genitor cell regulators. erogeneous cell morphologies with different levels of GFP and exhibited nonuniform growth across the flasks, suggesting that some cells proliferated more rapidly than others. However, in later time series | RNA-Seq | immortalization | Drosophila passages the cells appeared more homogeneous, suggesting that a single or a few cell types predominated. We successfully derived he highly coordinated expression of genes functioning in seven cell lines (R1–R7) from independent primary cultures. All Tcommon processes is a widespread phenomenon from bac- seven lines showed similar passage kinetics before they reached a terial operons (1) to eukaryotic synexpression groups (2). Because stable state, and different phases could be distinguished based on of the strong correlation between cofunction and coexpression (2), inferring gene function based on covariation of expression profiles Significance (called “guilt by association”) is a powerful approach in functional genomics. Importantly, because biological systems are dynamic, Cell line derivation is a complex process and a major challenge recording gene expression over a time series rather than deter- outside of mammalian systems. Here we take an unbiased ge- mining a static single measurement can greatly facilitate the char- nomic approach to characterize a Drosophila embryonic culture acterization of coregulated genes in a particular process. system allowing the efficient derivation of cell lines of still un- Using a Drosophila embryonic culture system, cell lines can be known origins. We reveal that commonly immortalized cells are derived efficiently from primary cell cultures established from related to adult muscle precursors (AMPs), a stem cell-like pop- V12 embryos expressing constitutively active Ras (3). This process ulation contributing to adult muscles and sharing properties with is progressive, with the cell lines reaching a stable state within vertebrate satellite cells. Remarkably, the cells retain the ability to approximately 6 mo. This system provides a unique opportunity differentiate terminally in vitro. We also confirm in vivo a pre- to apply a time-series profiling approach to discover synexpres- viously unidentified regulator of AMP proliferation predicted by sion groups in essential biological processes involved in cell im- our analysis. Our study reveals the potential of this genetically mortalization such as cell-cycle regulation, epigenetic regulation, controlled progenitor culture system to provide key missing re- and cellular differentiation. Furthermore, this unbiased tran- sources to the Drosophila toolbox for cell-based assays. scriptomic approach can provide insights into the unknown ori- gins, regulators, and properties of the immortalized cells. Author contributions: M.-L.D., D.F., A.S., G.J.H., and N.P. designed research; M.-L.D., D.F., Here, we perform the first, to our knowledge, in-depth genomic K.S., and A.S. performed research; M.-L.D., D.F., and Y.H. analyzed data; and M.-L.D. and and temporal characterization of five Drosophila embryonic cell N.P. wrote the paper with contributions from D.F., A.S., and G.J.H. lines during their establishment. Analysis of differential expression Reviewers: P.C., Indiana University; G.K., Lawrence Berkeley National Laboratory; and between early and late time points of the cultures indicated that R.P., Swiss Federal Institute of Technology in Zurich. most cell lines reached a similar stable state reminiscent of neuro- The authors declare no conflict of interest. genic and myogenic progenitor types. To uncover groups of func- Data deposition: Data have been deposited in the National Center for Biotechnology tionally related genes, we applied systematic synexpression network Information Gene Expression Omnibus (GEO), www.ncbi.nlm.nih.gov/geo/info/submission. analyses clustering genes with correlated expression profile dy- html (accession no. GSE73354). namics, using high-resolution time-series profiling datasets. By an- 1To whom correspondence may be addressed. Email: [email protected] alyzing the transcriptional signature of a module associated with or [email protected]. the transcription factor twist (twi), we found that the immortalized This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cells are related to adult muscle precursors (AMPs), a stem cell-like 1073/pnas.1517729112/-/DCSupplemental. 12974–12979 | PNAS | October 20, 2015 | vol. 112 | no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1517729112 (Fig. S2B). Despite some differences, comparisons of the up- Cell lines regulated genes among the lines revealed global similarities, based Immortalization on enrichments of GO categories (described in the next sections), in the R1, R3, R4, and R7 cell lines, with partial differences with the R5 cell line (Fig. 3B), as is consistent with the PCA analysis. Embryonic Primary culture A Proliferative State Associated with the E2 Promoter Binding Factor/ act>RasV12 Retinoblastoma Protein Pathway. GO category enrichment revealed that cell-cycle and cell-division genes represent the most common Transcriptional time series (RNA-Seq, Affymetrix Arrays) significantly up-regulated genes in all RasV12 lines (Fig. 3B and Fig. S3). Interestingly, this set contains many known gene targets of the E2 promoter binding factor/retinoblastoma protein (E2F/RB) Characterization of the lines after immortalization pathway (highlighted in Fig. S3)(9–11) that plays a central role in Up-regulated genes at late stage
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