Ongoing transposition in cell culture reveals the phylogeny of diverse Drosophila S2 sub-lines.

Cultured cells are widely used in molecular biology despite poor understanding of how cell line genomes change in vitro over time. Previous work has shown that Drosophila cultured cells have a higher transposable element (TE) content than whole flies, but whether this increase in TE content resulted from an initial burst of transposition during cell line establishment or ongoing transposition in cell culture remains unclear. Here we sequence the genomes of 25 sub-lines of Drosophila S2 cells and show that TE insertions provide abundant markers for the phylogenetic reconstruction of diverse sub-lines in a model animal cell culture system. Analysis of DNA copy number evolution across S2 sub-lines revealed dramatically different patterns of genome organization that support the overall evolutionary history reconstructed using TE insertions. Analysis of TE insertion site occupancy and ancestral states support a model of ongoing transposition dominated by episodic activity of a small number of retrotransposon families. Our work demonstrates that substantial genome evolution occurs during long-term Drosophila cell culture, which may impact the reproducibility of experiments that do not control for sub-line identity.

A novel transposable element based authentication protocol for Drosophila cell lines

Drosophila cell lines are used by researchers to investigate various cell biological phenomena. It is crucial to exercise good cell culture practice. Poor handling can lead to both inter- and intraspecies cross-contamination. Prolonged culturing can lead to introduction of large- and small-scale genomic changes. These factors, therefore, make it imperative that methods to authenticate Drosophila cell lines are developed to ensure reproducibility. Mammalian cell line authentication is reliant on short tandem repeat (STR) profiling, however the relatively low STR mutation rate in D. melanogaster at the individual level is likely to preclude the value of this technique. In contrast, transposable elements (TE) are highly polymorphic among individual flies and abundant in Drosophila cell lines. Therefore, we investigated the utility of TE insertions as markers to discriminate Drosophila cell lines derived from the same or different donor genotypes, divergent sub-lines of the same cell line, and from other insect cell lines. We developed a PCR-based next-generation sequencing protocol to cluster cell lines based on the genome-wide distribution of a limited number of diagnostic TE families. We determined the distribution of five TE families in S2R+, S2-DRSC, S2-DGRC, Kc167, ML-DmBG3-c2, mbn2, CME W1 Cl.8+, and OSS Drosophila cell lines. Two independent downstream analyses of the NGS data yielded similar clustering of these cell lines. Double-blind testing of the protocol reliably identified various Drosophila cell lines. In addition, our data indicate minimal changes with respect to the genome-wide distribution of these five TE families when cells are passaged for at least 50 times. The protocol developed can accurately identify and distinguish the numerous Drosophila cell lines available to the research community, thereby aiding reproducible Drosophila cell culture research.

The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila orthologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, the only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

A novel transposable element based authentication protocol for Drosophila cell lines

Drosophila cell lines are used by researchers to investigate various cell biological phenomena. It is crucial to exercise good cell culture practice. Poor handling can lead to both inter- and intraspecies cross-contamination. Prolonged culturing can lead to introduction of large- and small-scale genomic changes. These factors, therefore, make it imperative that methods to authenticate Drosophila cell lines are developed to ensure reproducibility. Mammalian cell line authentication is reliant on short tandem repeat (STR) profiling, however the relatively low STR mutation rate in D. melanogaster at the individual level is likely to preclude the value of this technique. In contrast, transposable elements (TE) are highly polymorphic among individual flies and abundant in Drosophila cell lines. Therefore, we investigated the utility of TE insertions as markers to discriminate Drosophila cell lines derived from the same or different donor genotypes, divergent sub-lines of the same cell line, and from other insect cell lines. We developed a PCR-based next-generation sequencing protocol to cluster cell lines based on the genome-wide distribution of a limited number of diagnostic TE families. We determined the distribution of five TE families in S2R+, S2-DRSC, S2-DGRC, Kc167, ML-DmBG3-c2, mbn2, CME W1 Cl.8+, and OSS Drosophila cell lines. Two independent downstream analyses of the NGS data yielded similar clustering of these cell lines. Double-blind testing of the protocol reliably identified various Drosophila cell lines. In addition, our data indicate minimal changes with respect to the genome-wide distribution of these five TE families when cells are passaged for at least 50 times. The protocol developed can accurately identify and distinguish the numerous Drosophila cell lines available to the research community, thereby aiding reproducible Drosophila cell culture research.

Transposable element profiles reveal cell line identity and loss of heterozygosity in Drosophila cell culture

Cell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabelling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here we leverage the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of LTR retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome sequencing data (called ngs_te_mapper2), which revealed loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.

Cell image segmentation by using feedback and convolutional LSTM

Human brain is known to have a layered structure and perform not only feedforward process from lower layer to upper layer, but also feedback process from upper layer to lower layer. Neural network is a mathematical model of the function of neurons, and several models are proposed until now. Although neural network imitates the human brain, everyone uses only feedforward process and direct feedback process from upper layer to lower layer is not used in prediction process. Therefore, in this paper, we propose Feedback U-Net using convolutional LSTM. Our model is a segmentation model using convolutional LSTM and feedback process. The output of U-Net at the first round is fed back to the input, and our method re-considers the segmentation result at the second round. By using convolutional LSTM, the features are extracted well based on the features extracted at the first round. On both of the Drosophila cell image and Mouse cell image datasets, our model outperformed conventional U-Net which uses only feedforward process.

Interplay between cell proliferation and recruitment controls the duration of growth and final size of the Drosophila wing

How organs robustly attain a final size despite perturbations in cell growth and proliferation rates is a fundamental question in developmental biology. Since organ growth is an exponential process driven mainly by cell proliferation, even small variations in cell proliferation rates, when integrated over a relatively long time, will lead to large differences in size, unless intrinsic control mechanisms compensate for these variations. Here we use a mathematical model to consider the hypothesis that in the developing wing of Drosophila, cell recruitment, a process in which undifferentiated neighboring cells are incorporated into the wing primordium, determines the time in which growth is arrested in this system. Under this assumption, our model shows that perturbations in proliferation rates of wing-committed cells are compensated by an inversely proportional duration of growth. This mechanism ensures that the final size of the wing is robust in a range of cell proliferation rates. Furthermore, we predict that growth control is lost when fluctuations in cell proliferation affects both wing-committed and recruitable cells. Our model suggests that cell recruitment may act as a temporal controller of growth to buffer fluctuations in cell proliferation rates, offering a solution to a long-standing problem in the field.

The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila homologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and spermatocytes, the only two Drosophila cell types bearing motile cilia/flagella. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

Generation of Drosophila attP containing cell lines using CRISPR-Cas9

The generation of Drosophila stable cell lines have become invaluable for complementing in vivo experiments and as tools for genetic screens. Recent advances utilizing attP/PhiC31 integrase system has permitted the creation of Drosophila cells in which recombination mediated cassette exchange (RMCE) can be utilized to generate stably integrated transgenic cell lines that contain a single copy of the transgene at the desired locus. Current techniques, besides being laborious and introducing extraneous elements, are limited to a handful of cell lines of embryonic origin. Nonetheless, with well over 100 Drosophila cell lines available, including an ever-increasing number CRISPR/Cas9 modified cell lines, a more universal methodology is needed to generate a stably integrated transgenic line from any one of the available Drosophila melanogaster cell lines. Here we describe a toolkit and procedure that combines CRISPR/Cas9 and the PhiC31 integrase system. We have generated and isolated single cell clones containing an Actin5C::dsRed cassette flanked by attP sites into the genome of Kc167 and S2R+ cell lines that mimic the in vivo attP sites located at 25C6 and 99F8 of the Drosophila genome. Furthermore, we tested the functionality of the attP docking sites utilizing two independent GFP expressing constructs flanked by attB sites that permit RMCE and therefore the insertion of any DNA of interest. Lastly, to demonstrate the universality of our methodology and existing constructs, we have successfully integrated the Actin5C::dsRed cassette flanked by attP sites into two different CNS cell lines, ML-DmBG2-c2 and ML-DmBG3-c2. Overall, the reagents and methodology reported here permit the efficient generation of stable transgenic cassettes with minimal change in the cellular genomes in existing D. melanogaster cell lines.

Transposable element profiles reveal cell line identity and loss of heterozygosity in Drosophila cell culture

ABSTRACTCell culture systems allow key insights into biological mechanisms yet suffer from irreproducible outcomes in part because of cross-contamination or mislabelling of cell lines. Cell line misidentification can be mitigated by the use of genotyping protocols, which have been developed for human cell lines but are lacking for many important model species. Here we leverage the classical observation that transposable elements (TEs) proliferate in cultured Drosophila cells to demonstrate that genome-wide TE insertion profiles can reveal the identity and provenance of Drosophila cell lines. We identify multiple cases where TE profiles clarify the origin of Drosophila cell lines (Sg4, mbn2, and OSS_E) relative to published reports, and also provide evidence that insertions from only a subset of LTR retrotransposon families are necessary to mark Drosophila cell line identity. We also develop a new bioinformatics approach to detect TE insertions and estimate intra-sample allele frequencies in legacy whole-genome shotgun sequencing data (called ngs_te_mapper2), which revealed copy-neutral loss of heterozygosity as a mechanism shaping the unique TE profiles that identify Drosophila cell lines. Our work contributes to the general understanding of the forces impacting metazoan genomes as they evolve in cell culture and paves the way for high-throughput protocols that use TE insertions to authenticate cell lines in Drosophila and other organisms.