Spatiotemporal Patterns of Gene Expression During Development of a Complex Colony Morphology
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ABSTRACT: Clonal communities of single celled organisms, such as bacterial or fungal colonies and biofilms, are spatially structured, with subdomains of cells experiencing differing environmental conditions. In the development of such communities, cell specialization is not only important to respond and adapt to the local environment but has the potential to increase the fitness of the clonal community through division of labor. Here, we examine colony development in a yeast strain (F13) that produces colonies with a highly structured “ruffled” phenotype in the colony periphery and an unstructured “smooth” phenotype in the colony center. We demonstrate that in the F13 genetic background deletions of transcription factors can either increase (dig1 deletion, sfl1 deletion) or decrease (tec1deletion) the degree of colony structure. We identify genes responding additively and non-additively to the genotype and spatiotemporal factors and cluster these genes into a number of different expression patterns, including patterns that correlate closely with the degree of colony structure in each sample and include genes with known roles in the development of colony structure. Individual deletion of 26 genes sampled from different clusters identified 5 with strong effects on colony morphology (BUD8, CIS3, FLO11, MSB2 and SFG1), all of which eliminated or greatly reduced the structure of the F13 outer region.
Project description:Transcriptome comparison of cells from 4 and 7 day-old microcolonies of wild Saccharomyces cerevisiae BR-F strain, 4 and 7 day-old microcolonies of feral BR-RF strain and 4- and 7 day-old microcolonies of domesticated BR-S strain. All colonies grown on solid complex media with glycerol as carbon source. The aim of the study was to identify genes required for fluffy (structured) colony formation as well as the genes specific for certain phenotypic variant. BR-F is wild strain isolated from natural habitat and forms structured colonies when grown on media with non-fermentable carbon source. BR-S strain arose by phenotypic switch from the original wild BR-F strain during the cultivation of BR-F strain under rich and favourable conditions (process of so-called domestication), forms smooth colonies. BR-RF strain is derived from the domesticated BR-S strain under adverse conditions and restores the formation of structured colonies and other properties of original wild BR-F strain.
Project description:Transcriptome comparison of cells from 4 and 7 day-old microcolonies of wild Saccharomyces cerevisiae BR-F strain, 4 and 7 day-old microcolonies of feral BR-RF strain and 4- and 7 day-old microcolonies of domesticated BR-S strain. All colonies grown on solid complex media with glycerol as carbon source. The aim of the study was to identify genes required for fluffy (structured) colony formation as well as the genes specific for certain phenotypic variant. BR-F is wild strain isolated from natural habitat and forms structured colonies when grown on media with non-fermentable carbon source. BR-S strain arose by phenotypic switch from the original wild BR-F strain during the cultivation of BR-F strain under rich and favourable conditions (process of so-called domestication), forms smooth colonies. BR-RF strain is derived from the domesticated BR-S strain under adverse conditions and restores the formation of structured colonies and other properties of original wild BR-F strain. Comparison of transcriptomes of cells from BR-F colonies vs cells from BR-RF colonies (see samples BR-FxBR-RF...), comparison of transcriptomes of cells from BR-F colonies vs cells from BR-S colonies (see samples BR-FxBR-S...) and comparison of transcriptomes of cells from BR-RF colonies vs cells from BR-S colonies (see samples BR-RFxBR-S...). Comparison of each couple performed with 4 day-old colonies and with 7 day-old colonies. 2 biological replicates for each time point from total 3 technical replicates (for first biological replicate see ...rep1, ...rep2 files, second biological replicate ...rep3 file). Dye-swap was performed between first two replicates (...rep1, ...rep2). In total six samples for each couple. Spotted ORFs microarray slides contain double genome of S. cerevisiae.
Project description:Human multipotent stromal cells readily form single-cell-derived colonies when plated at clonal densities. However, the colonies are heterogeneous because cells from a colony form new colonies that vary in size and differentiation potential when replated at clonal densities. The experiments here tested the hypothesis that cells in the inner regions of colonies are partially differentiated, but the differentiation is reversible. Cells were separately isolated from the dense inner (IN) regions and less-dense outer regions (OUT) of single-cell-derived colonies. Cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation. IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when replated at clonal densities and assayed for rates of propagation and clonogenicity. Also, colonies formed by IN cells were similar to colonies formed by OUT cells because they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30-75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded. The results demonstrated that cells in the IN region of single-cell-derived colonies are partially differentiated, but the differentiation can be reversed by replating the cells at clonal densities. Experiment Overall Design: In this study heterogeneity of human multipotent stromal cells in single-cell-derived colonies was examined. Cells were grown as colonies on slides for 12 days and cells from inside (IN) and outside (OUT) of colonies were isolated with laser capture microdissection. Cells from two colonies were pooled for each sample. This study consists of 3 biological replicates for each colony region (3 for IN and 3 for OUT). All 6 samples were run on Affymetrix microarrays after amplification and labeling using Nugen Ovation technology.
Project description:Human multipotent stromal cells readily form single-cell-derived colonies when plated at clonal densities. However, the colonies are heterogeneous because cells from a colony form new colonies that vary in size and differentiation potential when replated at clonal densities. The experiments here tested the hypothesis that cells in the inner regions of colonies are partially differentiated, but the differentiation is reversible. Cells were separately isolated from the dense inner (IN) regions and less-dense outer regions (OUT) of single-cell-derived colonies. Cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation. IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when replated at clonal densities and assayed for rates of propagation and clonogenicity. Also, colonies formed by IN cells were similar to colonies formed by OUT cells because they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30-75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded. The results demonstrated that cells in the IN region of single-cell-derived colonies are partially differentiated, but the differentiation can be reversed by replating the cells at clonal densities.
Project description:Purpose:To dissect the mechanisms underlying altered gene expression in aneuploids, we measured transcript abundance in colonies of haploid yeast strain F45 and derived strains, including strains disomic for chromosomes XV and XVI, using RNA-seq. F45 colonies display complex “fluffy” morphologies, while the disomic colonies are smooth, resembling laboratory strains Methods: RNA-seq analysis was carried out on RNA isolated from fully developed S. cerevisiae colonies, grown on solid medium for four days, either in triplicate or quadruplicate. Stranded, paired-end sequencing was carried out in two batches. In the first batch 2x51 bp sequencing was carried out on an Illumina Hiseq2000 and in the second batch 2x75 bp sequencing was carried out on an Illumina NextSeq. Readpairs were aligned using Bowtie2 (version 2.1.0)with the parameters [-N 1 -I 50 -X 450 -p 6 --reorder -x -S] and allowing 1 mismatch per read. Differential transcription was detected and quantified using EdgeR (v. 3.6.8) Results: Our two disomes displayed similar transcriptional profiles, a phenomenon not driven by their shared smooth colony morphology nor specified purely by the karyotype. Surprisingly, the environmental stress response (ESR) was induced in euploid F45, relative to the two disomes, rather than vice-versa. We also identified genes whose expression reflected a non-linear interaction between the copy number of a transcriptional regulatory gene on chromosome XVI, DIG1, and the copy number of other chromosome XVI genes. DIG1 and the remaining chromosome XVI genes also demonstrated distinct contributions to the effect of the chromosome XVI disome on ESR gene expression. Conclusions: Expression changes in aneuploids reflect a mixture of effects shared between different aneuploidies, including stress responses, and effects unique to perturbing the copy number of particular chromosomes, including non-linear copy number interactions between genes. The balance between these two phenomena is likely to be genotype and environment specific.
Project description:Bacillus subtilis has been extensively used as a model for molecular studies on biofilm formation. These studies encompassed the development of complex macro-colonies on agar, the formation of pellicles at the air-liquid interface, and lately the formation of submerged architectural biofilms at the solid-liquid interface. Beside similarities, these multicellular communities also display considerable heterogeneity at the structural, chemical and biological levels. Here we use RNA-seq to analyze nine different spatio-physiological conditions, including the three biofilm populations (colony, pellicle, and submerged).
Project description:Here, we introduce CRaft-ID (CRISPR-based microRaft followed by gRNA identification) to enable the screening of image-based phenotypes such as subcellular protein localization from a pool of lenti-CRISPR bulk infected cells. Clonal colonies selected from microRaft arrays are isolated after phenotyping for a targeted-sequencing library preparation to identify the infected sgRNA in each colony. sgRNA representation in the pool of cells is measured with a targeted-sequencing library preparation from the bulk sample.
Project description:We report the development and application of isogenic colony sequencing to profile heterogeneity among yeast colonies. We profiled transcriptomes of the opaque-white switching in C. albicans colonies and an ARO4 mutagenesis library in S. cerevisiae colonies.
Project description:Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). However, successfully reprogrammed cells are highly proliferative and escape from cellular senescence - it is therefore conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency and size was higher when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs had indistinguishable in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs – this is of relevance for standardization and automation of cell culture procedures
Project description:Cellular processes are subject to variability, or noise, yet mechanisms that promote cell-to-cell uniformity are poorly understood. We have identified such a role for Dig1, a redundant (with Dig2) MAPK-responsive inhibitor of the S. cerevisiae mating pathway transcription factor Ste12. Cells lacking Dig1, but not Dig2, exhibited increased variability in outputs of the mating pathway. dig1∆ mutants also displayed a Ste12-dependent defect in growth in the absence of mating pheromone and a quantitative defect in the process of mating, itself. In cells expressing two reporter genes driven by the same promoter, both intrinsic/uncorrelated and extrinsic/correlated noise were found to increase. Remarkably, the extrinsic noise phenotype in cells lacking Dig1 correlates with the aggregation of target genes: we observed subnuclear foci of Ste12 specifically in dig1∆ cells and, using a newly-developed method to immunoprecipitate a single locus from crosslinked chromatin, we found that Dig1 inhibits long-range interactions between Ste12 target genes in vivo. Dig1 may shield binding surfaces on Ste12 whose unmasking leads to inappropriate gene associations and increased gene expression noise. These studies reveal how investigations of variability modulation mechanisms can yield unexpected biological insights. The FUS1 locus is tagged with an array of lac operators. Wild type and dig1∆ cells containing this tagged FUS1 locus and expressing a mCherry-LacI were grown up for ChIP-chip. The FUS1 locus was selectively immunoprecipitated using an anti-DsRed antibody.