Dataset Information

Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture

ABSTRACT: Purpose: To establish a molecular fingerprint for optoWnt-induced differentiation, we measured global transcriptional changes using bulk-population RNA-seq of optoWnt and WT hESCs after 48 hrs of light stimulation. Illuminated WT cells served as a phototoxicity control, and unilluminated optoWnt cells controlled for potential CRISPR/Cas9 knock-in effects, cell perturbation due to optoWnt expression, and dark-state Wnt pathway activation. We also measured feedback between optoWnt/WT populations during co-culture (1:1) by FACS sorting for each population after 48 hrs of growth. Methods: Bulk RNA-seq of hESCs using Illumina TruSeq Stranded mRNA Library Prep Kit and sequenced on HiSeq 4000 Results: Principal component analysis (PCA) showed clustering of biological triplicates for each condition and strong transcriptional changes upon optoWnt stimulation that account for 95% gene variance among samples. Dark and illuminated monoculture WT cells clustered together, and differential analysis of monocultures showed minimal gene expression differences, demonstrating minimal phototoxicity effects after 48 hrs of continuous 0.8 µW/mm-2 blue light stimulation. In contrast, optoWnt stimulation induced a broad transcriptional effect, with ~5,500 differentially expressed genes between the dark and illuminated conditions. Direct B-catenin target genes, such as CDX1, DKK1, T, and TBX3, are among the most differentially expressed genes, all with a log fold change (LFC) of ~ 9 – 13. Lastly, WT cells grown in illuminated co-culture with optoWnt cells showed differential gene expression and upregulation of TGF-B target genes when compared with WT cells grown in monoculture or in dark co-cultures, indicating signaling feedback between the optoWnt and WT populations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE137289 | GEO | 2023/07/01

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:In the unicellular cyanobacterium, Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock in continuous light (LL) conditions. Here, we employed high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild type, kaiABC-null and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild type strain more than 30% of transcripts significantly oscillated in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was nullified in kaiABC-null strains. Although KaiC has been proposed to globally repress gene expression, our analysis revealed that dawn expressing genes were upregulated by kaiC-overexpression, such that the clock was arrested at subjective dawn. Transfer of cells to continuous dark (DD) conditions from LL immediately suppressed expression of most of genes, while the clock keeps time even in the absence of transcriptional feedback. Thus, the Synechococcus genome seems primarily regulated by the light/dark cycles and dramatically modified by the protein-based circadian oscillator. Keywords: timecourse data (~48 hours under continuous light or darkness) from Synechococcus elongatus PCC 7942 (wild type, kaiABC-null, and inducible kaiC-overexpressor) strains Wild type (WT), kaiABC-null (Dkai), and inducible kaiC-overexpressor (oxC) S. elongatus PCC 7942 strains were analyzed under continuous light (LL), continuous dark (DD) using Affymetrix high-density oligonucleotide microarrays (GeneChip CustomExpress Arrays) representing predicted 2,515 protein-coding genes on the genome of Synechococcus elongatus PCC 6301, which can be used also to the almost homologous strain, S. elongatus PCC 7942: Two independent experiments in WT under LL (hours 0 to 52) and DD (hours 0 to 48); Two independent experiments in Dkai under LL (hours 0 to 48); Two independent experiments in oxC under LL in the presence or absence of an inducer, IPTG, from hour 25 to 33 in LL; a single experiment in WT under DD in the presence of an transcriptional inhibitor, rifampicin.

Dataset Information

Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure