Rad21 is a subunit of cohesin. The main function of cohesin is to hold replicated chromosomes together until cells divide, but it also plays a role in gene expression. To find out which genes might be regulated by cohesin, a study was conducted to look for global changes in gene expression in zebrafish embryos lacking cohesin component Rad21.
Positive regulation of c-Myc by cohesin is direct, and evolutionarily conserved.
Specimen part, TimeView Samples
In addition to satisfying the metabolic demands of cells, mitochondrial metabolism helps regulate immune cell function. To date, such cell-intrinsic metabolic-immunologic cross-talk has only been described operating in cells of the immune system. Here we show that epidermal cells utilize fatty acid -oxidation to fuel their contribution to the immune response during cutaneous inflammation. By live imaging metabolic and immunological processes within intact zebrafish embryos during cutaneous inflammation, we uncover a mechanism where elevated -oxidation-fueled mitochondria-derived reactive oxygen species within epidermal cells helps guide matrix metalloproteinase-driven leukocyte recruitment. This mechanism requires the activity of a zebrafish homolog of the mammalian mitochondrial enzyme, Immunoresponsive gene 1. This study describes the first example of metabolic reprogramming operating within a non-immune cell type to help control its contribution to the immune response. Targeting of this metabolic-immunologic interface within keratinocytes may prove useful in treating inflammatory dermatoses.
Epidermal cells help coordinate leukocyte migration during inflammation through fatty acid-fuelled matrix metalloproteinase production.
Specimen part, TreatmentView Samples
Mitochondrial DNA (mtDNA) mutations cause inherited diseases and are implicated in the pathogenesis of common late-onset disorders, but it is not clear how they arise and propagate in the humans. Here we show that mtDNA mutations are present in primordial germ cells (PGCs) within healthy female human embryos. Close scrutiny revealed the signature of selection against non-synonymous variants in the protein-coding region, tRNA gene variants, and variants in specific regions of the non-coding D-loop. In isolated single PGCs we saw a profound reduction in the cellular mtDNA content, with discrete mitochondria containing ~5 mtDNA molecules during early germline development. Single cell deep mtDNA sequencing showed rare variants reaching higher heteroplasmy levels in later PGCs, consistent with the observed genetic bottleneck, and predicting >80% levels within isolated organelles. Genome-wide RNA-seq showed a progressive upregulation of genes involving mtDNA replication and transcription, linked to a transition from glycolytic to oxidative metabolism. The metabolic shift exposes deleterious mutations to selection at the organellar level during early germ cell development. In this way, the genetic bottleneck prevents the relentless accumulation of mtDNA mutations in the human population predicted by Muller's ratchet. Mutations escaping this mechanism will, however, show massive shifts in heteroplasmy levels within one human generation, explaining the extreme phenotypic variation seen in human pedigrees with inherited mtDNA disorders. Overall design: RNA-Seq and NGS analysis to investigate transcriptomes and mtDNA sequences of fetal hPGCs
Segregation of mitochondrial DNA heteroplasmy through a developmental genetic bottleneck in human embryos.
No sample metadata fieldsView Samples
How cells acquire their fate is a fundamental question in both developmental and regenerative biology. Multipotent progenitors undergo gradual cell fate restriction in response to temporal and positional cues from the microenvironment, the nature of which is far from being clear. In the case of the lymphatic system, venous endothelial cells are thought to give rise to lymphatic vessels, through a process of trans-differentiation. Upon expression of a set of transcription factors, venous cells acquire a lymphatic fate, and bud out to generate the lymphatic vasculature. In this work we challenge this view and show that while lymphatic endothelial cells (LECs) do arise in the Cardinal Vein (CV), they do so from a previously uncharacterized pool of multipotent angioblasts. Using lymphatic-specific transgenic zebrafish, in combination with endothelial photoconvertible reporters, and long-term live imaging, we demonstrate that these multipotent angioblasts can generate not only lymphatic, but also arterious, and venous fates. We further reveal that the underlying endoderm serves as a source of Wnt5b, which acts as a lymphatic inductive signal, promoting the angioblast-to-lymphatic transition. Moreover, Wnt5b induced lymphatic specification in human embryonic stem cells- derived vascular progenitors, suggesting that this process is evolutionary conserved. Our results uncover a novel mechanism of lymphatic vessel formation, whereby multipotent angioblasts and not venous endothelial cells give rise to the lymphatic endothelium, and provide the first characterization of their inductive niche. More broadly, our findings highlight the CV as a plastic and heterogeneous structure containing different cell populations, analogous to the hematopoietic niche in the aortic floor. Overall design: Following Kaede photoconversion of dorsal or ventral halves of the PCV in Tg(fli1:gal4;uasKaede) embryos at 24 hpf, 6Â embryos per group were used for FACS isolation of Kaede photconverted (red) ECs.
Lymphatic vessels arise from specialized angioblasts within a venous niche.
No sample metadata fieldsView Samples
A highly metastatic breast cancer cell line, 4T1, was used to generate stable Wnt5a expressing and vector only control cells. Cells were generated using lentivirus infection and selection with blasticidin. Expression of Wnt5a was confirmed using western blot. Cell behaviour was characterized. Wnt5a expressing cells exhibited reduced migration in a transwell assay and reduced metastasis in a tail vein injection assay. Growth was not significantly affected.
WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells.
Cell lineView Samples
To investigate differential gene expression that might account for the differing glomerular phenotype of NPHS2-Cre +/+ mice when compared with wild-type control, including altered GBM thickness, loss of normal foot process morphology, and decrease in podocyte number, RNA sequencing analysis was performed on glomeruli extracted from both NPHS2-Cre +/+ and wild-type control mice. Overall design: Following isolation of glomeruli using Dynabeads from NPHS2-Cre +/+ and wild-type control mice (n=2 biological replicates per genotype, singly isolated), total RNA was extracted and RNA samples were submited for sample preparation and sequencing.
Podocyte-specific expression of Cre recombinase promotes glomerular basement membrane thickening.
Sex, Age, Specimen part, Cell line, SubjectView Samples
It is well known that both recipient cells and donor nuclei demonstrate a mitotic advantage as observed in the traditional reprogramming with somatic cell nuclear transfer (SCNT). However, It is not known whether a specific mitotic factor plays a critical role in reprogramming. Here we identify an isoform of human bromodomain-containing 3 (BRD3), BRD3R (BRD3 with Reprogramming activity), as a reprogramming factor. BRD3R positively regulates mitosis during reprogramming, upregulates a large set of mitotic genes at early stages of reprogramming, and associates with mitotic chromatin. Interestingly, a set of the mitotic genes upregulated by BRD3R constitutes a pluripotent molecular signature. The two BRD3 isoforms display differential binding to acetylated histones. Our results suggest a molecular interpretation for the mitotic advantage in reprogramming, and show that mitosis may be a driving force of reprogramming. Overall design: Human BJ cells transduced with lentiviral particles of the conventional reprogramming factors (OCT3/4, SOX2 and KLF4) were used as controls. Two types of controls were used: 1) BJ transduced with OSK (OCT4, SOX2 and KFL4) viruses; 2) BJ cells transduced with OSK plus GFP viruses. Experimental treatment was BJ cells transduced with OSK plus BRD3R viruses. RNA was extracted from cells at day 3 of reprogramming because the reprogramming cells are still homogeneous and transgenes are well expressed at this time point.
The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis.
No sample metadata fieldsView Samples
We report a simultaneous comparison of striatal mRNA levels by RNA sequencing mice with graded levels of HD-like abnormalities Overall design: Examination of 4 different mouse lines
Allelic series of Huntington's disease knock-in mice reveals expression discorrelates.
Specimen part, SubjectView Samples
Analysis of the genes and cellular signalling cascades mediating the response of SCN slices to vasoactive intestinal peptide (VIP). Primary goal was to find novel genes that may be involved in circadian phase shifting for further study. Promoter analysis of significantly regulated genes and gene ontology analysis would provide information into pathways VIP acts through in the SCN.
Vasoactive intestinal peptide controls the suprachiasmatic circadian clock network via ERK1/2 and DUSP4 signalling.
Specimen partView Samples