MicroRNA-126 is a regulator of platelet-supported thrombin generation.

Circulating microRNA (miRNA) expression profiles correlate with platelet reactivity. MiR-126 is a promising candidates in this regard. We generated a transgenic zebrafish line with thrombocyte-specific overexpression of miR-126. Laser injury of the posterior cardinal vein of 5 day-old larvae was performed with or without antithrombotic pre-treatment. Platelet-like structures (PLS) derived from human megakaryocytes transfected with miR-126 were also evaluated for procoagulant activity. Finally, we studied the correlation between miR-126 level and thrombin generation markers in a cohort of stable cardiovascular patients. Control zebrafish developed small thrombocyte-rich thrombi at the site of vessel injury, without vessel occlusion. The miR-126 transgenic line developed an occluding thrombus in 75% (95% CI: 51-91%) of larvae. Pre-treatment with the direct thrombin inhibitor argatroban, but not aspirin, prevented vessel occlusion in the transgenic line (0% occlusion, 95%CI: 0-18%). Upon activation, human PLS showed an increased procoagulant profile after miR-126 transfection compared to control. Finally, the plasma levels of miR-126, but not a control platelet-derived miRNA, correlated with markers of in vivo thrombin generation in a cohort of 185 cardiovascular patients. Our results from three complementary approaches support a key role for miR-126 in platelet-supported thrombin generation and open new avenues in the tailoring of antithrombotic treatment.

Tetrapod V1R-like ora genes in an early-diverging ray-finned fish species: the canonical six ora gene repertoire of teleost fish resulted from gene loss in a larger ancestral repertoire.

BACKGROUND: Chemical senses serve a multitude of essential functions across the animal kingdom. Vertebrates employ four GPCR families to detect odors, among them the v1r/ora gene family. The V1R family is known to evolve rapidly in the lobe-finned lineage giving rise to tetrapods, but the homologous ORA family consists of just six highly conserved genes in teleost fish, with direct orthologs in the lobe-finned fish coelacanth. Thus, the teleost repertoire of six canonical ora genes was assumed to be the ancestral feature before the divergence of ray-finned and lobe-finned fish. So far, this hypothesis has not been tested with earlier diverging ray-finned fish. RESULTS: We have newly identified the complete ora gene repertoires of five teleost species, and of spotted gar, a basal ray-finned fish, using thorough data mining and extensive phylogenetic analysis. The genomes of eight further teleost species were re-analyzed for their ORA repertoires. We report that direct orthologs of the six canonical ora genes (ora1-6) were present in all newly analyzed species, with faithfully preserved exon/intron structure and mostly preserved genomic arrangement in symmetric pairs for ora1-4. In four teleost species including medaka and cave fish we observe species-specific gene duplication events. Thus, the ora gene repertoire in teleost fish is not quite as strictly conserved as previously assumed. In fact, the examination of non-synonymous vs. synonymous substitution rates (dN/dS) shows pronounced negative selection in five of the six ora genes, but also rare occurrence of positive selection in ora3 and ora6. Surprisingly, spotted gar possesses beyond the six canonical genes three additional genes, ora7-8b, orthologous to coelacanth genes v1r07-10. No orthologs for these genes were found in teleosts and cartilaginous fish. CONCLUSIONS: Early diverging ray-finned fish such as the spotted gar possess several v1r-like genes previously assumed to be restricted to the lobe-finned lineage, but now found to be already present in the most recent common ancestor of lobe- and ray-finned fish. Thus, the presence of just six canonical ora genes in many teleost species is not the ancestral feature of the ray-finned lineage, but caused by loss of two ancestral genes in teleosts.

Generation of a Zebrafish Knock-In Model Recapitulating Childhood ETV6::RUNX1-Positive B-Cell Precursor Acute Lymphoblastic Leukemia.

Approximately 25% of children with B-cell precursor acute lymphoblastic leukemia (pB-ALL) harbor the t(12;21)(p13;q22) translocation, leading to the ETV6::RUNX1 (E::R) fusion gene. This translocation occurs in utero, but the disease is much less common than the prevalence of the fusion in newborns, suggesting that secondary mutations are required for overt leukemia. The role of these secondary mutations remains unclear and may contribute to treatment resistance and disease recurrence. We developed a zebrafish model for E::R leukemia using CRISPR/Cas9 to introduce the human RUNX1 gene into zebrafish etv6 intron 5, resulting in E::R fusion gene expression controlled by the endogenous etv6 promoter. As seen by GFP fluorescence at a single-cell level, the model correctly expressed the fusion protein in the right places in zebrafish embryos. The E::R fusion expression induced an expansion of the progenitor cell pool and led to a low 2% frequency of leukemia. The introduction of targeted pax5 and cdkn2a/b gene mutations, mimicking secondary mutations, in the E::R line significantly increased the incidence in leukemia. Transcriptomics revealed that the E::R;pax5mut leukemias exclusively represented B-lineage disease. This novel E::R zebrafish model faithfully recapitulates human disease and offers a valuable tool for a more detailed analysis of disease biology in this subtype.

Polycomb Repressive Complex 2 Regulates Genes Necessary for Intestinal Microfold Cell (M Cell) Development.

BACKGROUND & AIMS: Microfold cells (M cells) are immunosurveillance epithelial cells located in the Peyer's patches (PPs) in the intestine and are responsible for monitoring and transcytosis of antigens, microorganisms, and pathogens. Mature M cells use the receptor glycoprotein 2 (GP2) to aid in transcytosis. Recent studies have shown transcription factors, Spi-B and SRY-Box Transcription Factor 8 (Sox8). are necessary for M-cell differentiation, but not sufficient. An exhaustive set of factors sufficient for differentiation and development of a mature GP2+ M cell remains elusive. Our aim was to understand the role of polycomb repressive complex 2 (PRC2) as an epigenetic regulator of M-cell development. Estrogen-related-receptor γ (Esrrg), identified as a PRC2-regulated gene, was studied in depth, in addition to its relationship with Spi-B and Sox8. METHODS: Comparative chromatin immunoprecipitation and global run-on sequencing analysis of mouse intestinal organoids were performed in stem condition, enterocyte conditions, and receptor activator of nuclear factor κ B ligand-induced M-cell condition. Esrrg, which was identified as one of the PRC2-regulated transcription factors, was studied in wild-type mice and knocked out in intestinal organoids using guide RNA's. Sox8 null mice were used to study Esrrg and its relation to Sox8. RESULTS: chromatin immunoprecipitation and global run-on sequencing analysis showed 12 novel PRC2 regulated transcription factors, PRC2-regulated Esrrg is a novel M-cell-specific transcription factor acting on a receptor activator of nuclear factor κB ligand-receptor activator of nuclear factor κB-induced nuclear factor-κB pathway, upstream of Sox8, and necessary but not sufficient for a mature M-cell marker of Gp2 expression. CONCLUSIONS: PRC2 regulates a significant set of genes in M cells including Esrrg, which is critical for M-cell development and differentiation. Loss of Esrrg led to an immature M-cell phenotype lacking in Sox8 and Gp2 expression. Transcript profiling: the data have been deposited in the NCBI Gene Expression Omnibus database (GSE157629).

Functional Validation of microRNA-126-3p as a Platelet Reactivity Regulator Using Human Haematopoietic Stem Cells.

BACKGROUND: Platelets are an abundant source of micro-ribonucleic acids (miRNAs) that may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, have been noted as potential biomarkers of platelet reactivity and the recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain, and the functional validation of candidate miRNAs on human-derived cells is lacking. OBJECTIVE: This article functionally validates miR-126-3p as a regulator of platelet reactivity in platelet-like structures (PLS) derived from human haematopoietic stem cells. MATERIALS AND METHODS: CD34+-derived megakaryocytes were transfected with miR-126-3p and differentiated in PLS. PLS reactivity was assessed using perfusion in a fibrinogen-coated flow chamber. miR-126-3p's selected gene targets were validated using quantitative polymerase chain reaction, protein quantification and a reporter gene assay. RESULTS: CD34+-derived megakaryocytes transfected with miR-126-3p generated PLS exhibiting 156% more reactivity than the control. These functional data were in line with those obtained analysing CD62P expression. Moreover, miR-126-3p transfection was associated with the down-regulation of a disintegrin and metalloproteinase-9 (ADAM9) messenger RNA (mRNA), a validated target of miR-126-3p, and of Plexin B2 (PLXNB2) mRNA and protein, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results to miR-126-3p transfection. Finally, using a reporter gene assay, we validated PLXNB2 as a direct target of miR-126-3p. CONCLUSION: We functionally validated miR-126-3p as a regulator of platelet reactivity in PLS derived from human haematopoietic stem cells. Moreover, PLXNB2 was validated as a new gene target of miR-126-3p in human cells, suggesting that miR-126-3p mediates its effect on platelets, at least in part, through actin dynamics regulation.

Kappe neurons, a novel population of olfactory sensory neurons.

Perception of olfactory stimuli is mediated by distinct populations of olfactory sensory neurons, each with a characteristic set of morphological as well as functional parameters. Beyond two large populations of ciliated and microvillous neurons, a third population, crypt neurons, has been identified in teleost and cartilaginous fishes. We report here a novel, fourth olfactory sensory neuron population in zebrafish, which we named kappe neurons for their characteristic shape. Kappe neurons are identified by their Go-like immunoreactivity, and show a distinct spatial distribution within the olfactory epithelium, similar to, but significantly different from that of crypt neurons. Furthermore, kappe neurons project to a single identified target glomerulus within the olfactory bulb, mdg5 of the mediodorsal cluster, whereas crypt neurons are known to project exclusively to the mdg2 glomerulus. Kappe neurons are negative for established markers of ciliated, microvillous and crypt neurons, but appear to have microvilli. Kappe neurons constitute the fourth type of olfactory sensory neurons reported in teleost fishes and their existence suggests that encoding of olfactory stimuli may require a higher complexity than hitherto assumed already in the peripheral olfactory system.