Under pressure: integrated endothelial cell response to hydrostatic and shear stresses.

Blood flow within the vasculature is a critical determinant of endothelial cell (EC) identity and functionality, yet the intricate interplay of various hemodynamic forces and their collective impact on endothelial and vascular responses are not fully understood. Specifically, the role of hydrostatic pressure in the EC flow response is understudied, despite its known significance in vascular development and disease. To address this gap, we developed in vitro models to investigate how pressure influences EC responses to flow.

Differentiation latency and dormancy signatures define fetal liver HSCs at single cell resolution.

Decoding the gene regulatory mechanisms mediating self-renewal of hematopoietic stem cells (HSCs) during their amplification in the fetal liver (FL) is relevant for advancing therapeutic applications aiming to expand transplantable HSCs, a long-standing challenge. Here, to explore intrinsic and extrinsic regulation of self-renewal in FL-HSCs at the single cell level, we engineered a culture platform designed to recapitulate the FL endothelial niche, which supports the amplification of serially engraftable HSCs ex vivo. Leveraging this platform in combination with single cell index flow cytometry, serial transplantation assays, and single cell RNA-sequencing, we elucidated previously unrecognized heterogeneity in immunophenotypically defined FL-HSCs and demonstrated that differentiation latency and transcriptional signatures of biosynthetic dormancy are distinguishing properties of self-renewing FL-HSCs with capacity for serial, long-term multilineage hematopoietic reconstitution.

Engineering a niche supporting hematopoietic stem cell development using integrated single-cell transcriptomics.

Hematopoietic stem cells (HSCs) develop from hemogenic endothelium within embryonic arterial vessels such as the aorta of the aorta-gonad-mesonephros region (AGM). To identify the signals responsible for HSC formation, here we use single cell RNA-sequencing to simultaneously analyze the transcriptional profiles of AGM-derived cells transitioning from hemogenic endothelium to HSCs, and AGM-derived endothelial cells which provide signals sufficient to support HSC maturation and self-renewal. Pseudotemporal ordering reveals dynamics of gene expression during the hemogenic endothelium to HSC transition, identifying surface receptors specifically expressed on developing HSCs.

Multipotent progenitors and hematopoietic stem cells arise independently from hemogenic endothelium in the mouse embryo.

During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSCs). Although previous studies have shown that yolk-sac-derived erythromyeloid progenitors and HSCs emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from common HE. In this study, we demonstrate by clonal assays and single-cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSCs.

WNT5A from the fetal liver vascular niche supports human fetal liver hematopoiesis.

The human fetal liver is a critical organ for prenatal hematopoiesis, the study of which offers insights into niche signals that regulate the fates of hematopoietic stem and progenitor cells (HSPCs) during fetal development. Here, we demonstrate that human fetal liver endothelium uniquely supports the maturation and expansion of multilineage HSPCs. Specifically, co-culture of fetal liver-derived immature CD43CD45 hematopoietic cells with human fetal liver endothelial cells (ECs) led to a profound increase in the numbers of phenotypic CD45CD34 HSPCs and multilineage colony-forming progenitors generated in vitro, when compared to co-culture with ECs derived from other organs.

Location, Location, Location: How Vascular Specialization Influences Hematopoietic Fates During Development.

During embryonic development, sequential waves of hematopoiesis give rise to blood-forming cells with diverse lineage potentials and self-renewal properties. This process must accomplish two important yet divergent goals: the rapid generation of differentiated blood cells to meet the needs of the developing embryo and the production of a reservoir of hematopoietic stem cells to provide for life-long hematopoiesis in the adult. Vascular beds in distinct anatomical sites of extraembryonic tissues and the embryo proper provide the necessary conditions to support these divergent objectives, suggesting a critical role for specialized vascular niche cells in regulating disparate blood cell fates during development.

YTHDF2 destabilizes mA-modified neural-specific RNAs to restrain differentiation in induced pluripotent stem cells.

-methyladenosine (mA) is an abundant post-transcriptional modification that can impact RNA fate via interactions with mA-specific RNA binding proteins. Despite accumulating evidence that mA plays an important role in modulating pluripotency, the influence of mA reader proteins in pluripotency is less clear. Here, we report that YTHDF2, an mA reader associated with mRNA degradation, is highly expressed in induced pluripotent stem cells (iPSCs) and down-regulated during neural differentiation.

Zika virus noncoding sfRNAs sequester multiple host-derived RNA-binding proteins and modulate mRNA decay and splicing during infection.

Insect-borne flaviviruses produce a 300-500-base long noncoding RNA, termed subgenomic flavivirus RNA (sfRNA), by stalling the cellular 5'-3'-exoribonuclease 1 (XRN1) via structures located in their 3' UTRs. In this study, we demonstrate that sfRNA production by Zika virus represses XRN1 analogous to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay with human choriocarcinoma (JAR) cells, we demonstrate that the sfRNAs from both dengue type 2 and Zika viruses interact with a common set of 21 RNA-binding proteins that contribute to the regulation of post-transcriptional processes in the cell, including splicing, RNA stability, and translation.

Small changes, big implications: The impact of mA RNA methylation on gene expression in pluripotency and development.

In order to maintain a state of self-renewal, yet retain the ability to rapidly differentiate in response to external signals, pluripotent cells exert tight control over gene expression at many levels. Recent studies have suggested that N6-methyladenosine (mA) RNA methylation, one of the most abundant post-transcriptional modifications, is important for both pluripotency and differentiation. In this review, we summarize the current state of the mA field, with emphasis on the impact of writers, erasers and readers of mA on RNA metabolism and stem cell biology.

Sequences encoding C2H2 zinc fingers inhibit polyadenylation and mRNA export in human cells.

The large C2H2-Zinc Finger (C2H2-ZNF) gene family has rapidly expanded in primates through gene duplication. There is consequently considerable sequence homology between family members at both the nucleotide and amino acid level, allowing for coordinated regulation and shared functions. Here we show that multiple C2H2-ZNF mRNAs experience differential polyadenylation resulting in populations with short and long poly(A) tails.

The Interplay between the RNA Decay and Translation Machinery in Eukaryotes.

RNA decay plays a major role in regulating gene expression and is tightly networked with other aspects of gene expression to effectively coordinate post-transcriptional regulation. The goal of this work is to provide an overview of the major factors and pathways of general messenger RNA (mRNA) decay in eukaryotic cells, and then discuss the effective interplay of this cytoplasmic process with the protein synthesis machinery. Given the transcript-specific and fluid nature of mRNA stability in response to changing cellular conditions, understanding the fundamental networking between RNA decay and translation will provide a foundation for a complete mechanistic understanding of this important aspect of cell biology.

Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability.

Changes in the rate of mRNA decay are closely coordinated with transcriptional changes and together these events have profound effects on gene expression during development and disease. Traditional approaches to assess mRNA decay have relied on inhibition of transcription, which can alter mRNA decay rates and confound interpretation. More recently, metabolic labeling combined with chemical modification and fractionation of labeled RNAs has allowed the isolation of nascent transcripts and the subsequent calculation of mRNA decay rates.

The CELF1 RNA-Binding Protein Regulates Decay of Signal Recognition Particle mRNAs and Limits Secretion in Mouse Myoblasts.

We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and in vitro binding assays using recombinant CELF1, we now provide evidence that CELF1 directly binds the mRNAs encoding each of the subunits of the SRP. Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts.