Tracking early mammalian organogenesis - prediction and validation of differentiation trajectories at whole organism scale.

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.

GATA2 mitotic bookmarking is required for definitive haematopoiesis.

In mitosis, most transcription factors detach from chromatin, but some are retained and bookmark genomic sites. Mitotic bookmarking has been implicated in lineage inheritance, pluripotency and reprogramming. However, the biological significance of this mechanism in vivo remains unclear.

Dynamic Runx1 chromatin boundaries affect gene expression in hematopoietic development.

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures.

The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition.

Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence.

Ezh2 is essential for the generation of functional yolk sac derived erythro-myeloid progenitors.

Yolk sac (YS) hematopoiesis is critical for the survival of the embryo and a major source of tissue-resident macrophages that persist into adulthood. Yet, the transcriptional and epigenetic regulation of YS hematopoiesis remains poorly characterized. Here we report that the epigenetic regulator Ezh2 is essential for YS hematopoiesis but dispensable for subsequent aorta-gonad-mesonephros (AGM) blood development.

A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the () gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs).

The genome-wide impact of trisomy 21 on DNA methylation and its implications for hematopoiesis.

Down syndrome is associated with genome-wide perturbation of gene expression, which may be mediated by epigenetic changes. We perform an epigenome-wide association study on neonatal bloodspots comparing 196 newborns with Down syndrome and 439 newborns without Down syndrome, adjusting for cell-type heterogeneity, which identifies 652 epigenome-wide significant CpGs (P < 7.67 × 10) and 1,052 differentially methylated regions.

The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors.

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM.

Microhomologies are prevalent at Cas9-induced larger deletions.

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike.

Cell-extrinsic hematopoietic impact of Ezh2 inactivation in fetal liver endothelial cells.

Despite the well-established cell-intrinsic role of epigenetic factors in normal and malignant hematopoiesis, their cell-extrinsic role remains largely unexplored. Herein we investigated the hematopoietic impact of inactivating , a key component of polycomb repressive complex 2 (PRC2), in the fetal liver (FL) vascular niche. Hematopoietic specific (-iCre) inactivation enhanced FL hematopoietic stem cell (HSC) expansion with normal FL erythropoiesis.

Disruption of the aortic wall by coelomic lining-derived mesenchymal cells accompanies the onset of aortic hematopoiesis.

In vertebrates, definitive hematopoietic stem cells (HSCs) first emerge in the ventral wall of the aorta in the Aorta-Gonad-Mesonephros (AGM) region of the embryo, where they differentiate from a specialized type of endothelium termed Hemogenic Endothelium (HE). While the transition from HE to hematopoietic tissue has received much experimental attention, much less is known regarding generation of HE itself. The current study investigates the emergence of the HE in the chick embryo aorta.

The Role of Runx1 in Embryonic Blood Cell Formation.

The de novo generation of hematopoietic stem and progenitor cells (HSPC) occurs solely during embryogenesis from a population of epithelial cells called hemogenic endothelium (HE). During midgestation HE cells in multiple intra- and extraembryonic vascular beds leave the vessel wall as they transition into HSPCs in a process termed the endothelial to hematopoietic transition (EHT). Runx1 expression in HE cells orchestrates the transcriptional switch necessary for the transdifferentiation of endothelial cells into functional HSPCs.

Transcriptional regulation of Hhex in hematopoiesis and hematopoietic stem cell ontogeny.

Hematopoietic stem cells (HSCs) emerge during development via an endothelial-to-hematopoietic transition from hemogenic endothelium of the dorsal aorta (DA). Using in situ hybridization and analysis of a knock-in RedStar reporter, we show that the transcriptional regulator Hhex is expressed in endothelium of the dorsal aorta (DA) and in clusters of putative HSCs as they are specified during murine development. We exploited this observation, using the Hhex locus to define cis regulatory elements, enhancers and interacting transcription factors that are both necessary and sufficient to support gene expression in the emerging HSC.

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors.

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling.

Hematopoietic Reprogramming In Vitro Informs In Vivo Identification of Hemogenic Precursors to Definitive Hematopoietic Stem Cells.

Definitive hematopoiesis emerges via an endothelial-to-hematopoietic transition in the embryo and placenta; however, the precursor cells to hemogenic endothelium are not defined phenotypically. We previously demonstrated that the induction of hematopoietic progenitors from fibroblasts progresses through hemogenic precursors that are Prom1(+)Sca1(+)CD34(+)CD45(-) (PS34CD45(-)). Guided by these studies, we analyzed mouse placentas and identified a population with this phenotype.

An international effort to cure a global health problem: A report on the 19th Hemoglobin Switching Conference.

Every 2 years since 1978, an international group of scientists, physicians, and other researchers meet to discuss the latest developments in the underlying etiology, mechanisms of action, and developmental acquisition of cellular and systemic defects exhibited and elicited by the most common inherited human disorders, the hemoglobinopathies. The 19th Hemoglobin Switching Conference, held in September 2014 at St. John's College in Oxford, once again exceeded all expectations by describing cutting edge research in cellular, molecular, developmental, and genomic advances focused on these diseases.

SMAD1 and SMAD5 Expression Is Coordinately Regulated by FLI1 and GATA2 during Endothelial Development.

The bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin, in vitro reporter assay, and in vivo transgenic data to identify and validate Smad1+63 and the Smad5 promoter as tissue-specific cis-regulatory elements that are active in the developing endothelium.

Single-cell analyses of regulatory network perturbations using enhancer-targeting TALEs suggest novel roles for PU.1 during haematopoietic specification.

Transcription factors (TFs) act within wider regulatory networks to control cell identity and fate. Numerous TFs, including Scl (Tal1) and PU.1 (Spi1), are known regulators of developmental and adult haematopoiesis, but how they act within wider TF networks is still poorly understood.

Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level.

Haematopoietic stem cells (HSCs) are the founding cells of the adult haematopoietic system, born during ontogeny from a specialized subset of endothelium, the haemogenic endothelium (HE) via an endothelial-to-haematopoietic transition (EHT). Although recently imaged in real time, the underlying mechanism of EHT is still poorly understood. We have generated a Runx1 +23 enhancer-reporter transgenic mouse (23GFP) for the prospective isolation of HE throughout embryonic development.

A short history of hemogenic endothelium.

Definitive hematopoietic cells are generated de novo during ontogeny from a specialized subset of endothelium, the so-called hemogenic endothelium. In this review we give a brief overview of the identification of hemogenic endothelium, explore its links with the HSC lineage, and summarize recent insights into the nature of hemogenic endothelium and the microenvironmental and intrinsic regulators contributing to its transition into blood. Ultimately, a better understanding of the processes controlling the transition of endothelium into blood will advance the generation and expansion of hematopoietic stem cells for therapeutic purposes.

Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells.

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor.

RUNX1 is a key target in t(4;11) leukemias that contributes to gene activation through an AF4-MLL complex interaction.

The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood.

Dlk1 is a negative regulator of emerging hematopoietic stem and progenitor cells.

The first mouse adult-repopulating hematopoietic stem cells emerge in the aorta-gonad-mesonephros region at embryonic day (E) 10.5. Their numbers in this region increase thereafter and begin to decline at E12.