Search

Cell fate is established through coordinated gene expression programs in individual cells. Regulatory networks that include the Gata2 transcription factor play central roles in hematopoietic fate establishment. Although Gata2 is essential to the embryonic development and function of hematopoietic stem cells that form the adult hierarchy, little is known about the in vivo expression dynamics of Gata2 in single cells. Here, we examine Gata2 expression in single aortic cells as they establish hematopoietic fate in Gata2Venus mouse embryos. Time-lapse imaging reveals rapid pulsatile level changes in Gata2 reporter expression in cells undergoing endothelial-to-hematopoietic transition. Moreover, Gata2 reporter pulsatile expression is dramatically altered in Gata2+/- aortic cells, which undergo fewer transitions and are reduced in hematopoietic potential. Our novel finding of dynamic pulsatile expression of Gata2 suggests a highly unstable genetic state in single cells concomitant with their transition to hematopoietic fate. This reinforces the notion that threshold levels of Gata2 influence fate establishment and has implications for transcription factor-related hematologic dysfunctions.

Hematopoietic stem cells (HSC), the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM) region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment.

Rad23a and Rad23b proteins are linked to nucleotide excision DNA repair (NER) via association with the DNA damage recognition protein xeroderma pigmentosum group C (XPC) are and known to be implicated in protein turnover by the

Knowledge of the key transcription factors that drive hematopoietic stem cell (HSC) generation is of particular importance for current hematopoietic regenerative approaches and reprogramming strategies. Whereas GATA2 has long been implicated as a hematopoietic transcription factor and its dysregulated expression is associated with human immunodeficiency syndromes and vascular integrity, it is as yet unknown how GATA2 functions in the generation of HSCs. HSCs are generated from endothelial cells of the major embryonic vasculature (aorta, vitelline, and umbilical arteries) and are found in intra-aortic hematopoietic clusters. In this study, we find that GATA2 function is essential for the generation of HSCs during the stage of endothelial-to-hematopoietic cell transition. Specific deletion of Gata2 in Vec (Vascular Endothelial Cadherin)-expressing endothelial cells results in a deficiency of long-term repopulating HSCs and intra-aortic cluster cells. By specific deletion of Gata2 in Vav-expressing hematopoietic cells (after HSC generation), we further show that GATA2 is essential for HSC survival. This is in contrast to the known activity of the RUNX1 transcription factor, which functions only in the generation of HSCs, and highlights the unique requirement for GATA2 function in HSCs throughout all developmental stages.

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

We present a time-saving and objective flow cytometric immunofluorescence assay for the simultaneous detection of antibodies against platelets, granulocytes or lymphocytes using a reconstituted mixture of these cell populations. Platelets, granulocytes and lymphocytes could be distinguished on the basis of their forward (FSC) and sideways (SSC) light scattering properties plotted on scales of 4 log orders. After setting FSC/SSC gates around the platelets, granulocytes and lymphocytes, the reactivity of the sera with the cell populations was determined by histogram analyses of immunofluorescence for each gate. The flow cytometric assay of reconstituted cell mixtures showed a strong, positive correlation with a reference microscopic immunofluorescence assay of separate cell suspensions. The reproducible procedures for the isolation and staining of the cells and the electronic stability of the flow cytometer permitted the use of the same gate and marker settings throughout the experiments. Consequently, the entire analysis of data stored in list mode could be performed using a keystroke, so that time consuming and subjective manual analyses were avoided.