Evidence for novel fate of Flk1+ progenitor: contribution to muscle lineage

Flk1 is one of the specific cell surface receptors for vascular endothelial growth factor and one of the most specific markers highlighting the earliest stage of hematopoietic and vascular lineages. However, recent new evidence suggests that these Flk1(+) mesodermal progenitor cells also contribute to muscle lineages. All evidence is based on the experiments using in vitro differentiation and in vivo transplantation systems. Although this approach revealed a differentiation potential range of Flk1(+) cells that is wider than previously expected, it fails to determine whether Flk1(+) cells contribute to muscle lineage as part of the normal developmental process. To obtain direct evidence for the fate of Flk1(+) cells in development, we used a knock-in mouse line where Cre is expressed in Flk1(+) cells. Studies with these Cre lines provide direct evidence that Flk1(+) cells are progenitors for muscles, in addition to hematopoietic and vascular endothelial cells.     

Induction of hematopoietic and endothelial cell program orchestrated by ETS transcription factor ER71/ETV2

The ETS factor ETV2 (aka ER71) is essential for the generation of the blood and vascular system, as ETV2 deficiency leads to a complete block in blood and endothelial cell formation and embryonic lethality in the mouse. However, the ETV2-mediated gene regulatory network and signaling governing hematopoietic and endothelial cell development are poorly understood. Here, we map ETV2 global binding sites and carry out in vitro differentiation of embryonic stem cells, and germ line and conditional knockout mouse studies to uncover mechanisms involved in the hemangiogenic fate commitment from mesoderm. We show that ETV2 binds to enhancers that specify hematopoietic and endothelial cell lineages. We find that the hemangiogenic progenitor population in the developing embryo can be identified as FLK1^highPDGFRα⁻. Notably, these hemangiogenic progenitors are exclusively sensitive to ETV2-dependent FLK1 signaling. Importantly, ETV2 turns on other Ets genes, thereby establishing an ETS hierarchy. Consequently, the hematopoietic and endothelial cell program initiated by ETV2 is maintained partly by other ETS factors through an ETS switching mechanism. These findings highlight the critical role that transient ETV2 expression plays in the regulation of hematopoietic and endothelial cell lineage specification and stability.     

Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.     

Variability of Inducible Expression across the Hematopoietic System of Tetracycline Transactivator Transgenic Mice

The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell types of several commonly used transgenic tet transactivator mouse strains. We find that even in strains where the tet transactivator is expressed from a nominally ubiquitous promoter, the efficiency of tet-regulated expression can be highly variable between hematopoietic lineages and between differentiation stages within a lineage. In some cases tet-regulated reporter expression differs markedly between cells within a discrete, immunophenotypically defined population, suggesting mosaic transactivator expression. A recently developed CAG-rtTA3 transgenic mouse displays intense and efficient reporter expression in most blood cell types, establishing this strain as a highly effective tool for probing hematopoietic development and disease. These findings have important implications for interpreting tet-regulated hematopoietic phenotypes in mice, and identify mouse strains that provide optimal tet-regulated expression in particular hematopoietic progenitor cell types and mature blood lineages.     

Endocardial cells are a distinct endothelial lineage derived from Flk1+ multipotent cardiovascular progenitors

Identification of multipotent cardiac progenitors has provided important insights into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, characterization of the endocardial lineage has been limited by the lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line capable of labeling this specific endothelial subpopulation at the earliest stages of cardiac development. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation in vitro recapitulates the close temporal–spatial relationship observed between myocardium and endocardium seen in vivo. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, these studies emphasize that the endocardium is a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor.     

A stop-EGFP transgenic mouse to detect clonal cell lineages generated by mutation

The investigation of cell lineages and clonal organization in tissues is facilitated by techniques that allow labelling of clonal cell lineages. Here, we describe a novel transgenic mouse that allows clonal cell lineages to be traced in virtually any tissue. A green fluorescent cell lineage is generated by a random mutation at an enhanced green fluorescent protein gene that carries a premature stop codon, ensuring clonality. The transgenic system allows efficient detection of mutations and stem-cell fate mapping in the epidermis using live mice, as well as in the kidney and liver post-mortem. Cell lineages that descended from single epidermal stem cells were found to be capable of generating three adjacent corneocytes using the system, providing evidence for horizontal migration of epidermal cells between epidermal proliferative units (EPUs), in contrast to the classical EPU model. The transgenic mouse system is expected to provide a novel tool for stem-cell lineage studies.     

Spry1 is expressed in hemangioblasts and negatively regulates primitive hematopoiesis and endothelial cell function

Background

Development of the hematopoietic and endothelial lineages derives from a common mesodermal precursor, the Flk1⁺ hemangioblast. However, the signaling pathways that regulate the development of hematopoietic and endothelial cells from this common progenitor cell remains incompletely understood. Using mouse models with a conditional Spry1 transgene, and a Spry1 knockout mouse, we investigated the role of Spry1 in the development of the endothelial and hematopoietic lineages during development.

Methodology/Principal Findings

Quantitative RT-PCR analysis demonstrates that Spry1, Spry2, and Spry4 are expressed in Flk1⁺ hemangioblasts in vivo, and decline significantly in c-Kit⁺ and CD41⁺ hematopoietic progenitors, while expression is maintained in developing endothelial cells. Tie2-Cre-mediated over-expression of Spry1 results in embryonic lethality. At E9.5 Spry1;Tie2-Cre embryos show near normal endothelial cell development and vessel patterning but have reduced hematopoiesis. FACS analysis shows a reduction of primitive hematopoietic progenitors and erythroblastic cells in Spry1;Tie2-Cre embryos compared to controls. Colony forming assays confirm the hematopoietic defects in Spry1;Tie2-Cre transgenic embryos. Immunostaining shows a significant reduction of CD41 or CD71 and dpERK co-stained cells in Spry1;Tie2-Cre embryos compared to controls, whereas the number of VEC⁺ and dpERK co-stained cells is comparable. Compared to controls, Spry1;Tie2-Cre embryos also show a decrease in proliferation and an increase in apoptosis. Furthermore, loss of Spry1 results in an increase of CD41⁺ and CD71⁺ cells at E9.5 compared with controls.

Conclusions/Significance

These data indicate that primitive hematopoietic cells derive from Tie2-expressing hemangioblasts and that Spry1 over expression inhibits primitive hematopoietic progenitor and erythroblastic cell development and expansion while having no obvious effect on endothelial cell development.     

The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells

The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2‐expressing populations using Tie2‐Cre;Rosa26R‐EYFP mice. In Tie2‐Cre;Rosa26R‐EYFP mice, analysis of bone marrow cells showed Cre‐mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ～ 84% of each lineage was EYFP⁺, and nearly all cells that come from Tie2‐expressing lineages are CD45⁺, confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2⁺ origin. Our findings of high levels of Tie2‐Cre recombination in the hematopoietic lineage have implications for the use of the Tie2‐Cre mouse as a lineage‐restricted driver strain. genesis 48:563–567, 2010. © 2010 Wiley‐Liss, Inc.     

Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.