Katrin Ottersbach

Hematopoietic Stem Cells Localize to the Endothelial Cell Layer in the Midgestation Mouse Aorta

The emergence of the first adult hematopoietic stem cells (HSCs) during mammalian ontogeny has been under intense investigation. It is as yet unresolved whether these first HSCs are derived from intraembryonic hemangioblasts, hemogenic endothelial cells, or other progenitors. Thus, to examine the spatial generation of functional HSCs within the mouse embryo, we used the well-known HSC marker, Sca-1, and a transgenic approach with an Ly-6A (Sca-1) GFP marker gene. Our results show that this transgene marker is expressed in all functional HSCs in the midgestation aorta. Immunohistology of aorta-gonads-mesonephros (AGM) regions show that GFP(+) cells are specifically localized to the endothelial layer lining the wall of the dorsal aorta but not to the mesenchyme, strongly suggesting that HSC activity arises within a few cells within the endothelium of the major vasculature.

GATA-2 plays two functionally distinct roles during the ontogeny of hematopoietic stem cells.

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs), whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac, fetal liver, and adult bone marrow. However, HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also, cytotoxic drug-induced regeneration studies show a clear GATA-2 dose–related proliferation defect in adult bone marrow. Thus, GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow.

Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early hematopoietic development

Conservation of the vertebrate body plan has been attributed to the evolutionary stability of gene-regulatory networks (GRNs). We describe a regulatory circuit made up of Gata2, Fli1, and Scl/Tal1 and their enhancers, Gata2-3, Fli1+12, and Scl+19, that operates during specification of hematopoiesis in the mouse embryo. We show that the Fli1+12 enhancer, like the Gata2-3 and Scl+19 enhancers, targets hematopoietic stem cells (HSCs) and relies on a combination of Ets, Gata, and E-Box motifs. We show that the Gata2-3 enhancer also uses a similar cluster of motifs and that Gata2, Fli1, and Scl are expressed in embryonic day-11.5 dorsal aorta where HSCs originate and in fetal liver where they multiply. The three HSC enhancers in these tissues and in ES cell-derived hemangioblast equivalents are bound by each of these transcription factors (TFs) and form a fully connected triad that constitutes a previously undescribed example of both this network motif in mammalian development and a GRN kernel operating during the specification of a mammalian stem cell.

Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

Summary Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.

JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia

The JAK2 V617F mutation is found in most patients with a myeloproliferative neoplasm and is sufficient to produce a myeloproliferative phenotype in murine retroviral transplantation or transgenic models. However, several lines of evidence suggest that disease phenotype is influenced by the level of mutant JAK2 signaling, and we have therefore generated a conditional knock-in mouse in which a human JAK2 V617F is expressed under the control of the mouse Jak2 locus. Human and murine Jak2 transcripts are expressed at similar levels, and mice develop modest increases in hemoglobin and platelet levels reminiscent of human JAK2 V617F-positive essential thrombocythemia. The phenotype is transplantable and accompanied by increased terminal erythroid and megakaryocyte differentiation together with increased numbers of clonogenic progenitors, including erythropoietin-independent erythroid colonies. Unexpectedly, JAK2(V617F) mice develop reduced numbers of lineage(-)Sca-1(+)c-Kit(+) cells, which exhibit increased DNA damage, reduced apoptosis, and reduced cell cycling. Moreover, competitive bone marrow transplantation studies demonstrated impaired hematopoietic stem cell function in JAK2(V617F) mice. These results suggest that the chronicity of human myeloproliferative neoplasms may reflect a balance between impaired hematopoietic stem cell function and the accumulation of additional mutations.

Embryonic stromal clones reveal developmental regulators of definitive hematopoietic stem cells

Hematopoietic stem cell (HSC) self-renewal and differentiation is regulated by cellular and molecular interactions with the surrounding microenvironment. During ontogeny, the aorta–gonad–mesonephros (AGM) region autonomously generates the first HSCs and serves as the first HSC-supportive microenvironment. Because the molecular identity of the AGM microenvironment is as yet unclear, we examined two closely related AGM stromal clones that differentially support HSCs. Expression analyses identified three putative HSC regulatory factors, β-NGF (a neurotrophic factor), MIP-1γ (a C–C chemokine family member) and Bmp4 (a TGF-β family member). We show here that these three factors, when added to AGM explant cultures, enhance the in vivo repopulating ability of AGM HSCs. The effects of Bmp4 on AGM HSCs were further studied because this factor acts at the mesodermal and primitive erythropoietic stages in the mouse embryo. In this report, we show that enriched E11 AGM HSCs express Bmp receptors and can be inhibited in their activity by gremlin, a Bmp antagonist. Moreover, our results reveal a focal point of Bmp4 expression in the mesenchyme underlying HSC containing aortic clusters at E11. We suggest that Bmp4 plays a relatively late role in the regulation of HSCs as they emerge in the midgestation AGM.

The Ly‐6A (Sca‐1) GFP Transgene is Expressed in all Adult Mouse Hematopoietic Stem Cells

The Sca‐1 cell surface glycoprotein is used routinely as a marker of adult hematopoietic stem cells (HSCs), allowing a >100‐fold enrichment of these rare cells from the bone marrow of the adult mouse. The Sca‐1 protein is encoded by the Ly‐6A/E gene, a small 4‐exon gene that is tightly controlled in its expression in HSCs and several hematopoietic cell types. For the ability to sort and localize HSCs directly from the mouse, we initiated a transgenic approach in which we created Ly‐6A (Sca‐1) green fluorescent protein (GFP) transgenic mice. We show here that a 14‐kb Ly‐6A expression cassette directs the transcription of the GFP marker gene in all functional repopulating HSCs in the adult bone marrow. A >100‐fold enrichment of HSCs occurred by sorting for the GFP‐expressing cells. Furthermore, as shown by fluorescence‐activated cell sorting and histologic analysis of several hematopoietic tissues, the GFP transgene expression pattern generally corresponded to that of Sca‐1. Thus, the Ly‐6A GFP transgene facilitates the enrichment of HSCs and presents the likelihood of identifying HSCs in situ.

Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver

Transcription factors such as Scl/Tal1, Lmo2, and Runx1 are essential for the development of hematopoietic stem cells (HSCs). However, the precise mechanisms by which these factors interact to form transcriptional networks, as well as the identity of the genes downstream of these regulatory cascades, remain largely unknown. To this end, we generated an Scl(-/-) yolk sac cell line to identify candidate Scl target genes by global expression profiling after reintroduction of a TAT-Scl fusion protein. Bioinformatics analysis resulted in the identification of 9 candidate Scl target transcription factor genes, including Runx1 and Runx3. Chromatin immunoprecipitation confirmed that both Runx genes are direct targets of Scl in the fetal liver and that Runx1 is also occupied by Scl in the yolk sac. Furthermore, binding of an Scl-Lmo2-Gata2 complex was demonstrated to occur on the regions flanking the conserved E-boxes of the Runx1 loci and was shown to transactivate the Runx1 element. Together, our data provide a key component of the transcriptional network of early hematopoiesis by identifying downstream targets of Scl that can explain key aspects of the early Scl(-/-) phenotype.

Ventral embryonic tissues and Hedgehog proteins induce early AGM hematopoietic stem cell development

Hematopoiesis is initiated in several distinct tissues in the mouse conceptus. The aorta-gonad-mesonephros (AGM) region is of particular interest, as it autonomously generates the first adult type hematopoietic stem cells (HSCs). The ventral position of hematopoietic clusters closely associated with the aorta of most vertebrate embryos suggests a polarity in the specification of AGM HSCs. Since positional information plays an important role in the embryonic development of several tissue systems, we tested whether AGM HSC induction is influenced by the surrounding dorsal and ventral tissues. Our explant culture results at early and late embryonic day 10 show that ventral tissues induce and increase AGM HSC activity, whereas dorsal tissues decrease it. Chimeric explant cultures with genetically distinguishable AGM and ventral tissues show that the increase in HSC activity is not from ventral tissue-derived HSCs, precursors or primordial germ cells (as was previously suggested). Rather, it is due to instructive signaling from ventral tissues. Furthermore, we identify Hedgehog protein(s) as an HSC inducing signal.

Signaling from the Sympathetic Nervous System Regulates Hematopoietic Stem Cell Emergence during Embryogenesis

Summary The first adult-repopulating hematopoietic stem cells (HSCs) emerge in the aorta-gonads-mesonephros (AGM) region of the embryo. We have recently identified the transcription factor Gata3 as being upregulated in this tissue specifically at the time of HSC emergence. We now demonstrate that the production of functional and phenotypic HSCs in the AGM is impaired in the absence of Gata3. Furthermore, we show that this effect on HSC generation is secondary to the role of Gata3 in the production of catecholamines, the mediators of the sympathetic nervous system (SNS), thus making these molecules key components of the AGM HSC niche. These findings demonstrate that the recently described functional interplay between the hematopoietic system and the SNS extends to the earliest stages of their codevelopment and highlight the fact that HSC development needs to be viewed in the context of the development of other organs.