Marella de Bruijn

Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors

Most haematopoietic cells renew from adult haematopoietic stem cells (HSCs), however, macrophages in adult tissues can self-maintain independently of HSCs. Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSCs, and fetal macrophages can develop independently of Myb, a transcription factor required for HSC, and can persist in adult tissues. Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC-derived progenitors are still unclear. Here we show in mice that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells) and lung (alveolar macrophages) originate from a Tie2+ (also known as Tek) cellular pathway generating Csf1r+ erythro-myeloid progenitors (EMPs) distinct from HSCs. EMPs develop in the yolk sac at embryonic day (E) 8.5, migrate and colonize the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes. Kupffer cells, microglia and Langerhans cells are only marginally replaced in one-year-old mice, whereas alveolar macrophages may be progressively replaced in ageing mice. Our fate-mapping experiments identify, in the fetal liver, a sequence of yolk sac EMP-derived and HSC-derived haematopoiesis, and identify yolk sac EMPs as a common origin for tissue macrophages.

Combinatorial Transcriptional Control In Blood Stem/Progenitor Cells: Genome-wide Analysis of Ten Major Transcriptional Regulators

Combinatorial transcription factor (TF) interactions control cellular phenotypes and, therefore, underpin stem cell formation, maintenance, and differentiation. Here, we report the genome-wide binding patterns and combinatorial interactions for ten key regulators of blood stem/progenitor cells (SCL/TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1, and GFI1B), thus providing the most comprehensive TF data set for any adult stem/progenitor cell type to date. Genome-wide computational analysis of complex binding patterns, followed by functional validation, revealed the following: first, a previously unrecognized combinatorial interaction between a heptad of TFs (SCL, LYL1, LMO2, GATA2, RUNX1, ERG, and FLI-1). Second, we implicate direct protein-protein interactions between four key regulators (RUNX1, GATA2, SCL, and ERG) in stabilizing complex binding to DNA. Third, Runx1(+/-)::Gata2(+/-) compound heterozygous mice are not viable with severe hematopoietic defects at midgestation. Taken together, this study demonstrates the power of genome-wide analysis in generating novel functional insights into the transcriptional control of stem and progenitor cells.

Core-binding factors in hematopoiesis and immune function.

Core binding factors are heterodimeric transcription factors containing a DNA binding Runx1, Runx2, or Runx3 subunit, along with a non DNA binding CBFβ subunit. All four subunits are required at one or more stages of hematopoiesis. This review describes the role of Runx1 and CBFβ in the initiation of hematopoiesis in the embryo, and in the emergence of hematopoietic stem cells. We also discuss the later stages of hematopoiesis for which members of the core binding factor family are required, as well as the recently described roles for these proteins in autoimmunity.

Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis

Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks. Here we have determined the expression of a network of 18 key haematopoietic transcription factors in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow. We demonstrate that different stem/progenitor populations are characterized by distinctive transcription factor expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated transcription factor pairings, including previously unrecognized relationships between Gata2, Gfi1 and Gfi1b. Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b. Single-cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease.

The SCL transcriptional network and BMP signaling pathway interact to regulate RUNX1 activity

Hematopoietic stem cell (HSC) development is regulated by several signaling pathways and a number of key transcription factors, which include Scl/Tal1, Runx1, and members of the Smad family. However, it remains unclear how these various determinants interact. Using a genome-wide computational screen based on the well characterized Scl +19 HSC enhancer, we have identified a related Smad6 enhancer that also targets expression to blood and endothelial cells in transgenic mice. Smad6, Bmp4, and Runx1 transcripts are concentrated along the ventral aspect of the E10.5 dorsal aorta in the aorta–gonad–mesonephros region from which HSCs originate. Moreover, Smad6, an inhibitor of Bmp4 signaling, binds and inhibits Runx1 activity, whereas Smad1, a positive mediator of Bmp4 signaling, transactivates the Runx1 promoter. Taken together, our results integrate three key determinants of HSC development; the Scl transcriptional network, Runx1 activity, and the Bmp4/Smad signaling pathway.

Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I–hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes. Mouse-to-human genomic comparisons illuminate conserved transcriptional programs despite regulatory rewiring. Rewiring the gene regulatory landscape DNAse I hypersensitive sites (DHSs) correlate with genomic locations that control where messenger RNA is to be produced. DHSs differ, depending on the cell type, developmental stage, and species. Viestra et al. compared mouse and human genome-wide DHS maps. Approximately one-third of the DHSs are conserved between the species, which separated approximately 550 million years ago. Most DHSs fell into tissue-specific cohorts; however, these were generally not conserved between the human and mouse. It seems that the majority of DHSs evolve because of changes in the sequence that gradually change how the region is regulated. Science, this issue p. 1007

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. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.

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. Here we perform functional analysis of over 1,800 and transcriptional analysis of 268 single 23GFP(+) HE cells to explore the onset of EHT at the single-cell level. We show that initiation of the haematopoietic programme occurs in cells still embedded in the endothelial layer, and is accompanied by a previously unrecognized early loss of endothelial potential before HSCs emerge. Our data therefore provide important insights on the timeline of early haematopoietic commitment.

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.