Regulation of hematopoietic development in the aorta-gonad-mesonephros region mediated by Lnk adaptor protein

Development of hematopoietic cells in the aorta-gonad-mesonephros (AGM) region in the midgestation mouse embryo involves a multistep process, sequentially changing from endothelial cell-like cells, including hemangioblasts, into hematopoietic stem cells, progenitors, and/or lineage-committed cells. An adaptor molecule, Lnk, is known to negatively control the production of pro- and pre-B cells and hematopoietic progenitor cells in adult bone marrow. Here we show a role of Lnk in hematopoietic development in the AGM region. Lnk was predominantly expressed in the endothelial cells lining the dorsal aorta at embryonic day 11.5 (E11.5). Overexpression of Lnk in the primary culture of the AGM region at E11.5 suppressed the emergence of CD45+ hematopoietic cells. Point mutation in the SH2 domain of Lnk, which abolishes the binding capability of Lnk to c-Kit upon stimulation with stem cell factor (SCF), led to loss of Lnk-dependent inhibition of hematopoietic cell development in AGM cultures, suggesting Lnk-mediated inhibition of the SCF/c-Kit signaling pathway. In cultured AGM cells from Lnk homozygous mutant mouse embryos, the number of emerged CD45+ cells was 2.5-fold larger than that from heterozygous littermates. Furthermore, aorta cells of E11.5 Lnk homozygous mutant mice also showed enhanced hematopoietic colony-forming activity. Thus, Lnk is a negative regulator of hematopoiesis in the AGM region.     

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45(low) c-Kit(+) cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45(low) c-Kit(-) cells that showed a granulocyte morphology; CD45(high) c-Kit(low/-) that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45(low) c-Kit(+) cells from the AGM culture had the abilities to reproduce CD45(low) c-Kit(+) cells and differentiate into CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) cells, whereas CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) did not produce CD45(low) c-Kit(+) cells. Furthermore, CD45(low) c-Kit(+) cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45(low) c-Kit(+) cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.     

CD45(low)c-Kit(high) cells have hematopoietic properties in the mouse aorta-gonad-mesonephros region

Long-term reconstituting hematopoietic stem cells first arise from the aorta of the aorta-gonad-mesonephros (AGM) region in a mouse embryo. We have previously reported that in cultures of the dispersed AGM region, CD45(low)c-Kit(+) cells possess the ability to reconstitute multilineage hematopoietic cells, but investigations are needed to show that this is not a cultured artifact and to clarify when and how this population is present. Based on the expression profile of CD45 and c-Kit in freshly dissociated AGM cells from embryonic day 9.5 (E9.5) to E12.5 and aorta cells in the AGM from E13.5 to E15.5, we defined six cell populations (CD45(-)c-Kit(-), CD45(-)c-Kit(low), CD45(-)c-Kit(high), CD45(low)c-Kit(high), CD45(high)c-Kit(high), and CD45(high)c-Kit(very low)). Among these six populations, CD45(low)c-Kit(high) cells were most able to form hematopoietic cell colonies, but their ability decreased after E11.5 and was undetectable at E13.5 and later. The CD45(low)c-Kit(high) cells showed multipotency in vitro. We demonstrated further enrichment of hematopoietic activity in the Hoechst dye-effluxing side population among the CD45(low)c-Kit(high) cells. Here, we determined that CD45(low)c-Kit(high) cells arise from the lateral plate mesoderm using embryonic stem cell-derived differentiation system. In conclusion, CD45(low)c-Kit(high) cells are the major hematopoietic cells of mouse AGM.     

CD41+ cmyb+ precursors colonize the zebrafish pronephros by a novel migration route to initiate adult hematopoiesis.

Development of the vertebrate blood lineages is complex, with multiple waves of hematopoietic precursors arising in different embryonic locations. Monopotent, or primitive, precursors first give rise to embryonic macrophages or erythrocytes. Multipotent, or definitive, precursors are subsequently generated to produce the adult hematopoietic lineages. In both the zebrafish and the mouse, the first definitive precursors are committed erythromyeloid progenitors (EMPs) that lack lymphoid differentiation potential. We have previously shown that zebrafish EMPs arise in the posterior blood island independently from hematopoietic stem cells (HSCs). In this report, we demonstrate that a fourth wave of hematopoietic precursors arises slightly later in the zebrafish aorta/gonad/mesonephros (AGM) equivalent. We have identified and prospectively isolated these cells by CD41 (itga2b) and cmyb expression. Unlike EMPs, CD41(+) AGM cells colonize the thymus to generate rag2(+) T lymphocyte precursors. Timelapse imaging and lineage tracing analyses demonstrate that AGM-derived precursors use a previously undescribed migration pathway along the pronephric tubules to initiate adult hematopoiesis in the developing kidney, the teleostean equivalent of mammalian bone marrow. Finally, we have analyzed the gene expression profiles of EMPs and AGM precursors to better understand the molecular cues that pattern the first definitive hematopoietic cells in the embryo. Together, these studies suggest that expression of CD41 and cmyb marks nascent HSCs in the zebrafish AGM, and provide the means to further dissect HSC generation and function in the early vertebrate embryo.     

Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin(+) CD41(-) CD45(-) endothelial cells and CD41(+) CD45(-) non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin(+) endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41(+) progenitors lacked the B lymphopoietic potential. VE-cadherin(+) endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41(+) progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41(+) progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41(+) progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.     

Negative regulation of hematopoiesis by the fused in myeloproliferative disorders gene product

The t(8;13) translocation, found in a rare and aggressive type of stem cell myeloproliferative disorder, leads to the generation of a fusion protein between the N-terminal gene product of fused in myeloproliferative disorders (FIM)/ZNF198 and the fibroblast growth factor receptor 1 (FGFR1) kinase domain. The chimeric protein was reported to have constitutively activated tyrosine kinase activity. However, little is known about a role of FIM in hematopoietic cell regulation. Here we show that FIM protein is ubiquitously expressed in mouse embryonic tissues but much less in hematopoietic cells. We also show that forced expression of FIM inhibits the emergence of hematopoietic cells in the cultured mouse aorta-gonad-mesonephros (AGM) region on embryonic day (E) 11.5, where definitive hematopoiesis is first found during embryogenesis. These results suggest that the expression level of FIM determines the development of hematopoiesis during mouse ontogeny.     

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.     

Dual role of Mpl receptor during the establishment of definitive hematopoiesis

Cytokine signaling pathways are important in promoting hematopoietic stem cell (HSC) self-renewal, proliferation and differentiation. Mpl receptor and its ligand, TPO, have been shown to play an essential role in the early steps of adult hematopoiesis. We previously demonstrated that the cytoplasmic domain of Mpl promotes hematopoietic commitment of embryonic stem cells in vitro, and postulated that Mpl could be important in the establishment of definitive hematopoiesis. To answer this question, we investigated the temporal expression of Mpl during mouse development by in situ hybridization. We found Mpl expression in the HSCs clusters emerging in the AGM region, and in the fetal liver (FL) as early as E10.5. Using Mpl(-/-) mice, the functional relevance of Mpl expression was tested by comparing the hematopoietic progenitor (HP) content, long-term hematopoietic reconstitution (LTR) abilities and HSC content of control and Mpl(-/-) embryos at different times of development. In the AGM, we observed delayed production of HSCs endowed with normal LTR but presenting a self-renewal defect. During FL development, we detected a decrease in HP and HSC potential associated with a defect in amplification and self-renewal/survival of the lin(-) AA4.1(+) Sca1(+) population of HSCs. These results underline the dual role of Mpl in the generation and expansion of HSCs during establishment of definitive hematopoiesis.     

Definitive hematopoiesis requires the mixed-lineage leukemia gene

The Mixed-Lineage Leukemia (MLL) gene encodes a Trithorax-related chromatin-modifying protooncogene that positively regulates Hox genes. In addition to their well-characterized roles in axial patterning, Trithorax and Polycomb family proteins perform less-understood functions in vertebrate hematopoiesis. To define the role of MLL in the development of the hematopoietic system, we examined the potential of cells lacking MLL. Mll-deficient cells could not develop into lymphocytes in adult RAG-2 chimeric animals. Similarly, in vitro differentiation of B cells required MLL. In chimeric embryos, Mll-deficient cells failed to contribute to fetal liver hematopoietic stem cell/progenitor populations. Moreover, we show that aorta-gonad-mesonephros (AGM) cells from Mll-deficient embryos lacked hematopoietic stem cell (HSC) activity despite their ability to generate hematopoietic progeny in vitro. These results demonstrate an intrinsic requirement for MLL in definitive hematopoiesis, where it is essential for the generation of HSCs in the embryo.     

Role of Oncostatin M in hematopoiesis and liver development

Definitive hematopoietic stem cells (HSCs) first appear in the aorta/gonad/mesonephros (AGM) region and migrate to the fetal liver where they massively produce hematopoietic cells before establishing hematopoiesis in the bone marrow at a perinatal stage. In the AGM region, Oncostatin M (OSM) enhances the development of both hematopoietic and endothelial cells by possibly stimulating their common precursors, so-called hemangioblasts. During development of HSCs in the AGM region, the liver primodium is formed at the foregut and accepts HSCs. While fetal hepatic cells function as hematopoietic microenvironment for expansion of hematopoietic cells during mid to late gestation, they do not possess most of the metabolic functions of adult liver. Along with the expansion of hematopoietic cells in fetal liver, OSM is produced by hematopoietic cells and induces differentiation of fetal hepatic cells, conferring various metabolic activities of adult liver. Matured hepatic cells then lose the ability to support hematopoiesis. Thus, OSM appears to coordinate the development of liver and hematopoiesis in the fetus.     

HIPK2 mediates M1 polarization of microglial cells via STAT3: A new mechanism of depression-related neuroinflammation

This study aimed to investigate the role of protein kinase HIPK2 in depression and its associated mechanism. The chronic unpredictable mild stress (CUSM) model was constructed to simulate mice with depression to detect the mouse behaviors. Moreover, by using mouse microglial cells BV2 as the model. After conditional knockdown of HIPK2, the depressive behavior disorder of mice was improved, meanwhile, neuroinflammation was alleviated, and the M1 cell proportion was reduced. Similar results were obtained after applying the HIPK2 inhibitor tBID or ASO-HIPK2 treatment. HIPK2 was overexpressed in BV2 cells, which promoted M1 polarization of cells, while tBID suppressed the effect of HIPK2 and reduced the M1 polarized level in BV2 cells. Pull-down assay results indicated that HIPK2 bound to STAT3 and promoted STAT3 phosphorylation. We found that HIPK2 can bind to STAT3 to promote its phosphorylation, which accelerates M1 polarization of microglial cells, aggravates the depressive neuroinflammation, and leads to abnormal behaviors. HIPK2 is promising as the new therapeutic target of depression.     

Ontogeny of the mouse hematopoietic system

In vertebrates the extraembryonic mesoderm of the yolk sac (YS) is the first site during embryogenesis where morphologically discernible hematopoiesis may be found. Later hematopoiesis shifts into the embryo proper, first to the liver, the major fetal hematopoietic site, then to definitive hematopoietic territories, the spleen and bone marrow. It is widely accepted that in the mouse this picture reflects the migration of pluripotent hematopoietic stem cells (HSC) from the YS accompanied by subsequent colonization of the hematopoietic tissues during embryogenesis. However, there is no conclusive evidence showing unequivocally the initiating role of the YS in murine adult hematopoiesis. Recently, we have demonstrated the important role of embryo body tissues in the development of CFU-S before the establishment of definitive hematopoiesis in the fetal liver. This finding suggests that the early development of the hematopoietic system in the mouse is more complex than has been previously proposed and we consider here the early hematopoietic events in the developing mouse embryo.     

The placenta is a niche for hematopoietic stem cells

The hematopoietic system develops during embryogenesis at temporally and anatomically restricted sites. The anatomical origin of definitive HSCs is not fully resolved, and little is known about how the different fetal hematopoietic microenvironments direct HSC development. Here, we show that the mouse placenta functions as a hematopoietic organ that harbors a large pool of pluripotent HSCs during midgestation. The onset of HSC activity in the placenta parallels that of the AGM (aorta-gonad-mesonephros) region starting at E10.5-E11.0. However, the placental HSC pool expands until E12.5-E13.5 and contains >15-fold more HSCs than the AGM. The expansion of the CD34(+)c-kit(+) HSC pool in the placenta occurs prior to and during the initial expansion of HSCs in the fetal liver. Importantly, the placental HSC pool is not explained by rare circulating HSCs, which appear later. These data support an important, but unappreciated, role for the placenta in establishing the mammalian definitive hematopoietic system.