Cavefish cope with environmental hypoxia by developing more erythrocytes and overexpression of hypoxia inducible genes

Dark caves lacking primary productivity can expose subterranean animals to hypoxia. We used the surface-dwelling (surface fish) and cave-dwelling (cavefish) morphs of Astyanax mexicanus as a model for understanding the mechanisms of hypoxia tolerance in the cave environment. Primitive hematopoiesis, which is restricted to the posterior lateral mesoderm in other teleosts, also occurs in the anterior lateral mesoderm in Astyanax, potentially pre-adapting surface fish for hypoxic cave colonization. Cavefish have enlarged both hematopoietic domains and develop more erythrocytes than surface fish, which are required for normal development in both morphs. Laboratory induced hypoxia suppresses growth in surface fish but not in cavefish. Both morphs respond to hypoxia by overexpressing hypoxia-inducible factor 1 (hif1) pathway genes, and some hif1 genes are constitutively upregulated in normoxic cavefish to similar levels as in hypoxic surface fish. We conclude that cavefish cope with hypoxia by increasing erythrocyte development and constitutive hif1 gene overexpression.

Low-Dose Pesticides Alter Primary Human Bone Marrow Mesenchymal Stem/Stromal Cells through ALDH2 Inhibition

(1) Background: The impact of occupational exposure to high doses of pesticides on hematologic disorders is widely studied. Yet, lifelong exposure to low doses of pesticides, and more particularly their cocktail effect, although poorly known, could also participate to the development of such hematological diseases as myelodysplastic syndrome (MDS) in elderly patients. (2) Methods: In this study, a cocktail of seven pesticides frequently present in water and food (maneb, mancozeb, iprodione, imazalil, chlorpyrifos ethyl, diazinon and dimethoate), as determined by the European Food Safety Authority, were selected. Their in vitro effects at low-doses on primary BM-MSCs from healthy volunteers were examined. (3) Results: Exposure of normal BM-MSCs to pesticides for 21 days inhibited cell proliferation and promoted DNA damage and senescence. Concomitantly, these cells presented a decrease in aldehyde dehydrogenase 2 (ALDH2: mRNA, protein and enzymatic activity) and an increase in acetaldehyde levels. Pharmacological inhibition of ALDH2 with disulfiram recapitulated the alterations induced by exposure to low doses of pesticides. Moreover, BM-MSCs capacity to support primitive hematopoiesis was significantly altered. Similar biological abnormalities were found in primary BM-MSCs derived from MDS patients. (4) Conclusions: these results suggest that ALDH2 could participate in the pathophysiology of MDS in elderly people long exposed to low doses of pesticides.

Heightened activation of embryonic megakaryocytes causes aneurysms in the developing brain of mice lacking podoplanin

During early embryonic development in mammals, including humans and mice, megakaryocytes first originate from primitive hematopoiesis in the yolk sac. These embryonic megakaryocytes (eMk) circulate in the vasculature with unclear function. Here we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on megakaryocytes/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to lower diencephalon in mutant mouse embryos than wild-type littermates prior to hemorrhage. We found that PDPN counteracted the collagen I-induced secretion of angiopoietin-1 from fetal megakaryocytes, which coincided with enhanced TIE2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.

Tissue-Resident Macrophage Development and Function

Tissue-resident macrophages have been associated with important and diverse biological processes such as native immunity, tissue homeostasis and angiogenesis during development and postnatally. Thus, it is critical to understand the origins and functions of tissue-resident macrophages, as well as mechanisms underlying their regulation. It is now well accepted that murine macrophages are produced during three consecutive waves of hematopoietic development. The first wave of macrophage formation takes place during primitive hematopoiesis, which occurs in the yolk sac, and gives rise to primitive erythroid, megakaryocyte and macrophage progenitors. These “primitive” macrophage progenitors ultimately give rise to microglia in the adult brain. The second wave, which also occurs in the yolk sac, generates multipotent erythro-myeloid progenitors (EMP), which give rise to tissue-resident macrophages. Tissue-resident macrophages derived from EMP reside in diverse niches of different tissues except the brain, and demonstrate tissue-specific functions therein. The third wave of macrophages derives from hematopoietic stem cells (HSC) that are formed in the aorta-gonad-mesonephros (AGM) region of the embryo and migrate to, and colonize, the fetal liver. These HSC-derived macrophages are a long-lived pool that will last throughout adulthood. In this review, we discuss the developmental origins of tissue-resident macrophages, their molecular regulation in specific tissues, and their impact on embryonic development and postnatal homeostasis.

The versican-hyaluronan complex provides an essential extracellular matrix niche for Flk1+ hematoendothelial progenitors

Little is known about extracellular matrix (ECM) contributions to formation of the earliest cell lineages in the embryo. Here, we show that the proteoglycan versican and glycosaminoglycan hyaluronan are associated with emerging Flk1+ hematoendothelial progenitors at gastrulation. The mouse versican mutant Vcanhdf lacks yolk sac vasculature, with attenuated yolk sac hematopoiesis. CRISPR/Cas9-mediated Vcan inactivation in mouse embryonic stem cells reduced vascular endothelial and hematopoietic differentiation in embryoid bodies, which generated fewer blood colonies, and had an impaired angiogenic response to VEGF165. HA was severely depleted in Vcanhdf embryos, with corresponding increase in the HA-depolymerase TMEM2. Conversely, HA-deficient mouse embryos also had vasculogenic suppression but with increased versican proteolysis. VEGF165 and Indian hedgehog, crucial vasculogenic factors, utilized the versican-HA matrix, specifically versican chondroitin sulfate chains, for binding. Versican-HA ECM is an obligate requirement for vasculogenesis and primitive hematopoiesis, acts as an vasculogenic factor-enriching microniche for Flk1+ progenitors from their origin at gastrulation.

Definitive EMP and Pre-HSC Emerge in Myb-Null Murine Embryos and Retain Macrophage Potential

Myb-null murine embryos lack definitive erythropoiesis but can produce primitive erythroid cells, allowing their survival to embryonic day 15 (E15) (Mucenski et al., Cell, 1991). Myb expression has been used to detect emerging HSC and is required for HSC maintenance (Lieu et al., PNAS 2009; North et al., Cell 2009). These data have led to the model that myb function is required for definitive hematopoiesis. Interestingly, macrophages and megakaryocytes are still detected in myb-null embryos and, as both of these lineages are also components of primitive hematopoiesis, it is proposed that these cells are not definitive in origin. Myb-independent macrophages infiltrate fetal tissues and have been implicated as a self-renewing source for several adult tissue-resident macrophage populations (Schulz et al., Nature 2012; Gomez Perdiguero et al., Glia 2013; Hoeffel et al., Immunity 2015). We tested the hypothesis that definitive hematopoiesis is entirely myb-dependent by examining two distinct sources of definitive erythroid/myeloid potential: 1) HSCs that emerge from hemogenic endothelium, including the AGM region at E10.5 in mice and 2) HSC-independent definitive EMP that emerge after primitive hematopoiesis from yolk sac hemogenic endothelium beginning at E8.25 (Frame et al., Stem Cells 2016). By E9.5, EMP can be prospectively isolated based on immunophenotype and contain all the erythroid/myeloid progenitor activity present at this time (McGrath et al., Cell Reports 2015). Surprisingly, we found normal numbers of immunophenotypic EMP in E9.5 Myb-null yolk sacs, and immunohistochemical analysis confirmed their emergence from Runx1+ hemogenic endothelium. At E10.5, reduced numbers of Myb-null EMP were found not only in the yolk sac, but also in the bloodstream and the liver. This decrease correlated with fewer hemogenic clusters in the yolk sacs of Myb-null embryos, as well as alterations in their cell-cycle status. The presence of significant numbers of immunophenotypic EMP suggests they could serve as an alternate source of Myb-null macrophages. Clonal analysis of sorted EMP confirmed that Myb function is necessary for erythroid and granulocyte progenitors, but Myb-null EMP retain normal plating efficiencies for macrophage progenitors. Indeed, Myb-null EMP generate only macrophages in liquid culture, lacking not only erythroid and granulocyte cells, but also Ly6C+ monocytes. Consistent with these results, RNA sequencing analysis of Myb-null versus wildtype EMP demonstrated decreased expression of genes in pathways associated with cellular growth, as well as erythroid and granulocyte fates. We further determined that Myb is not required for the emergence of immunophenotypic pre-HSC in the AGM region of E10.5 embryos. In addition, there were normal numbers of clusters in the aorta of E10.5 myb-null embryos. We were also able to detect lineage-, Kit+ (LK) cells in E14.5 livers as previously reported (Sumner et al., Oncogene 2000). While LK numbers were reduced, the Sca1+ (LSK) subset was present in normal numbers. Like EMP, sorted Myb-null E10.5 pre-HSC, as well as E14.5 liver LK and LSK, lacked erythroid or granulocyte CFC activity, but retained normal CFC-M plating efficiencies. In addition, Ly6C+ monocytes were not observed in liquid cultures of sorted Myb-null E10.5 pre-HSC, which produced only macrophages in vitro, or in Myb-null E14.5 livers. Together these data indicate that Myb is not required for hematopoietic emergence of definitive EMP or HSC, but does facilitate the expansion of these definitive stem/progenitor cells and is required for erythroid and granulocyte differentiation. Additionally, EMP and HSC contain Myb-independent macrophage potential, which does not appear to differentiate from a monocyte intermediate. Disclosures Palis: Rubius Therapeutics: Consultancy.