Critical requirement of VEGF-C in transition to fetal erythropoiesis

Key Points Vegfc is essential for mobilization, maturation, and enucleation of primitive erythroblasts. Vegfc deletion compromises liver colonization by erythro-myeloid progenitors and subsequent macrophage/erythroid expansion.

Csnk2β Knockout during Hematopoiesis Results in Lethality at Mid/Late Gestation Mostly Due to Impaired Fetal Erythropoiesis

Background. CK2, a serine-threonine kinase composed of two catalytic (α) and two regulatory (β) subunits, has been clearly involved in several hematologic malignancies. This kinase regulates the PTEN/PI3K/AKT, Wnt/βcatenin, Hedgehog, JAK-STAT, cMyc and NF-κB signalling cascades, all of which are known to be of critical importance for hematopoietic stem cell (HSC) biology and normal hematopoiesis. However, the role played by CK2 during blood cell development has remained as yet unexplored. Aims and methods. CK2 function in hematopoiesis was investigated generating conditional knockout mice for CK2β by crossing Csnk2β-Flox/Flox mice with Vav1-CRE transgenic mice. Inactivation of Csnk2β started from 9.5 dpc during embryonic development. Histo-cytological methods, FACS analysis, colony-forming assays (CFA), signal transduction studies by western blotting and RT-PCR were employed to characterize the cellular and molecular phenotype. High throughput RNAseq analysis was also performed on purified Ter119-positive erythroid cells from Csnk2β knockout and Csnk2β control mice to identify differentially expressed CK2-dependent transcriptional targets. Results. Csnk2β knockout in hematopoiesis resulted lethal at mid-late gestation. Rarely some pups were found dead at birth. Macroscopic and phenotypic analysis during gestation revealed the appearance of pale and hydropic fetuses after 12.5 dpc. The majority of pups showed teleangiectasic vessels and haemorrhages. Fetal livers appeared smaller and paler. Cytological analysis and CFA studies unveiled a great depletion of hematopoietic elements belonging to both the erythroid, megakaryocytic and granulocytic-monocytic precursors. A more thorough analysis of the erythroid phenotype revealed that Csnk2β loss caused impairment/loss of red cell maturation at two developmental stages: the earlier stages of Megakaryocyte-Erythroid Precursors (MEP) and pro-erythroblasts and the later stages of terminal maturation (orthocromatic erythroblasts towards reticulocytes). Expression analysis of proteins/genes belonging to known hematopoietic and erythroid-regulating pathways showed perturbations in cell cycle regulatory molecules, cellular apoptosis, a marked reduction of total and phosphorylated Akt in Ser473 and Ser129, a decrease of GATA1 protein levels and a decrease of Hedgehog/Wnt target genes such as Gli-1 and Cyclin D1. Erythropoietin-dependent AKT activation and GATA1 phosphorylation was impaired by Csnk2β loss. Moreover, starting at 14.5 dpc, blood cells displayed a massive p53-dependent response, marked by high levels of p21 and a progressive clear apopototic phenotype. At 17.5 dpc residual hematopoietic cells in the fetal liver were represented by dying erythroid cells, immature myelo-monocytic precursors (expressing high CD11b and low Gr1 levels on the surface) and B-cells displaying an aberrant phenotype with low intensity of expression of B220 and CD19 on the surface. High throughput RNAseq analysis of Ter119-expressing fetal liver cells (erythroid lineage) obtained from 14.5 dpc pups revealed the upregulation of 145 transcripts and the downregulation of 68 transcripts. Among the most increased transcripts were the transcription factors Jun/AP1 and stress-related intermediaries and embryonal globin ε and ζ chains. Among the most decreased transcripts were sugar transporters, glycoproteins CD36 and CD59a, Duffy Blood Group Atypical Chemokine Receptor and component members. Conclusions. We found that Csnk2β plays a critical role in mouse blood development by regulating definitive hematopoiesis of all the hematopoietic cell lineages; however, Csnk2β was needed for the early and late erythropoiesis whilst its loss could be compatible with a certain extent of immature/altered myelo-monocytic and B cell development. Among the pathways found targeted by Csnk2β loss were the PI3K/Akt and the p53-p21 cascades. Our data also suggest that Csnk2β might have a role in the proper activation of the erythroid master regulator GATA1. Moreover, RNAseq analysis revealed that this kinase might have a broader impact during erythroid cell maturation by regulating the activity of critical stress related transcription factors, of molecules regulating energy-managing cellular processes and of mechanisms controlling the switch from embryonal to fetal erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Pregnancy-Secreted Acid Phosphatase, Uteroferrin, Enhances Fetal Erythropoiesis

Uteroferrin (UF) is a progesterone-induced acid phosphatase produced by uterine glandular epithelia in mammals during pregnancy and targeted to sites of hematopoiesis throughout pregnancy. The expression pattern of UF is coordinated with early fetal hematopoietic development in the yolk sac and then liver, spleen, and bone to prevent anemia in fetuses. Our previous studies suggested that UF exerts stimulatory impacts on hematopoietic progenitor cells. However, the precise role and thereby the mechanism of action of UF on hematopoiesis have not been investigated previously. Here, we report that UF is a potent regulator that can greatly enhance fetal erythropoiesis. Using primary fetal liver hematopoietic cells, we observed a synergistic stimulatory effect of UF with erythropoietin and other growth factors on both burst-forming unit-erythroid and colony-forming unit-erythroid formation. Further, we demonstrated that UF enhanced erythropoiesis at terminal stages using an in vitro culture system. Surveying genes that are crucial for erythrocyte formation at various stages revealed that UF, along with erythropoietin, up-regulated transcription factors required for terminal erythrocyte differentiation and genes required for synthesis of hemoglobin. Collectively, our results demonstrate that UF is a cytokine secreted by uterine glands in response to progesterone that promotes fetal erythropoiesis at various stages of pregnancy, including burst-forming unit-erythroid and colony-forming unit-erythroid progenitor cells and terminal stages of differentiation of hematopoietic cells in the erythroid lineage.