Novel insights on the role of CXCL12-CXCR4 signaling in hematopoietic development

The CXCL12-CXCR4 axis plays a key role in inflammation and embryonic/fetal hematopoiesis. Although the importance of this pathway in hematopoietic development has been established, significant gaps remain in understanding the specific functions of CXCR4 different ligands in early hematopoietic progenitor dynamics. Additionally, High Mobility Group Box 1 (HMGB1), known for its role in inflammation and tissue regeneration, can bind CXCL12, forming a heterocomplex signaling through CXCR4. The role of HMGB1-CXCR4 in embryonic hematopoiesis is still largely unexplored. To precisely map the expression of CXCL12 at early stages of hematopoiesis, we took advantage of the CXCL12+/eGFP knock-in transgenic mouse line. This line also allows to analyze the hematopoietic phenotype of homozygous CXCL12eGFP/eGFP embryos lacking CXCL12. We assessed CXCL12 expressing cells in primary and secondary hematopoietic sites at embryonic day 10.5 (E10.5) through whole-mount immunofluorescence (IFs). In parallel, we performed flow cytometric analysis of Fetal Liver (FL) and Peripheral Blood (PB) CXCL12-null embryos at E12.5 and at E14.5. Furthermore, to dissect the timing of CXCR4 requirement during fetal hematopoiesis we adopted a conditional knockout strategy employing Cdh5-CreERT2;R26LSL-tdTomato;Cxcr4fl/fl transgenic mice. In midgestation embryos (E10.5) we detected strong CXCL12 expression in the vitelline artery, which we have previously observed to give-rise to fetal-restricted multipotent progenitors. Accordingly, in FLs lacking CXCL12 we observed a significant reduction in the total number of LSK+CD48+CD150- multipotent progenitors (MPPs) as well as in other hematopoietic progenitor cells (Ter119-CD45+Kit+). HMGB1 knockout E14.5 FLs showed a similar phenotype, exhibiting a significant reduction in absolute number and frequencies of MPPs and Ter119-CD45+Kit+, along with severe reduction of myeloid cells in the PB. Interestingly, at E12.5 we observed an even more severe phenotype, with a notable reduction in the total number of FL cells. These results were supported by ex vivo colony assays highlighting a severe reduction of hematopoietic progenitors in embryos lacking HMGB1. Finally, preliminary data showed that the deletion of CXCR4 during the hematopoietic stem cell (HSC)-dependent wave impairs immunophenotypic HSCs number and frequency. Additional functional and molecular characterization of these models is ongoing. Taken together, our results show that the lack of CXCR4 ligands results in an impairment in the development of HSCs-independent hematopoietic progenitors, thus suggesting an important role of the CXCL12-CXCR4 axis in this progenitor cell type, which was recently described to be one of the primary contributors to fetal hematopoiesis. This project will shed new light in CXCL12- CXCR4-HMGB1 function during hematopoietic development. Our results bear relevance for protocols aimed at ex vivo HSC generation as well as for neonatal and pediatric leukemias.