Vav1 Deficiency Differentially Affects Engraftment of Fetal Hematopoietic Organs During Development Compared with Adult Marrow After Transplantation

Abstract 1212

Rac GTPases function as molecular switches, which integrate extracellular signals that are critical for hematopoietic stem cell (HSC) engraftment and bone marrow repopulation during transplantation. We have recently reported that loss of the Vav1 protein, a known hematopoietic-specific activator of Rac, drastically reduces engraftment of adult HSCs without compromising steady-state hematopoiesis in adult mice (Sanchez-Aguilera et al., PNAS 2011). This suggests that HSC migration during ontogeny is unaffected by Vav1 loss and implies that GTPase regulation of HSC migration is distinctly different during embryonic migration versus (vs.) migration required during transplant-mediated engraftment. To verify this, we have examined the role of Vav1 in fetal hematopoietic cells. In the developing embryo, Vav1 expression is first detected at E11.5 and increases to E14.5 (Bustelo et al., Cell Growth Differ 1993). At this developmental age, fetal liver (FL) is the major hematopoietic organ in the embryo. We found no difference between Vav1^−/− and WT animals in the number of immunophenotypically defined HSCs (LSK SLAM or c-Kit⁺CD34⁺) in the FL isolated from E13.5 embryo (4.4 +/− 0.6% LSK-SLAM in the WT group vs. 7.6 +/− 1.4 in the Vav1^−/−group). Vav1^−/− FL cells also formed normal numbers of hematopoietic progenitor colonies in methylcellulose (42.01 +/−3.2 in the WT group vs. 37.63 +/− 2.5 in the Vav1^−/−group). We then compared the number of LSK SLAM cells in the BM of Vav1^−/− newborn mice to WT, as well as their ability to form colonies in methylcellulose, and did not detect any difference between the two genotypes. Together, these data suggest that during hematopoietic development, the initial colonization of the FL and subsequent transition to BM hematopoiesis are not affected by deletion of Vav1. To determine whether Vav1 is required for engraftment of FL cells after transplantation, we performed competitive transplantation experiments. We found that Vav1^−/− E13.5 FL cells failed to provide long term engraftment to lethally irradiated syngeneic recipients, as measured by peripheral blood (PB) chimerism at 4, 8 and 16 weeks after transplantation (an average of 0.08%, 0.42% and 0.12% donor-derived CD45.2 positive cells in the PB of Vav mice at the three time points analyzed vs. 42.1%, 49.5% and 66.35% in the WT group; p<0.0001, p=0.0049, p=0.0016, respectively). In addition, Vav1^−/− FL cells failed to contribute to PB reconstitution in newborn syngeneic recipients that were sublethally irradiated or born from pregnant mothers conditioned with busulfan treatment. These data confirm the differential requirement for Vav1 during fetal physiological organ engraftment compared to HSC transplant. Next, we sought to determine the mechanisms underlying the lack of engraftment of Vav1^−/− fetal cells during transplantation. Given that the Vav1-Rac axis has been shown to contribute to the activation of the JNK pathway (Crespo et al., Oncogene 1996), which is induced by pro-inflammatory cytokines (Kyaris et al., Nature 1994) produced during bone marrow ablation, we tested whether Vav1^−/− cells were hypersensitive to pro-inflammatory cytokines. Vav1^−/− cells produced normal numbers of progenitor-derived colonies in vitro in the presence of increasing doses of TNFα and IFNγ. Similarly, the lack of engraftment of Vav1^−/−HSC was not rescued upon transplantation into W/Wv mice, which do not require any myelablation for effective engraftment of WT HSC. These results indicate that the lack of engraftment of Vav1 cells is not due to an increased sensitivity to pro-inflammatory cytokines.

In conclusion, our data suggest that distinct mechanisms regulate the physiological migration of FL cells to the BM during development and their ability to reconstitute the hematopoietic system of recipients during transplantation. We believe that understanding the basis of these mechanisms is critical for transplant biology and regenerative medicine.