Rac1 Regulates Migration of Yolk Sac Hematopoietic Progenitors into the Blood, a Process Essential for Subsequent Seeding of Fetal Liver Hematopoiesis.

Abstract

Hematopoiesis originates in the mouse embryo at gastrulation with the formation of an initial wave of primitive erythrocytes in the yolk sac (YS) of the extra embryonic tissue. During embryonic development the anatomic site of hematopoiesis changes and hematopoietic stem cells (HSC) are postulated to migrate and seed successive embryonic sites. The origin of definitive hematopoiesis, which has been postulated to be either the YS or the PAS/AGM region, remains controversial, and both sites have previously been shown to contribute to the seeding of HSC/P to the fetal liver (FL). We utilized a genetic approach to study molecular pathways involved in the migration of definitive hematopoietic progenitor cells between the YS and the embryo (AGM and FL). We have recently shown that Rac1 regulates the engraftment phenotype of adult HSC/P (Cancelas et al. Nat Med 2005). In the present study, we induced deletion of Rac1 in a hematopoietic tissue-specific manner by crossing Rac1 flox/null mice with a Vav1-Cre transgenic mouse (Croker B et al. Immunity 2004). Using RosaLacZ reporter mice, we first documented the presence of Vav1Cre-induced recombination in mice in the extraembryonic tissue as early as E 7.5, and in an anatomical distribution concordant with emergence of primitive hematopoiesis. Flow cytometric analysis of single cells derived from the embryo body and from the YS of E 9.5 double transgenic (RosaGFP, Vav1Cre) embryos demonstrated complete recombination in definitive hematopoietic progenitors defined as CD41hiFlk1dim. These data demonstrate that Vav1Cre transgene can induce recombination of genomic flox sequences in both primitive as well as definitive hematopoietic cells early in embryonic development. In crosses of Vav1Cre, Rac1null/wt and Rac1flox/wt mice, no triple transgenic (Vav1Cre, Rac1flox/null) mice survived past E14.5 (0/29, p < 0.02). We next studied hematopoiesis in the developing embryos by enumerating hematopoietic progenitor content. At E9.5, CFU content of YS of Vav1Cre, Rac1flox/null embryos was equivalent to littermate control (Table). When compared with littermate controls at E10.5, even in the presence of normal YS hematopoiesis, we observed significantly decreased numbers of circulating CFU in the blood of Vav1Cre, Rac1flox/null embryos, suggesting defective migration of these cells out of the YS. At E10.5, the content of CFU in FL and embryo proper (after removal of FL and blood) was significantly reduced in Vav1Cre, Rac1flox/null embryos. The defect in FL hematopoiesis was more pronounced at E11.5 and E12.5. Consistent with these results, FL of E11.5 Vav1Cre, Rac1flox/null embryos were pale and had markedly reduced cellularity when compared with littermate controls (0.11x106±0.08x106 vs 1.2x106±0.3x106, n ≥ 5, p < 0.01), demonstrating a failure of fetal liver hematopoiesis.These data demonstrate that Rac1 regulates the migration of HSC/P from the YS into the blood and suggest that this migration is essential for subsequent seeding of both the embryo proper and the FL.TissueGenotypeE9.5E10.5E11.5E12.5YScontrol1024±298719±167--TgRac1 flox/null1011±257722±178--Bloodcontrol-459±116--TgRac1 flox/null-253±74 *--FLcontrol-432±1127496±209612889±2512TgRac1 flox/null-39±32 *235±103 *103±125 *Embryo propercontrol-825±214--TgRac1 flox/null-74±62 *--Data are presented as mean±SD, * - p<0.01