The Rho GTPase Rac1 Is Not Required for Definitive Hematopoietic Specification in ES-Derived Hematopoiesis.

Rac1 is a Rho GTPase involved in integrating signaling pathways that regulate numerous cellular processes including adhesion, migration, proliferation and HSC engraftment. Homozygous deletion of Rac1 is lethal in the murine embryo prior to E9.5 and Rac1^−/− embryos demonstrate defective gastrulation associated with reduced epiblast adhesion and motility. We have recently demonstrated using lineage-specific conditional deletion that Rac1 insufficiency results in severely impaired hematopoiesis in the embryonic sites of hematopoiesis (AGM, aortic clusters and fetal liver) in the setting of normal hematopoietic development in the yolk sac (YS) and reduced HSC and progenitors in the fetal circulation. This data appears to support the controversial hypothesis that YS derived HSC seed embryonic sites, but an alternative explanation is that Rac1 is essential for some aspect of the induction of intraembryonic hematopoiesis in situ. Another possibility is that Vav1-Cre-mediated excision of Rac1 occurs prior to the onset of hematopoiesis in the embryo proper but not early enough to affect yolk sac hematopoiesis. To test whether Rac1 insufficiency perturbs the normal early differentiation of hematopoietic cells in vitro, we used a lentivirus expressing a Rac1-specific shRNA to knock down expression in an ES line previously characterized to have good hemogenic potential. We observed that the de novo knockdown of Rac1 expression appeared to have no impact upon derivation of hematopoietic progenitors. To demonstrate that this was not the result of inefficient knockdown of Rac1, we derived Rac1^−/− ES lines from blastomeres resulting from the mating of Rac1^+/− mice. Rac1^−/− ES lines were produced in normal Mendelian ratios (4 Rac1^+/+: 9 Rac1^+/−: 3 Rac1^−/−) and did not demonstrate any evidence of abnormal expansion on murine embryonic fibroblasts. In order to assess the impact of Rac1 deficiency on the hemogenic potential of ES cells, standard in vitro differentiation via embryoid body formation was utilized. Neither Rac1 haploinsufficiency nor complete absence of Rac1 had any impact on the production of CD41⁺/c-Kit⁺ hematopoietic progenitors within embryoid bodies (Table 1). Furthermore, colony forming assays demonstrated that Rac1 insufficiency did not alter the relative frequency of hematopoietic progenitor compartments (Table 2). We conclude that in the absence of a requirement for vascular migration of HSC, Rac1 is not required for the specification of definitive hematopoiesis. These data, together with our previously published in vivo data continue to support the hypothesis that HSC migration from the YS to the embryo may be required for development of hematopoiesis in the embryo proper.

No relevant conflicts of interest to declare.