Involvement Of Smad5 Gene In Regulation Of Embryonic Hematopoiesis

The hematopoietic and vascular systems of the mouse arise from extraembryonic mesoderm that migrate through primitive streak to the presumptive yolk sac between days 6.5 and 7.0 of gestation. Commitment to the hematopoietic and endothelial lineages within the yolk sac results in the development of discrete structures known as blood islands. Over the next 12 hours, its central cells give rise to the primitive erythrocytes, a population of large nucleated red blood cells that produce embryonic globins. Subsequent transition of hematopoietic activity from yolk sac to fetal liver defines the switch from the single-lineage primitive erythropoiesis to definitive multilineage hematopoiesis. However, the mechanisms regulating mesoderm commitment to hematopoietic lineages remain poorly understood. Recently, findings from studies using xenopus animal cap induction assays, mouse knockouts and in vitro ES cells differentiation models have implicated the contributions of specific growth factors and transcription factors to this process.The transforming growth factor-β (TGF-β) superfamily, a large group of highly conserved growth factors including TGF-βs, activins and BMPs, regulate a wide variety of cellular functions such as proliferation, differentiation, apoptosis and migration. Signals from these growth factors are transduced by a group of Smad proteins. To date, there are nine vertebrate Smads, including the receptor-activated Smads (R-Smads), Smads1-3, 5 and 8, the common mediator Smad4 and Smad4β, and the inhibitory Smads, Smad6 and 7. Signaling is initiated when the ligand induces assembly of a heteromeric complex of type Ⅱ and type ? TGFβ receptors. Then R-Smads are directly phosphorylated by activated typeⅠTGF-β receptors. Upon phosphorylation, R-Smads interact with Smad4 to form heteromeric complexes that translocated to the nucleus, where they associate with DNA-binding partners or transcription factors and then regulate the transcriptional response of the target genes. Previous studies have strongly shown that TGF-β can either in vivo or in vitro preferentially inhibit proliferation of long-term culture initiation cells (LTC-IC) and highproliferative colony-forming cells (HPP-CFC), but had no effect on mature CFC. Although original in vitro investigations have suggested that Smad2 and 3 act downstream of the TGF-β, recent finding demonstrates that Smad5, conventionally thought to respond to BMP signals, is also capable of transducing the inhibitory signal of TGF-β on hematopoietic progenitor cells from adult bone marrow.Targeted disruption of Smad5 gene results in multiple defects and embryonic lethality at E10.5-E11.5. As preliminary analysis have shown that Smad5-/- yolk sacs contained increased numbers of CFU-GM, it is highly probable that TGF-β may also regulate embryonic hematopoiesis via Smad5 transduced signals. the CFU-E at E9.5 represents late precursors of definitive erythropoiesis. Considering the selective modulation of TGF-β on primitive hematopoietic progenitors from bone marrow and cord blood, the appropriate choose of the precursors to be focused may be critical. HPP-CFC, detected first at early somite stages (E8.25) in mouse yolk sac and ES cells-derived EBs, is the earliest multipotential hematopoietic precursors that can be cultured in vitro in the absence of stromal cells. While CFU-GM represents relative mature stage of hematopoietic differentiation, accordingly, the specific concern on high proliferative potential colony forming cells (HPP-CFC) is more relevant. In addition, due to the defective microenvironment including angiogenesis defect and mesenchyme apoptosis in early Smad5-/- embryos, direct analysis of the embryonic hematopoiesis in these embryos would not give reliable results. The in vitro hematopoietic differentiation of embryonic stem (ES) cells has been shown to recapitulate the early embryonic hematopoiesis while circumvents the drawbacks of a defect microenvironment in some mutant embryos. In this study, we capitalize on this system and analyz...