The role of apoptosis in the development of AGM hematopoietic stem cells revealed by Bcl-2 overexpression.

Apoptosis is an essential process in embryonic tissue remodeling and adult tissue homeostasis. Within the adult hematopoietic system, it allows for tight regulation of hematopoietic cell subsets. Previously, it was shown that B-cell leukemia 2 (Bcl-2) overexpression in the adult increases the viability and activity of hematopoietic cells under normal and/or stressful conditions. However, a role for apoptosis in the embryonic hematopoietic system has not yet been established. Since the first hematopoietic stem cells (HSCs) are generated within the aortagonad-mesonephros (AGM; an actively remodeling tissue) region beginning at embryonic day 10.5, we examined this tissue for expression of apoptosis-related genes and ongoing apoptosis. Here, we show expression of several proapoptotic and antiapoptotic genes in the AGM. We also generated transgenic mice overexpressing Bcl-2 under the control of the transcriptional regulatory elements of the HSC marker stem cell antigen-1 (Sca-1), to test for the role of cell survival in the regulation of AGM HSCs. We provide evidence for increased numbers and viability of Sca-1(+) cells in the AGM and subdissected midgestation aortas, the site where HSCs are localized. Most important, our in vivo transplantation data show that Bcl-2 overexpression increases AGM and fetal liver HSC activity, strongly suggesting that apoptosis plays a role in HSC development.

Stromal cell lines from mouse aorta-gonads-mesonephros subregions are potent supporters of hematopoietic stem cell activity.

The aorta-gonads-mesonephros (AGM) region autonomously generates the first adult repopulating hematopoietic stem cells (HSCs) in the mouse embryo. HSC activity is initially localized to the dorsal aorta and mesenchyme (AM) and vitelline and umbilical arteries. Thereafter, HSC activity is found in the urogenital ridges (UGs), yolk sac, and liver. As increasing numbers of HSCs are generated, it is thought that these sites provide supportive microenvironments in which HSCs are harbored until the bone marrow microenvironment is established. However, little is known about the supportive cells within these midgestational sites, and particularly which microenvironment is most supportive for HSC growth and maintenance. Thus, to better understand the cells and molecules involved in hematopoietic support in the midgestation embryo, more than 100 stromal cell lines and clones were established from these sites. Numerous stromal clones were found to maintain hematopoietic progenitors and HSCs to a similar degree as, or better than, previously described murine stromal lines. Both the AM and UG subregions of the AGM produced many supportive clones, with the most highly HSC-supportive clone being derived from the UGs. Interestingly, the liver at this stage yielded only few supportive stromal clones. These results strongly suggest that during midgestation, not only the AM but also the UG subregion provides a potent microenvironment for growth and maintenance of the first HSCs.