The P.I. intends to identify the developmental origin of the hematopoietic stem cell which can repopulate the hematopoietic system of adult mice. The proposal is based on solid preliminary observations made by the P.I. that the earliest stage and site of repopulating stem cells in the mouse is in the AGM region of day 10 mouse embryos. repopulating cells appear in the yolk sac and fetal liver subsequent to day 10. Since circulation is well established by day 10, hypotheses holding that stem cells arise de novo in the AGM region and/or seed there and elsewhere by entering the circulation are viable. This proposal tests that hypothesis.
Aim 1 will make use of organ culture to identify the origin of the earliest repopulating stem cells. This use of organ culture removes the complications of circulation from the analysis, and preliminary experiments show that organs can be cultured from 8 to 11 day embryos. Yolk sac, liver primordium, and the AGM region will be examined and limiting dilution will be used to estimate the number of CFU-S and repopulating stem cells. Verification of multilineage repopulation will be provided by genetic markers and if necessary retroviral marking. Both procedures have been used in the past by the P.I. for related studies. Once the origin of repopulating stem cells is established, the P.I. proposes to investigate the emergence of repopulating stem cells in transgenic mice which are deficient in GATA-2, and 3. These animals have defects in definitive hematopoiesis. Finally, the P.I. will examine the cell surface characteristics of AGM stem cells. These studies will be aided by transgenic mice which express lac Z under the control of the Sca-1 promoter, aiding in the identification and further characterization of these cells.
In aim 2, the P.I. will examine the microenvironment in the yolk sac for its ability to support AGM derived HSC. The use of organ culture will allow the isolation of HSC free yolk sac cells on which various normal and mutant HSCs will be grown. The pattern of cytokine and cytokine receptor gene expression, as well as TGFb family members will be examined in stromal cells using RT-PCR. This information will be compared to immunohistochemistry of embryo tissues which has been done previously or will be done in parallel with the RT-PCR analysis.
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