An important unresolved question is how the programs to generate primitive (embryonic) and definitive (adult) erythroid cells differ. This has clinical relevance for sickle cell anemia, which can be ameliorated by reactivation of a primitive erythroid program. A novel protocol has been developed to isolate stage-specific mouse primary erythroid cells using laser capture microdissection (LCM), followed by microarray expression assays. By identifying genes that are differentially expressed in primitive and definitive cells, we will generate testable hypotheses about genes that control erythroid development. The raw microarray data will be widely disseminated, and we will test the functions of prioritized candidate genes. The feasibility and reproducibility of these techniques has been established using the rarest type of erythroid cells, purified from mouse embryonic day 9 (E9) yolk sac tissue sections. In mouse expression arrays, 98 genes are expressed significantly higher in E9 erythroid than in control E9 epithelial cells. In our Preliminary Studies and the work of others, candidate regulators of primitive erythropoiesis include Rnd2, Slit 2, reelin and LMO2, and members of the BMP, VEGF and retinoid signaling pathways.
Aim 1 is to complete the genetic profile of mouse erythroid development, using RNA from highly purified, temporally-staged cells from fetal liver and bone marrow in hybridizations to mouse oligonucleotide microarrays. This will complement our data for embryonic yolk sac. Differentially expressed transcription factor, chromatin regulatory, cell surface receptor, cell growth and signaling genes will be of high priority for study. Among the statistical analyses, supervised learning methods will identify genes that behave similarly to the developmental^ controlled globin genes. The observed erythroid- and developmental stage-specific expression of selected, high priority candidate genes will be verified using quantitative RT-PCR, and testable hypotheses about the control mechanisms of primitive and definitive erythropoiesis will be generated.
In Aim 2, genes differentially expressed due to the absence of KLF2, a transcription factor essential for primitive erythropoiesis, are being identified. RNA from LCM-isolated erythroid cells from KLF2-/- E9 yolk sacs is being used in microarray assays to compare the gene expression profile to that of wild-type cells.
In Aim 3, the hypotheses generated about genes controlling primitive erythropoiesis will be tested using existing knockout mice and knockdowns in erythroid cell lines.
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