Genome Expression Profiling of the First Hematopoietic Progenitors of the Mouse Embryo.

Abstract 2536

Poster Board II-513

The transcriptional networks that regulate lineage commitment and expansion of the earliest hematopoietic progenitors in the mammalian embryo have not been well studied, due to a lack of methods for isolating these cells. We have begun to address this problem by purifying the first hematopoietic-committed progenitors in the mouse embryo based on expression of a human ε-globin::H2B-EGFP transgene that is expressed exclusively within the primitive erythroid (EryP) lineage, as early as embryonic day (E) 7.5. EryP are the first lineage-specific cell type to form in the embryo. They arise in large numbers from yolk sac-derived progenitors at the end of gastrulation, enter the circulation as nucleated cells soon thereafter, and continue to mature in a stepwise, synchronous fashion until they enucleate. The early and lineage specific expression of the GFP reporter allowed us to isolate not only circulating EryP (E9.5-E11.5) but also a population from dispersed E7.5-8.5 embryos that is enriched in EryP progenitors. Genome expression profiling allowed us to define the transcriptome from each stage of development and revealed highly dynamic changes during the progression from progenitor to maturing erythroblast. Hierarchical clustering analysis was used to organize genes on the basis of overall similarity in expression patterns; six major cluster patterns were identified. Genes within these clusters comprised distinct functional classes. For example, particularly prominent increases in expression were detected for genes involved in ribosome biogenesis, translation, chromosome condensation, and autophagy. Genes that were downregulated included those involved in DNA replication, cell cycle, and nucleolar and organelle biogenesis. We have focused on the emergence of EryP in the yolk sac. Expression of Gata2 is high in the progenitor population at E7.5 and decreases dramatically by E8.5. In contrast, Gata1, Scl, and Eklf are all upregulated during maturation of EryP progenitors, suggesting that these transcription factors have distinct functions during primitive erythropoiesis. Consistent with expression of the GFP reporter as early as E7.5, we find that endogenous mouse embryonic globin genes are also expressed at this stage. Therefore, globin gene expression is an early feature of EryP development. Analysis of promoters of differentially expressed genes allowed us to identify candidate transcriptional regulators, some of which have not previously been implicated in erythroid development. This is the first lineage specific transcription profiling of a differentiating hematopoietic cell type in the early mouse embryo. While we have focused on the development of EryP, insights from this study should have broader relevance to the definitive erythroid lineage.