Regulation of Primitive Erythroid Progenitor Development

Abstract 1211

Primitive erythroid cells (EryP) are the first hematopoietic cell type to form in the mammalian embryo. Their progenitors are found in the mouse yolk sac from embryonic day (E) ∼E7.5–8.5 and, as circulation initiates, they begin to differentiate, enter the bloodstream and continue to mature in a stepwise, synchronous fashion until their enucleation several days later. We purified these first hematopoietic-committed progenitors from staged embryos based on the expression of a nuclear (histone H2B fusion) GFP transgene that is expressed specifically within the EryP lineage as early as E6.75. Their very high proliferative capacity can be easily followed using the H2B-GFP reporter and with vital dyes, and we are using these and other approaches to study their cell cycle properties. We have now generated analogous mouse lines expressing different color fluorescent reporters (CFP, mCherry), to permit crosses with other GFP or YFP expressing mouse lines so that simultaneous imaging of (for example) EryP and endothelial cell development can be followed in real time. Genome-wide expression profiling (Isern et al., Blood 117:4924–4934, 2011) allowed us to define the transcriptome from each stage of development of FACS-sorted (GFP+) cells, including progenitor stages, and revealed highly dynamic changes during the progression from progenitor to maturing erythroblast. We focused on the emergence of EryP progenitors in the yolk sac and on the transition to circulation stage, when progenitor activity is lost and a peak is observed in the number of genes whose expression changes. Promoter analysis of differentially expressed genes allowed us to identify candidate transcriptional regulators, some of which have not previously been implicated in erythroid development (e.g. Nkx3.1, known previously as a regulator of prostate stem cells). We have found that the Wnt signaling pathway is active in EryP progenitors; studies to evaluate the function of this pathway are in progress. EryP progenitors express genes associated with anaerobic glucose metabolism (the Warburg effect), a phenotype characteristic of cancer and other rapidly proliferating cells. Interestingly, we have identified three clusters enriched in genes related to mitochondrial structure/function; genes in these clusters are sequentially expressed. Analysis of these changes may provide insights into whether the glycolytic profile of EryP reflects the energy needs of these cells or a more unique feature of primitive erythropoiesis. Currently we are using computational methods to identify transcription factor (ChEA, ChIP Enrichment Analysis) and kinase (KEA, Kinase Enrichment Analysis) networks that may play a role in the regulation of primitive erythroid development. This is the first lineage specific transcription profiling of a differentiating cell type in the early mouse embryo and will provide a strong basis for future work on normal erythropoiesis throughout ontogeny. It may also help guide efforts to direct the differentiation of stem/progenitor and cells of other lineages to an erythroid cell fate.