Reduced Erg Dosage Perturbs Fetal and Adult Hematopoiesis

Abstract 1189

Erg is an ETS family transcription factor playing a central role in both normal development and in disease. Using a forward genetic screen, a potential role of Erg in definitive hematopoiesis and self-renewal of adult hematopoietic stem cells (HSC) was demonstrated previously: mice heterozygous for a mutant Erg allele (Erg^mld2) carrying a point mutation had significantly reduced HSCs with impaired repopulating capacity (Nat Immunol. 9:810–9). However, whether this hematopoietic defect is due to a reduced Erg dosage or due to a dominant negative effect of this mutant allele is unknown. In particular, little is known about how Erg controls normal development and how changes in Erg dosage contribute to disease, especially at the stem cell level.

Using reverse genetics, we generated an Erg knockdown allele (Erg^kd, ∼80% knockdown) by inserting a transcriptional stop cassette in the Erg locus. Erg^kd/kd animals die around E10.5-E11.5 due to defects in endothelial and hematopoietic cells. In Erg^kd/+ mice, ∼40% reduction of Erg dosage perturbs both fetal liver and bone marrow (BM) hematopoiesis. A significantly reduced proportion and also absolute numbers of lin-Sca-1+c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) was observed, although in clonogenic in vitro assays, the distribution of individual myeloid colonies was comparable between Erg^kd/+ and wild type (WT). Furthermore, in accordance with the reduced number of platelet counts in the peripheral blood of Erg^kd/+ mice, the proportion of megakaryocytic progenitors within the lin-Sca-1-c-Kit+CD41+ gate was significantly reduced. In megakaryocytic colony assays, we documented smaller sizes of Erg^kd/+ colonies with reduced acetylcholinesterase activity, suggesting that the platelet phenotype stems from a defective differentiation in the megakaryocytic lineage.

The stop cassette in the Erg^kd allele is flanked by loxP sites and Cre-mediated excision of this cassette restores Erg expression to WT level. Tie2-Cre-mediated restoration of Erg in hematopoietic and endothelial cells rescues the lethal phenotype in Erg^kd/kd embryos. To test whether the hematopoietic defect in Erg^kd/+ mice is due to haploinsufficiency of Erg, we used Mx1-Cre to restore Erg in adult hematopoietic cells. In Mx1-Cre;Erg^kd/+;R26-L-S-L-YFP (R26Y) mice, leaky expression of Cre leads to restoration of Erg and simultaneous activation of the YFP reporter (R26Y) within the same cell. By generating genetic mosaics where only a fraction of cells (YFP+ cells) had undergone recombination, we found a competition between hematopoietic cells expressing different Erg levels in both peripheral blood and BM. In particular, the majority of cells in the BM LSK population were YFP+ (thus Erg-restored). Erg-rescued YFP+ BM LSK cells also formed more colonies in semi-solid medium compared to YFP- BM LSK cells from the same animal. These data demonstrate that Erg-restored HSPCs outcompete Erg^kd/+ HSPCs for contributing to adult hematopoiesis in vivo. In order to compare gene expression profiles of HSPCs expressing different Erg levels, we sorted YFP+ and YFP- LSK cells from Mx1-Cre;Erg^kd/+;R26Y mice. Intriguingly, we found many pathways related to stem cells (e.g., Wnt signaling, Hedgehog signaling, Insulin receptor signaling, the human HSC signature from Nat Med. 17:1086–93) enriched in YFP- mutant LSK cells, rather than in YFP+ LSK. This unexpected observation was further confirmed by retrospective analysis of the published microarray data from Erg^mld2/+ LSK cells. We reason that this may be largely due to impaired differentiation of HSCs so that primitive long-term HSCs (LT-HSCs) are over-represented in the mutant LSK population.

In conclusion, our study suggests the major requirement for Erg during development is in hematopoietic and endothelial cells. ∼40% reduction of Erg gene dosage affects contribution of HSCs to both fetal and adult hematopoiesis, partly due to impaired differentiation from LT-HSCs to more differentiated hematopoietic cells.