Analysis of a LacZ Knock-In Allele of MDS1 Indicates a Critical Role in Hematopoietic Development.

The MDS1-EVI1 locus is of considerable interest due to its role in myeloid malignancies and dysplasias. It is well established now that the locus has two different transcription start sites (TSS) located 0.5 Mb apart, and these have the capacity to encode different isoforms, which variably contain zinc finger DNA binding domains and a SET-like domain that may have histone modifying ability. In order to better understand the biological role of this locus, we knocked in a lacZ allele into the Mds1 (upstream) TSS by homologous recombination in ES cells and created mice harboring this modified allele (K. Lezon-Geyda, S. Lin, G. Steele-Perkins et al, in preparation). By staining for beta-galactosidase activity, we documented the distribution of Mds1 activity during embryonic development and in the adult. During development, five major organ systems showed expression: musculoskeletal, renal, cardiac, neural, and hematopoietic, and in the latter three, there was a striking and highly specific spatiotemporal pattern of expression suggesting that Mds1-Evi1 plays important regulatory roles. In the developing heart, staining was seen in the anterior heart field specifically during the formation of the cardiac outflow tract, with significant spatiotemporal overlap with Mef2c, which encodes an important cardiac transcriptional regulatory protein. Thereafter, expression in the heart is very low. Beta-gal staining in the hematopoietic system in the embryo is limited to the clusters of nascent hematopoietic progenitors that develop at day 9.5 p.c. in the ventrolateral wall of the dorsal aorta and bud into the vascular lumen. Strikingly, we see no staining in other endothelium, nor in the fetal liver of 12.5–14.5 day embryos, wherein the majority of fetal hematopoiesis takes place. In adult bone marrow, there is beta-gal activity exclusively in the lin− c-kit+ Sca1+ progenitor population, with all of the beta-gal-positive cells being in the progenitor pool, and nearly all of the progenitor cells staining. While homozygous mice are viable, they are small, kyphotic, and have a shortened lifespan. Morphologic and quantitative analysis of the peripheral blood failed to reveal any significant abnormality. To assess the function of the hematopoietic system more rigorously, competitive repopulations of homozygous Mds1-deficient marrow progenitors with wildtype progenitors were performed. Within several weeks after transplant, the Mds1−/− cells were undetectable in the recipients, revealing that the homozygous Mds1-null bone marrow progenitors are deficient in their repopulating ability. To identify what function Mds1-Evi1 plays in hematopoetic cells, we used shRNA to suppress its expression in the myeloid cell lines 32Dcl3 and DA-1. This revealed an increase in steady state levels of cell death, as documented by histone release, TUNEL staining, and caspase activation. These data suggest that a primary role for the Mds1-Evi1 locus in hematopoietic cells is to promote their survival, thus allowing normal expansion at the progenitor stage.