Abstract
Overexpression of miRNA, miR-24, in mouse hematopoietic progenitors increases monocytic/ granulocytic differentiation and inhibits B cell development. To determine if endogenous miR-24 is required for hematopoiesis, we antagonized miR-24 in mouse embryonic stem cells (ESCs) and performed in vitro differentiations. Suppression of miR-24 resulted in an inability to produce blood and hematopoietic progenitors (HPCs) from ESCs. The phenotype is not a general defect in mesoderm production since we observe production of nascent mesoderm as well as mesoderm derived cardiac muscle and endothelial cells. Results from blast colony forming cell (BL-CFC) assays demonstrate that miR-24 is not required for generation of the hemangioblast, the mesoderm progenitor that gives rise to blood and endothelial cells. However, expression of the transcription factors Runx1 and Scl is greatly reduced, suggesting an impaired ability of the hemangioblast to differentiate. Lastly, we observed that known miR-24 target, Trib3, is upregulated in the miR-24 antagonized embryoid bodies (EBs). Overexpression of Trib3 alone in ESCs was able to decrease HPC production, though not as great as seen with miR-24 knockdown. These results demonstrate an essential role for miR-24 in the hematopoietic differentiation of ESCs. Although many miRNAs have been implicated in regulation of hematopoiesis, this is the first miRNA observed to be required for the specification of mammalian blood progenitors from early mesoderm.
Highlights
MicroRNAs are small (~22 nucleotide) RNA molecules that regulate gene expression post-transcriptionally
The embryonic stem cells (ESCs) differentiation system has been valuable for dissecting the molecular regulation of the development of mesoderm into hematopoietic progenitor cells (HPCs)
Extracellular signals regulate a complex network of transcription factors to direct embryonic hematopoietic development
Summary
MicroRNAs (miRNAs) are small (~22 nucleotide) RNA molecules that regulate gene expression post-transcriptionally. The first direct evidence demonstrating the existence of the hypothesized hemangioblast came from work with ESCs. Keller and colleagues identified a progenitor from ESC derived embryoid bodies (EBs) that formed a blast colony in methylcellulose, which contained clonal blood and endothelial cells [10]. They termed this progenitor the blast colony-forming cell (BL-CFC), which was proposed to be the in vitro equivalent of the hemangioblast These progenitors were enriched in the EB population of cells that coexpresses the mesoderm specific transcription factor T (Brachyury), and the tyrosine kinase receptor Flk1 [11]. In the aortic-gonad-mesenephros (AGM) region of the mouse embryo, blood has been shown to arise from differentiated endothelial cells termed the hemogenic endothelium, which appears independent of a hemangioblast cell [13, 14]. During differentiation of ES derived BL-CFCs, it has been observed that BL-CFC cells form a hemogenic endothelium intermediate during the development of HPCs [5]
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