The study of hematopoiesis is essential for understanding the etiology of blood diseases, and for many years has served as a paradigm for answering fundamental questions concerning genetic regulation of stem and progenitor cells, and lineage fate. The goal of this proposal is to continue and expand our current research on the molecular control of hematopoiesis by the bone morphogenetic protein (BMP) signaling pathway, an important regulator of embryonic patterning. BMPs are members of a large superfamily of signaling molecules, and exert many of their biological effects via activation of a subset of downstream SMAD transcription factors. Our recent studies using a mouse embryonic stem (ES) cell model have shown that Smad1 exerts temporally distinct regulatory roles on hematopoietic potential, stimulating hematopoiesis prior to commitment of precursor cells before later inhibiting hematopoietic expansion. I plan to build on these studies to identify and examine key regulatory gene networks using both mouse ES cell and zebrafish (Danio rerio) model systems. For this purpose, I will pursue three separate aims, which will converge on their analysis of the BMP pathway and the validation of relevant gene targets identified by various established and emerging genomic technologies and databases. Experiments in the first Aim will investigate the individual and combinatorial contributions of the BMP effectors Smad1 and Smad5 to hematopoietic lineage commitment and expansion using conditional gene knockdown strategies in mouse ES cells. In the second Aim, I will use the zebrafish model to probe the hematopoietic functions of previously identified targets of Smad1 and Smad5, with an eye toward defining their roles in hematopoietic transitions that progress as the site of active hematopoiesis migrates within the developing embryo. I will complement these findings with murine ES cell studies to validate direct SMAD binding targets via chromatin immunoprecipitation. Finally, in the third Aim I will use small inhibitor molecules in ES cells and the zebrafish to examine regulatory roles of BMP signaling on embryonic hematopoiesis upstream of SMAD activation, with particular attention to SMAD- independent processes, to define novel mechanisms of BMP-mediated control of specific blood lineages.
This project addresses the genetic regulatory mechanisms underlying hematopoiesis, an essential developmental process that is impaired in many chronic and acute blood diseases. It proposes to extend understanding of the targets and modulators of the BMP pathway, a major signaling pathway implicated in several developmental processes and disease states. I expect these findings to inform strategies for future identification of palliative therapies for hematopoietic disorders.