Embryologic studies have shown that extrinsic signals produced by non-hematopoietic tissues are required for blood development, but the identity of the signaling molecule (s) that transmit this information are unknown. We have shown that Bone morphogenetic protein (BMP) function is required in ectodermal cells to generate a secondary signal (s) that act across gem layers to enable blood progenitors to commit to the erythroid pathway and to protect pathways from apoptosis. Furthermore, calmodulin-dependent kinase IV (CaM KIV) is required to negatively regulate BMP mediated transcriptional responses during hematopoiesis. Our data support a model in which CaM KIV inhibits BMP signaling by activating a substrate that binds to CREB binding protein (CBP). This binding disrupts interactions between limiting amounts of CBP and an unidentified hematopoietic-specific DNA binding protein that functions in concert with BMP-specific transcription factors. Preliminary data suggest that this DNA binding protein is GATA-2. We will test this hypothesis and will begin to identify intrinsic factors that signal downstream of this transcription factor network by completing the following aims: 1. Preliminary data show that loss of GATA-2 in ectodermal cells of Xenopus embryos phenocopies hematopoietic defects caused by misregulation of BMP activity. We will use epistasis and biochemical analysis to determine whether these function in the same or parallel pathways. 2. We have identified stem cell factor as an ectodermally derived cytokine that is required for blood formation and have used a microarray approach to identify genes that may be transcriptionally regulated by BMP/ CaM KIV signals in ectodermal cells. We will use quantitative approaches to validate these targets and will begin to test their function in blood development in Xenopus embryos as time allows. The proposed studies will set the stage for further analysis of interactions between BMP/CamKIV and GATA-2 in transcriptional regulation of extrinsic signals required for blood development. In addition, we will identify novel components of the blood development cascade for future studies. Understanding how normal blood development occurs is crucial to understanding and preventing disease resulting from misregulation of these pathways.
