Identification of extrinsic signals in blood formation

胚胎学研究表明，非造血组织产生的外部信号是血液发育所必需的，但传递该信息的信号分子的身份尚不清楚。我们已经证明，骨形态发生蛋白（BMP）功能在外胚层细胞中是必需的，以产生二级信号，该信号跨宝石层作用，使血液祖细胞参与红系途径并保护途径免于凋亡。此外，在造血过程中，钙调素依赖性激酶IV （calmodulin-dependent kinase IV, CaM KIV）需要负向调节BMP介导的转录反应。我们的数据支持CaM KIV通过激活与CREB结合蛋白（CBP）结合的底物来抑制BMP信号传导的模型。这种结合破坏了有限数量的CBP与一种未识别的造血特异性DNA结合蛋白（与bmp特异性转录因子协同工作）之间的相互作用。初步数据表明该DNA结合蛋白为GATA-2。我们将检验这一假设，并通过完成以下目标，开始识别该转录因子网络下游信号的内在因子：1。初步数据显示，爪蟾胚胎外胚层细胞中GATA-2的缺失会导致BMP活性调控不当导致的造血缺陷。我们将使用上位性和生化分析来确定它们是否在相同或平行的途径中起作用。2. 我们已经确定干细胞因子是一种外胚层来源的细胞因子，是血液形成所必需的，并使用微阵列方法鉴定了外胚层细胞中可能受BMP/ CaM KIV信号转录调节的基因。我们将使用定量方法来验证这些靶点，并在时间允许的情况下开始测试它们在爪蟾胚胎血液发育中的功能。建议的研究将为进一步的分析奠定基础

DESCRIPTION (provided by applicant): 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.