Identification of extrinsic signals in blood formation

描述（由申请人提供）：胚胎学研究表明，血液发育需要非造血组织产生的外源性信号，但传递该信息的信号分子的身份尚不清楚。我们已经表明，骨形态发生蛋白（BMP）的功能是外胚层细胞所必需的，以产生一个或多个次级信号，该信号作用于整个宝石层，使血液祖细胞能够致力于红细胞途径，并保护途径免于凋亡。此外，钙调蛋白依赖性激酶IV（CaM KIV）需要负调控BMP介导的造血过程中的转录反应。我们的数据支持一种模型，其中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。这种结合破坏了有限数量的CBP和未鉴定的造血特异性DNA结合蛋白之间的相互作用，该蛋白与BMP特异性转录因子一起发挥作用。初步数据表明，这种DNA结合蛋白是加塔-2。我们将测试这一假设，并将开始，以确定内在因素，信号下游的转录因子网络完成以下目标：1。初步数据显示，爪蟾胚胎外胚层细胞中加塔-2的缺失与BMP活性失调引起的造血缺陷表型相似。我们将使用上位性和生化分析来确定这些功能是否在相同或平行的途径。2.我们已经确定了干细胞因子作为一种外胚层衍生的细胞因子，这是血液形成所需的，并已使用微阵列方法来确定可能是由BMP/ CaM KIV信号在外胚层细胞转录调控的基因。我们将使用定量的方法来验证这些目标，并将开始测试它们在非洲爪蟾胚胎血液发育中的功能。拟议的研究将为进一步分析 BMP/CamKIV和加塔-2在血液发育所需的外部信号的转录调节中的相互作用。此外，我们将确定血液发展级联的新成分，以供未来研究。了解正常的血液发育是如何发生的，对于理解和预防这些途径的失调导致的疾病至关重要。

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.