Fox Transcription Factors in Development of Pulmonary Capillaries

描述（由申请人提供）：叉头盒（Fox）转录因子Foxml和Foxfl在发育中的肺内皮细胞中表达，并在肺发育中起重要作用。这些Fox基因中的任何一个的缺乏都与外周肺毛细血管形成的严重缺陷以及导致胚胎死亡的许多器官的异常有关。我们的假设是，Foxml和Foxfl调节肺血管发育过程中内皮细胞增殖和分化的关键基因的转录。为了检验这一假设，我们将使用Tie 2启动子驱动的他莫昔芬诱导型Cre重组酶（Tie 2-Cre-ERT 2）转基因来介导内皮细胞中Foxfl或Foxml LoxP/LoxP（fl/fl）靶向等位基因的发育阶段特异性缺失。在目的1中，我们描述了使用Tie 2-Cre-ERT 2 Foxml fl/fl胚胎肺和来自这些胚胎的培养的肺内皮细胞来确定Foxml是否是肺血管发育期间内皮细胞增殖和分化所需的计划。在目标2中，我们将开发含有Foxfl fl/fl靶向等位基因的小鼠，并使用Tie 2-Cre-ERT 2转基因在肺发育的不同阶段介导Foxfl fl/fl的时间内皮特异性缺失。我们将使用Tie 2-Cre-ERT 2 Foxfl fl/fl小鼠来确定Foxfl是否直接调节内皮细胞分化，并鉴定需要Foxfl功能来引发适当的血管肺发育和调节Foxfl靶基因的时间段。我们已经表明，Foxfl调节参与Notch-2信号传导的基因的表达，并且Notch-2途径对于肺微血管的正常发育至关重要。在目标3中，我们提出产生具有内皮特异性Tie-2启动子驱动的Notch-2转基因的小鼠。这些Tie-2 Notch-2小鼠将与Foxfl小鼠一起繁殖，以确定胚胎Foxfl肺中增加的Notch-2受体水平是否将恢复肺泡微血管发育的正常状态并预防Foxfl新生小鼠的围产期肺出血。完成拟议的研究将使我们能够确定Foxfl和Foxml蛋白在肺发育过程中内皮细胞中的作用。这些Fox蛋白还可以在人类儿科肺部疾病的遗传筛查、诊断和治疗中提供新的靶标，所述人类儿科肺部疾病包括围产期肺出血和早产儿呼吸窘迫综合征。

DESCRIPTION (provided by applicant): Forkhead Box (Fox) transcription factors Foxml and Foxfl are expressed in developing pulmonary endothelial cells and play an essential role in lung development. Deficiency of either one of these Fox genes is associated with severe defects in formation of peripheral pulmonary capillaries as well as abnormalities in many organs causing embryonic lethality. Our hypothesis is that Foxml and Foxfl regulate transcription of genes critical for proliferation and differentiation of endothelial cells during pulmonary vascular development. In order to test this hypothesis, we will use the Tie2 promoter driven Tamoxifen-inducible Cre recombinase (Tie2-Cre-ERT2) transgene to mediate developmental-stage-specific deletion of either Foxfl or Foxml LoxP/LoxP (fl/fl) targeted allele in endothelial cells. In Aim 1, we describe plans to use Tie2-Cre-ERT2 Foxml fl/fl embryonic lungs and cultured pulmonary endothelial cells derived from these embryos to determine whether Foxml is required for endothelial cell proliferation and differentiation during lung vascular development. In Aim 2, we will develop a mouse containing a Foxfl fl/fl targeted allele and use the Tie2-Cre- ERT2 transgene to mediate temporal endothelial-specific deletion of the Foxfl fl/fl at different stages of lung development. We will use Tie2-Cre-ERT2 Foxfl fl/fl mice to determine whether Foxfl directly regulates endothelial cell differentiation and identify a time period during which Foxfl function is required to elicit proper vascular lung development and to regulate Foxfl target genes. We have shown that Foxfl regulates expression of genes involved in Notch-2 signaling and that the Notch-2 pathway is critical for normal development of lung microvasculature. In Aim 3, we propose to generate mice with the endothelial-specific Tie-2 promoter driven Notch-2 transgene. These Tie-2 Notch-2 mice will be bred with Foxfl mice to determine whether increased Notch-2 receptor levels in embryonic Foxfl lung will restore normalcy to the development of alveolar microvasculature and prevent perinatal pulmonary hemorrhage in Foxfl newborn mice. Completion of the proposed studies will enable us to determine the role of Foxfl and Foxml proteins in endothelial cells during lung development. These Fox proteins may also provide novel targets in genetic screening, diagnosis and treatment of human pediatric lung diseases including perinatal pulmonary hemorrhage and respiratory distress syndrome of premature born babies.