Fox Transcription Factors in Development of Pulmonary Capillaries

描述(申请人提供)：Forkhead Box(Fox)转录因子Foxm1和Foxf1在发育中的肺内皮细胞中表达，在肺发育中发挥重要作用。这两个Fox基因中的任何一个的缺失都与周围肺毛细血管形成的严重缺陷以及许多器官的异常有关，从而导致胚胎死亡。我们的假设是，在肺血管发育过程中，Foxm1和Foxfl调控内皮细胞增殖和分化的关键基因的转录。为了验证这一假设，我们将使用Tie2启动子驱动的三苯氧胺诱导的Cre重组酶(Tie2-Cre-ERT2)转基因来介导内皮细胞中Foxf1或Foxm1loxP/loxP(f1/f1)靶向等位基因的发育阶段特异性缺失。在目标1中，我们描述了使用Tie2-Cre-ERT2 Foxmlf1/f1胚胎肺和来自这些胚胎的培养的肺内皮细胞来确定在肺血管发育过程中是否需要Foxm1来促进内皮细胞的增殖和分化。在目标2中，我们将开发一只含有Foxfl fl/fl靶向等位基因的小鼠，并使用Tie2-Cre-ERT2转基因基因来介导Foxfl fl/fl在肺发育不同阶段的时间内皮细胞特异性缺失。我们将使用Tie2-Cre-ERT2 Foxfl fl/fl小鼠来确定Foxfl是否直接调控内皮细胞分化，并确定Foxf1功能需要在什么时间段引发适当的血管肺发育和调节Foxfl靶基因。我们已经证明，Foxfl调节与Notch-2信号转导有关的基因的表达，并且Notch-2通路对于肺微血管的正常发育至关重要。在目标3中，我们建议用内皮特异性Tie-2启动子驱动的Notch-2转基因来产生小鼠。这些Tie-2 Notch-2小鼠将与Foxfl小鼠杂交，以确定胚胎Foxfl肺中Notch-2受体水平的增加是否会恢复Foxfl新生小鼠肺泡微血管发育的正常，并防止围产期肺出血。拟议研究的完成将使我们能够确定Foxf1和Foxm1蛋白在肺发育过程中内皮细胞中的作用。这些Fox蛋白还可能为围产期肺出血和早产儿呼吸窘迫综合征等儿童肺部疾病的基因筛查、诊断和治疗提供新的靶点。

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.