Mechanisms of Early Kidney Development

描述（由申请人提供）：干细胞技术的最新进展为细胞疗法和组织工程有朝一日可能取代因发育不良或疾病而丧失的肾功能提供了希望。组织工程的前提是了解胚胎肾诱导和发育的机制。原肾直接起源于中间中胚层（IM），代表脊椎动物肾脏诱导的第一阶段。近轴中胚层（PM），它产生体节，诱导原肾，但PM诱导原肾的细胞和分子机制尚未确定。体细胞发生和原肾的诱导与血管发育密切相关。在早期胚胎中，前肾位于主后静脉和主动脉之间，PM含有成血管细胞。成血管细胞来源的信号诱导胚胎心脏，胰腺和肝脏，这表明这可能是一个通用的诱导机制。初步研究表明，抑制血管生成降低了IM中肾脏标志物pax-2的表达，并表明血管破裂的时机很重要。我们推测，常驻成血管细胞分泌的分子调节近轴中胚层的原肾诱导。我们知道，成血管细胞满足原肾诱导剂的时间和空间标准。在目标1中，我们将使用高分辨率命运映射来定义与原肾诱导相关的血管前体的时间和空间外观。延时视频显微镜将跟踪PM、IM成血管细胞和血管的发育。我们将确定异位诱导的体节和原肾的形成是否与异位血管有关。这些研究的结果将有助于我们了解成血管细胞，PM和IM之间的相互作用的细节，在其规格。目的2将确定PM诱导原肾是否需要血管前体，从而满足诱导剂的第二个标准。我们将确定从缺乏血管的VEGFR 2（-/-）小鼠获得的PM是否可以在有能力（能够形成原肾）的鹌鹑中胚层中诱导原肾。将可溶性VEGF-R1注射到鹌鹑中胚层中以破坏血管发生，并其次破坏原肾诱导。目标3将确定血管是否足以诱导原肾，从而满足诱导剂的第三个标准。我们将研究异位或过度血管形成对原肾诱导的影响，我们将重组胚胎动脉和静脉与胶原凝胶培养中未诱导的中胚层，以确定原肾诱导是否特异于特定的血管类型，或者是血管和成血管细胞的一般特征。本提案中描述的实验将联合收割机结合鸟类模型的实验操作的简易性和突变小鼠的遗传能力，以确定血管形成、体节发生和原肾诱导之间的分子关系。

DESCRIPTION (provided by applicant): Recent advances in stem cell technology offer hope that cell therapy and tissue engineering may one day replace renal function lost as a consequence of maldevelopment or disease. Prerequisite to tissue engineering is the understanding of the mechanisms underlying embryonic kidney induction and development. The pronephros arises directly from intermediate mesoderm (IM) and represents the first stage of vertebrate kidney induction. Paraxial mesoderm (PM), which gives rise to somites, induces the pronephros, but the cellular and molecular mechanisms underlying pronephros induction by the PM have not been defined. Somitogenesis and pronephros induction are closely linked to vascular development. The pronephros lies sandwiched between the post-cardinal vein and the aorta in the early embryo, and the PM contains angioblasts. Angioblast-derived signals induce the embryonic heart, pancreas, and liver, suggesting that this may be a general inductive mechanism. Pilot studies showed that inhibition of vasculogenesis decreased expression of the kidney marker, pax-2, in the IM, and suggested that the timing of vascular disruption is important. We hypothesize that molecules secreted by resident angioblasts regulate pronephros induction by the paraxial mesoderm. We know that angioblasts meet the temporal and spatial criteria for a pronephros inducer. In Aim 1, we will define the temporal and spatial appearance of blood vessel precursors in relation to pronephros induction using high-resolution fate mapping. Time-lapse videomicroscopy will follow the development of PM, IM angioblasts, and blood vessels. We will determine whether ectopically induced somites and pronephroi form in association with ectopic blood vessels. Results of these studies will help us understand the details of interactions between angioblasts, PM and IM during their specification. Aim 2 will determine if blood vessel precursors are required for pronephros induction by PM, thereby fulfilling the second criterion for an inducer. We will determine if PM obtained from VEGFR2 (-/-) mice, which lack blood vessels, can induce the pronephros in competent (able to form pronephros) quail mesoderm. Soluble VEGF-R1 will be injected into quail mesoderm to disrupt vasculogenesis, and secondarily, pronephros induction. Aim 3 will determine if blood vessels are sufficient for pronephros induction and thus fulfill the third criterion for an inducer. We will examine the effect of ectopic or excessive blood vessel formation on pronephros induction, and we will recombine embryonic arteries and veins with uninduced competent mesoderm in collagen gel cultures to determine if pronephros induction is specific to a particular vascular type, or is a general feature of blood vessels and angioblasts. The experiments described in this proposal will combine the ease of experimental manipulation of the avian model with the genetic power of mutant mice to define the molecular relationships between blood vessel formation, somitogenesis and pronephros induction.