Genetic Control of Ureter and Kidney Development

描述(申请人提供)：对决定肾脏大小和肾单位数量的发育机制知之甚少。为什么肾脏会增长到特定的大小和复杂性，然后就停止了？祖细胞的分裂数量是否有内在的限制，或者器官的大小是否受到其他机制的调节，如外部信号？在这个修订申请中，我们提出了新的实验来解决这些问题。两种主要的肾细胞系是来自输尿管芽的集合管上皮细胞和来自后肾间充质的肾单位上皮细胞。表达Ret受体的细胞位于输尿管芽的顶端，是集合管的主要祖细胞，而表达Ret配体GDNF的后肾间充质细胞是肾单位上皮的祖细胞。我们将使用在Ret或GDNF基因控制下表达可诱导形式的Cre重组酶(Cre-ERT2)的小鼠。这些小鼠将与一种有条件地表达细胞毒性基因DTA的菌株杂交，这种菌株只在表达活性Cre的细胞及其后代中表达。通过用他莫昔芬诱导Cre活性，我们将选择性地减少在肾脏发育的特定阶段输尿管芽或后肾间充质中的祖细胞数量。然后我们将分析活体和器官培养中的肾脏，以询问这是否会改变输尿管芽生长和肾脏形成的比率，以及最终的肾脏大小和肾单位数量。如果祖细胞被编程为分裂有限的次数，那么破坏其中的一小部分应该会导致器官大小的减小。或者，如果这些细胞有过剩的增殖能力，并受到外部信号的调节，器官可能会恢复并达到正常大小。输尿管芽细胞或肾祖细胞的数量，或两者都有，可能会限制肾脏的生长速度和最终大小。我们的实验还解决了基本问题，即顶端细胞数量是否可能决定输尿管芽的伸长和分枝速度，以及类似地，肾单位祖细胞的数量如何可能影响肾单位的形成速度和大小。了解肾脏如何达到其正常大小和肾单位数目具有非常重要的临床意义，因为肾单位数目减少可能有利于肾脏疾病和高血压的进展。拟议的研究扩大了我们最初项目的范围(其目标是通过生长因子、酪氨酸激酶受体和细胞内信号机制调节肾脏发育)，以包括祖细胞在肾脏生长和发育中的作用的研究。它将通过提供资金聘请新的博士后研究人员以及购买额外的用品和服务来创造就业机会。 公共卫生相关性：了解肾脏如何达到正常大小具有重要的临床意义，因为胎儿发育过程中器官生长缺陷可能会导致肾单位(过滤单位)的数量减少。这反过来可能会促进肾脏疾病和高血压的进展。这项建议研究了控制肾脏如何生长到正确大小的机制，以及如何形成适当数量的肾单位。

DESCRIPTION (provided by applicant): Little is known about the developmental mechanisms that determine the size of the kidney and the number of nephrons. Why does the kidney grow to a particular size and complexity, and then stop? Is there an intrinsic limitation in the number of divisions of progenitor cells, or is organ size regulated by some other mechanism, such as external signals? In this Revision application, we propose new experiments to address these questions. Two major renal cell lineages are the collecting duct epithelia, which derive from the ureteric bud, and the nephron epithelia, which derive from the metanephric mesenchyme. Cells expressing the Ret receptor, located at the tips of the ureteric bud, are the major progenitors of collecting ducts, while metanephric mesenchyme cells expressing the Ret- ligand GDNF are the progenitors of nephron epithelia. We will use mice expressing an inducible form of Cre recombinase (Cre-ERT2) under the control of the Ret or Gdnf genes. These mice will be crossed with a strain that conditionally expresses a cytotoxic gene, DTA, only in cells that express active Cre, and in their descendants. By inducing Cre activity with Tamoxifen, we will selectively reduce the number of the progenitor cells in the ureteric bud or metanephric mesenchyme, at specific stages of kidney development. We will then analyze the kidneys in vivo and in organ culture, to ask if this alters the rates of ureteric bud growth and nephrogenesis, and the final kidney size and nephron number. If the progen- itors are programmed to divide a limited number of times, then destroying a fraction of them should result in decreased organ size. Alternatively, if these cells have excess proliferative capacity and are regulated by external signals, the organ may recover and reach its normal size. Either the number of ureteric bud or nephron progenitor cells, or both, might limit the growth rate and final size of the kidney. Our experiments also address the basic question of whether tip cell number might determine the rates of elongation and branching of the ureteric bud, and similarly, how the number of nephron progenitors might affect the rate of formation and the size of nephrons. Understanding how the kidney achieves its normal size and nephron number has very important clinical implications, as reduced nephron number may favor the progression of renal diseases and hypertension. The proposed studies expand the scope of our original project (whose Aims concern the regulation of kidney development by growth factors, tyrosine kinase receptors, and intracellular signaling mechanisms) to include studies on the role of progenitor cells in kidney growth and development. It will allow for job creation by providing the funds to hire new postdoctoral researchers, and to purchase additional supplies and services. PUBLIC HEALTH RELEVANCE: Understanding how the kidney achieves its normal size has important clinical implications, as defects in organ growth during fetal development can lead to a reduction in the number of nephrons, the filtering units. This, in turn, may promote the progression of renal diseases and hypertension. This proposal investigates the mechanisms that control how the kidney grows to the correct size, and how the proper number of nephrons is formed.