Role of the cdx homeobox genes in the regulation of wt1 and nephron development

描述（由申请人提供）： 先天性肾畸形是儿童慢性肾功能衰竭的主要原因。目前，我们对控制肾单位（肾脏的关键功能单位）形成的发育途径了解甚少。我们正在使用斑马鱼来阐明胚胎途径，支配肾单位细分为功能不同的部分。斑马鱼胚胎是这些研究的理想对象，因为躯干两侧的中间中胚层形成两个简单的线性肾单位。从功能基因组学的方法，我们已经分离出一些肾转录因子和溶质转运蛋白基因。这些基因的表达模式将斑马鱼肾单位细分为不同的近端和远端节段，非常像哺乳动物肾单位。转录因子的组合定义了每个片段，表明“肾转录因子编码”决定了片段的身份。在同源框转录因子cdx 1a和cdx 4基因缺陷的胚胎中，这种编码被破坏，cdx 1a和cdx 4基因是hox基因的主要调节因子。双缺陷胚胎显示异常的hox表达模式，肾脏位置的后移，以及远端小管的缺失。此外，cdx突变体表现出Wilms肿瘤抑制因子-1a（wt 1a）的表达增加，wt 1a是一种与哺乳动物后肾发育和足细胞分化有关的转录因子。cdx基因在与中间中胚层相邻的树干（近轴中胚层）中表达。raldh 2的表达，编码参与视黄酸（RA）合成的酶，扩展在近轴中胚层的cdx缺陷的胚胎。敲低cdx 4突变体中的Raldh 2能够挽救肾脏定位缺陷，而在野生型中，它下调wt 1a表达。这些结果表明，异常的RA信号是负责的cdx突变体的肾脏缺陷，并导致我们检查RA在肾单位分割的作用。使用Raldh 2的化学拮抗剂DEAB，我们发现在肾发生的多个阶段需要RA来诱导足细胞和近端小管，并抑制远端小管的命运。基于这些数据，我们假设cdx-hox通路作用于raldh 2的上游以限制RA的合成，并且RA通过上调诸如wt 1a的基因来诱导足细胞和近端小管的命运。本提案的目的是检验这一假设。我们将描述cdx和RA缺陷动物的细胞和分子肾单位缺陷，并使用救援实验和敲除研究来确定cdx，hox和raldh 2基因之间的上位关系。使用RA的化学和分子拮抗剂，以及RA响应的转基因报告系，我们将确定何时何地需要RA信号来诱导近端肾单位的命运。这些研究将建立cdx-hox和RA通路在控制肾单位分割中的联系，为肾脏出生缺陷提供新的线索，并将有助于开发治疗或预防慢性肾功能衰竭的新疗法。影响肾脏的出生缺陷是婴儿和儿童慢性肾功能衰竭的主要原因，但目前，我们对控制肾脏形成的基因知之甚少。我们已经发现，维甲酸，维生素A的衍生物，在调节正常肾脏发育中起着重要作用。这项工作推进了我们对肾脏如何在胚胎中产生的理解，并可能导致新的治疗靶点来治疗或预防慢性肾衰竭。

DESCRIPTION (provided by applicant): Congenital kidney malformations are the leading cause of chronic renal failure in children. At present, we have a poor understanding of the developmental pathways that control the formation of the nephron, the key functional unit of the kidney. We are using zebrafish to elucidate the embryonic pathways that govern the subdivision of the nephron into functionally distinct segments. The zebrafish embryo is ideal for these studies as the intermediate mesoderm on either side of the trunk forms two simple linear nephrons. From a functional genomics approach we have isolated a number of renal transcription factor and solute transporter genes. The expression patterns of these genes subdivide the zebrafish nephron into distinct proximal and distal segments much like the mammalian nephron. Combinations of transcription factors define each segment suggesting a `renal transcription factor code' determines segment identity. This code is disrupted in embryos deficient in the homeobox transcription factor genes cdx1a and cdx4, which act as master regulators of the hox genes. Doubly-deficient embryos display abnormal hox expression patterns, a posterior shift in the position of the kidney, and a loss of distal tubules. In addition, cdx mutants exhibit expanded expression of Wilms' tumor suppressor-1a (wt1a), a transcription factor implicated in metanephros development and podocyte differentiation in mammals. The cdx genes are expressed in the trunk (paraxial mesoderm) adjacent to the intermediate mesoderm. Expression of raldh2, encoding an enzyme involved in retinoic acid (RA) synthesis, is expanded in the paraxial mesoderm of cdx-deficient embryos. Knock-down of Raldh2 in cdx4 mutants is able to rescue the kidney positioning defect, whereas in wild-types it down-regulates wt1a expression. These results suggest that aberrant RA signaling is responsible for the renal defects in cdx mutants and led us to examine the role of RA in nephron segmentation. Using DEAB, a chemical antagonist of Raldh2, we found that RA is required at multiple stages of nephrogenesis to induce podocytes and the proximal tubules, and to suppress distal tubule fates. Based on these data we hypothesize that the cdx-hox pathway acts upstream of raldh2 to restrict RA synthesis and that RA induces podocytes and proximal tubule fates by up-regulating genes such as wt1a. The aims of this proposal are designed to test this hypothesis. We will characterize the cellular and molecular nephron defects in cdx- and RA-deficient animals and use rescue experiments and knock-down studies to determine the epistatic relationships between the cdx, hox, and raldh2 genes. Using chemical and molecular antagonists of RA, as well as a RA-responsive transgenic reporter line, we will determine where and when RA signaling is required to induce proximal nephron fates. These studies will establish a link between the cdx-hox and RA pathways in the control of nephron segmentation, shed new light on renal birth defects, and will be useful for developing new therapies to treat, or prevent, chronic renal failure.Birth defects that affect the kidneys are the leading cause of chronic renal failure in infants and children, however at present, we have a poor understanding of the genes that control kidney formation. We have found that retinoic acid, a derivative of Vitamin A, plays a central role in regulating normal renal development. This work advances our understanding of how kidneys arise in the embryo and may be lead to new therapeutic targets to treat or prevent chronic renal failure.