APC and Retinoids in Zebrafish Enterocyte Development

描述(申请人提供)：APC或其调控靶点β-连环蛋白的突变被认为通过促进增殖和阻止结肠癌细胞的适当分化而导致结肠癌。然而，我们对控制结肠细胞分化的机制的了解是有限的。最近的研究表明，人类结肠腺瘤和结肠癌都严重缺乏维甲酸生物合成酶。此外，将野生型APC重新导入缺乏APC的结肠癌细胞系，可诱导视黄醇脱氢酶L，并增加维甲酸的产量。这些观察表明了一种新的模型，其中APC通过控制维甲酸的生物合成来促进肠细胞分化。这项建议中概述的研究将以斑马鱼为模型系统，研究正常肠细胞中APC和维甲酸生物合成之间的遗传关系。初步数据显示，在斑马鱼胚胎中，zAPC或zRDHB的吗啉基因敲除会导致已知需要维甲酸的结构的缺陷，这与这些基因在RA依赖途径中的作用是一致的。此外，APC或zRDHB变种鱼发育的肠道缺乏柱状上皮细胞，并且不表达分化标记肠道脂肪酸结合蛋白(I-FABP)。用维甲酸处理APC或zRDHB变异胚胎可以挽救缺陷的表型，首次将zAPC置于RA的上游，并暗示RA参与了肠道分化。因此，这些初步数据有力地支持了维甲酸在斑马鱼肠细胞发育中的关键作用，并提供了将维甲酸和HOX基因定位在APC下游的遗传证据。该项目的长期目标是通过了解APC突变后最早的细胞扰动，促进结肠腺瘤形成的新预防措施的开发。

DESCRIPTION (provided by applicant): Mutations in APC, or in its regulatory target, beta-catenin, are thought to cause colon neoplasms by promoting proliferation and preventing proper differentiation of colonocytes. However, our understanding of the mechanisms that control colonocyte differentiation is limited. Recent studies have shown that human colon adenomas and carcinomas show a profound deficiency of retinoic acid biosynthetic enzymes. Furthermore, re-introduction of wild type APC into an APC-deficient colon cancer cell line induced retinol dehydrogenase L and increased retinoic acid production. These observations suggest a novel model wherein APC promotes enterocyte differentiation by controlling retinoic acid biosynthesis. The studies outlined in this proposal will examine the genetic relationship between APC and retinoic acid biosynthesis in normal enterocytes using zebrafish as a model system. Preliminary data show that morpholino knockdown of either zAPC or zRDHB in zebrafish embryos results in defects in structures known to require retinoic acid, consistent with a role for these genes in RA-dependent pathways. In addition, APC or zRDHB morphant fish develop intestines that lack columnar epithelial cells and fail to express the differentiation marker intestinal fatty acid binding protein (i-FABP). Treatment of either APC or zRDHB morphant embryos with retinoic acid rescued the defective phenotypes, for the first time placing zAPC upstream of RA and implicating RA in intestinal differentiation. These preliminary data, therefore, strongly support a critical role for retinoic acid in zebrafish enterocyte development and provide genetic evidence placing retinoic acid and hox genes downstream of APC. The long term goal of this project is to facilitate the development of new preventive measures for colon adenoma formation by understanding the earliest cellular perturbations that follow APC mutation.