APC and Retinoids in Zebrafish Enterocyte Development

描述（由申请人提供）：APC或其调节靶点β-连环蛋白的突变被认为通过促进增殖和阻止结肠细胞的适当分化而引起结肠肿瘤。然而，我们对控制结肠细胞分化的机制的理解是有限的。最近的研究表明，人类结肠腺瘤和癌显示出视黄酸生物合成酶的严重缺乏。此外，将野生型APC重新引入APC缺陷的结肠癌细胞系中诱导视黄醇脱氢酶L并增加视黄酸的产生。这些观察结果表明一种新的模型，其中APC通过控制视黄酸生物合成促进肠上皮细胞分化。本研究将以斑马鱼为模型系统，研究APC与正常肠上皮细胞中视黄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的吗啉代敲低导致已知需要视黄酸的结构缺陷，这与这些基因在RA依赖性途径中的作用一致。此外，APC或zRDHB变形鱼发育的肠缺乏柱状上皮细胞，并且不能表达分化标志物肠脂肪酸结合蛋白（i-FABP）。用维甲酸处理APC或zRDHB morphant胚胎挽救了缺陷表型，首次将zAPC置于RA上游并暗示RA在肠分化中。因此，这些初步的数据，强烈支持视黄酸在斑马鱼肠上皮细胞发育中的关键作用，并提供遗传证据，将视黄酸和hox基因下游的APC。本项目的长期目标是通过了解APC突变后最早的细胞扰动来促进结肠腺瘤形成的新预防措施的发展。

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.