Roth K A, Rubin D C, Birkenmeier E H, Gordon J I
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1991 Mar 25;266(9):5949-54.
The intestinal epithelium establishes and maintains a precise spatial organization despite its continuous and rapid renewal. We have used transgenic mice containing liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to begin to define the molecular mechanisms which are responsible for appropriate regional and cell-specific expression of genes in the gut. Multilabel immunocytochemical methods were employed to characterize the patterns of expression of two transgenes in the enteroendocrine and enterocytic populations of late gestation fetal mice at the time of initial cytodifferentiation of the gastrointestinal epithelium (fetal days 16-19). Surveys of the enteroendocrine cell population using a panel of antibodies directed against 11 neuroendocrine products revealed that these cells are scarce prior to fetal day 17, show a progressive increase in number through day 19, and while the relative proportion of subpopulations (defined by their principal peptide product) are somewhat different than in adults, their geographic distribution along the duodenal to colonic and intervillus(crypt) to villus axes are very similar to that encountered in adult (2-5 month old) mice. Immunoreactive L-FABP is first detectable at fetal day 17 and at this time of first appearance shows an adult pattern of regional enterocytic expression: i.e. it is present in cells overlying nascent villi but not those in the intervillus zone, it is highest in proximal small bowel, declines distally, and is absent from colonocytes. Colocalization studies indicate that L-FABP is not present in enteroendocrine cells during fetal life. Mapping studies indicate that nucleotides -596 to +21 of the rat L-FABP gene are sufficient to reproduce an appropriate temporal, cellular, and regional pattern of reporter (hGH) expression in fetal transgenic mice (with the exception that a subset(s) of enteroendocrine cells, typically containing immunoreactive gastric inhibitory peptide, support transgene but not L-FABP expression). This is in marked contrast to adult transgenic mice where inappropriate hGH accumulation occurs in crypt-associated epithelial cells, in colonocytes, and in many enteroendocrine populations. These studies indicate the importance of considering developmental stage when interpreting the results of any mapping study of cis-acting elements that regulate cell-specific and regional expression of genes in the perpetually renewing intestinal epithelium. Moreover, they also raise the possibility of using transgenes to define fundamental temporal changes in the gut's epithelial cell populations.
尽管肠上皮持续快速更新，但仍能建立并维持精确的空间组织。我们利用含有肝脂肪酸结合蛋白/人生长激素（L-FABP/hGH）融合基因的转基因小鼠，开始确定负责肠道中基因适当区域和细胞特异性表达的分子机制。采用多标记免疫细胞化学方法，在胃肠道上皮细胞开始分化时（胎儿期第16 - 19天），对妊娠晚期胎儿小鼠的肠内分泌细胞群和肠细胞群中两个转基因的表达模式进行了表征。使用一组针对11种神经内分泌产物的抗体对肠内分泌细胞群进行调查发现，在胎儿期第17天之前这些细胞很少，到第19天数量逐渐增加，虽然亚群（由其主要肽产物定义）的相对比例与成年小鼠略有不同，但其沿十二指肠到结肠以及绒毛间（隐窝）到绒毛轴的地理分布与成年（2 - 5月龄）小鼠非常相似。免疫反应性L-FABP在胎儿期第17天首次可检测到，首次出现时呈现出成年期区域肠细胞表达模式：即在新生绒毛上方的细胞中存在，而绒毛间区域的细胞中不存在，在近端小肠中含量最高，向远端递减，结肠细胞中不存在。共定位研究表明，在胎儿期，L-FABP不存在于肠内分泌细胞中。图谱研究表明，大鼠L-FABP基因的核苷酸-596至+21足以在胎儿转基因小鼠中重现报告基因（hGH）表达的适当时间、细胞和区域模式（除了一部分通常含有免疫反应性胃抑制肽的肠内分泌细胞亚群支持转基因表达但不支持L-FABP表达）。这与成年转基因小鼠形成鲜明对比，在成年转基因小鼠中，hGH在隐窝相关上皮细胞、结肠细胞和许多肠内分泌细胞群中不适当积累。这些研究表明，在解释任何调控不断更新的肠上皮细胞中基因细胞特异性和区域表达的顺式作用元件图谱研究结果时，考虑发育阶段非常重要。此外，它们还增加了利用转基因来定义肠道上皮细胞群基本时间变化的可能性。
