TRANSGENIC MURINE MODELS TO STUDY HIRSCHSPRUNGS DISEASE

先天性巨结肠症(HD)，先天性远端神经元缺失 肠道，是一种潜在的致命的出生缺陷，困扰着 5000个活生生的人类中就有1个。HD被认为是失败的后果 神经节细胞前体细胞(神经母细胞)在后肠的定植 源自神经脊线。这些过程调节着 肠神经母细胞的迁移、增殖和分化 不了解，但可能涉及以下各项之间的动态交互 细胞和周围的肠间充质。致命斑点(Ls)， 花椒致死(Sl)和显性巨结肠(Dom)是三个等位基因。 与小鼠的HD有关。我希望了解更多关于发病机制的知识。 通过对这些小鼠模型的研究，对HD进行了研究。在拟议的研究中， 肠神经母细胞、它们的微环境以及它们之间的相互作用 其中两个将通过转基因D/β/H-1的表达进行检测。 NlacZ，作为野生型小鼠和ls/ls、sl/sl和 DOM/+胚胎。转基因的使用是一种新的方法，它允许 肠神经母细胞“未分化”的阳性鉴定 关键阶段的完整肠道和组织切片 当肠道被定植时的胚胎发生。一种组合 免疫组织化学、超微结构、放射自显影技术将 用于定义形态和生化特征，这些特征 区分肠神经母细胞，并确定可能的差异 这些细胞或周围的间质介于野生型，ls/ls，sl/sl， 和DOM/+胚胎。Sl/sl和dom/+胚胎缺陷的性质 将通过检测通过聚集产生的嵌合小鼠来进行检查 转基因突变胚胎与非转基因野生型胚胎。我们 最近发表的关于ls/ls-野生型嵌合体的研究 证明了由ls突变引起的主要缺陷不是 细胞自主性(迁移的神经母细胞固有的)，但 环境(环境中的外在异常 神经母细胞迁移)。环境操纵对环境的影响 神经母细胞在野生型和突变型胚胎肠道中的定植 植入肾被膜下也将进行调查 目的是寻找“拯救”突变表型的生长条件。 这些研究的累积结果将提供对 HD的发病机制和未来识别特定基因的研究方向 或者是环境因素导致了人类的HD。

Hirschsprung's disease (HD), congenital absence of neurons in the distal intestinal tract, is a potentially lethal birth defect that afflicts 1/5000 liveborn humans. HD is thought to be a consequence of failed colonization of the hindgut by ganglion cell precursors (neuroblasts) derived from the neural crest. The processes which regulate the migration, proliferation, and differentiation of enteric neuroblasts are not understood, but probably involve dynamic interactions between these cells and the surrounding intestinal mesenchyme. Lethal spotted (ls), piebald lethal (sl), and dominant megacolon (Dom) are three alleles associated with HD in mice. I hope to learn more about the pathogenesis of HD through study of these murine models. In the proposed studies, enteric neuroblasts, their microenvironment, and interactions between the two will be examined by using expression of the transgene, D/beta/H- nlacZ, as a marker of neuroblasts in wild-type mice and ls/ls, sl/sl, and Dom/+ embryos. Use of the transgene is a novel approach which permits positive identification of "undifferentiated" enteric neuroblasts in intact gut and histological sections during the critical stages of embryogenesis when the gut is colonized. A combination of immunohistochemical, ultrastructural, autoradiographic techniques will be applied to define morphological and biochemical features which distinguish enteric neuroblasts and to identify possible differences in these cells or surrounding mesenchyme between wild-type, ls/ls, sl/sl, and Dom/+ embryos. The nature of the defect in sl/sl and Dom/+ embryos will be examined by examining chimeric mice produced by aggregating transgenic mutant embryos with non-transgenic wild-type embryos. We recently published such studies of ls/ls-wild type chimeras which demonstrated that the primary defect caused by the ls mutation is not cell autonomous (intrinsic to migrating neuroblasts), but is environmental (extrinsic anomalies in the milieu through which neuroblasts migrate). The effects of environmental manipulations on neuroblast colonization in wild-type and mutant embryonic intestines engrafted under the renal capsule will also be investigated with the intent of finding growth conditions that "rescue" the mutant phenotype. The cumulative results of these studies will provide insight into the pathogenesis of HD and direct future studies to identify specific genetic or environmental factors responsible for HD in humans.