FLP-MEDIATED CELL LINEAGE ANALYSIS IN THE EMBRYO

这个项目的总体目标是开发一个二进制 基于重组的系统对体细胞谱系进行命运定位和操纵 并利用这一新工具来揭示机制 神经脊细胞谱系的潜在建立。一个中心问题 在哺乳动物发育中--了解血统和环境 相互作用来确定表型-一直受到无法 追踪特定细胞在原位的命运并观察其分布 他们的后代在整个怀孕期间都在胚胎中。如此精确的血统 测绘是了解儿童发育决定因素的先决条件 细胞的增殖、分化和迁移。了解这些知识 机制是理解先天性心脏病起源的基础 畸形。 我们在转基因小鼠中设计了一种原位谱系标记系统。 这应该标记出可遗传细胞中的特定细胞群 在胚胎发育过程中自主、非稀释的方式。 此外，该系统还可以与相应的 重组以执行体内谱系/组织特异性突变。我们 利用在酵母中发现的切除重组系统 这些目的：重组酶FLP催化重组 FLP重组靶点(FRT)的直接重复切除 介入的DNA。自1993年开展这项计划以来，我们有：(1) 构建了FLP和通用目标(FRT中断)的模块化集合 LacZ)载体；(2)展示了有效的FLP激活靶标 胚胎干细胞中的转基因；(3)产生单独的FLP和 靶向(FRT干扰的LacZ)转基因小鼠系；和(4)证明 FLP介导的FLPx靶杂交胚胎中的重组。我们现在是 在活体中充分描述这个系统的独特地位 用于标记细胞谱系和定向修改 小鼠基因组。 因为神经脊细胞广泛迁移并分化形成 多种细胞类型，它们是研究血统和 环境相互作用决定细胞的命运。神经脊线错乱 细胞发育与许多先天畸形有关 包括神经管、肢体、头颅、肠神经节和心脏 缺陷、耳聋和胸腺发育不全。一个主要悬而未决的问题是 这群细胞何时以及如何产生这样的表型 多样性。为了达到所提出的目标，我们已经培育出转基因小鼠 FLP在背侧中枢的表达作为激活神经中LacZ的手段 纹章的始祖。我们将：(1)利用这个标记系统来绘制地图 小鼠神经脊；(2)识别异同 小鼠的地图和雏鸟的地图，后者提供了我们的大部分 当前知识；(3)从对细胞命运的描述转向分析 通过对突变胚胎的谱系研究来了解潜在的机制。 斑点(Pax-3)致死的致病机制 斑点(IS)和W(c-kit)表型将使我们深入了解 平行人类综合征的发病机制(例如，Waardenburg综合征和 先天性巨结肠)。

The overall objectives of this project are to develop a binary recombination-based system to fate map and manipulate somatic lineages in the mouse embryo, and to utilize this new tool to reveal mechanisms underlying establishment of neural crest cell lineages. A central issue in mammalian development -understanding how lineage and environment interact to determine phenotype - has been limited by the inability to follow the fate of specific cells in situ and to observe the distribution of their progeny in the embryo throughout gestation. Such precise lineage mapping is prerequisite to understanding developmental determinants of cell proliferation, differentiation, and migration. Knowledge of these mechanisms is fundamental to understanding the origins of congenital malformations. We have engineered an in situ lineage marking system in transgenic mice that should mark specific populations of cells in a heritable, cell autonomous, non-diluting fashion during embryonic development. Additionally, this system can be used in conjunction with homologous recombination to perform lineage/tissue-specific in vivo mutagenesis. We have exploited an excisional recombination system found in yeast for these purposes: the recombinase FLP catalyzes recombination between direct repeats of FLP recombination targets (FRTs) excising the intervening DNA. Since initiating this project in 1993 we have: (1) constructed a modular set of FLP and universal target (FRT-disrupted lacZ) vectors; (2) demonstrated efficient FLP-activation of the target transgene in embryonic stem (ES) cells; (3) generated separate FLP and target (FRT-disrupted lacZ) transgenic mouse lines; and (4) demonstrated FLP-mediated recombination in embryos from a FLPxtarget cross. We are now in the unique position to fully characterize this system in vivo as a tool for marking cell lineages and for directed modifications of the mouse genome. Because neural crest cells migrate extensively and differentiate to form a variety of cell types they are optimal to study how lineage and environment interact to determine cell fate. Derangements of neural crest cell development are implicated in numerous congenital malformations including neural tube, limb, cranial, enteric ganglion and cardiac defects, deafness, and thymic agenesis. A major unanswered question is when and how does this population of cells generate such phenotypic diversity. Toward the proposed aims, we have generated transgenic mice expressing FLP in the dorsal CNS as a means to activate lacZ in neural crest progenitors. We will: (1) utilize this marking system to map the murine neural crest; (2) identify similarities and differences between the murine map and that of the chick, the latter providing much of our current knowledge; (3) move from descriptions of cell fate to analysis of underlying mechanisms through lineage studies in mutant embryos. Mechanisms of pathogenesis associated with Splotch (Pax-3), lethal spotting (Is), and W (c-kit) phenotypes will give insight into the pathogenesis of parallel human syndromes (e.g. Waardenburg Syndrome and Hirschsprung's disease).