A Lineage Analysis of Cells in the Vertebrate Limb

描述(由申请人提供)： 尽管在理解组织脊椎动物肢体发育的细胞间信号方面已经取得了巨大的进展，但对于产生各种肢体组织的细胞之间的谱系关系，或者产生组织信号的细胞的不同命运，几乎一无所知。确定这样的血统关系对于理解肢体形态发生是至关重要的。在这里，提出了一个双重战略。首先，来自肢体侧板中胚层(LPM)的单个细胞将通过标记寡核苷酸标记的复杂逆转录病毒文库进行追踪，以询问单个细胞及其后代何时与特定的细胞命运和空间位置有关。LPM产生了肢体骨骼、结缔组织、平滑肌肉和真皮，但这些种群如何以及何时分化尚不清楚。其次，将追踪极化活动区(ZPA)中细胞的后代。ZPA细胞表达Sonic Hedgehog(Shh)，这是一种分泌信号，沿着A/P轴极化肢体。使用Cre重组酶系统将在表达Shh的ZPA的所有后代中产生永久的、可遗传的LacZ表达。包括ZPA在内的后肢芽中的一些细胞经历了凋亡，而另一些细胞则参与了手指结构的形成。ZPA的后代是否可以重新编程基因表达模式并对肢体结构做出贡献尚不清楚。如果是真的，识别Shh表达的这种动态变化将对肢体模式的机制产生深远的影响。

DESCRIPTION (provided by applicant): Although there has been enormous progress toward understanding the intercellular signals that organize the developing vertebrate limb, practically nothing is known about the lineage relationships between cells that give rise to the various limb tissues, or of the differential fates of cells which produce the organizing signals. Defining such lineage relationships is essential to understanding limb morphogenesis. Here, a twofold strategy is proposed. First, individual cells derived from the limb lateral plate mesoderm (LPM) will be traced, by labeling with a complex library of oligonucleotide-tagged retroviruses, to ask when individual cells and their descendents become committed to particular cell fates and spatial locations. The LPM gives rise to the limb skeleton, connective tissue, smooth muscle and dermis, but how and when these populations diverge is unknown. Second, the progeny of cells in the Zone of Polarizing Activity (ZPA) will be traced. ZPA cells express Sonic Hedgehog (Shh), a secreted signal that polarizes the limb along its A/P axis. Permanent, heritable LacZ expression will be generated in all descendents of the Shh-expressing ZPA using the Cre-recombinase system. Some cells in the posterior limb bud, a region that includes the ZPA, undergo apoptosis while others contribute to digit structures. Whether descendents of the ZPA can reprogram gene expression patterns and contribute to limb structures is unknown. If true, the identification of such dynamic shifts in Shh expression would have profound implications for the mechanism of limb patterning.