A Lineage Analysis of Cells in the Vertebrate Limb

描述（由申请人提供）： 尽管在理解组织发育中的脊椎动物肢体的细胞间信号方面已经取得了巨大的进展，但实际上对产生各种肢体组织的细胞之间的谱系关系或产生组织信号的细胞的不同命运一无所知。定义这种谱系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）的单个细胞将被追踪，通过标记一个复杂的库的抗病毒肽标记的逆转录病毒，问当单个细胞和它们的后代成为致力于特定的细胞命运和空间位置。LPM产生肢体骨骼、结缔组织、平滑肌和真皮，但这些群体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，可使肢体沿着其A/P轴极化。使用Cre重组酶系统，在Shh表达ZPA的所有后代中产生永久的、可遗传的LacZ表达。后肢芽（包括ZPA的区域）中的一些细胞经历细胞凋亡，而其他细胞则有助于手指结构。ZPA的后代是否可以重新编程基因表达模式并有助于肢体结构尚不清楚。如果这是真的，那么Shh表达的这种动态变化的识别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.