Riley B B, Savage M P, Simandl B K, Olwin B B, Fallon J F
Department of Anatomy, University of Wisconsin-Madison 53706.
Development. 1993 May;118(1):95-104. doi: 10.1242/dev.118.1.95.
To investigate the role of fibroblast growth factor-2 (basic fibroblast growth factor) in chick limb development, we constructed a replication-defective spleen necrosis virus to ectopically express fibroblast growth factor-2 in stage 20-22 chick limb bud. Because infecting cells in vivo proved to be inefficient, limb bud cells were dissociated, infected in vitro, and then grafted back into host limbs. This procedure caused duplications of anterior skeletal elements, including proximal humerus, distal radius, and digits 2 and 3. Eighty-nine percent of host wings receiving infected grafts at their anterior borders had duplications of one or more of these elements. The frequency of duplication declined dramatically when infected cells were grafted to progressively more posterior sites of host limb buds, and grafting to the posterior border had no effect at all. Several techniques were used to determine the role of infected tissue in forming skeletal duplications. First, staining with an fibroblast growth factor-2 specific monoclonal antibody showed higher than endogenous levels of fibroblast growth factor-2 expression associated with extra elements. Second, the host/donor composition of duplicated elements was determined by simultaneously infecting donor cells with viruses encoding fibroblast growth factor-2 or beta-galactosidase; donor tissue was then visualized by X-gal staining. Patterns of ectopic fibroblast growth factor-2 expression and X-gal staining confirmed the presence of infected donor tissue near duplicated structures, but the duplicated skeletal elements themselves showed very little staining. Similar results were obtained in duplications caused by infected quail wing bud cells grafted to the chick wing bud. These observations suggest that fibroblast growth factor-2-expressing donor tissue induced host tissue to form normally patterned extra elements. In support of this conclusion, implanting beads containing fibroblast growth factor-2 caused partial duplications of digit 2. These data provide the first direct evidence that fibroblast growth factor-2 plays a role in patterning in the limb bud.
为了研究成纤维细胞生长因子2（碱性成纤维细胞生长因子）在鸡胚肢体发育中的作用，我们构建了一种复制缺陷型脾坏死病毒，以便在第20 - 22期鸡胚肢体芽中异位表达成纤维细胞生长因子2。由于在体内感染细胞效率低下，于是将肢体芽细胞解离，在体外进行感染，然后再移植回宿主肢体。这一操作导致了前侧骨骼元件的重复，包括近端肱骨、远端桡骨以及第2和第3指。在其前边界接受感染移植的宿主翅膀中，89%出现了这些元件中一个或多个的重复。当将感染细胞移植到宿主肢体芽越来越靠后的位置时，重复的频率显著下降，而移植到后边界则完全没有影响。我们采用了多种技术来确定感染组织在形成骨骼重复中的作用。首先，用成纤维细胞生长因子2特异性单克隆抗体染色显示，与额外元件相关的成纤维细胞生长因子2表达水平高于内源性水平。其次，通过用编码成纤维细胞生长因子2或β - 半乳糖苷酶的病毒同时感染供体细胞来确定重复元件的宿主/供体组成；然后通过X - gal染色观察供体组织。异位成纤维细胞生长因子2表达和X - gal染色模式证实了在重复结构附近存在感染的供体组织，但重复的骨骼元件本身染色很少。将感染的鹌鹑翅膀芽细胞移植到鸡翅膀芽所导致的重复中也获得了类似结果。这些观察结果表明，表达成纤维细胞生长因子2的供体组织诱导宿主组织形成了正常模式的额外元件。为支持这一结论，植入含有成纤维细胞生长因子2的珠子导致了第2指的部分重复。这些数据首次直接证明了成纤维细胞生长因子2在肢体芽模式形成中发挥作用。
