Yang Y, Drossopoulou G, Chuang P T, Duprez D, Marti E, Bumcrot D, Vargesson N, Clarke J, Niswander L, McMahon A, Tickle C
Department of Molecular and Cellular Biology, The Biolabs, Harvard University, Cambridge, Massachusetts 02138, USA.
Development. 1997 Nov;124(21):4393-404. doi: 10.1242/dev.124.21.4393.
Anteroposterior polarity in the vertebrate limb is thought to be regulated in response to signals derived from a specialized region of distal posterior mesenchyme, the zone of polarizing activity. Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity and appears to mediate the action of the zone of polarizing activity. Here we have manipulated Shh signal in the limb to assess whether it acts as a long-range signal to directly pattern all the digits. Firstly, we demonstrate that alterations in digit development are dependent upon the dose of Shh applied. DiI-labeling experiments indicate that cells giving rise to the extra digits lie within a 300 microm radius of a Shh bead and that the most posterior digits come from cells that lie very close to the bead. A response to Shh involves a 12-16 hour period in which no irreversible changes in digit pattern occur. Increasing the time of exposure to Shh leads to specification of additional digits, firstly digit 2, then 3, then 4. Cell marking experiments demonstrate that cells giving rise to posterior digits are first specified as anterior digits and later adopt a more posterior character. To monitor the direct range of Shh signalling, we developed sensitive assays for localizing Shh by attaching alkaline phosphatase to Shh and introducing cells expressing these forms into the limb bud. These experiments demonstrate that long-range diffusion across the anteroposterior axis of the limb is possible. However, despite a dramatic difference in their diffusibility in the limb mesenchyme, the two forms of alkaline phosphatase-tagged Shh proteins share similar polarizing activity. Moreover, Shh-N (aminoterminal peptide of Shh)-coated beads and Shh-expressing cells also exhibit similar patterning activity despite a significant difference in the diffusibility of Shh from these two sources. Finally, we demonstrate that when Shh-N is attached to an integral membrane protein, cells transfected with this anchored signal also induce mirror-image pattern duplications in a dose-dependent fashion similar to the zone of polarizing activity itself. These data suggest that it is unlikely that Shh itself signals digit formation at a distance. Beads soaked in Shh-N do not induce Shh in anterior limb mesenchyme ruling out direct propagation of a Shh signal. However, Shh induces dose-dependent expression of Bmp genes in anterior mesenchyme at the start of the promotion phase. Taken together, these results argue that the dose-dependent effects of Shh in the regulation of anteroposterior pattern in the limb may be mediated by some other signal(s). BMPs are plausible candidates.
脊椎动物肢体的前后极性被认为是对源自远端后间充质一个特殊区域（极化活性区）的信号作出反应而受到调控。音猬因子（Shh）在极化活性区表达，并且似乎介导了极化活性区的作用。在此，我们在肢体中操纵Shh信号，以评估它是否作为一种长距离信号直接决定所有指（趾）的形态。首先，我们证明指（趾）发育的改变取决于所施加的Shh剂量。DiI标记实验表明，产生额外指（趾）的细胞位于距Shh珠子300微米半径范围内，并且最靠后的指（趾）来自非常靠近珠子的细胞。对Shh的反应涉及一个12 - 16小时的时间段，在此期间指（趾）形态不会发生不可逆的变化。增加暴露于Shh的时间会导致额外指（趾）的形成，首先是第2指（趾），然后是第3指（趾），接着是第4指（趾）。细胞标记实验表明，产生靠后指（趾）的细胞最初被指定为靠前的指（趾），随后才具有更靠后的特征。为了监测Shh信号传导的直接范围，我们开发了灵敏的检测方法，通过将碱性磷酸酶连接到Shh上并将表达这些形式的细胞引入肢芽来定位Shh。这些实验表明，沿肢体前后轴进行长距离扩散是可能的。然而，尽管两种形式的碱性磷酸酶标记的Shh蛋白在肢体间充质中的扩散性存在显著差异，但它们具有相似的极化活性。此外，尽管来自这两种来源的Shh的扩散性存在显著差异，但用Shh - N（Shh的氨基末端肽）包被的珠子和表达Shh的细胞也表现出相似的形态发生活性。最后，我们证明当Shh - N连接到一种整合膜蛋白上时，转染了这种锚定信号的细胞也以剂量依赖的方式诱导镜像模式重复，类似于极化活性区本身。这些数据表明，Shh本身不太可能在远距离发出指（趾）形成的信号。浸泡在Shh - N中的珠子不会在前肢间充质中诱导Shh表达，排除了Shh信号的直接传播。然而，在促进阶段开始时，Shh在前间充质中诱导Bmp基因的剂量依赖性表达。综上所述，这些结果表明，Shh在肢体前后模式调控中的剂量依赖性效应可能是由某些其他信号介导的。骨形态发生蛋白（BMPs）是合理的候选信号。
