López-Martínez A, Chang D T, Chiang C, Porter J A, Ros M A, Simandl B K, Beachy P A, Fallon J F
Department of Anatomy and Neuroscience, University of Wisconsin, Madison 53706, USA.
Curr Biol. 1995 Jul 1;5(7):791-6. doi: 10.1016/s0960-9822(95)00156-4.
Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH.
We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud.
Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.
音猬因子（Shh）是果蝇体节极性基因刺猬因子（hh）在脊椎动物中的同源物，与鸡胚肢体发育的模式形成有关。Shh沿后肢芽边缘的局限性表达以及表达Shh的异源细胞具有类似于后肢芽边缘极化区细胞的肢体极化活性的观察结果表明了这种作用。然而，尚未证明单独的音猬因子蛋白（SHH）足以进行肢体模式形成。SHH已被证明在体外会进行自催化切割，产生两种较小的产物。因此，确定SHH在发育中的肢体中是否发生加工以及这种加工如何影响SHH的功能是很有意义的。
我们证明SHH在发育中的鸡胚肢体中会进行蛋白水解加工。表达与单个切割产物相对应的SHH蛋白变体的细胞移植表明，诱导模式化基因表达和在肢芽上形成形态模式的能力仅限于SHH蛋白水解切割的氨基末端产物（SHH-N）。我们还证明，从植入的珠子中释放的细菌合成并纯化的SHH-N足以产生肢体模式形成活性。最后，我们表明内源性氨基末端切割产物紧密定位于肢芽的后缘。
我们的数据表明，在SHH自催化产生的两种切割产物中，移植的异源细胞中表达的或纯化形式提供的SHH-N足以在发育中的肢体上形成模式。此外，内源性氨基末端SHH切割产物在鸡胚肢芽后缘的局限性定位使得其模式形成活性不太可能是由于它在前后轴上以广泛的梯度分布所致。与观察到的定位更一致的模型是，氨基末端SHH切割产物通过在极化区或其附近的局部诱导发挥其模式形成作用，启动一系列基因表达，最终扩展到整个发育中的肢体。
