The role of spatially controlled cell proliferation in limb bud morphogenesis.
The role of spatially controlled cell proliferation in limb bud morphogenesis.
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DOI：
10.1371/journal.pbio.1000420
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发表时间：
2010-07-13
期刊：
影响因子：
9.8
通讯作者：
Sharpe J
中科院分区：
文献类型：
--
作者：
Boehm B;Westerberg H;Lesnicar-Pucko G;Raja S;Rautschka M;Cotterell J;Swoger J;Sharpe J
定向细胞行为可能在发育过程中的肢芽伸长中发挥比之前“基于生长的形态发生”假说更重要的作用。尽管脊椎动物肢芽作为空间模式形成和细胞规范的模型系统已经被研究了几十年，但相比之下，其向远端伸长的细胞基础一直是一个相对被忽视的话题。传统观点认为，沿着肢体存在各向同性增殖的梯度，远端的增殖率较高，而朝向身体的增殖率较低，并且这种梯度是生长背后的驱动力。在这里，我们通过将定量经验数据集与计算机模型相结合来测试这一假设，以评估空间控制的增殖率在定向肢芽生长过程中的潜在作用。特别是，我们为小鼠后肢生成了两个新的经验数据集——形状变化的数值描述和细胞周期时间的定量 3D 图——并将它们与组织生长的新 3D 有限元模型相结合。通过开发参数优化方法（探索组织生长的空间模式），我们的计算机模拟表明，观察到的增殖率分布在控制远端延伸的肢体形状方面没有显着作用，并表明定向细胞活动可能是肢芽生长背后的驱动力。这一理论预测促使我们寻找肢芽间质细胞定向方向的证据，因此我们发现了一种惊人的高度分支和延伸的细胞形状，由动态伸展和缩回的丝状伪足、高尔基体位置的远端定向偏差以及细胞分裂方向的偏差组成。因此，我们提供了理论和经验证据，表明肢芽伸长是通过定向细胞活动而不是增殖率的PD梯度来实现的。尽管脊椎动物肢芽作为细胞命运空间控制的经典模型系统已经被研究了几十年，但对肢芽如何物理延长的问题的研究却少得多。一种特殊的假说在该领域占据主导地位，称为增殖梯度假说或基于生长的形态发生。这表明伸长是通过刺激远端细胞（距离身体最远）比近端细胞分裂得更快来实现的。重要的是，这个假设并没有提出任何类型的定向或定向细胞行为——非定向增殖的高远端率被认为是足够的——事实上，一些二维计算机模拟已经在计算机中重现了这个概念。然而，迄今为止，来自肢芽的定量数据尚未纳入这些模型中。在这里，我们将计算机模拟扩展到 3D，并纳入了形状变化和增殖率的定量数据。这些新的模拟表明，非定向增殖的梯度无法解释肢芽伸长。因此，我们通过实验测试了定向细胞行为的证据，确实发现细胞形状、高尔基体定向和细胞分裂在肢芽生长过程中都表现出非随机偏差。我们的数据与增殖梯度假设相反，表明定向细胞行为对于驱动伸长很重要。
Oriented cell behaviors likely have a more important role in limb bud elongation during development than previously suggested by the “growth-based morphogenesis” hypothesis. Although the vertebrate limb bud has been studied for decades as a model system for spatial pattern formation and cell specification, the cellular basis of its distally oriented elongation has been a relatively neglected topic by comparison. The conventional view is that a gradient of isotropic proliferation exists along the limb, with high proliferation rates at the distal tip and lower rates towards the body, and that this gradient is the driving force behind outgrowth. Here we test this hypothesis by combining quantitative empirical data sets with computer modelling to assess the potential role of spatially controlled proliferation rates in the process of directional limb bud outgrowth. In particular, we generate two new empirical data sets for the mouse hind limb—a numerical description of shape change and a quantitative 3D map of cell cycle times—and combine these with a new 3D finite element model of tissue growth. By developing a parameter optimization approach (which explores spatial patterns of tissue growth) our computer simulations reveal that the observed distribution of proliferation rates plays no significant role in controlling the distally extending limb shape, and suggests that directional cell activities are likely to be the driving force behind limb bud outgrowth. This theoretical prediction prompted us to search for evidence of directional cell orientations in the limb bud mesenchyme, and we thus discovered a striking highly branched and extended cell shape composed of dynamically extending and retracting filopodia, a distally oriented bias in Golgi position, and also a bias in the orientation of cell division. We therefore provide both theoretical and empirical evidence that limb bud elongation is achieved by directional cell activities, rather than a PD gradient of proliferation rates. Although the vertebrate limb bud has been studied for decades as a classical model system for the spatial control of cell fates, the question of how the limb bud physically elongates has been much less studied. One particular hypothesis has been dominant in the field, known either as the proliferation gradient hypothesis or growth-based morphogenesis. This states that elongation is achieved by distal cells (furthest from the body) being stimulated to divide faster than proximal cells. Importantly, this hypothesis does not propose any kind of oriented or directional cell behaviours—high distal rates of non-oriented proliferation are considered to be sufficient—and indeed several 2D computer simulations have reproduced this concept in silico. However, thus far quantitative data from the limb bud has not been incorporated into these models. Here, we extended computer simulations into 3D and incorporated quantitative data on both shape changes and proliferation rates. These new simulations demonstrated that gradients of non-oriented proliferation are unable to explain limb bud elongation. We thus experimentally tested for evidence of oriented cell behaviours and indeed found that the cell shape, Golgi orientation, and cell divisions all display a non-random bias during limb bud outgrowth. Our data run contrary to the proliferation gradient hypothesis, indicating instead that oriented cell behaviours are important for driving elongation.
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