Molecular Mechanisms of Patterning the Limb Proximodistal Axis and Quantitative Studies Addressing its Scalability over Five-Fold Changes in Size.

动物如何在发育过程中形成模式和衡量器官大小是发育细胞生物学的一个基本问题，这也与用于再生应用的工程组织有关。脊椎动物的肢芽是一个重要的模型，用来研究上臂、小臂和手是如何沿着近端/远端(PD)轴适当比例出现的，但目前还没有一个一致的模型。Axolotl肢体再生为解决这一问题提供了独特的实验优势。肢体再生通过与发育中相同的HOXA基因诱导序列进行，但比其他脊椎动物更慢，它发生在大小不同5倍的动物的胚泡中。在这里，我们使用异种细胞移植、主要信号通路的体内和体外扰动以及CRISPR介导的新的顶端上皮基因的敲除来测试主要的肢体模式模型，并确定控制HOXA11和HOXA13诱导的分子通路。然后，我们使用这些信息来测试关于肢体图案如何在肢体视野大小的5倍范围内缩放的三个假设。

How animals pattern and scale organ size during development is a fundamental problem in developmental cell biology that also has relevance for engineering tissues for regenerative applications. The vertebrate limb bud is an important model to examine how an appropriately proportioned upper arm, lower arm and hand along the proximal/distal (PD) axis arises but a consensus model does not yet exist. Axolotl limb regeneration provides unique experimental advantages to address this problem. Limb regeneration occurs via the same sequence of HOXA gene induction as in development but more slowly than other vertebrates and it occurs in blastemas from animals >5-fold different in size. Here we use heterochronic cell transplantation, in vivo and ex vivo perturbation of major signalling pathways and CRISPR-mediated knockout of novel apical epithelial genes to test the major limb patterning models and identify the molecular pathways governing HOXA11 and HOXA13 induction. We then use this information to test three hypotheses of how limb patterning scales over a 5-fold range in limb field size.