Signal scaling during limb regeneration of different sized animals

Axolotl在胚胎后的所有阶段再生完整肢体的能力，从2厘米的幼虫到20厘米的成年，为了解形态生成系统如何随着系统/组织的大小而变化提供了一个极好的机会。截肢导致胚泡的形成，胚泡中含有间充质祖细胞，表达与在Axolotl和其他脊椎动物的胚胎肢芽中发现的发育因子相同的发育因子。重要的是，再生的轴突胚泡的大小与身体大小成比例，强烈表明发育因子的作用范围也与身体大小成比例。这一建议的中心目标是将定量成像与分子信号因子的定量图谱相结合，以实现数学建模，最终目标是了解不同大小动物的轴突肢体胚芽中信号尺度的空间域。我们将对肢体信号如何适应不同胚泡大小的两个替代假说进行实验测试和数学建模。假设1：信号梯度是由有效扩散建立的。在小胚泡和大胚泡中，有效扩散系数、降解率或对信号的敏感性存在大小相关的调制。假设2：信号场是通过传播波建立的，其中胚泡细胞充当信号传播的中继单位。为了区分假设1和假设2，并阐明它们的机制基础，我们将：1)测量小型和大型胚泡中信号因子的有效扩散系数和对信号感知的敏感性2)创建报告并分析可能驱动信号传播的潜在信号传播波3)生成形态梯度缩放的数学模型，推导可测试的预测，并设计歧视性实验4)最后，我们将通过实施TomoSeq结合功能分析来解决哪些分子调控肢体形态因子的表达和活性区域的大小。

The ability of the axolotl to regenerate a complete limb at all post-embryonic stages, ranging from the 2-cm larvae to the 20-cm adult, provides a remarkable opportunity to understand how morphogen systems scale with system/tissue size. Limb amputation results in the formation of a blastema, which contains mesenchymal progenitors that express the same developmental factors as those found in the embryonic limb bud of the axolotl and other vertebrates. Importantly, the size of the regenerating axolotl blastema scales with body size, strongly suggesting that the range of action of developmental factors also scale with body size. The central aim of this proposal is to combine quantitative imaging with quantitative profiling of molecular signalling factors to enable mathematical modelling, with the ultimate goal to understand whether spatial domains of signaling scale in the axolotl limb blastema of different sized animals. We will experimentally test and perform mathematical modelling of two alternative hypotheses for how limb signalling can adapt to different blastema sizes. Hypothesis 1: Signalling gradients are established by effective diffusion. There is size-dependent modulation of effective diffusion coefficients, degradation rates, or sensitivity to signaling in small versus large blastemas. Hypothesis 2: Signaling fields are established by propagating waves, where blastema cells serve as relay units for signal propagation. To discriminate between hypotheses 1 and 2, and elucidate their mechanistic basis, we will: 1) Measure effective diffusion coefficients and sensitivity to signal perception of signaling factors in small and large blastemas 2) Create reporters and assay potential propagating waves of signaling that could drive signal propagation 3) Generate mathematical models of morphogen gradient scaling, derive testable predictions, and design discriminatory experiments 4) Finally, we will address which molecules regulate the size of expression and activity domains of the limb morphogens by implementing TomoSeq combined with functional assays.