Signal scaling during limb regeneration of different sized animals

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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.