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Abstract

Building precise, robust patterns and structures from an initially homogeneous state is fundamental to developmental biology. Digit patterning is a representative example of a periodic pattern in development. Previous studies have shown that a reaction–diffusion (Turing) system, in which diffusible activators and inhibitors interact, is the most likely explanation of how the spatial pattern of the digits is formed. Although self-organisation mechanisms such as the Turing system successfully recapitulate many aspects of digit patterning, critical questions remain regarding its timing and behaviour. First I addressed the question of timing, or how long reaction-diffusion plays a role in the developing digits. I perturbed the digit patterning process of embryonic limbs by inserting beads that contain morphogens involved in the reaction-diffusion mechanism. Then I quantified the degree of pattern change, or plasticity of the patterning, from limbs harvested at different developmental timing throughout the digit patterning stage. For quantification, I developed a custom image analytic pipeline that extracts relevant topology and represents the difference between perturbed and unperturbed patterns. Modelling the plasticity profile over the digit patterning process, through extensive interplay of experiments and modelling, revealed that plasticity during digit patterning decreases in a sigmoidal manner. Transcriptomics analysis that matches with the sigmoidal decrease observed in expression patterns further identified gene candidates that could be critical to the digit patterning. Further, the timing of reaction-diffusion is discussed in the context of the tissue movements, revealing that Sox9 digit patterning happens significantly earlier than cell density changes. The second part aims at improving our understanding about which pathways and components of the pathways are involved in the digit forming Turing network. Previously identified digit patterning Turing network, such as BSW model, abstracts the entire Wnt and Bmp signalling pathways’ activities into each node. Thus there is insufficient knowledge on the mechanistic role of Wnt signalling mediated Sox9 repression. To further clarify detailed mechanisms of the Turing network, I used an unbiased screening approach to systematically perturb digit patterning using small molecule inhibitors, ligands, and peptides at different doses in systems such as limb culture and micromass. Out of multiple steps critical to Wnt signalling, including Wnt production, Wnt receptor interaction, Wnt canonical pathway cytosolic interactions, and Wnt canonical pathway transcriptional interactions, I identified that inhibition of Wnt production and Wnt transcriptional component inhibition category most effectively disrupt digit patterning. I also identified candidate ligands such as sFRP1 and Dkk1 as potential extracellular Wnt inhibitors that effectively change digit patterning upon application. These results provide the first quantitative insight into the duration of the reaction-diffusion based mechanism in a biological system, and how a screening approach complemented with data driven modelling can complement and clarify workings of a reaction diffusion based system. Further work in improving our knowledge on the Turing system with tissue growth, cell movements, and ectodermal-mesenchymal interaction will eventually allow generation of a complete organogenesis simulation model.