Gene regulatory networks underlying most common developmental systems have been characterized in increasingly granular detail using mRNA expression as a primary readout. Yet, mRNA expression levels are poorly correlated with protein levels. A large component of the unexplained variation in protein levels is thought to be attributable to translation efficiency, the number of proteins synthesized per mRNA. Translationally regulated genes can be obtained by identifying actively translated mRNAs by ribosome profiling. In combination with conventional gene expression techniques, this approach provides a method for calculating the translational efficiency of mRNAs. However, this approach requires large numbers of cells (more than 1 million) that are unfeasible for application to most in vivo developmental systems. One of us recently pioneered the development of a novel microfluidic approach for ribosome profiling termed Ribo-ITP that delivers high coverage and high-resolution ribosome occupancy measurements from ultra-low input samples (~100 cells). This method enables the identification of translationally regulated genes even in dynamic developmental systems with limited cellular material. We propose to apply this new approach to determine the mechanisms regulating the initial phase of limb outgrowth. This process is mediated by FGF10, which is essential for the epithelial to mesenchymal transformation (EMT) of the lateral plate mesoderm as well as for subsequent limb bud outgrowth. We hypothesize that limb bud EMT and FGF10-responsive gene regulatory networks are translationally regulated. In specific aim 1, we will determine the contribution of translational regulation to limb bud epithelial to mesenchymal transformation (EMT). In specific aim 2, we will identify transcriptional and translational networks mediated by FGF10, which is necessary and sufficient for mediating limb bud initiation via poorly understood mechanisms. At the completion of these studies, we will have characterized the process of EMT in limb bud initiation and determined the contribution of translational control to this process. Furthermore, we will have identified the FGF10-mediated transcriptional network of genes, and determined if they are translationally regulated, substantially advancing our understanding of limb bud initiation. Collectively, this proposal has the potential to have a transformative impact on our understanding of how translational regulation influences vertebrate organogenesis.