Small RNAs are key regulatory molecules and it was recently shown that gradients formed by mobile small RNAs contribute significantly to developmental patterning. Organ polarity is an excellent model to study this newly recognized role of small RNAs. In developing leaves, dorsoventral determinants are expressed in robust and sharply defined domains on the upper (adaxial) or lower (abaxial) side of the leaf, resulting in stable positioning of the adaxial-abaxial boundary. This polar expression pattern is generated via two small RNAs which move intercellularly from their site of biogenesis and accumulate in opposing gradients across developing leaves, restricting the expression of adaxial and abaxial determinants to one side. The fact that these small RNAs generate discretely defined expression domains of their targets suggests a dose-dependent, perhaps morphogen-like read-out of small RNA gradients. Mathematical modeling supports such a scenario and predicts that small RNA gradients resulting from mobility are uniquely suited to generate sharply defined target gene expression boundaries. The experiments outlined in this proposal will test the mechanism of small RNA gradient read-out and the role these gradients play in sharpening target gene expression domains by examining target gene expression in plants where small RNA gradients were perturbed. In addition, I will test whether small RNA gradients provide robustness to leaf development by generating a stable adaxial-abaxial boundary by measuring variability in leaf parameters in plants with perturbed small RNA gradients under different stress conditions. I will test whether the two opposing small RNA gradients of the leaf are interconnected by investigating the regulation of small RNA production. Finally, I will address the question whether differential small RNA stability contributes to gradient formation by comparing small RNA half-lives in the adaxial and abaxial domains of the leaf.