Study of the functional role of microRNAs in the regulatory networks underlying vertebrate eye development

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. They exert diverse functions in controlling normal tissue and organ development and physiology. Many miRNAs show spatially and temporally restricted expression patterns during vertebrate eye development but the roles of individual miRNAs in controlling this process remain however, largely unknown. The aim of my thesis was to shed further light on the role of specific miRNAs in regulating basic processes of ocular development mainly by exploiting the medakafish (Oryzias latipes) model system. In particular, I focused my attention on the miRNA subfamily constituted by miR-181a and miR-181b, which are expressed in the Inner Nuclear Layer (INL) and Ganglion Cell Layer (GCL) of the vertebrate retina. Morpholinomediated combined knockdown of miR-181a/b function in medakafish results in a specific retinal phenotype characterized by the reduction of Inner Plexiform Layer (IPL) thickness, without any apparent reduction in the number of retinal cells. To dissect this phenotype further, I studied the consequences of miR- 181a/b ablation in two medakafish transgenic lines, namely GFP-Six3.2 and GFP-Ath5, in which GFP expression can be specifically visualized in amacrine and retinal ganglion cells (RGCs), respectively. This analysis revealed that miR-181a/b exert a role in the specification and growth of amacrine and RGC axons. The above alterations translate into an impairment of retinal circuits assembly and to visual function defects, as assessed by the evaluation of the Optokinetic Response (OKR) behavioral test. Using a combination of bioinformatic, as well as on in vitro and in vivo experimental approaches, I identified ERK2, a kinase member of the MAPK signaling cascade, as one of the direct targets of these two microRNAs. I demonstrated that the function of miR-181a/b in growth cone cytoskeleton remodeling during retinal development are largely mediated by ERK2 targeting and by the modulation of its downstream signaling cascade. Moreover I provide, for the first time, in vivo evidence of an antagonism between the TGF-b pathway and the ERK2 cascade in the regulation of retinal axon specification and growth, which is exerted via TGF-b regulation of miR- 181a/b levels. These data expand our knowledge on the role of miRNAs in eye patterning in vertebrates, and demonstrate that miR-181a/b-targeting of ERK2 and the consequent modulation of the MAPK cascade, in concert with TGF-b-action, play important roles in the signaling network that define the correct wiring and assembly of functional retina neural circuits.