Adapting in ovo RNAi as a tool to study gene function during neural circuit formation in the cerebellum

The cerebellum is not only essential for motor control but also involved in higher cognitive functions and emotion processing. Its involvement in neurodevelopmental disorders, such as autism spectrum disorders and attention deficit hyperactivity disorder, has enhanced interest in molecular and cellular mechanisms of cerebellar development. We previously established ex ovo RNAi as a tool to study temporally controlled gene function during neural circuit formation in the embryonic chicken cerebellum in vivo. However, ex ovo RNAi requires more skills and is less efficient than in ovo RNAi, a technique that we established for the assessment of gene function in the spinal cord and the peripheral nervous system. To provide an easier method than ex ovo RNAi, we adapted our in ovo RNAi protocol to make it useful for studies of cerebellar development. Chicken embryos are injected and electroporated in the egg with plasmids and/or double-stranded RNA to manipulate gene expression in the developing cerebellum. Despite the early manipulation of chicken embryos, our protocol allows for cell type-specific loss- or gain-of-function analyses in the developing cerebellum. In contrast to the ex ovo protocol, the adapted version of our in ovo RNAi protocol is easier to learn and increases survival rates, thus improving efficiency. To validate our method, we replicated phenotypes from our ex ovo analysis of Endoglycan function. Endoglycan was shown to regulate adhesive strength. In the developing cerebellum, Endoglycan is required for Purkinje cell migration. This in turn impacts granule cell proliferation and growth of cerebellar lobes. These phenotypes are reproduced with our adapted in ovo RNAi protocol. The adapted protocol provides a convenient tool to study the function of disease-associated genes during cerebellum development with improved efficiency. • Accessibility in egg makes chicken embryos an ideal model for developmental studies • Cerebellar development is completed in 3 weeks in chicken • In vivo analysis of gene function in tissue and cell-type specific manner • Rapid analysis of gene function without the need for time-consuming breeding