Expression and function of microRNAs during Xenopus development

MicroRNAs are approximately 22-nucleotide non-coding RNAs that are important regulators of diverse biological processes. I have developed and adapted methods to study the precise function of individual microRNAs during embryonic development of the African frog, Xenopus. I began by developing an in situ hybridization protocol to study the spatiotemporal expression patterns of microRNAs during embryogenesis. Using digoxigenin-labeled probes complementary to the primary microRNA sequence I was able to determine tissue-specific expression patterns. These included numerous conserved expression patterns between Xenopus and other vertebrates, including miR-9 and miR-124 (central nervous system), miR-1 and miR-133 (muscle), and miR-10c (posterior mesoderm), as well as novel expression patterns for conserved microRNAs such as miR-23b and miR-24a. This method can also distinguish the unique expression patterns for different members of a microRNA family. From this analysis, I selected two microRNAs for further functional studies. miR-24a is expressed in the neural retina during its development, and blocking the function of miR-24a with an antisense morpholino results in a small eye phenotype. I show that this reduction in eye size is not due to changes in patterning, specification, differentiation, or proliferation, but is due instead to an increase in programmed cell death (apoptosis). I have identified two genes important for apoptosis, caspase9 and apaf1, that are regulated targets of miR-24a. Caspase9 protein levels are increased when miR-24a is knocked down, caspase9 inhibitors can specifically rescue the knockdown phenotype, and miR-24a is able to rescue caspase9-induced apoptosis. These data strongly suggest that miR-24a is required in the developing neural retina to repress apoptosis by regulating caspase9 and apaf1.The second microRNA that I have done extensive functional studies on is miR-133b, which is expressed in somitic mesoderm and developing hypaxial myoblasts. Knockdown of miR-133b causes a reduction in markers of hypaxial muscle differentiation without affecting specification, migration, or proliferation. At late stages, embryos lacking miR-133b function have increased levels of apoptosis in hypaxial domains and a severe reduction in body wall and head muscles derived from hypaxial myoblasts. Overexpression of miR-133b causes premature differentiation but has no effect on myoblast proliferation. Animal caps injected with the myogenic factor myoD or activin can induce miR-133b and may be useful as a secondary experimental system to identify miR-133b targets. Several computationally predicted targets are identified and discussed.While computational predictions have become the standard method for identifying potential microRNA targets, the algorithms used to make these predictions are flawed in many ways. To ensure that I was investigating biologically relevant targets, I have attempted several different biochemical purification techniques aimed at isolating microRNA:mRNA duplexes in vivo. I have had little success with microRNA-directed RT-PCR or digoxigenin-labeled pre-microRNA immunopurification, but I am encouraged by results using biotin-labeled mature microRNAs for purification. Future refinements to this protocol must be completed before a screen for miR-133b targets is initiated