Early Expression And Role Of Unc5 Gene In The Forebrain Of Zebrafish

UNC5 family proteins are axon guidance trans-membrane receptors which were discovered in C. elegans. There have been characterized four orthologous of C. elegans UNC-5, including UNC5A, Unc5b, Unc5c and Unc5D in vertebrates. As one kind of important receptors, Unc5 family proteins mediate long-distance and short-distance repulsion signals for the axonal growth cones response to their ligand, Netrin. The UNC5 family proteins may also play critical roles in angiogenesis and carcinogenesis.Here we showed the expression pattern of zebrafish specific Unc5 family receptors and tried to figure out its impacts on forebrain development which expressed in these regions. We have determined the temporal and spatial expression patterns of Unc5 gene family members (Unc5a, Unc5c, Unc5da and Unc5db) by RT-PCR and in situ hybridization. RT-PCR results showed that all transcripts were expressed maternally. While Unc5c transcript was detected at all the time points between shield stage and 48 hours post fertilization (hpf), Unc5a, Unc5da and Unc5db showed expression at 24hpf and later time points. In situ hybridization analysis revealed that Unc5a, Unc5da and Unc5db transcripts were expressed in the telencephalon, parts of thalamus and hindbrain between 24hpf and 48hpf. The expression patterns of Unc5a-Unc5da and Unc5a-Unc5db in the telencephalon showed substantial overlap by fluorescent in situ hybridization. Unc5c showed expression in the roof plate, the hindbrain and the mouth region.Anterior commissure (AC) is axon tracts which cross the anterior midline to connect two embryonic telencephalon, located in rostral surface of zebrafish forebrain. Knocking down Unc5a function by injecting Unc5a morpholino antisense oligonucleotides into embryos of transgenic zebrafish may cause aberrant development of anterior commissure. After Kaede protein photo-conversion, results show the change of anterior commissure formation. The AC has clear aberrant branches of axon pathfinding that parts of axon tract kept their original ways to connect two sides of telencephalon, however, other axon tracts chose deeper and more dorsal path to extend. After immunohistochemistry, the glia cells around anterior commissure altered their distribution and showed similar appearance of branches like the axon tracts. The results of knocking down Unc5a and Unc5da have a further change of neuron migration in the midline. Our results provide a starting point to uncovering roles of Unc5 gene family in zebrafish forebrain development and axonal outgrowth or guidance.