Characterization of a novel interaction between Islr2 and Vasorin and its role in retinal axon routing at the vertebrate optic chiasm

Abstract

A striking feature of embryonic axon tracts is their high level of guidance stereotypy. To isolate new candidate molecules with putative functions in retinal axon pathfinding we focused on the extracellular leucine-rich repeat (eLRR) protein family in zebrafish. eLRR proteins are known to convey specialized, dynamic functions at the axon and growth cone membranes. The compilation of an eLRR gene catalogue and the analysis of their expression patterns allowed the identification of islr2 as a candidate putatively involved in early guidance decisions undertaken by retinal axons. We used the zebrafish to model Islr2 behaviour in vivo, taking advantage of the rapid development of its retinotectal projection and amenability to imaging. islr2sa82 mutants display the presence of ectopic ipsilateral projections, although zebrafish normally have a completely crossed retinal axon projection. These data suggest that islr2 facilitates retinal axon crossing to the opposite side of the brain. Taking advantage of a novel protein-protein interaction screening method, dedicated to detecting low affinity binding events, we identified Vasorin A and B as specific Islr2 binding partners. We generated immunoreagents in order to describe these proteins’ localization in zebrafish embryos and found that their expression domains are closely associated to the retinal axon pathway. Vasnb, in particular, is expressed by a small population of cells at the anterior ventral diencephalic midline, a location reminiscent of the glial knot. The knot is a structure identified in mouse and chick which was anatomically thought to redirect retinal axon fibers at the midline. vasna/vasnb single and double knockout animals, however, do not show overt phenotypes in the optic nerves traversing the chiasm. In conclusion, we propose that other midline factors act through Islr2 allowing retinal axons to cross the midline. They compensate for the absence of Vasna and Vasnb function in vivo, a scenario that is not surprising when considering the high level of signalling redundancy characteristic of the optic chiasm. In a parallel project, we created transgenic zebrafish lines expressing chromobodies in an inducible fashion. Chromobodies are single-domain antibodies derived from heavy-chain IgG2 and IgG3 of camelids. Actin- and PCNA-directed chromobodies can be expressed intracellularly, allowing to trace endogenous targets without the need of protein tagging or overexpression. This technique can be applied in live zebrafish embryos, introducing intracellular immunoreagents as a complementary technique for protein localization studies. Using Actin-CB we were able to follow detailed cytoskeletal dynamics during rapid cellular behaviours in living animals. Additionally, PCNA-CB allows accurate tracing of DNA replication forks foci in the nucleus, making it possible to discern cell cycle progression in vivo. These tools surprisingly do not cause morphological or developmental defects throughout the duration of early development and can be expressed to adulthood. This is the first report of the applicability of the chromobody technology to living vertebrates.