Navigating axons respond to environmental guidance signals, but can also follow axons that have gone before -pioneer axons. Pioneers have been studied extensively in simple systems, but the role of axon-axon interactions remains largely unexplored in large vertebrate axon tracts, where cohorts of identical axons could potentially use isotypic interactions to guide each other through multiple choice points. Furthermore, the relative importance of axon-axon interactions compared with axon-autonomous receptor function has not been assessed. Here, we test the role of axon-axon interactions in retinotectal development, by devising a technique to selectively remove or replace early-born retinal ganglion cells (RGCs). We find that early RGCs are both necessary and sufficient for later axons to exit the eye. Furthermore, introducing misrouted axons by transplantation reveals that guidance from eye to tectum relies heavily on interactions between axons, including both pioneer-follower and community effects. We conclude that axon-axon interactions and ligand-receptor signaling have co-equal roles, cooperating to ensure the fidelity of axon guidance in developing vertebrate tracts.KEY WORDS: Robo2, astray, ath5, atoh7, Morpholino, Fasciculation, Transplant, Cell-autonomy, Zebrafish Development 135, 2865Development 135, -2871Development 135, (2008 DEVELOPMENT 2866 they make at most fleeting contacts with tPOC axons (Burrill and Easter, 1995), and embryological manipulations that remove tPOC axons do not affect retinal axon guidance (Cornel and Holt, 1992). Thus, retinal axons do not require heterotypic pioneers. Despite the extensive retinotectal literature, there has been only one functional test of whether retinal pioneer axons might guide later retinal axons (i.e. through an isotypic interaction). In Xenopus, dorsocentral RGCs are the first to send out their axons. There was no effect on the guidance of later retinal axons when heterochronic transplants were used to delay the outgrowth of dorsocentral RGCs (Holt, 1984), suggesting that retinal pioneers are not required. Here, we re-examine the role of retinal pioneers in guidance both within the eye and after exiting it. We use genetic and embryological manipulations both to remove early RGCs, and to replace them with cells that lack the Robo2 guidance receptor. We find that isotypic pioneers in fact play multiple roles during the formation of this archetypal vertebrate tract. MATERIALS AND METHODS TransgenicsAll zebrafish were of the Tü or TL strains. The transgenic lines and mutant alleles used were: Tg(isl2b: GFP) zc7 , Tg(isl2b:mCherry-CAAX) zc23 or zc25 (both of similar brightness), s356t (Xiao et al., 2005), and the null ast allele ast ti272z (Fricke et al., 2001). As ast is homozygous adult viable, ast embryos were generated by incrossing homozygote parents. Embryos were raised at 28.5°C in 0.1 mM phenylthiourea and staged according to time postfertilization and morphology (Kimmel et al., 1995). Experimental procedures followed NIH guidelines and were app...

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