Novel mutations affecting axon guidance in zebrafish and a role for plexin signalling in the guidance of trigeminal and facial nerve axons

Hideomi Tanaka Ryu Maeda Wataru Shoji Hironori Wada Ichiro Masai Toshiyuki Shiraki Megumi Kobayashi Ryoko Nakayama Hitoshi Okamoto ) In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord. INTRODUCTION The neuromuscular connection is one of the simplest model systems available for analysis of the mechanisms underlying establishment of functional neural circuits. This system has been used in many studies in vertebrates and invertebrates. Gene knockdown analyses and tissue- and cell-transplantation studies in mouse, chick and zebrafish have revealed that the differentiation and specification of each motoneuron type that innervates target muscles is regulated by the combined temporal expression of transcription factors. These include the LIM-homeodomain-type transcription factors Islet1 (Isl1), Islet2 (Isl2) and Lhx3, and the bHLH-type transcription factors neurogenin 2 and Neuro-M (Appel et al., 1995; Eisen, 1991; Inoue et al., 1994; Landmesser, 2001; Lee and Pfaff, 2003; Segawa et al., 2001; Thaler et al., 2002; Tokumoto et al., 1995; Tsuchida et al., 1994; Uemura et al., 2005). In invertebrates, screening for Drosophila mutants showing defects in the axonal pathfinding of motoneurons to specific target muscles has led to the identification of specific ligands and receptors in the neuromuscular connection system, including Semaphorin (Sema)/Plexin (Plex), Slit/Robo, Netrin/Frazzled (a Drosophila homologue of mouse Dcc), and receptor protein tyrosine phosphatases (Desai et al., 1996; Johnson and Van Vactor, 2003; Kidd et al., 1999; Kolodkin et al., 1993; Kolodziej et al., 1996; Winberg et al., 1998). Cell adhesion molecules, extracellular matrix molecules, and glycosylated derivatives of these molecules, also participate in the axonal pathfinding process (Birely et al., 2005; Kantor et al., 2004; Keshishian et al., 1996; Schneider and Granato, 2006). The role of these molecules in the regulation of motoneuron axonal pathfinding, the temporal and spatial regulation of these molecules as an integrated system, and the mechanisms underlying their molecular interactions remain unclear. The jaw muscles of zebrafish embryos are composed of a small number of identifiable muscles derived from the first and second branchial arches (BA1 and BA2) (Fig. 1A,B) (Higashijima et al., 1997; Higashijima et al., 2000; Schilling and Kimmel, 1994; Schilling and Kimmel, 1997). These muscles are innervated by the axons of the anterior and posterior trigeminal motoneurons (Va and Vp) that originate from rhombomeres 2, 3 (r2 and r3), and also by the axons of the facial motoneurons (VII) that originate from r4 (Fig. 1C). The present study shows the stereotypical stepwise outgrowth pattern of the Vp and VII motor axons to target muscles. This was achieved in the Isl1-GFP transgenic strain (Higashijima et al., 2000) using time-lapse observations of axonal pathfinding behaviour of the Vp and VII motoneurons in the lower jaw region of BA1 and BA2, together with laser-mediated cell ablation and single-cell labelling of the Vp motoneurons. The identification of the genetic loci regulating the stereotypical axonal pathfinding of the Vp and VII motoneurons was achieved by screening mutants. Seven distinct mutant loci leading to specific disruption of the different steps of the axonal pathfinding processes were identified. These mutants were classified into four groups according to the developmental stage in which the axons of the Vp and VII motoneurons began to show abnormal behaviour in the lower jaw region. In addition to abnormal motor axon pathfinding, some mutants displayed other neural defects. In the mutant vermicelli (vmc), the thick bundle of the Vp and VII motor axons defasciculated into thin branches, with each axon behaving randomly after separation from the common pathway shared with sensory axons. We identified a defect in the gene encoding the zebrafish orthologue of plxna3 and demonstrated that Plxna3mediated Sema3a1 signalling is required for the outgrowth of the Vp and VII motoneurons. MATERIALS AND METHODS Animals Zebrafish (Danio rerio) were maintained according to standard procedures (Westerfield, 2000). The Isl1-GFP and -actin-GFP lines, registered as Tg(CM-isl1:GFP)rw0 and Tg( -actin:GFP), respectively, in the Zebrafish National BioResource Center of Japan, http://www.shigen.nig.ac.jp/zebra/ (Higashijima et al., 1997; Higashijima et al., 2000), are derived from the RIKEN Wako (RW) wild-type strain. The WIK strain was used for genetic mapping. The embryos were staged according to Kimmel et al. (Kimmel et al., 1995). To prevent pigmentation, embryos were raised in fish water containing 0.0015% N-phenylthiourea (PTU; Nakarai) from approximately 12 hours postfertilisation (hpf) (Burrill and Easter, 1994). Mutagenesis Mutagenesis was carried out as described previously (Solnica-Krezel et al., 1994; Wada et al., 2005). Mutations were induced in the male germ cells of Isl1-GFP fish using N-ethyl-N-nitrosourea (ENU; Sigma). To isolate the mutants showing defects in axonal pathfinding of the Vp and VII motoneurons, the embryos from the F2 pairwise crosses were fixed at 72 hpf using trichloroacetic acid (Wako) and stained with anti-acetylated -tubulin antibody, as described below. The axonal projection patterns of the Vp and VII motoneurons were examined under a dissecting microscope (SMZ1500; Nikon). Of the 1816 haploid genomes (1171 families) screened, three alleles of the vermicelli locus (vmcrw260, vmcrw314 and vmcrw413), one allele of the keep off locus ( (...truncated)