PlexinA3 restricts spinal exit points and branching of trunk motor nerves in embryonic zebrafish

Abstract

The pioneering primary motor axons in the zebrafish trunk are guided by multiple cues along their pathways. Plexins are receptor components for semaphorins that influence motor axon growth and path finding. We cloned plexinA3 in zebrafish and localized plexinA3 mRNA in primary motor neurons during axon outgrowth. Antisense morpholino knock-down led to substantial errors in motor axon growth. Errors comprised aberrant branching of primary motor nerves as well as additional exit points of axons from the spinal cord. Excessively branched and supernumerary nerves were found in both ventral and dorsal pathways of motor axons. The trunk environment and several other types of axons, including trigeminal axons, were not detectably affected by plexinA3 knock-down. RNA overexpression rescued all morpholino effects. Synergistic effects of combined morpholino injections indicate interactions of plexinA3 with semaphorin3A homologs. Thus, plexinA3 is a crucial receptor for axon guidance cues in primary motor neurons.

Figure 1.

Structural features and identity of plexinA3 in zebrafish. A, Domain structure of plexinA3. SEMA, Semaphorin domain; PLEXIN, plexin domain; MRS, Met-related sequence. B, Multiple comparisons in a phylogenetic tree group zebrafish plexinA3 with plexinA3 homologs in other vertebrates. Drosophila plexinA was added as an outgroup. The scale bar represents 10 substitutions per 100 aa. z, Zebrafish; m, mouse; h, human; x, Xenopus; d, Drosophila.

Figure 2.

Expression and function of plexinA3 in primary motor neurons. A, B, A schematic representation of the outgrowth of primary motor axons, which is preceded by the CaP axon at ∼18 hpf, is given in a lateral view of one trunk segment in A. B, The distribution of semaphorin ligands relative to the ventral motor axon pathway. C–H, In situ hybridization of plexinA3 mRNA shows expression in clusters of GFP-immunopositive motor neurons of hb9:GFP transgenic fish in lateral views at 24 hpf. C–E, A caudal region in which CaP axons (D, arrowheads) are just growing out. The arrowhead in C and E depicts a more dorsal, GFP-immunonegative cell that shows strong expression of plexinA3 mRNA. At higher magnification in F–H, two adjacent intensely plexinA3 mRNA-positive neurons in a more rostral segment are depicted. These are likely the CaP (right cell) and MiP (left cell) motor neurons, judging by the trajectories of the GFP-positive MiP (arrow) and CaP (arrowhead) axons. I–N, Lateral views at midtrunk levels of anti-tubulin-labeled whole-mounted 24 hpf embryos are shown. In uninjected embryos (I) or those injected with 1 mm plexinA3, 5 mm morpholino (5 mm; L), single unbranched motor nerves (arrows in I and L) grow ventrally out of the spinal cord. Injection of 1 mm plexinA3 morpholino1 (MO1) induces branching (arrow in J) or a second spinal exit point for motor nerves per hemisegment (arrows indicate additional nerves in M). Injection of 1 mm plexinA3 morpholino2 (MO2) also induced aberrant branching (K, arrows) of the ventral motor nerve and additional nerves exiting the spinal cord (N, arrows). O, P, Axons in the dorsal MiP pathway are visualized in hb9:GFP transgenic fish in selected confocal image stacks at 31 hpf, indicating normal growth in uninjected embryos (O, arrowheads), and excessive branching (curved arrow in P) and supernumerary nerves (straight arrow in P) in 1 mm plexinA3 morpholino2-injected embryos. The asterisk in P indicates an additional nerve exit point with a dorsal and ventral nerve branch exiting the spinal cord. Arrowheads in P point to normal appearance of axons in the dorsal motor axon pathway. Rostral is left in A to P. Scale bars (in E) C–E, 25 μm; (in H) F–H, 12.5 μm; (in N) I–N, 25 μm; (in P) O, P, 25 μm.

Figure 3.

Combining plexinA3 with semaphorin morpholinos has synergistic effects. 5 mm, Control morpholino based on plexin morpholino 1 with five mismatched bases. For concentrations, see Results. *p < 0.05; ***p < 0.001.