The Journal of Neuroscience, May 2, 2007, ():
PlexinA3 Restricts Spinal Exit Points and Branching of Trunk Motor Nerves in Embryonic Zebrafish
J. Neurosci. Feldner et al.
27: 4978
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplementary Fig. 1 Zebrafish plexinA3 shares sequence homologies with plexinA3 of other vertebrate species. The deduced amino acid sequence of zebrafish plexinA3 is aligned with mouse and human plexinA3. Black and gray shading represents identical and similar amino acids, respectively. sema = semaphorin domain, MRS = Met Related Sequence, IPT = Immunoglobulin-like fold shared by Plexins and Transcription factors, TM = transmembrane region, plexin = plexin domain.
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Supplementary Fig. 2 Expression pattern of plexinA3 mRNA and normal growth of trigeminal axons under plexinA3 knock down conditions. Lateral views of whole-mounted 24 hpf embryos are shown, rostral is left. A,B: PlexinA3 mRNA is present in the telencephalon (tel), epiphysis (epi), tegmentum (teg), hindbrain neurons (hb) in the head (A), as well as in spinal cord (sp) and motor neurons (mn) (B). Additional expression is found in the tip of the tail (arrow in B). Yolk droplets (arrowhead in B) show non-specific staining. C-E: There is no difference in the differentiation of trigeminal ganglia in 1 mM control morpholino injected animals (C) and 1mM plexinA3 morpholino2 injected animals (D). Numbers of trigeminal axons are not significantly altered by injection of specific morpholinos compared to control morpholinos (1mM) (E). Bar in D = 100 µm.
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Supplementary Fig. 3 The spinal cord and the extra-spinal environment of motor axons are not altered by morpholino injections. Lateral views of whole-mounted 24 hpf embryos at mid-trunk levels are shown (rostral is left). PlexinA3 morpholino1 was injected at 1 mM (B,D,F,H,J,L). A-D: Labeling with an antibody to chondroitin sulfates labels vertical myosepta (vm) (A,B), the surface of the notochord (nc) and spinal floor plate (fp) (C,D) in uninjected (A,C) and morpholino injected embryos (B, D). E,F: Labeling Rohon-Beard (RhB) and motor neurons (mn) with an antibody to islet-1/2 indicates comparable numbers and locations of these cell types in uninjected embryos (E) and those injected with morpholino (F). G,H: Labeling of commissural primary ascending interneurons in the spinal cord with the 3A10 antibody indicates normal positioning of somata (CoPA) and contralateral axons (arrowheads), which eventually join the dorsal longitudinal fascicle (DLF) in uninjected embryos (G) and in morpholino-injected embryos (H). I,J: Double-labeling of tenascin-C (red) at the horizontal myoseptum and ventral motor axons (green) labeled with an anti-HNK-1 antibody reveals normal punctate expression of tenascin-C at the horizontal myoseptum and strong expression in vertical mysepta of uninjected (I) and morpholino-injected embryos (J). The arrow in J indicates abnormal branching of a ventral motor nerve at the level of the horizontal myoseptum. K,L: Simultaneous labeling of muscle pioneer cells (mp) at the horizontal myoseptum with an antibody to engrailed, and ventral motor axons with an anti-tubulin antibody, did not show systematic differences between uninjected (K) and morpholino injected embryos (L). Arrow in L indicates aberrant branching of a ventral motor nerve. Bar in D = 25 µm for A-D; bar in H = 25 µm for E-H; bar in L = 12.5 µm for I-L.
