Ccdc80-l1 Is involved in axon pathfinding of zebrafish motoneurons

Abstract

Axon pathfinding is a subfield of neural development by which neurons send out axons to reach the correct targets. In particular, motoneurons extend their axons toward skeletal muscles, leading to spontaneous motor activity. In this study, we identified the zebrafish Ccdc80 and Ccdc80-like1 (Ccdc80-l1) proteins in silico on the basis of their high aminoacidic sequence identity with the human CCDC80 (Coiled-Coil Domain Containing 80). We focused on ccdc80-l1 gene that is expressed in nervous and non-nervous tissues, in particular in territories correlated with axonal migration, such as adaxial cells and muscle pioneers. Loss of ccdc80-l1 in zebrafish embryos induced motility issues, although somitogenesis and myogenesis were not impaired. Our results strongly suggest that ccdc80-l1 is involved in axon guidance of primary and secondary motoneurons populations, but not in their proper formation. ccdc80-l1 has a differential role as regards the development of ventral and dorsal motoneurons, and this is consistent with the asymmetric distribution of the transcript. The axonal migration defects observed in ccdc80-l1 loss-of-function embryos are similar to the phenotype of several mutants with altered Hedgehog activity. Indeed, we reported that ccdc80-l1 expression is positively regulated by the Hedgehog pathway in adaxial cells and muscle pioneers. These findings strongly indicate ccdc80-l1 as a down-stream effector of the Hedgehog pathway.

Figure 1. Analysis of chromosomal organization of the three ccdc80 zebrafish homologs across vertebrates.

Each ccdc80 gene is shown as a reference locus. Genes annotated as paralogs (no surrounding line) or orthologs (with a surrounding line) by the Ensembl database share the same color, blue lines beneath individual tracks indicate that orientations of gene blocks and are inverted with respect to their genomic annotation. For zebrafish ccdc80 (chr. 9), ccdc80-l1 (chr. 6) and ccdc80-l2 (chr. 21), only ccdc80 shows notable synteny with other vertebrates. The figure was derived from the output of the Genomicus website (version 57.01).

Figure 2. Expression of ccdc80-l1 analyzed by RT-PCR and WISH.

(A) RT-PCR performed on different embryonic stages and adult tissues; the expression of ccdc80-l1 and β-actin are shown. Lanes are: ladder (lane 1), ovary (lane 2), 2–4 cells stage (lane 3), 64–1000 cells stage (lane 4), 30% epiboly (lane 5), 60–70% epiboly (lane 6), somitogenesis (lane 7), 24 hpf (lane 8), 30 hpf (lane 9), 48 hpf (lane 10), 72 hpf (lane 11), adult muscle (lane 12) and negative control (lane 13) in the absence of cDNA. (B–J) WISH performed on zebrafish embryos at several stage of development. (B, C) During somitogenesis ccdc80-l1 was expressed by cranial ganglia (cg), dorsal dermis (asterisk), adaxial cells and muscle pioneers at the level of the horizontal myoseptum (arrow). (D) ccdc80-l1 expression in a transverse section of the trunk of an embryo at 12 somites stage (arrows). (E–H) At 24 hpf, the hybridization signal was detectable in cranial ganglia (cg), dermis (asterisk), adaxial cells (arrow) and ventral somites (arrowhead). (F) Higher magnification of the tail at 24 hpf. (G) Transversal section of an embryo at 24 hpf. (H) Transversal section showing that at 24 hpf ccdc80-l1 hydridization signal co-localized with the nuclear labeling of 4D9 antibody, corresponding to the engrailed-positive muscle pioneers population (open arrowhead). (I, J) At 36 hpf, the signal of ccdc80-l1 probe was detected in cranial ganglia (cg), migrated adaxial cells (arrow), dorsal dermis (asterisk) and caudal vein plexus region (cvp). (K, L) At 48 hpf, ccdc80-l1 was detected in dorsal dermis (asterisk), external adaxial cells (arrows in K) and caudal vein plexus region (cvp in L). (B, E, F, I) Lateral views; dorsal is up, anterior is left; (C) dorsal view, anterior is left; (D,G, H, J–L) transversal sections, dorsal is up.

Figure 3. Analysis of myogenic markers expression and muscle pioneers in ccdc80-l1 morphant embryos.

(A–D) The myogenic markers myod (A, B) and myog (C, D) were correctly expressed both in control and morphants embryos at 10 s stage (A, B) and 24 hpf (C, D), respectively. (E, F) The MF20 antibody staining showed that both slow and fast twitch fibers were correctly formed and distributed in control and in knocked-down embryos at 24 hpf. (G, H) At the same developmental stage, muscle pioneers resulted unaffected after ccdc80-l1 loss-of-function, as shown by the labeling with 4D9 antibody (anti-engrailed) (arrows). (A, B) Dorsal views, anterior is left; (C–H) lateral views of the tails, dorsal is up and anterior is left.

Figure 4. Analysis of motoneurons morphology by means of znp1- and zn-5-immunohistochemistry.

(A, B) At 48 hpf, using 12 ng/embryo of morpholino, both ventral (arrows) and dorsal axons (arrowheads) were mis-orientated and over-branched in morphants (B) in comparison to control embryos (A). (C) Statistical analysis showing the percentages of the different phenotypes (affected ventral axons, dorsal axons or both) occurring in control embryos and in morphants, when different doses of ccdc80-l1-MO were injected (12 ng/embryo and 8 ng/embryo). Using a lower dose of morpholino (8 ng/embryo), we observed that in a significant percentage of embryos only ventral axons were defective. (D–G) Immunohistochemistry performed at 26 hpf (D, E) and 30 hpf (F, G) confirmed that loss-of-ccdc80-l1-function affects both CaPs (arrows) and MiPs (arrowheads) axonal migration. (H, I) The same analysis performed at 48 hpf using zn-5 antibody revealed that also SMNs axonal migration is impaired in morphants (arrows in I) in comparison to control embryos (H). (A, B; D–I) Lateral flat-mount preparation was applied for a better visualization of the motoneurons. Lateral views of the trunk region overhanging the yolk extension, dorsal is up and anterior is left.

Figure 5. ccdc80-l1 is positively regulated by shh.

(A–C) ccdc80-l1 expression in somites and myoseptum resulted strongly inhibited in embryos treated with 5 µM cyclopamine (asterisks in B), in comparison to control embryos at the same developmental stage (A). By converse, over-expression of shh led to an up-regulation of ccdc80-l1 in muscular territories (C). Expression in cranial ganglia (cg) was never perturbed. (D–F) ccdc80-l1 resulted slightly down-regulated in the muscles of heterozygous syu ^+/− mutants (E) in comparison to wild type siblings (D). A strikingly down-regulation was observed in homozygous syu ^−/− mutants (F). (A–C) Dorsal flat-mount preparations, anterior is up. (D–F) Lateral views of the tails, anterior is left.