ccdc80-l1 Is Involved in Axon Pathfinding of Zebrafish Motoneurons

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

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

<p><i>(A, B)</i> At 48 hpf, using 12 ng/embryo of morpholino, both ventral (arrows) and dorsal axons (arrowheads) were mis-orientated and over-branched in morphants <i>(B)</i> in comparison to control embryos <i>(A)</i>. <i>(C)</i> 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 <i>ccdc80-l1</i>-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. <i>(D–G)</i> Immunohistochemistry performed at 26 hpf <i>(D, E)</i> and 30 hpf <i>(F, G)</i> confirmed that loss-of-<i>ccdc80-l1</i>-function affects both CaPs (arrows) and MiPs (arrowheads) axonal migration. <i>(H, I)</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>I</i>) in comparison to control embryos <i>(H). (A, B; D–I)</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.</p

Loss-of-<i>ccdc80-l1</i>-function impairs PMNs axonal migration.

<p>Embryos injected with 12 ng/embryo of <i>ccdc80-l1</i>-MO were observed also at 26 hpf and 30 hpf. At these stages, affected embryos were 54,5% and 62%, respectively. The percentages of the different phenotypes are listed.</p

The phenotype of <i>ccdc80-l1</i>-MO-injected embryos is dose-dependent.

<p>The percentage of embryos displaying axonal defects decreased from 84% to 64% when a lower dose of morpholino was used. Both ventral and dorsal axonal pathfinding resulted impaired in the 69% of affected embryos when 12 ng/embryo of morpholino were used. After the injection of the lower dose of <i>ccdc80-l1</i>-MO (8 ng/embryo), 27% of affected embryos showed alteration of both ventral and dorsal axons, whereas the 37% displayed only ventral defective axons and dorsal axons alone were never affected.</p

Analysis of chromosomal organization of the three <i>ccdc80</i> zebrafish homologs across vertebrates.

<p>Each <i>ccdc80</i> 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 <i>ccdc80</i> (chr. 9), <i>ccdc80-l1</i> (chr. 6) and <i>ccdc80-l2</i> (chr. 21), only <i>ccdc80</i> shows notable synteny with other vertebrates. The figure was derived from the output of the Genomicus website (version 57.01).</p

<i>ccdc80-l1</i> is positively regulated by <i>shh</i>.

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

Analysis of myogenic markers expression and muscle pioneers in <i>ccdc80-l1</i> morphant embryos.

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

Percentages of identity and similarity among human and zebrafish Ccdc80 homologs.

<p>The table shows the scores obtained after alignments between the aminoacidic sequences of zebrafish and human CCDC80 homologs. Alignments were performed with Stretcher-P tool.</p