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

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

Crucial role of zebrafish in hypothalamic catecholaminergic neurons development-0

Epiboly (lane 2), 50% epiboly (lane 3), 80% epiboly (lane 4), tail bud (lane 5), 8 somites (lane 6), 15 somites (lane 7), 24 hpf (lane 8), 72 hpf (lane 9), 5 dpf (lane 10) and negative control (lane 11) in the absence of cDNA. ) RT-PCR performed on different adult organs: DNA ladder (L), testis (lane 1), overy (lane 2), gills (lane 3), gut (lane 4), eye (lane 5), brain (lane 6) and liver (lane 7). Arrowhead indicates the size of the -specific PCR product (620 bp). WISH the first signals appeared at 2 s in the otic placode (arrowhead). at 15 s the signal is detected in the lens placode (arrowhead), and somites (inset). at 24 hpf is expressed the hypothalamus (asterisc), the pituitary (black arrowhead), the pretectal segment (prosomere 1) (white arrow), as well as segmentally arranged cells of the hindbrain (black arrow). transverse section through the forebrain of a 24 hpf stage zebrafish embryo shows the signal in the lens (black arrowhead). at 24 hpf additional signals are present in the liver primordium (arrow), and posterior lateral line primordium (arrowheads). later during development, (48 hpf) expression is detected in distinct domains in the liver (arrow) and pancreas (arrowhead), while a further signal appeares in the retina (white arrow). transverse section through the forebrain of a 7 dpf stage zebrafish larva shows the signals in the retina inner nuclear layer (arrow) and in the pretectal nuclei (arrowhead). Lateral views are shown. Frontal view is shown. Dorsal views are shown. Anterior is always to the left. Scale bars indicate 100 μm () or 200 μm ().<p><b>Copyright information:</b></p><p>Taken from "Crucial role of zebrafish in hypothalamic catecholaminergic neurons development"</p><p>http://www.biomedcentral.com/1471-213X/8/27</p><p>BMC Developmental Biology 2008;8():27-27.</p><p>Published online 10 Mar 2008</p><p>PMCID:PMC2288594.</p><p></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

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

Crucial role of zebrafish in hypothalamic catecholaminergic neurons development-3

Ls in control and overexpressed /embryos at 36 hpf. The most numerous class in the group of GFP mRNA injected control embryos presented 8 CA hypothalamic neurons, and only 3 embryos presented more than 11 TH hypothalamic positive cells (n = 54). The most numerous class in the group of the overexpressed /embryos (n = 64) presented 10 CA neurons, and 20 embryos showed more than 11 TH hypothalamic positive cells. Immunostaining with TH antibody shows ectopic TH positive cells on the yolk surface ectoderm of double injected embryos (arrowheads), while these cells are not present on the yolk of control embryos. Ectopic TH positive cell on the yolk surface ectoderm. Scale bars indicate 50 μm.<p><b>Copyright information:</b></p><p>Taken from "Crucial role of zebrafish in hypothalamic catecholaminergic neurons development"</p><p>http://www.biomedcentral.com/1471-213X/8/27</p><p>BMC Developmental Biology 2008;8():27-27.</p><p>Published online 10 Mar 2008</p><p>PMCID:PMC2288594.</p><p></p

Crucial role of zebrafish in hypothalamic catecholaminergic neurons development-1

), dorsal view. Eyes or lens have been removed for better lateral viewing. () WISH combined with TH immunohistochemistry. Anti-TH antibody labels the PT and hypothalamic CA neurons at 36 hpf. Colabelling with is evident in a fraction of TH-positive neuroblasts in the hypothalamus (arrowheads), as also confirmed by the longitudinal section of the embryo (). microinjection of MO lowers the number of TH-labelled CA neurons in the hypothalamus in comparison to standard control injected embryos . coinjection of mRNA and MO rescued the morphant phenotype. () Quantitative real time RT-PCR. TH-specific mRNA is almost five-fold decreased following MO injection. The result represents at least three independent experiments, and 18S was used as an internal control. The following abbreviations are used: posterior tuberculum (PT), pituitary (Pit), hypothalamus (Hy), standard control morpholino oligonucleotide (stdr MO). Scale bars indicate 10 μm or 20 μm .<p><b>Copyright information:</b></p><p>Taken from "Crucial role of zebrafish in hypothalamic catecholaminergic neurons development"</p><p>http://www.biomedcentral.com/1471-213X/8/27</p><p>BMC Developmental Biology 2008;8():27-27.</p><p>Published online 10 Mar 2008</p><p>PMCID:PMC2288594.</p><p></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

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