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Ccdc80-l1 Is involved in axon pathfinding of zebrafish motoneurons.

Brusegan C, Pistocchi A, Frassine A, Della Noce I, Schepis F, Cotelli F - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia, Università degli Studi di Milano, Milan, Italy.

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.

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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.
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pone-0031851-g005: 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.

Mentions: Both muscle and motoneurons induction is finely regulated by levels and range of shh expression [9], [48], [49]. Due to ccdc80-l1 expression in adaxial cells and muscle pioneers, we decided to investigate the existence of a ccdc80-l1 up-stream regulation Hedgehog-mediated. We modulated shh activity by exposing embryos to cyclopamine, that inhibits the Hedgehog transducer Smoothened (smo) [9], [50]. To avoid the complete loss of the territories in which ccdc80-l1 is expressed, we chose a concentration of cyclopamine (5 µM) by which muscle pioneers and adaxial cells-derived slow fibers are unaffected, as already described [9] and as we demonstrated by the proper expression of their markers engrailed, myod and smyhc1 respectively. (Fig. S5). A striking down-regulation of ccdc80-l1 expression was observed in 72% of the treated embryos in comparison to controls (N = 32) (Fig. 5A, 5B). Interestingly, this down-regulation was detectable only at the level of myoseptum and somites, whereas the cephalic territories in which ccdc80-l1 is expressed were not involved. A similar down-regulation was observed in syu mutants, carriers of a deletion in the gene sonic-you encoding for shh (Fig. 5D–F) [16]. ccdc80-l1 signal in adaxial cells was extremely weak or absent in the 35% of mutants, and slightly down-regulated in the 40% of observed embryos (N = 20). Moreover, the overexpression of shh by means of the injection of the full-length transcript (300 pg/embryo), led to the opposite phenotype with an increasing of ccdc80-l1 expression in the somites of the 71% of the injected embryos (N = 31) (Fig. 5C). On the contrary, ccdc80-l1 loss-of-function did not affect shh expression (Fig. S6). Therefore, these findings suggest that ccdc80-l1 is a down-stream target of the Hedgehog pathway.


Ccdc80-l1 Is involved in axon pathfinding of zebrafish motoneurons.

Brusegan C, Pistocchi A, Frassine A, Della Noce I, Schepis F, Cotelli F - PLoS ONE (2012)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285184&req=5

pone-0031851-g005: 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.
Mentions: Both muscle and motoneurons induction is finely regulated by levels and range of shh expression [9], [48], [49]. Due to ccdc80-l1 expression in adaxial cells and muscle pioneers, we decided to investigate the existence of a ccdc80-l1 up-stream regulation Hedgehog-mediated. We modulated shh activity by exposing embryos to cyclopamine, that inhibits the Hedgehog transducer Smoothened (smo) [9], [50]. To avoid the complete loss of the territories in which ccdc80-l1 is expressed, we chose a concentration of cyclopamine (5 µM) by which muscle pioneers and adaxial cells-derived slow fibers are unaffected, as already described [9] and as we demonstrated by the proper expression of their markers engrailed, myod and smyhc1 respectively. (Fig. S5). A striking down-regulation of ccdc80-l1 expression was observed in 72% of the treated embryos in comparison to controls (N = 32) (Fig. 5A, 5B). Interestingly, this down-regulation was detectable only at the level of myoseptum and somites, whereas the cephalic territories in which ccdc80-l1 is expressed were not involved. A similar down-regulation was observed in syu mutants, carriers of a deletion in the gene sonic-you encoding for shh (Fig. 5D–F) [16]. ccdc80-l1 signal in adaxial cells was extremely weak or absent in the 35% of mutants, and slightly down-regulated in the 40% of observed embryos (N = 20). Moreover, the overexpression of shh by means of the injection of the full-length transcript (300 pg/embryo), led to the opposite phenotype with an increasing of ccdc80-l1 expression in the somites of the 71% of the injected embryos (N = 31) (Fig. 5C). On the contrary, ccdc80-l1 loss-of-function did not affect shh expression (Fig. S6). Therefore, these findings suggest that ccdc80-l1 is a down-stream target of the Hedgehog pathway.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biologia, Università degli Studi di Milano, Milan, Italy.

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.

Show MeSH
Related in: MedlinePlus