The ccdc80 and ccdc80-like1 (ccdc80-l1) genes were isolated in zebrafish in silico, on the basis of their high aminoacidic sequence identity with the human CCDC80 protein (coiled-coil domain containing 80). In human, CCDC80 is involved in several carcinomas, and during recent years it has been proposed as an onco-suppressor gene, increasing the interest in the comprehension of its functions. During my Ph.D., I have been studying the expression patterns and the functions of its zebrafish homologs, in order to gain insights into the processes and the molecular mechanisms in which they are involved. I took advantage of zebrafish as a model system allowing the application of common genetic and biological experimental assays such as PCR, whole mount in situ hybridization and microinjection technique. I investigated ccdc80 and ccdc80-l1 functions during zebrafish embryonic development, finding that they show very different expression patterns and roles. ccdc80 is expressed in the notochord during somitogenesis up to 48 hour post fertilization (hpf). At this stage, it is expressed also in the heart. Instead, at the same developmental stages, ccdc80-l1 is expressed in cranial ganglia, adaxial cells, muscle pioneers and dorsal dermis. These patterns are suggestive of different roles, in fact, the genes are involved in distinct developmental processes, such as somitogenesis and axonal pathfinding, respectively. Functional analysis were obtained performing loss- and gain-of-function experiments. The results clearly demonstrated that manipulation of Ccdc80 protein levels during embryonic development, both increasing and decreasing them, leads to a severe impairment of somites, metameric structures from which several tissues derive, such as muscle. Nevertheless, this phenotype seemed to be recovered at 24 hpf, since at this stage somites no longer showed the same morphological alterations observed previously. By converse, notwithstanding its expression in muscular tissues, loss-of-ccdc80-l1 does not impair somites formation, leading instead to motoneurons axonal migrations defects, capable to affect embryonic motility. Given the external development of the zebrafish embryo, motility is required very soon for prey and escaping predators, and muscle and motoneurons are equally required for a proper motor behavior. Communication and interactions between these two tissues are fundamental for proper muscles innervation and transmission of nervous inputs to be translated in muscular contractions. ccdc80 and ccdc80-l1 may derive from an ancestor gene involved in these processes and, during evolution, may have developed different functions, but still bound to a key process for embryonic viability in the external environment.

CCDC80 AND CCDC80-L1: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF TWO NOVEL GENES INVOLVED IN ZEBRAFISH (DANIO RERIO) DEVELOPMENT / C. Brusegan ; tutor: F. Cotelli ; coordinatore: M. Bolognesi. Universita' degli Studi di Milano, 2012 Jan 23. 24. ciclo, Anno Accademico 2011. [10.13130/brusegan-chiara_phd2012-01-23].

CCDC80 AND CCDC80-L1: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF TWO NOVEL GENES INVOLVED IN ZEBRAFISH (DANIO RERIO) DEVELOPMENT

C. Brusegan
2012

Abstract

The ccdc80 and ccdc80-like1 (ccdc80-l1) genes were isolated in zebrafish in silico, on the basis of their high aminoacidic sequence identity with the human CCDC80 protein (coiled-coil domain containing 80). In human, CCDC80 is involved in several carcinomas, and during recent years it has been proposed as an onco-suppressor gene, increasing the interest in the comprehension of its functions. During my Ph.D., I have been studying the expression patterns and the functions of its zebrafish homologs, in order to gain insights into the processes and the molecular mechanisms in which they are involved. I took advantage of zebrafish as a model system allowing the application of common genetic and biological experimental assays such as PCR, whole mount in situ hybridization and microinjection technique. I investigated ccdc80 and ccdc80-l1 functions during zebrafish embryonic development, finding that they show very different expression patterns and roles. ccdc80 is expressed in the notochord during somitogenesis up to 48 hour post fertilization (hpf). At this stage, it is expressed also in the heart. Instead, at the same developmental stages, ccdc80-l1 is expressed in cranial ganglia, adaxial cells, muscle pioneers and dorsal dermis. These patterns are suggestive of different roles, in fact, the genes are involved in distinct developmental processes, such as somitogenesis and axonal pathfinding, respectively. Functional analysis were obtained performing loss- and gain-of-function experiments. The results clearly demonstrated that manipulation of Ccdc80 protein levels during embryonic development, both increasing and decreasing them, leads to a severe impairment of somites, metameric structures from which several tissues derive, such as muscle. Nevertheless, this phenotype seemed to be recovered at 24 hpf, since at this stage somites no longer showed the same morphological alterations observed previously. By converse, notwithstanding its expression in muscular tissues, loss-of-ccdc80-l1 does not impair somites formation, leading instead to motoneurons axonal migrations defects, capable to affect embryonic motility. Given the external development of the zebrafish embryo, motility is required very soon for prey and escaping predators, and muscle and motoneurons are equally required for a proper motor behavior. Communication and interactions between these two tissues are fundamental for proper muscles innervation and transmission of nervous inputs to be translated in muscular contractions. ccdc80 and ccdc80-l1 may derive from an ancestor gene involved in these processes and, during evolution, may have developed different functions, but still bound to a key process for embryonic viability in the external environment.
23-gen-2012
Settore BIO/06 - Anatomia Comparata e Citologia
Settore BIO/11 - Biologia Molecolare
COTELLI, FRANCO
BOLOGNESI, MARTINO
Doctoral Thesis
CCDC80 AND CCDC80-L1: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF TWO NOVEL GENES INVOLVED IN ZEBRAFISH (DANIO RERIO) DEVELOPMENT / C. Brusegan ; tutor: F. Cotelli ; coordinatore: M. Bolognesi. Universita' degli Studi di Milano, 2012 Jan 23. 24. ciclo, Anno Accademico 2011. [10.13130/brusegan-chiara_phd2012-01-23].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/168377
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