CaracterizaciĆ³n del gen pelado durante el desarrollo embrionario de pez cebra
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Fecha
2017
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es
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Universidad AndrƩs Bello
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Licencia CC
Licencia CC
Resumen
El gen pelado se ha identificado en los organismos invertebrados
Drosophila melanogaster y Caenorhabditis elegans;Ķ¾ se encuentra conservado
hasta mamĆferos superiores como humanos. Estudios funcionales realizados en
ambos invertebrados mediante el knockout del gen, han reportado que su funciĆ³n
estarĆa relacionada al proceso de neurogĆ©nesis En C.elegans, se ha observado
que su falta de funciĆ³n afecta el correcto proceso de neurogĆ©nesis, lo cual tambiĆ©n
se ha observado en D. melanogaster. Con el objetivo de hacer extensiva la
caracterizaciĆ³n de este gen a organismos vertebrados, un trabajo previo a esta
tesis, evaluĆ³ la posible funciĆ³n de pelado en el proceso de neurogĆ©nesis del pez
cebra. Para ello, utilizĆ³ como modelo la lĆnea lateral y se determinĆ³ que la falta de
funciĆ³n de pelado no afecta el proceso de neurogĆ©nesis en la lĆnea lateral, sin
embargo, provoca defectos en su formaciĆ³n.
Esta tesis propuso dilucidar la funciĆ³n del gen pelado sobre el primordio
migratorio del pez cebra, estructura encargada de dar origen a la lĆnea lateral
posterior. Para caracterizar la funciĆ³n de este gen, utilizamos morfolinos
antisentidos en embriones silvestres y en lĆneas transgĆ©nicas que presentan
marcado con fluorescencia las diferentes estructuras de la lĆnea lateral. Nuestros
resultados muestran que pelado se expresa desde 0hpf hasta estadios larvarios,
en particular en el cerebro, tubo neural ventral y notocorda. El knockdown de
pelado, genera una disminuciĆ³n del tamaƱo del primordio migratorio, dado por una
disminuciĆ³n de la proliferaciĆ³n celular y un aumento de la muerte celular en la
misma estructura. Sin embargo, el fenotipo del knockdown de pelado es reversible
al bloquear la actividad de p53. De esta forma, el fenotipo de la falta de funciĆ³n de
pelado mediante la microinyecciĆ³n de morfolinos, puede estar modulando la
muerte celular a travĆ©s de p53 o bien, ser inespecĆfico. En el Ćŗltimo caso, Ć©sta
tesis serĆa el primer trabajo que demostrarĆa que el uso de morfolinos podrĆa
inducir la activaciĆ³n de p53 de forma gradual, haciĆ©ndolo un excelente modelo de
evaluaciĆ³n pre-Āeliminar de morfolinos que no alcanzan el umbral suficiente de
activaciĆ³n de p53 que genere defectos crĆ”neo-Āfaciales, pero que si activarĆan de
forma inespecĆfica la muerte celular en menor medida.
The pelado gene has been identified in both, Drosophila melanogaster and Caenorhabditis elegans;Ķ¾ it is conserved until human. Functional studies performed on both invertebrates through gene knockout have reported that their function would be related to neurogenesis. In D. melanogaster, the knockout result present structural defects in the photoreceptors that form the eye, and in C. elegans, problems in the growth and migration of the axons of their motor neurons are observed. Aiming to extend the characterization of this gene to vertebrate organisms, a work prior to this thesis, evaluated the possible pelado function on the neurogenesis of Danio rerio (zebrafish). For this, the lateral line (mechanosensory organ) was used as model and it was determined that the knockdown of pelado does not affect the neurogenesis process in the lateral line, however, causes defects on itās formation. This thesis proposed to elucidate the function of pelado gene over the migratory primordium in zebrafish, structure responsible of the formation of the posterior lateral line. To characterize the function of this gene, we used antisense morpholinos in wild embryos and in transgenic lines that fluorescently labeled the different structures of the lateral line (primordium and neuromasts). Our results show that pelado is expressed from 0hpf to larval stages, particularly in the brain, ventral neural tube and notochord. The knockdown of pelado, generates a smaller migratory primordium, given by a diminution of the cellular proliferation and an increase of the cellular death in the same structure. However, the knockdow phenotype of pelado is reversible by blocking p53 activity. Thus, the phenotype of the knockdown of pelado through the microinjection of morpholino, may be an artifact associated with the technique, or pelado indeed modulates cell death through p53. In the latter case, this thesis would be the first work that would demonstrate that the use of morpholinos could induce the activation of p53 gradually, making it an excellent model of pre-Āelimination evaluation of morpholinos that do not reach the sufficient threshold of activation of p53 that produces craniofacial defects, but could activate nonspecific cell death to a lesser extent.
The pelado gene has been identified in both, Drosophila melanogaster and Caenorhabditis elegans;Ķ¾ it is conserved until human. Functional studies performed on both invertebrates through gene knockout have reported that their function would be related to neurogenesis. In D. melanogaster, the knockout result present structural defects in the photoreceptors that form the eye, and in C. elegans, problems in the growth and migration of the axons of their motor neurons are observed. Aiming to extend the characterization of this gene to vertebrate organisms, a work prior to this thesis, evaluated the possible pelado function on the neurogenesis of Danio rerio (zebrafish). For this, the lateral line (mechanosensory organ) was used as model and it was determined that the knockdown of pelado does not affect the neurogenesis process in the lateral line, however, causes defects on itās formation. This thesis proposed to elucidate the function of pelado gene over the migratory primordium in zebrafish, structure responsible of the formation of the posterior lateral line. To characterize the function of this gene, we used antisense morpholinos in wild embryos and in transgenic lines that fluorescently labeled the different structures of the lateral line (primordium and neuromasts). Our results show that pelado is expressed from 0hpf to larval stages, particularly in the brain, ventral neural tube and notochord. The knockdown of pelado, generates a smaller migratory primordium, given by a diminution of the cellular proliferation and an increase of the cellular death in the same structure. However, the knockdow phenotype of pelado is reversible by blocking p53 activity. Thus, the phenotype of the knockdown of pelado through the microinjection of morpholino, may be an artifact associated with the technique, or pelado indeed modulates cell death through p53. In the latter case, this thesis would be the first work that would demonstrate that the use of morpholinos could induce the activation of p53 gradually, making it an excellent model of pre-Āelimination evaluation of morpholinos that do not reach the sufficient threshold of activation of p53 that produces craniofacial defects, but could activate nonspecific cell death to a lesser extent.
Notas
Tesis (MagĆster en BiotecnologĆa)
Palabras clave
Pez Cebra, EmbriologĆa