Investigação de mecanismos genéticos associados às vias de formação de ossos intermusculares no Tambaqui (Colossoma macropomum)
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2020-11-27
Autores
Bronze, Emily
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Universidade Estadual Paulista (Unesp)
Resumo
Diversas espécies de peixes de importância na aquicultura mundial possuem ossos intermusculares (IBs) ou espinhas, como são popularmente conhecidos. Esses IBs são uma característica comercialmente indesejável por reduzir a palatabilidade dos peixes, sendo necessária sua remoção para a produção do filé, o que resulta em impactos econômicos e de frescor do produto. O tambaqui, Colossoma macropomum, é uma espécie dulcícola neotropical da ordem Characiformes que apresenta grande potencial para a aquicultura, mas que, no entanto, possui a desvantagem de gerar os IBs. Recentemente, numa população cultivada de tambaqui, foram reportados indivíduos com ausência de IBs, sugerindo que variantes genéticas e/ou epigenéticas estejam associadas à geração do fenótipo mutante. O objetivo deste trabalho foi investigar os mecanismos moleculares associados à formação dos ossos intermusculares, de modo a contribuir para a geração de animais com maior valor econômico agregado. Para isso foi analisada a expressão de genes codificadores e genes não codificadores associados às vias de formação dos IBs, além da caracterização temporal de formação dos IBs durante o desenvolvimento. Com base na literatura, foram inicialmente selecionados para estudo três genes candidatos à atuação nas vias de ossificação intramembranosa: bmp4, runx2 e mstn. Estes foram avaliados na F1 quanto à expressão no intervalo de crescimento que compreende desde o início até a completa formação dos IBs, nos comprimentos 18, 20, 23 e 26mm. Inicialmente foi realizada a desova com as matrizes que apresentaram o fenótipo mutante desejado (sem IBs) e um grupo controle (com IBs), seguido do cultivo dos alevinos e monitoramento dos mesmos para determinação do momento de início da ossificação dos IBs ou verificação de ausência destes. Posteriormente foi realizada a coleta de tecidos da região próxima ao pedúnculo caudal seguida da extração de RNA e síntese de cDNA desses indivíduos para subsequente análise de perfis de expressão gênica por qPCR. Concomitantemente, foi realizada a fixação e diafanização de alevinos em diferentes fases do desenvolvimento para a caracterização morfológica dos IBs. O RNA total foi isolado das matrizes mutante e controle para sequenciamento em larga escala de miRNAs (miRNA-Seq). Como resultados verificou-se que os IBs no tambaqui são formados a partir de 15 mm de comprimento total, anterior ao previamente reportado para outros teleósteos. A análise comparativa de expressão do bmp4 revelou diferença significativa entre mutantes e controle, havendo uma diminuição na expressão na F1 dos grupos mutantes, principalmente nos comprimentos considerados de ativa mineralização óssea (i.e., 20 e 23mm). O runx2b também apresentou diferença significativa entre mutantes e controle, porém o pico de diminuição de expressão ocorreu nos animais com 20mm de comprimento. Por outro lado, a mstn apresentou, um aumento significativo de expressão nos mutantes sem IBs na comparação aos animais controle, sugerindo possível envolvimento desses genes nas vias associadas a formação dos IBs. O miRNA-seq revelou a expressão de miRNAs importantes para a regulação de vias de formação óssea, incluindo os atuantes na regulação dos três genes codificantes alvo analisados, além da detecção de novos miRNAs exclusivamente expressos apenas nos tambaquis mutantes sem IBs. Este é um dos primeiros estudos onde avaliou-se a expressão de genes envolvidos na formação óssea em espécie de peixe neotropical, cujos espécimes foram obtidos pelo acasalamento de matrizes mutantes sem IBs. Adicionalmente trata-se da primeira análise do microRNAoma completo para investigação do fenótipo mutante sem ossos intermusculares. Esses dados inéditos gerados fornecem subsídios para trabalhos futuros visando a produção de linhagens de tambaqui sem IBs, com grande potencial para melhoria de produtividade na aquicultura.
Several species of fish with importance in aquaculture worldwide have intermuscular bones (IBs) or spines, as they are popularly known. These IBs are a commercially undesirable characteristic because they reduce the palatability of the fish, requiring their removal for production of the fillet, which results in economic and freshness impacts of the product. Tambaqui, Colossoma macropomum, is a neotropical sweet species of the order Characiformes that has great potential for aquaculture, but which has the disadvantage of generating IBs. Recently, in a cultivated population of tambaqui, individuals with absence of IBs have been reported, suggesting that genetic and/or epigenetic variants are associated with the generation of the mutant phenotype. The objective of this work was to investigate the molecular mechanisms associated with the formation of intermuscular bones, in order to contribute to the generation of animals with greater added economic value. For this purpose, the expression of coding genes and non-coding genes associated with the pathways of IBs formation was analyzed, in addition to the temporal characterization of the formation of IBs during development. Based on the literature, three candidate genes were initially selected for study in the intramembranous ossification pathways: bmp4, runx2 and mstn. These were evaluated for expression in the growth interval that comprises from the beginning until the complete formation of the IBs, in lengths 18,20,23 and 26mm. Initially, the spawning was carried out with the matrices that present the desired mutant phenotype (without IBs) and a control group (with IBs), followed by the cultivation of fingerlings and their monitoring to determine the time of ossification beginning of the IBs or absence of these. Subsequently, tissue was collected from the region close to the caudal peduncle followed by RNA extraction and cDNA synthesis of these individuals for subsequent analysis of gene expression profiles by qPCR. Concomitantly, the fixation and diaphanization of fingerlings at different stages of development was carried out for the morphological characterization of IBs. The total RNA was isolated from the mutant and control matrices for large-scale sequencing of miRNAs (miRNA-Seq). As a result, it was found that the IBs in tambaqui are formed from a total length of 15 mm, prior to previously reported. The comparative analysis of bmp4 expression revealed a significant difference between mutants and control, with a decrease in the expression of the mutante groups, mainly in the lengths considered to be active bone mineralization (i.e., 20 and 23mm). Runx2b also showed a significant difference between mutants and control, but the peak of decreased expression occurred in animals with 20mm in length. On the other hand, mstn showed a significant increase in expression in mutants without IBs compared to control animals, suggesting possible involvement of these genes in the pathways associated with the formation of IBS. MiRNA-seq revealed the expression of miRNAs important for the regulation of bone formation pathways, and others potentially active in the regulation of the three coding genes analyzed in RT-qPCR, in addition to the detection of new miRNAs exclusively expressed only in mutant tambaquis without IBs. This is the first study to evaluate the expression of genes involved in bone formation in neotropical species, whose pecimens originated from the mating of mutant matrices without IBs. Additionally, it is the first analysis of the complete microRNAoma to investigate the mutant phenotype without intermuscular bones. These unprecedented data provide subsidies for future work to produce tambaqui strains without IBs, with great potential for improving productivity in aquaculture.
Several species of fish with importance in aquaculture worldwide have intermuscular bones (IBs) or spines, as they are popularly known. These IBs are a commercially undesirable characteristic because they reduce the palatability of the fish, requiring their removal for production of the fillet, which results in economic and freshness impacts of the product. Tambaqui, Colossoma macropomum, is a neotropical sweet species of the order Characiformes that has great potential for aquaculture, but which has the disadvantage of generating IBs. Recently, in a cultivated population of tambaqui, individuals with absence of IBs have been reported, suggesting that genetic and/or epigenetic variants are associated with the generation of the mutant phenotype. The objective of this work was to investigate the molecular mechanisms associated with the formation of intermuscular bones, in order to contribute to the generation of animals with greater added economic value. For this purpose, the expression of coding genes and non-coding genes associated with the pathways of IBs formation was analyzed, in addition to the temporal characterization of the formation of IBs during development. Based on the literature, three candidate genes were initially selected for study in the intramembranous ossification pathways: bmp4, runx2 and mstn. These were evaluated for expression in the growth interval that comprises from the beginning until the complete formation of the IBs, in lengths 18,20,23 and 26mm. Initially, the spawning was carried out with the matrices that present the desired mutant phenotype (without IBs) and a control group (with IBs), followed by the cultivation of fingerlings and their monitoring to determine the time of ossification beginning of the IBs or absence of these. Subsequently, tissue was collected from the region close to the caudal peduncle followed by RNA extraction and cDNA synthesis of these individuals for subsequent analysis of gene expression profiles by qPCR. Concomitantly, the fixation and diaphanization of fingerlings at different stages of development was carried out for the morphological characterization of IBs. The total RNA was isolated from the mutant and control matrices for large-scale sequencing of miRNAs (miRNA-Seq). As a result, it was found that the IBs in tambaqui are formed from a total length of 15 mm, prior to previously reported. The comparative analysis of bmp4 expression revealed a significant difference between mutants and control, with a decrease in the expression of the mutante groups, mainly in the lengths considered to be active bone mineralization (i.e., 20 and 23mm). Runx2b also showed a significant difference between mutants and control, but the peak of decreased expression occurred in animals with 20mm in length. On the other hand, mstn showed a significant increase in expression in mutants without IBs compared to control animals, suggesting possible involvement of these genes in the pathways associated with the formation of IBS. MiRNA-seq revealed the expression of miRNAs important for the regulation of bone formation pathways, and others potentially active in the regulation of the three coding genes analyzed in RT-qPCR, in addition to the detection of new miRNAs exclusively expressed only in mutant tambaquis without IBs. This is the first study to evaluate the expression of genes involved in bone formation in neotropical species, whose pecimens originated from the mating of mutant matrices without IBs. Additionally, it is the first analysis of the complete microRNAoma to investigate the mutant phenotype without intermuscular bones. These unprecedented data provide subsidies for future work to produce tambaqui strains without IBs, with great potential for improving productivity in aquaculture.
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Palavras-chave
RT-qPCR, miRNA, Aquicultura