Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes.

We investigated the effects of embryonic temperature (ET) treatments (22, 26 and 31 degrees C) on the life-time recruitment of fast myotomal muscle fibres in zebrafish Danio rerio L. reared at 26/27 degrees C from hatching. Fast muscle fibres were produced until 25 mm total length (TL) at 22 degrees C ET, 28 mm TL at 26 degrees C ET and 23 mm TL at 31 degrees C ET. The final fibre number (FFN) showed an optimum at 26 degrees C ET (3600) and was 19% and 14% higher than for the 22 degrees C ET (3000) and 31 degrees C ET (3100) treatments, respectively. Further growth to the maximum TL of approximately 48 mm only involved fibre hypertrophy. Microarray experiments were used to determine global changes in microRNA (miRNA) and mRNA expression associated with the transition from the hyperplasic myotube-producing phenotype (M(+), 10-12 mm TL) to the hypertrophic growth phenotype (M(-), 28-31 mm TL) in fish reared at 26-27 degrees C over the whole life-cycle. The expression of miRNAs and mRNAs obtained from microarray experiments was validated by northern blotting and real-time qPCR in independent samples of fish with the M(+) and M(-) phenotype. Fourteen down-regulated and 15 up-regulated miRNAs were identified in the M(-) phenotype together with 34 down-regulated and 30 up-regulated mRNAs (>2-fold; P<0.05). The two most abundant categories of down-regulated genes in the M(-) phenotype encoded contractile proteins (23.5%) and sarcomeric structural/cytoskeletal proteins (14.7%). In contrast, the most highly represented up-regulated transcripts in the M(-) phenotype were energy metabolism (26.7%) and immune-related (20.0%) genes. The latter were mostly involved in cell-cell interactions and cytokine pathways and included beta-2-microglobulin precursor (b2m), an orthologue of complement component 4, invariant chain-like protein 1 (iclp), CD9 antigen-like (cd9l), and tyrosine kinase, non-receptor (tnk2). Five myosin heavy chain genes that were down-regulated in the M(-) phenotype formed part of a tandem repeat on chromosome 5 and were shown by in situ hybridisation to be specifically expressed in nascent myofibres. Seven up-regulated miRNAs in the M(-) phenotype showed reciprocal expression with seven mRNA targets identified in miRBase Targets version 5 (http://microrna.sanger.ac.uk/targets/v5/), including asporin (aspn) which was the target for four miRNAs. Eleven down-regulated miRNAs in the M(-) phenotype had predicted targets for seven up-regulated genes, including dre-miR-181c which had five predicted mRNA targets. These results provide evidence that miRNAs play a role in regulating the transition from the M(+) to the M(-) phenotype and identify some of the genes and regulatory interactions involved.

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