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°C) on the life-time recruitment of fast myotomal muscle fibres in zebrafish Danio rerio L. reared at 26/27°C from hatching. Fast muscle fibres were produced until 25 mm total length (TL) at 22°C ET, 28 mm TL at 26°C ET and 23 mm TL at 31°C ET. The final fibre number (FFN) showed an optimum at 26°C ET (3600) and was 19% and 14% higher than for the 22°C ET (3000) and 31°C ET (3100) treatments, respectively. Further growth to the maximum TL of ∼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°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 β-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.