An insight on the impact of teleost whole genome duplication on the regulation of the molecular networks controlling skeletal muscle growth

Abstract

Fish muscle growth is a complex process regulated by multiple pathways, resulting on the net accumulation of proteins and the activation of myogenic progenitor cells. Around 350-320 million years ago, teleost fish went through a specific whole genome duplication (WGD) that expanded the existent gene repertoire. Duplicated genes can be retained by different molecular mechanisms such as subfunctionalization, neofunctionalization or redundancy, each one with different functional implications. While the great majority of ohnolog genes have been identified in the teleost genomes, the effect of gene duplication in the fish physiology is still not well characterized. In the present study we studied the effect of WGD on the transcription of the duplicated components controlling muscle growth. We compared the expression of lineage-specific ohnologs related to myogenesis and protein balance in the fast-skeletal muscle of pacus (Piaractus mesopotamicus-Ostariophysi) and Nile tilapias (Oreochromis niloticus-Acanthopterygii) fasted for 4 days and refed for 3 days. We studied the expression of 20 ohnologs and found that in the great majority of cases, duplicated genes had similar expression profiles in response to fasting and refeeding, indicating that their functions during growth have been conserved during the period after the WGD. Our results suggest that redundancy might play a more important role in the retention of ohnologs of regulatory pathways than initially thought. Also, comparison to non-duplicated orthologs showed that it might not be uncommon for the duplicated genes to gain or loss new regulatory elements simultaneously. Overall, several of duplicated ohnologs have similar transcription profiles in response to pro-growth signals suggesting that evolution tends to conserve ohnolog regulation during muscle development and that in the majority of ohnologs related to muscle growth their functions might be very similar.

Fig 1. Response to fasting-refeeding protocol in pacus and Nile tilapias.

(A) Evolution of total body weight (g) in pacu (green line) and tilapia (red line) juveniles during 4 days of fasting (-4 to 0 day) and 3 of refeeding (0 to 3 day). Values are shown as Mean±SE (n = 6), different letters indicate significant differences between time points for each species (P<0.05). (B) Relative gene expression of F-box protein 32 (fbxo32) in pacu and tilapia fast skeletal muscle in response to fasting-refeeding expressed in Log10 scale. Values are shown as Mean±SE (n = 6), different letters indicate significant differences between time points for each species (P<0.05).

Fig 2. Lineage-specific ohnologs (LSOs) expression between skeletal muscle of pacus and Nile tilapias.

(A) Principal component analysis (PCA) showing LSOs expression data grouping according to fish species. Principal components were calculated using the SVD with imputation and Unit Variance Scaling method for row scaling. Prediction ellipses have 0.95 of confidence level. (B) Hierarchical clustering and non-hierarchical K-means clustering (K-means = 3) for pacus and tilapias LSOs and singletons transcription in response to fasting-refeeding. Red indicates high and blue indicates low expression values. Heatmap shows the 2^-ΔCt values of LSOs expression and One Minus Pearson Correlation was used as metric for clustering.

Fig 3. Lineage-specific ohnologs (LSOs) retained by redundancy.

Gene duplicates showing signs of redundancy as the main molecular mechanism of retention. LSOs and singletons expression of (A) myogenic differentiation (myod), (B) insulin like growth factor 2 mRNA binding protein 2 (igf2bp2), (C) phosphatidylinositol-4,5-bisphosphate 3 kinase catalytic subunit alpha (pik3ca), (D) phosphatidylinositol-5-phosphate 4 kinase type 2 alpha (pip4k2a), (E) myocyte enhancer factor 2D (mef2d), (F) eukaryotic translation initiation factor 3 subunit j (eif3j), (G) cdc42 binding protein kinase alpha (cdc42bpa) in pacu (green lines) and tilapia (red lines) skeletal muscle are represented as relative values, Mean±SE (n = 6).

Fig 4. Lineage-specific ohnologs (LSOs) retained by subfunctionalization or neofunctionalization.

Gene duplicates showing signs of subfunctionalization and neofunctionalization as the main molecular mechanisms of retention. LSOs and singletons expression of (A) transforming growth factor 3 (tgfb3), (B) insulin like growth factor binding protein 3 (igfbp3), (C) tripartite motif containing 63 (trim63), (D) insulin like growth factor 2 (igf2), (E) rptor independent companion of mtor complex 2 (rictor), (F) follistatin (fst), (G) transforming growth factor beta 1 (tgfb1), (H) growth factor receptor bound protein 2 (grb2), (I) raf-1 proto-oncogene, serine/threonine kinase (raf1), (J) activating transcription factor 4 (atf4), (K) akt serine/threonine kinase 2 (akt2), (L) ras related GTP binding c (rragc), (M) component of inhibitor of nuclear factor kappa B kinase complex (chuk) in pacu (green lines) and tilapia (red lines) skeletal muscle are represented as relative values, Mean±SE (n = 6).