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The Transcriptome of Metamorphosing Flatfish

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The Transcriptome of Metamorphosing Flatfish

Ricardo N Alves et al. BMC Genomics.

Abstract

Background: Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. Metamorphosis in vertebrates is driven by thyroid hormones (THs), but how they orchestrate the cellular, morphological and functional modifications associated with maturation to juvenile/adult states in flatfish is an enigma. Since THs act via thyroid receptors that are ligand activated transcription factors, we hypothesized that the maturation of tissues during metamorphosis should be preceded by significant modifications in the transcriptome. Targeting the unique metamorphosis of flatfish and taking advantage of the large size of Atlantic halibut (Hippoglossus hippoglossus) larvae, we determined the molecular basis of TH action using RNA sequencing.

Results: De novo assembly of sequences for larval head, skin and gastrointestinal tract (GI-tract) yielded 90,676, 65,530 and 38,426 contigs, respectively. More than 57 % of the assembled sequences were successfully annotated using a multi-step Blast approach. A unique set of biological processes and candidate genes were identified specifically associated with changes in morphology and function of the head, skin and GI-tract. Transcriptome dynamics during metamorphosis were mapped with SOLiD sequencing of whole larvae and revealed greater than 8,000 differentially expressed (DE) genes significantly (p < 0.05) up- or down-regulated in comparison with the juvenile stage. Candidate transcripts quantified by SOLiD and qPCR analysis were significantly (r = 0.843; p < 0.05) correlated. The majority (98 %) of DE genes during metamorphosis were not TH-responsive. TH-responsive transcripts clustered into 6 groups based on their expression pattern during metamorphosis and the majority of the 145 DE TH-responsive genes were down-regulated.

Conclusions: A transcriptome resource has been generated for metamorphosing Atlantic halibut and over 8,000 DE transcripts per stage were identified. Unique sets of biological processes and candidate genes were associated with changes in the head, skin and GI-tract during metamorphosis. A small proportion of DE transcripts were TH-responsive, suggesting that they trigger gene networks, signalling cascades and transcription factors, leading to the overt changes in tissue occurring during metamorphosis.

Keywords: Development; Flatfish; RNA sequencing; Thyroid hormone responsive; Tissue-remodelling; Transcriptome.

Figures

Fig. 1
Fig. 1
Atlantic halibut skin, gastrointestinal tract and head transcriptome annotation. a Venn diagram of common and unique tissue transcripts (using the transcript name); b Venn diagram representing the common and specific tissue gene ontology (GO) terms (using the unique GO terms); c Diagram representing the relative abundance of shared and tissue specific enriched GO terms by GO category (using the over/under-represented GO terms from the Fisher’s exact test)
Fig. 2
Fig. 2
Putative thyroid hormone (TH) responsive genes identified in the Atlantic halibut transcriptomes. Heat map of putative thyroid hormone (TH) responsive genes identified in the transcriptomes of skin, GI-tract and head of metamorphosing Atlantic halibut. The acronyms of TH-responsive genes are indicated and full names are given in Additional file 18
Fig. 3
Fig. 3
Differentially expressed transcripts between Atlantic halibut metamorphic stages and juveniles. Graphical representation of the relative number of DE transcripts (up- and down-regulated) identified when pro-metamorphic (stage 7), proclimax-metamorphic (stage 8) and metamorphic climax (9A, 9B and 9C) Atlantic halibut are compared with the juvenile post-metamorphic stage
Fig. 4
Fig. 4
Putative TH-responsive transcripts with differential expression between Atlantic halibut metamorphic stages and juveniles. Clustering of the putative thyroid hormone (TH) responsive transcripts with differential expression between metamorphic stages and juveniles of Atlantic halibut. a Heat map of the DE TH-responsive transcripts clustered by expression pattern. Transcript expression is represented as log2 of fold change for metamorphic stages versus juveniles; b Venn diagram revealing the number of DE TH-responsive transcripts that are shared between stages or that have a stage specific expression
Fig. 5
Fig. 5
Expression pattern of transcripts involved in the TH cascade during halibut metamorphosis. Schematic representation of the relative gene expression by qPCR of a thyroid hormones action (TRαA, TRαB, TRβ) and production (Tg); and b thyroid hormones transport (MCT8, MCT10) and regulation of the cellular availability of THs (DIO1, DIO2, DIO3) during Atlantic halibut metamorphosis. Results are presented as relative gene expression (arbitrary units). For detailed information and significance between stages for each transcript, please see Additional file 14
Fig. 6
Fig. 6
Correlation analysis between SOLiD and qPCR expression analysis. Correlation analyses between SOLiD and qPCR expression of transcripts with a constant expression and transcripts with a modified expression during Atlantic halibut metamorphosis. Comparison of normalized counts (SOLiD data) and relative gene expression profile (qPCR data) of six genes: apolipoprotein A-I (ApoAI), carboxypeptidase A2 (Cpa2), 40S ribosomal protein S30 (FAU), alpha-globin 1 (Gloα1), type I keratin isoform 2 (Krt1i2) and ribosomal protein L7 (RPL7). Different genes are represented by a specific symbol in the graph. Panel a Pearson Product Moment Correlation using metamorphic stages 5 to 9C (r = 0.843; p = 1.07 x 10−7). Panel b A Pearson Product Moment Correlation using metamorphic stages 5 to juvenile (r = 0.576; p = 1.23 x 10−4)

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