Alves et al. BMC Genomics (2016) 17:413 DOI 10.1186/s12864-016-2699-x RESEARCH ARTICLE Open Access The transcriptome of metamorphosing flatfish Ricardo N. Alves1†, Ana S. Gomes2†, Kurt Stueber3, Mbaye Tine3,6, M. A. S. Thorne4, H. Smáradóttir5, Richard Reinhard3, M. S. Clark4, Ivar Rønnestad2 and Deborah M. Power1* 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 Background habitat and feeding mode. The endocrine system and in Metamorphosis describes the “change in form” associated particular the thyroid hormones (THs), thyroxin (T4) and with the transition between life cycle stages in a wide range triiodothyronine (T3), play a central role in vertebrate of animal taxa [1–8]. This transition can be accompanied metamorphosis acting as transcription factors (TFs) when by modifications in morphology, physiology, behavior, they bind to their receptors. In amphibian metamorphosis, it is well established that THs directly or indirectly stimu- * Correspondence: [email protected] late apoptosis and resorption of larval tissue and also pro- †Equal contributors mote growth, differentiation and remodelling of tissues 1Comparative Endocrinology and Integrative Biology Group, Centro de that are crucial for the adult life form [8]. For instance, Ciências do Mar - CCMAR, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal THs are involved in the control of muscle fibre apoptosis Full list of author information is available at the end of the article in the amphibian tail during its regression and also © 2016 Alves et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Alves et al. BMC Genomics (2016) 17:413 Page 2 of 24 promote development of the limbs [9]. In amphibians the thyroid dependent [22, 30–32] and independent processes change in feeding habit from herbivore to carnivore during underpin this event. A complex task now lies ahead in es- the transition from tadpole to frog is associated with TH tablishing which developmental processes during meta- driven remodelling of the intestine that changes from a morphosis are fully TH dependent and which genetic long coiled tube into a complex differentiated organ pathways and endocrine systems cross-talk with THs. Fur- [10–12]. Similarly, THs modulate the change in the am- thermore, it remains to be established how profoundly dif- phibian integument from a simple to a stratified struc- ferent processes such as skin maturation, eye migration ture that is better adapted to terrestrial life [13]. and craniofacial remodelling or gastrointestinal tract (GI- Teleost fish also undergo a TH driven metamorphosis tract) development can be regulated by the same endo- that marks the larval to juvenile transition [14, 15]. crine factors. However, the term is more generally applied to the pro- One of the challenges of studying metamorphosis in found modifications associated with the change from bi- fish larvae is their relatively small dimension, which lateral symmetry to asymmetry during the larval-juvenile means pools of larvae rather than individuals or tissues transition of flatfish (pleuronectiformes) [7, 16–20]. In have generally been used which significantly reduces the flatfish metamorphosis the external morphology is dra- resolution of such studies. The advantage of the biggest matically transformed and they change from symmetric of flatfish, the Atlantic halibut, is the large size of the pelagic larvae to asymmetric benthic juveniles with both larvae and their slow metamorphosis (occurring over eyes on the upper, ocular side of the head (reviewed in approx. 58 days), which means it is possible to analyze Power et al. [7]). The external transformation in flatfish individuals or individual tissues. This is advantageous as morphology is accompanied by a plethora of changes in pools of larvae contain a mixture of tissues and fre- the structure and function of tissues and organs. Chem- quently contain individuals at different developmental ical disruption of the thyroid axis using thiourea or stages making resolution of tissue specific changes in methimazole (MMI) delays or stops stomach develop- transcripts and proteins during metamorphosis difficult ment in the Japanese flounder (Paralichthys olivaceus) or impossible. [21] and otolith mineralization in the Southern flounder The working hypothesis of the present study is that (Paralichthys lethostigma) [22]. The importance of THs since THs exert their action by binding to thyroid recep- during metamorphosis is further emphasized in other tors that are ligand activated TFs the overt change in flatfish species where they have been shown to be im- flatfish during metamorphosis will be preceded by sig- portant for the maturation of the muscle, stomach and nificant modifications in the transcriptome of responsive skin [23–29]. tissues. For this reason, large scale analysis of tissue- Flatfish have a high economic value and include species specific transcriptional changes in responsive tissue such as the Atlantic halibut (Hippoglossus hippoglossus), should provide insight into the underlying molecular common sole (Solea solea), Senegalese sole (S. Senegalesis), changes of tissue specific maturation. A 454 pyrose- turbot (Scophthalmus maximus) and the half-smooth quencing approach was used to survey the tissue specific tongue sole (Cynoglossus semilaevis). Overfishing and high transcriptomes in the skin, GI-tract and head of meta- consumer demand for flatfish has made them an interest- morphosing Atlantic halibut and to also generate a refer- ing target for aquaculture production and a better under- ence transcriptome. SOLiD technology was then used to standing of metamorphosis is of direct relevance for their map the transcriptional changes in individuals (n = 3/ efficient and successful production. Specific problems stage) at different stages of metamorphosis. Differentially linked to failures during metamorphosis include feeding expressed (DE) transcripts during metamorphosis were difficulties, reduced growth rate, arrested metamorphosis, identified by comparing the transcriptome at meta- abnormal pigmentation (albinism, ambicoloration or mo- morphic stages (stage 7, 8 and 9 [33]) with juvenile saicism), failed migration of the eye and skeletal deform- (benthic) stages. Subsequently, genes of the thyroid axis, ities (reviewed in Power et al. [7]). Control of hatchery TH-responsive transcripts and candidate genes that production requires an understanding of fish biology but underpin remodelling and maturation of tissues during also a comprehension of the mediators of metamorphosis, metamorphosis were identified and analyzed by quanti-