Role of Thyroid Hormone in Dynamic Variation of gdf6a Gene during Metamorphosis of Paralichthys olivaceus

The Japanese flounder (Paralichthys olivaceus) is a marine fish that undergoes a dramatic postembryonic metamorphosis, with the right eye shifting to the left and its lifestyle transitioning from planktonic to benthic. As the light environment of the habitat changes from bright to dim, its photoreceptor system also undergoes adaptive change. Growth differentiation factor 6a (Gdf6a) is a member of the BMP family, which plays a key role in regulating the dorsal–ventral pattern of the retina and photoreceptor fate, and the differentiation of different photoreceptors is also modulated by a thyroid hormone (TH) binding its receptor (TR). However, the relationship between gdf6a and TH and its role in the regulation of photoreceptors during flounder metamorphosis is still poorly understood. In this study, bioinformatics analysis showed that Gdf6a had a conserved TGFB structural domain and clusters with fishes. The expression analysis showed that the expression of gdf6a was highest in the eye tissue of adult flounder and tended to increase and then decrease during metamorphosis, reaching its highest levels at the peak of metamorphosis. Moreover, the expression of gdf6a increased in the early stages of metamorphosis after exogenous TH treatment, while it was inhibited after exogenous thiourea (a TH inhibitor, TU) treatment. To further investigate the targeting role of TH and gdf6a in the metamorphosis of flounder, the results of the Dual-Luciferase revealed that triiodothyronine (T3) may regulate the expression of gdf6a through TRβ. In conclusion, we speculate that TH influences the development of cone photoreceptors during the metamorphosis of the flounder by regulating the expression of gdf6a.


Introduction
Japanese flounder (Paralichthys olivaceus) is an economic marine fish, mainly distributed along the coasts of China, the Korean Peninsula, and Japan, which undergoes a drastic metamorphosis during postembryonic development.After metamorphosis, the right eye moves to the left side of the body, and the lifestyle changes from planktonic to benthic, so the light environment changes from bright to dim [1].With the change in the light environment, the visual system also undergoes adaptive changes.A previous study showed that opsin genes (Rh1, M/Lws, Rh2, Sws1, Sws2) of the flounder varied with the metamorphosis process, and rh1, sws2aβ, and lws were positively regulated by exogenous thyroid hormone (TH) binding to thyroid hormone receptors (TRs) [2].Opsin is mainly synthesized by photoreceptor cells, which include the cone cells and rod cells.The rod cells perceive weak light and are responsible for acquiring light information in dim light conditions, while the cone cells are responsible for distinguishing between structural and color information in images [3,4].It has been shown that both rod and cone cells are differentiated from retinal photoreceptor precursor cells (PPC) and regulated by multiple transcription factors during differentiation in higher animals [5].However, the molecular mechanisms underlying visual adaptation to the altered light environment during flounder metamorphosis are so far poorly understood.
Growth differentiation factor 6a (Gdf6a) is a member of the BMP family and belongs to the transforming growth factor beta (TGF-β) super-family [6].It is a key factor in regulating dorsal-ventral retinal patterns and photoreceptor fate [7,8].Abnormal photoreceptor morphology occurs in zebrafish gdf6a mutants, and their functional defects lead to photoreceptor degeneration [9].Gdf6a plays different roles in different subtypes of cone photoreceptors.For instance, the number of UV cones in gdf6a −/− zebrafish was normal, while the number of blue cones was reduced [10].T-box transcription factor 2b (Tbx2b) is a regulator of photoreceptor fate, specifically a key factor specifying the fate of UV cone photoreceptors [11].The deletion of gdf6a in tbx2b heterozygous mutant zebrafish resulted in a further reduction in the number of UV cones [12,13].
TH is present in almost all tissues and plays a key regulatory role in growth, metabolism, and cell differentiation [14].TH has two forms, triiodothyronine (T3) and thyroxine (T4), in which T3 plays a major role and produces the thyroid hormone's effects through the thyroid hormone receptor (TR) [15].TR is a member of the nuclear receptor superfamily, which binds to the thyroid hormone response element (TRE) in the promoter region of target genes, thereby regulating gene transcription [16].TH is associated with photoreceptor development, directing the expression of the correct genes in differentiated photoreceptors.Under the influence of TH, PPC produces cone cells at the expense of reducing another cell type.Thus, PPC may use thyroid hormones to direct the fate of photoreceptor cells [17].Related experiments have shown that TH promotes the differentiation of PPC to cone photoreceptors in rats, chickens, and humans [18,19].A study found that TR may affect the number of blue cones in zebrafish by influencing the expression of the gdf6a gene [12,20].
In this study, we constructed the spatiotemporal expression patterns of the gdf6a gene in flounder and analyzed the effect of exogenous TH on the expression level of gdf6a during metamorphosis to elucidate the important role of TH in regulating photoreceptor differentiation.By verifying the regulatory relationship between TH, TRs, and the gdf6a gene, the molecular mechanism of thyroid hormone regulation of retinal photoreceptor differentiation was initially revealed.

Bioinformatics Analysis of gdf6a Gene
The CDS region of the Paralichthys olivaceus gdf6a gene is 1353 bp, with ATG as the start codon and TAG as the stop codon, encoding 450 amino acids.As shown in Figure 1A, the amino acid sequence analysis showed that the N-terminal positions 1-28 of the flounder Gdf6 protein contained a signal peptide structure, positions 28-29 were cleavage sites, positions 7-29 had a transmembrane region, and positions 349-450 contained a TGFB structural domain.In Figure 1B, a comparison of the homology of the flounder Gdf6 amino acid sequence with other species shows that Gdf6 has a conserved TGFB structural domain and the highest homology between the flounder Gdf6 and Hippoglossus hippoglossus (97.56%), and the lowest homology with Xenopus laevis (53.88%).The phylogenetic tree of different species of Gdf6 was constructed using MEGA 5.0 software, and the results showed that flounder and Hippoglossus hippoglossus were most closely related and clustered together in the Osteichthyes, which follows the results of the amino acid homology analysis and is in line with the species evolutionary pattern (Figure 1C).

Expression of gdf6a in Adult Fish Tissues and Larval Eyes during Flounder Metamorphosis
The results of fluorescence quantitative PCR showed that the gdf6a gene was expressed in all tissues of the adult flounder, and the expression of gdf6a was highest in the eyes, followed by the brain, using the intestine as a reference (Figure 2A).As shown in Figure 2B, the expression of gdf6a in the eyes of flounder during metamorphosis was referenced to 40 dph and gradually increased throughout the metamorphosis process, peaking at 28 dph and then gradually decreasing.

Expression of gdf6a in Adult Fish Tissues and Larval Eyes during Flounder Metamorph
The results of fluorescence quantitative PCR showed that the gdf6a gene wa pressed in all tissues of the adult flounder, and the expression of gdf6a was highest i eyes, followed by the brain, using the intestine as a reference (Figure 2A).As show Figure 2B, the expression of gdf6a in the eyes of flounder during metamorphosis wa erenced to 40 dph and gradually increased throughout the metamorphosis process, p ing at 28 dph and then gradually decreasing.

Effects of Exogenous TH and TU on gdf6a Expression in the Eyes during Flounder Metamorphosis
As shown in Figure 3A, the NC group data at 40 dph were used as a reference.exogenous TH treatment, the expression levels of gdf6a in the eyes were significant creased at 20 dph, 24 dph, and 40 dph (p < 0.05), while the expression levels of gdf6a significantly decreased (p < 0.05) from 20 dph to 36 dph after exogenous TU treatme addition, in the rescue group experiment, the TU-treated flounders at 36 dph were t ferred to normal seawater (TU + NC) and TH-added seawater (TU + TH) to continue ing until 40 dph.As shown in Figure 3B, the expression levels of gdf6a in the eyes significantly higher in the rescue group (p < 0.05), using the 36 dph-TU group data reference.The above results indicate that TH affects the expression level of gdf6a i eyes of flounder during metamorphosis.

Effects of Exogenous TH and TU on gdf6a Expression in the Eyes during Flounder Metamorphosis
As shown in Figure 3A, the NC group data at 40 dph were used as a reference.After exogenous TH treatment, the expression levels of gdf6a in the eyes were significantly increased at 20 dph, 24 dph, and 40 dph (p < 0.05), while the expression levels of gdf6a were significantly decreased (p < 0.05) from 20 dph to 36 dph after exogenous TU treatment.In addition, in the rescue group experiment, the TU-treated flounders at 36 dph were transferred to normal seawater (TU + NC) and TH-added seawater (TU + TH) to continue rearing until 40 dph.As shown in Figure 3B, the expression levels of gdf6a in the eyes were significantly higher in the rescue group (p < 0.05), using the 36 dph-TU group data as a reference.The above results indicate that TH affects the expression level of gdf6a in the eyes of flounder during metamorphosis.(B) Expression levels of gdf6a in flounder eyes of the rescue group.NC group: cultured in normal seawater; TH group: cultured seawater supplemented with 0.1 mg/L of exogenous thyroid hormone (T3); TU group: cultured seawater supplemented with 30 mg/L of exogenous thiourea (TU).TUtreated flounders at 36 dph were transferred to normal seawater (TU + NC) and TH-added seawater (TU + TH) to continue rearing until 40 dph.All data are expressed using the mean ± standard deviation, where an asterisk indicates a significant difference from the contemporaneous NC group (A) or 36 dph-TU group (B) (p < 0.05).

Subcellular Localization
Predicted flounder gdf6a results using the subcellular localization online prediction site showed that 69.6% of the gdf6a was expressed in the nucleus, 13.0% in the cytoplasm, 13.0% in the mitochondria, and 4.3% in the plasma membrane.At the same time, 293T cells were transfected with pEGFP-N1 empty plasmid and pEGFP-gdf6a recombinant plasmid, respectively.As shown in Figure 4, it is verified that gdf6a is expressed in both the nucleus and cytoplasm of 293T cells.

Subcellular Localization
Predicted flounder gdf6a results using the subcellular localization online pred site showed that 69.6% of the gdf6a was expressed in the nucleus, 13.0% in the cytop 13.0% in the mitochondria, and 4.3% in the plasma membrane.At the same time, 293 were transfected with pEGFP-N1 empty plasmid and pEGFP-gdf6a recombinant pla respectively.As shown in Figure 4, it is verified that gdf6a is expressed in both the nu and cytoplasm of 293T cells.

T3 Mediates Targeting Regulation of the gdf6a Gene in Flounder through TRs
The TRE sites in the gdf6a promoter region were analyzed using the Transcr Factor Online Prediction website and according to the principle of action of TH.As s

T3 Mediates Targeting Regulation of the gdf6a Gene in Flounder through TRs
The TRE sites in the gdf6a promoter region were analyzed using the Transcription Factor Online Prediction website and according to the principle of action of TH.As shown in Figure 5A, the gdf6a promoter region has one T3R transcription factor binding site, three T3R-alpha transcription factor binding sites, one T3R-beta1 transcription factor binding site, and two potential TRE binding sites in the promoter region of gdf6a.The above results indicate that gdf6a has a very close relationship with TH.
As shown in Figure 5B, 293T cells were co-transfected with gdf6a pro (recombinant plasmid of gdf6a promoter region) and p3×Flag-TRs over-expressed plasmids, and exogenous T3 was added simultaneously.The Dual-Luciferase reporter gene validation showed that taking promoter activity of gdf6a as a reference, the luciferase activities were significantly elevated (p < 0.05) in both gdf6a pro + TRαA/TRαB/TRβ + T3 groups (Figure 5C-E).The luciferase activities were also significantly elevated in the gdf6a pro + TRαA/TRαB group (p < 0.05), while there was no significant difference in the gdf6a pro + TRβ group.The above results suggest that T3 may regulate the expression of gdf6a through TRβ.In addition, we constructed the promoter region plasmid of tbx2b and co-transfected it with the pEGFP-Gdf6a recombinant plasmid.As shown in Figure 5F, the Dual-Luciferase results indicated that, using the promoter activity of tbx2b as a reference, Gdf6a could significantly upregulate the transcription initiation activity of tbx2b (p < 0.05).
in Figure 5A, the gdf6a promoter region has one T3R transcription factor binding site, three T3R-alpha transcription factor binding sites, one T3R-beta1 transcription factor binding site, and two potential TRE binding sites in the promoter region of gdf6a.The above results indicate that gdf6a has a very close relationship with TH.As shown in Figure 5B, 293T cells were co-transfected with gdf6a pro (recombinant plasmid of gdf6a promoter region) and p3×Flag-TRs over-expressed plasmids, and exogenous T3 was added simultaneously.The Dual-Luciferase reporter gene validation showed that taking promoter activity of gdf6a as a reference, the luciferase activities were significantly elevated (p < 0.05) in both gdf6a pro + TRαA/TRαB/TRβ + T3 groups (Figure 5C-E).The luciferase activities were also significantly elevated in the gdf6a pro + TRαA/TRαB group (p < 0.05), while there was no significant difference in the gdf6a pro + TRβ group.The above results suggest that T3 may regulate the expression of gdf6a through TRβ.In addition, we constructed the promoter region plasmid of tbx2b and co-transfected it with the pEGFP-Gdf6a recombinant plasmid.As shown in Figure 5F, the Dual-Luciferase results indicated that, using the promoter activity of tbx2b as a reference, Gdf6a could significantly upregulate the transcription initiation activity of tbx2b (p < 0.05).

Discussion
The retinal photoreceptor system of dental flounder may also undergo adaptive changes as the light environment changes markedly during metamorphosis.gdf6a is thought to be required to initiate dorsal-ventral retinal patterning and lens development [7,8].In this paper, we analyzed the regulation of gdf6a gene expression by exogenous TH.The amino acid sequence analysis revealed that flounder Gdf6a has a conserved TGFB structural domain.The amino acid sequence of Gdf6 was compared with nine other species for homology and phylogenetic tree analysis, and it was found to have the highest

Discussion
The retinal photoreceptor system of dental flounder may also undergo adaptive changes as the light environment changes markedly during metamorphosis.gdf6a is thought to be required to initiate dorsal-ventral retinal patterning and lens development [7,8].In this paper, we analyzed the regulation of gdf6a gene expression by exogenous TH.The amino acid sequence analysis revealed that flounder Gdf6a has a conserved TGFB structural domain.The amino acid sequence of Gdf6 was compared with nine other species for homology and phylogenetic tree analysis, and it was found to have the highest homology with Hippoglossus hippoglossus and to be clustered with Osteichthyes, which follows the genetic law.Paralichthys olivaceus and Hippoglossus hippoglossus have similar appearance and habits, and both belong to the Pleuronectiformes, Osteichthyes.Similar studies have found that the amino acid sequence of GDF6 is highly conserved in vertebrates [21].The subcellular localization results showed that Gdf6a of the flounder was expressed in both the nucleus and cytoplasm of 293T cells, which was the same as the online prediction.And we found that the dental flounder gdf6a gene was expressed in the eye, brain, liver, intestine, stomach, kidney, and gonad tissues, but the highest expression was in the eye, which is consistent with its key role in eye development [22,23].Moreover, the expression level of gdf6a in the eye also changed during the metamorphosis of flounder, peaking at the peak of metamorphosis and then decreasing.Therefore, we believe that the differential expression of gdf6a in the eye is related to the metamorphosis process of flounder.
Previous studies found that TH was closely related to the metamorphosis process in flounder, with exogenous TH promoting metamorphosis and exogenous TU inhibiting the metamorphosis process [24].In this study, exogenous TH promoted the expression of gdf6a in the eyes of flounder at the early stage of metamorphosis, whereas exogenous TU inhibited the expression of gdf6a in the eyes throughout the metamorphosis period.In the rescue group, both normal seawater and seawater supplemented with T3 increased the expression level of gdf6a in the eyes of the TU group.Therefore, we believe that TH affects the expression of gdf6a in the eye of flounder.We analyzed the promoter region sequence of gdf6a through prediction and found that there are multiple TRE sites and T3R transcription factor binding sites in the promoter region of gdf6a.Subsequently, we further verified the regulatory relationship between TH, TRs, and gdf6a using the Dual-Luciferase reporter gene results.gdf6a pro showed significantly higher luciferase activity (p < 0.05) with the addition of T3 + TRαA/TRαB, indicating that exogenous T3 can exert a regulatory effect on gdf6a expression via TRαA and TRαB.The luciferase activity of gdf6a pro was also significantly increased when only TRαA or TRαB was added (p < 0.05).For this result, we have two hypotheses: one is the existence of endogenous T3 regulating the expression of gdf6a through TRαA and TRαB; the other is that TRαA and TRαB, as transcription factors, regulate the expression of gdf6a by binding to its promoter region.In contrast, the difference in luciferase activity of gdf6a pro was not significant when only TRβ was added, while the luciferase activity was significantly higher when T3 was added at the same time (p < 0.05).Combined with the above results, we speculate that T3 may directly regulate the expression of gdf6a through TRβ.Studies in mice have found that thyroid hormones regulate the expression of a large number of genes through TRs during retinal development [25].A similar study found that the knockdown of trβ alone did not affect blue cone fate in zebrafish, while blue cones were almost absent in the retina after the knockdown of trβ in gdf6a mutant zebrafish, further confirming the regulatory role of trβ in gdf6a gene expression [12].In addition, luciferase results showed that gdf6a promotes the expression of tbx2b.Similarly, it was found that over-expression of gdf6a in zebrafish similarly increased tbx2b expression in the retina [26][27][28].The above results indicate that gdf6a has a regulatory effect on the transcription of tbx2b.
In summary, the study found that gdf6a of flounder is closely related to its metamorphosis process and eye development.Meanwhile, exogenous TH affects the expression of gdf6a in the eye of flounder, and we found that exogenous T3 may directly regulate the expression of gdf6a through TRβ.In addition, we found that Gdf6a positively regulates its downstream gene tbx2b.We further speculate that TH regulates the expression of gdf6a to regulate the photoreceptor system of dental flounder to adapt to environmental changes during metamorphosis.

Sample Collection
All animal experiments in this study were conducted in strict accordance with the Laboratory Animals-Guidelines for Ethical Review of Animal Welfare of China (GB/T 35892-2018, https://openstd.samr.gov.cn/bzgk/gb/std_list,accessed on 11 November 2023).All experiments have been approved by the Animal Ethics Committee of Shanghai Ocean University (SHOU-DW-2021-060).
Flounder samples were obtained from the Beidaihe Central Experimental Station of the Chinese Academy of Fisheries Sciences and were uniformly cultured in filtered recirculating seawater.The water temperature was maintained at 17 ± 2 • C, salinity at 30‰, and natural light during the culture process.Larvae were fed with nutrient-fortified rotifers (Brachionus plicatilis) paired with Artemia salina larvae.Sample sex was not a consideration.All flounders were anesthetized with ms-222 (Sangon, Shanghai, China), euthanized by severing their heads, and rinsed with DEPC (Thermo, Waltham, MA, USA) water before sample collection.Three adult flounders of the same age (weighing 1.25 ± 0.10) were randomly selected from the culture water, and seven tissues were taken from each fish: eye, brain, liver, intestine, stomach, kidney, and gonad.Eye samples were collected from flounder larvae at 17 dph (days post-hatching), 24 dph, 28 dph, 32 dph, 36 dph, and 40 dph after emergence from the membrane at each metamorphic stage.Based on the results of the pre-test, the larvae at 15 dph were randomly divided into three groups with three replicates each.NC group: cultured in normal seawater (normal metamorphosis samples); TH group: cultured seawater supplemented with 0.1 mg/L of exogenous thyroid hormone (T3; Sangon, Shanghai, China); TU group: cultured seawater supplemented with 30 mg/L of exogenous thiourea (TU; Sangon, Shanghai, China).Sampling was then performed within 17-40 dph of metamorphosis, and three samples were taken from each tank for each group as biological replicates.
Rescue group: based on the results of the pre-test, flounders in the TU group at 36 dph were randomly divided into three groups with three replicates in each group and continued to be reared until 40 dph.TU group: the larvae continued to be cultured in seawater supplemented with 30 mg/L of exogenous TU; TU + NC group: the larvae were transferred to normal seawater and continued to be cultured; TU + TH group: the pups were transferred to seawater supplemented with 0.1 mg/L of exogenous T3 and continued to culture.The eye samples collected above were placed in 1.5 mL enzyme removal centrifuge tubes and immediately put into liquid nitrogen and then stored at −80 • C.

Bioinformatics Analysis of gdf6a
The amino acid sequence of the flounder Gdf6 (XP_019946621.1) protein was obtained from the NCBI database.The signal peptide and protein structure of Gdf6 protein were analyzed using SignalP-5.0and SMART protein structural domain database.Multiple comparisons and homology analyses of the amino acid sequences of the Gdf6 protein were performed using DNAMAN 8.0 software, and the phylogenetic tree was constructed using the neighbor-joining method of MEGA 5.0 software.

Quantitative Real-Time PCR
Total RNA was extracted from pre-collected samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.The integrity of RNA was assessed via agarose gel electrophoresis and RNA concentration was checked with a spectrophotometer NANODROP 2000C (Thermo, Waltham, MA, USA), 2.0 > A260/280 > 1.8.Total RNA (1 µg) from adult tissues was reverse transcribed using an RT-PCR kit (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer's instructions.
We obtained the sequence of the CDS region of gdf6a (gene ID: 109631977) from the NCBI website and designed primers (Table 1) using Primer Premier 5.0 software.Real-time fluorescence quantification was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA) and according to the instructions of ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, Jiangsu, China).Three biological replicates were available for each group, and 18S was selected as the internal reference gene to determine the relative expression of gdf6a mRNA using the 2 −∆∆CT method.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Animal Ethics Committee of Shanghai Ocean University (No.: SHOU-DW-2021-060), and strictly followed the "Guidelines for the Care and Use of Experimental Animals".
Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.Amino acid sequence analysis and phylogenetic tree of the gdf6a in Paralichthys olivaceus.(A) Amino acid sequence analysis of gdf6a.The red horizontal line indicates the signal peptide region, the black box indicates the transmembrane region, and the dashed line indicates the TGFB structural domain.(B) Comparison of amino acid sequences of multi-species gdf6a, the red dots represent the flounder in this study.(C) The phylogenetic tree of gdf6a.The amino acid sequences of

Figure 2 .
Figure 2. Tissue expression profile and temporal expression of gdf6a gene in flounder.(A) Ex sion levels of gdf6a in adult flounder tissues.(B) Expression levels of gdf6a in the eyes during fl der metamorphosis.All data are expressed using the mean ± standard deviation; different l indicate significant differences between data (p < 0.05).

Figure 2 .
Figure 2. Tissue expression profile and temporal expression of gdf6a gene in flounder.(A) Expression levels of gdf6a in adult flounder tissues.(B) Expression levels of gdf6a in the eyes during flounder metamorphosis.All data are expressed using the mean ± standard deviation; different letters indicate significant differences between data (p < 0.05).

Figure 3 .
Figure 3. Expression levels of gdf6a in the eyes of TH and TU-treated and rescue groups of flou (A) Expression levels of gdf6a in the eyes of exogenous TH and TU-treated flounder during morphosis.(B) Expression levels of gdf6a in flounder eyes of the rescue group.NC group: cul in normal seawater; TH group: cultured seawater supplemented with 0.1 mg/L of exogenou roid hormone (T3); TU group: cultured seawater supplemented with 30 mg/L of exogenous thi (TU).TU-treated flounders at 36 dph were transferred to normal seawater (TU + NC) and TH-a seawater (TU + TH) to continue rearing until 40 dph.All data are expressed using the mean ± s ard deviation, where an asterisk indicates a significant difference from the contemporaneou group (A) or 36 dph-TU group (B) (p < 0.05).

Figure 3 .
Figure 3. Expression levels of gdf6a in the eyes of TH and TU-treated and rescue groups of flounder.(A) Expression levels of gdf6a in the eyes of exogenous TH and TU-treated flounder during metamorphosis.

Figure 3 .
Figure 3. Expression levels of gdf6a in the eyes of TH and TU-treated and rescue groups of flo (A) Expression levels of gdf6a in the eyes of exogenous TH and TU-treated flounder during morphosis.(B) Expression levels of gdf6a in flounder eyes of the rescue group.NC group: cu in normal seawater; TH group: cultured seawater supplemented with 0.1 mg/L of exogenou roid hormone (T3); TU group: cultured seawater supplemented with 30 mg/L of exogenous th (TU).TU-treated flounders at 36 dph were transferred to normal seawater (TU + NC) and THseawater (TU + TH) to continue rearing until 40 dph.All data are expressed using the mean ± ard deviation, where an asterisk indicates a significant difference from the contemporaneo group (A) or 36 dph-TU group (B) (p < 0.05).

Figure 5 .
Figure 5. Analysis of the Dual-Luciferase results of gdf6a in flounder.(A) Analysis of TRs binding sites in the promoter region of gdf6a.(B) Experimental component of the Dual-Luciferase reporter gene.(C-E) T3 regulates the Dual-Luciferase activity of gdf6a via TRs.(F) Regulation of tbx2b of the Dual-Luciferase activity by gdf6a.Note: gdf6a pro and tbx2b pro represented the recombinant plasmid of gdf6a and tbx2b promoter region; N1 represented pEGFP-N1 empty plasmids; TRαA, TRαB, TRβ, and Gdf6a represented over-expressed plasmids of corresponding genes, respectively.All data are expressed using the mean ± standard deviation, and significant differences from the promoter activity group are indicated by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5 .
Figure 5. Analysis of the Dual-Luciferase results of gdf6a in flounder.(A) Analysis of TRs binding sites in the promoter region of gdf6a.(B) Experimental component of the Dual-Luciferase reporter gene.(C-E) T3 regulates the Dual-Luciferase activity of gdf6a via TRs.(F) Regulation of tbx2b of the Dual-Luciferase activity by gdf6a.Note: gdf6a pro and tbx2b pro represented the recombinant plasmid of gdf6a and tbx2b promoter region; N1 represented pEGFP-N1 empty plasmids; TRαA, TRαB, TRβ, and Gdf6a represented over-expressed plasmids of corresponding genes, respectively.All data are expressed using the mean ± standard deviation, and significant differences from the promoter activity group are indicated by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Table 1 .
Designed primers used in the experiment.