Wenteng Xu

The genome and transcriptome of Japanese flounder provide insights into flatfish asymmetry

Flatfish have the most extreme asymmetric body morphology of vertebrates. During metamorphosis, one eye migrates to the contralateral side of the skull, and this migration is accompanied by extensive craniofacial transformations and simultaneous development of lopsided body pigmentation. The evolution of this developmental and physiological innovation remains enigmatic. Comparative genomics of two flatfish and transcriptomic analyses during metamorphosis point to a role for thyroid hormone and retinoic acid signaling, as well as phototransduction pathways. We demonstrate that retinoic acid is critical in establishing asymmetric pigmentation and, via cross-talk with thyroid hormones, in modulating eye migration. The unexpected expression of the visual opsins from the phototransduction pathway in the skin translates illumination differences and generates retinoic acid gradients that underlie the generation of asymmetry. Identifying the genetic underpinning of this unique developmental process answers long-standing questions about the evolutionary origin of asymmetry, but it also provides insight into the mechanisms that control body shape in vertebrates.

Two Figla homologues have disparate functions during sex differentiation in half-smooth tongue sole (Cynoglossus semilaevis).

Figla is a germ-cell-specific transcription factor associated with ovary development and differentiation. In vertebrates, one transcriptional form of Figla is commonly found. However, besides the common form of this gene (named Figla_tv1), a new transcriptional form (named Figla_tv2) was identified in half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of Figla_tv1 was 1057 bp long with a 591-bp open reading frame encoding a predicted 196 amino acid protein, while Figla_tv2 encoded a 125 amino acid protein. Figla_tv1 and Figla_tv2 expression in various tissues was detected by qRT-PCR. Figla_tv1 was expressed mainly in ovary, skin and liver, while Figla_tv2 was expressed in all examined tissues. In the gonads, Figla_tv1 was expressed in ovary, while Figla_tv2 was predominately expressed in testis of pseudomales. Further, in situ hybridization located Figla_tv1 only in oocytes and Figla_tv2 mainly in germ cells of pseudomale testis. After knocking down Figla_tv2 in a pseudomale testis cell line, the expression of two steroid hormone-encoding genes, StAR and P450scc, was significantly up-regulated (P < 0.05). Our findings suggest that Figla_tv1 has a conserved function in folliculogenesis, as in other vertebrates, and that Figla_tv2 may have a role in the spermatogenesis of pseudomales by regulating the synthesis and metabolism of steroid hormones.

Ubiquitin ligase gene neurl3 plays a role in spermatogenesis of half-smooth tongue sole (Cynoglossus semilaevis) by regulating testis protein ubiquitination

E3 ubiquitin ligases are a large gene family that plays a diversity of roles in spermatogenesis. In this study, the functional characterization of a neuralized E3 ubiquitin protein ligase 3 (neurl3) revealed its potential participation in spermatogenesis. Firstly, we found that neurl3 exhibited male-biased transcription and that its translation was predominant in testis germ cells. The knockdown of neurl3 by RNA interference caused increased transcription of spermatogenesis-related genes. These results corroborate previous studies indicating a role for neurl3 in spermatogenesis. Moreover, the levels of neurl3 transcription and testis protein ubiquitination were closely correlated. Based on these findings, we speculate that neurl3 modulates testis protein ubiquitination in a dosage-dependent manner and that this influences spermatogenesis.

Proteome profiling reveals immune responses in Japanese flounder (Paralichthys olivaceus) infected with Edwardsiella tarda by iTRAQ analysis

Abstract The liver is an important organ for bacterial pathogen attack in fish. The differential proteomic response of the Japanese flounder liver to Edwardsiella tarda infection was examined using isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). A total of 3290 proteins were identified and classified into categories related to biological process (51.4%), molecular function (63.6%), and cellular component (57.7%). KEGG enrichment analysis indicated the complement and coagulation cascade pathways and the mineral absorption pathway were significantly enriched. Among the differentially expressed proteins, those involved in mediating complement cascade (e.g. complement component C7, C8, C9, complement factor H, complement factor Bf/C2) and mineral absorption (e.g. ferritin, STEAP‐4) were most significantly upregulated during infection. Subsequently, five significantly upregulated (C4, C8beta, ferritin middle subunit, PRDX4‐like and KRT18) and one significantly downregulated (transferrin) candidate immune proteins were validated by multiple reaction monitoring (MRM) assays. Furthermore, changes in expression of 15 proteins in the complement cascade and mineral absorption pathways were validated at the transcriptional level using quantitative real‐time PCR (qPCR). The transcriptional levels of four transcription factors (p21Ras, Rab‐31‐like, NF‐&kgr;B, STAT3) were also investigated by qPCR following infection with E. tarda. This study contributes to understanding the defense mechanisms of the liver in fish. HighlightsThe study identified the differently expressed liver proteins and two signal pathways of Japanese flounder under E. tarda infection by iTRAQ.Transcriptomic profiles analysis of fifteen proteins in complement cascade and mineral absorption were investigated.mRNA levels of four transcription factors confirmed the upregulated of RNA/DNA ratio in E. tarda infection.

Characterization and functional analysis of a novel C1q-domain-containing protein in Japanese flounder (Paralichthys olivaceus).

ABSTRACT The complement system is important in the innate immune response. C1q‐domain‐containing proteins have multiple functions and occur extensively in invertebrates and vertebrates. In this study, PoC1ql3 encoding a C1q‐domain‐containing protein in the Japanese flounder was identified. The 266‐amino‐acid polypeptide encoded, PoC1ql3, shares high sequence and structural similarity with orthologues in other fish and mammals. PoC1ql3 is abundantly expressed in the brain, but less in the blood, gills, and liver. Transcripts of PoC1ql3 were down‐regulated in the spleen and liver 6–24 h after bacterial infection, but were significantly up‐regulated after 48 h. Full‐length PoC1ql3 (C1ql3‐full) and its gC1q domain (C1ql3‐part) were both exerted anti‐Edwardsiella tarda activity. C1ql3‐part bound to lipopolysaccharide and peptidoglycan, and exerted antibacterial effects against E. tarda in vivo, suggesting that C1ql3 functions as a pathogen‐recognition receptor. Therefore, PoC1ql3 functions in the innate immune system, which would facilitate the investigation of the immune system in Japanese flounder. HighlightsCharacterization of a novel C1q‐domain‐containing protein of the Japanese flounder.PoC1ql3 was expressed not only in the brain, but also in many immune‐related tissues.C1ql3‐full and C1ql3‐part protein exerted antimicrobial function against E. tarda in vitro.The C1ql3‐part bound LPS and PGN as pattern‐recognition receptor.The C1ql3‐part inhibited the invasion of bacterium to fish kidney cells in vivo.

Quantitative trait loci detection of Edwardsiella tarda resistance in Japanese flounder Paralichthys olivaceus using bulked segregant analysis

In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese flounder (Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. olivaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis (BSA) and quantitative trait loci (QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333 (♀: F0768 ×♂: F0915) (Nomenclature rule: F+year+family number) were used to detect simple sequence repeats (SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers (Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group (LG)-1 exhibited a significant difference between DNA, pooled/bulked from the resistant and susceptible groups (P <0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of significance in LG1, where 17 and 18 SSR markers were identified, respectively. Each model found three resistance-related QTLs by composite interval mapping (CIM). These six QTLs, designated qE1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, qE-2 and qE-4, were located at the 66.7 cM region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese flounder in the future.

Molecular characterization and expression analysis of a novel r-spondin member ( rspo2l ) in Chinese tongue sole ( Cynoglossus semilaevis )

ABSTRACT Numerous studies suggest R‐spondins (Rspos) plays a role in mammalian sex development and differentiation by activating WNT signaling pathways. However, Rspos are frequently less reported in teleosts. In this study, a molecular characterization and expression analysis was conducted with a new rspondin member in the Chinese tongue sole, rspondin2‐like (rspo2l). The length of rspo2l cDNA is 1251 bp with 732 bp of coding sequence. A qRT‐PCR analysis revealed that the transcription of rspo2l was distributed in various tissues, with high transcription levels in the liver, skin, and gills which might indicate a possible role in immunity. We next examined a time‐course of transcription levels in four immune tissues (gill, liver, spleen, and kidney) after Vibrio harveyi challenge. It was found that rspo2l was up‐regulated in the gills, spleen, and kidney and down‐regulated in the liver, and the greatest responses occurred at 24 and 48 h after bacterial challenge. An assessment of &bgr;‐catenin, the key regulator of the canonical WNT signaling pathway, at different time points in four immune organs revealed that its transcription profile was similar to that of rspo2l after bacterial challenge. The results suggest that tongue sole rspo2l might play a role in immune responses after bacterial challenge, while the potential link with the WNT signaling pathway still requires further investigation. This is the first report about the involvement of rspondins in fish immune responses. HighlightsA new r‐spondin member, r‐spondin2‐like (rspo2l), was cloned and characterized in Chinese tongue sole.rspo2l transcription was detected in various tissues and the high levels were observed in skin and gill.rspo2l transcription showed obvious up‐regulation in gill, spleen, and kidney after Vibrio harveyi challenge.rspo2l might play a role in immune responses by cooperating with WNT signaling pathway.

Molecular cloning and expression analysis of the aqp1aa gene in half-smooth tongue sole (Cynoglossus semilaevis)

Aquaporin 1 (AQP1) is a member of the transmembrane water channel family of proteins with special structural features, and two AQP1 paralogous genes (aqp1aa and aqp1ab) are reported in teleosts. In the present study, the aqp1aa gene of half-smooth tongue sole (Cynoglossus semilaevis) was cloned and characterized. The full-length cDNA of aqp1aa is 1411 bp with a 786 bp open reading frame encoding a 261-amino acid putative protein with a characteristic structure consisting of 6 membrane-spanning α-helical domains and two highly conserved asparagine—proline—alanine motifs. Real-time quantitative PCR revealed that aqp1aa mRNA is expressed predominantly in the testis of males and pseudo-males, while its expression is low in the ovary and lowest in doublesex and mab-3-related transcription factor 1(DMRT1) knock out fish and triploid males. In situ hybridization indicated that aqp1aa mRNA is expressed mainly in the germ cells of males and pseudo-males, especially in spermatozoa and spermatids. These results suggest that the aqp1aa may play a role in spermatogenesis of C. semilaevis.

Expression analysis and characterization of zglp1 in the Chinese tongue sole (Cynoglossus semilaevis)

Zinc finger GATA like protein-1 (ZGLP1) is a nuclear zinc finger protein that regulates the interaction between somatic cells and germ cells during gonad developmental process in mammals. In this study, the zglp1 of Chinese tongue sole, Cynoglossus semilaevis (cysezglp1), was cloned and characterized for the first time in fish. Cysezglp1 had an open reading frame with five exons and was located to chromosome 9. The open reading frame of cysezglp1 consisted of 1692 nucleotides and encoded a 583 amino acid polypeptide. The predicted protein contained two zinc finger structures (Znf1 and Znf2), one of which was highly homologous to the GATA-type zinc finger domain. Multiple sequence alignment showed that Znf1 was conserved across different species while Znf2 was more divergent. Through quantitative Real-time PCR (qRT-PCR), we found that cysezglp1 was predominantly expressed in gonads, and the expression level of the ovary was significantly higher than that of the testis. We compared expression level in different embryonic stages and found that cysezglp1 mRNAs were mainly expressed in the fertilized egg to the cleavage stage, subsequently declining in the blastula stage. Cysezglp1 expression was not detected from the gastrulation stage onward. In the ovary, cysezglp1 expression was detected at 120 days after hatching and expression gradually increased with the maturation of the ovary. In situ hybridization showed that the cysezglp1 was mainly expressed in oocytes. Taken together, our results suggest that cysezglp1 may play an important role in the process of oogenesis in Chinese tongue sole.

Molecular characterization and expression analysis of strbp in Chinese tongue sole (Cynoglossus semilaevis)

Spermatid perinuclear RNA-binding protein (Strbp) is a kind of double-stranded (ds) RNA specific binding protein that plays important roles in mammalian spermatogenesis. In this study, we have isolated and characterized the strbp gene of Chinese tongue sole, Cynoglossus semilaevis, termed as CS-strbp. The CS-strbp genomic sequence contained fifteen exons and fourteen introns. Its cDNA was 2655 bp in length, encoding a 666-amino-acid protein with two conserved ds binding motifs. Using quantitative PCR, we found that CS-strbp mRNA exhibited sex-biased and tissue-specific distribution, predominantly expressed in the fertile male testis, though the expression levels varied throughout different developmental stages. Comparison of methylation profile in different sexual genotypes demonstrated the low methylation level of CS-strbp promoter in male and pseudo-male, which is consistent with the high expression levels in those genotypes. In situ hybridization revealed that CS-strbp mRNA mainly localized in male germ cells, especially in spermatids and spermatozoa. Given these findings, we postulate that CS-strbp might function in spermatogenesis of Chinese tongue sole.