Phylogeny, expression patterns and regulation of DNA Methyltransferases in early development of the flatfish, Solea senegalensis.

BACKGROUND: The identification of DNA methyltransferases (Dnmt) expression patterns during development and their regulation is important to understand the epigenetic mechanisms that modulate larval plasticity in marine fish. In this study, dnmt1 and dnmt3 paralogs were identified in the flatfish Solea senegalensis and expression patterns in early developmental stages and juveniles were determined. Additionally, the regulation of Dnmt transcription by a specific inhibitor (5-aza-2'-deoxycytidine) and temperature was evaluated. RESULTS: Five paralog genes of dnmt3, namely dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1 and dnmt3bb.2 and one gene for dnmt1 were identified. Phylogenetic analysis revealed that the dnmt gene family was highly conserved in teleosts and three fish-specific genes, dnmt3aa, dnmt3ba and dnmt3bb.2 have evolved. The spatio-temporal expression patterns of four dnmts (dnmt1, dnmt3aa, dnmt3ab and dnmt3bb.1) were different in early larval stages although all of them reduced expression with the age and were detected in neural organs and dnmt3aa appeared specific to somites. In juveniles, the four dnmt genes were expressed in brain and hematopoietic tissues such as kidney, spleen and gills. Treatment of sole embryos with 5-aza-2'-deoxycytidine down-regulated dntm1 and up-regulated dntm3aa. Moreover, in lecithotrophic larval stages, dnmt3aa and dnmt3ab were temperature sensitive and their expression was higher in larvae incubated at 16 °C relative to 20 °C. CONCLUSION: Five dnmt3 and one dnmt1 paralog were identified in sole and their distinct developmental and tissue-specific expression patterns indicate that they may have different roles during development. The inhibitor 5-aza-2'-deoxycytidine modified the transcript abundance of dntm1 and dntm3aa in embryos, which suggests that a regulatory feedback mechanism exists for these genes. The impact of thermal regime on expression levels of dnmt3aa and dnmt3ab in lecithotrophic larval stages suggests that these paralogs might be involved in thermal programing.

Molecular and functional characterization of seven Na+/K+-ATPase ß subunit paralogs in Senegalese sole (Solea senegalensis Kaup, 1858).

In the present work, seven genes encoding Na(+),K(+)-ATPase (NKA) ß-subunits in the teleost Solea senegalensis are described for the first time. Sequence analysis of the predicted polypeptides revealed a high degree of conservation with those of other vertebrate species and maintenance of important motifs involved in structure and function. Phylogenetic analysis clustered the seven genes into four main clades: ß1 (atp1b1a and atp1b1b), ß2 (atp1b2a and atp1b2b), ß3 (atp1b3a and atp1b3b) and ß4 (atp1b4). In juveniles, all paralogous transcripts were detected in the nine tissues examined albeit with different expression patterns. The most ubiquitous expressed gene was atp1b1a whereas atp1b1b was mainly detected in osmoregulatory organs (gill, kidney and intestine), and atp1b2a, atp1b2b, atp1b3a, atp1b3b and atp1b4 in brain. An expression analysis in three brain regions and pituitary revealed that ß1-type transcripts were more abundant in pituitary than the other ß paralogs with slight differences between brain regions. Quantification of mRNA abundance in gills after a salinity challenge showed an activation of atp1b1a and atp1b1b at high salinity water (60 ppt) and atp1b3a and atp1b3b in response to low salinity (5 ppt). Transcriptional analysis during larval development showed specific expression patterns for each paralog. Moreover, no differences in the expression profiles between larvae cultivated at 10 and 35 ppt were observed except for atp1b4 with higher mRNA levels at 10 than 35 ppt at 18 days post hatch. Whole-mount in situ hybridization analysis revealed that atp1b1b was mainly localized in gut, pronephric tubule, gill, otic vesicle, and chordacentrum of newly hatched larvae. All these data suggest distinct roles of NKA ß subunits in tissues, during development and osmoregulation with ß1 subunits involved in the adaptation to hyperosmotic conditions and ß3 subunits to hypoosmotic environments.

Molecular characterization and transcriptional regulation of the Na +/K+ ATPase α subunit isoforms during development and salinity challenge in a teleost fish, the Senegalese sole (Solea senegalensis).

In the present work, five genes encoding different Na(+),K(+) ATPase (NKA) α-isoforms in the teleost Solea senegalensis are described for the first time. Sequence analysis of predicted polypeptides revealed a high degree of conservation across teleosts and mammals. Phylogenetic analysis clustered the five genes into three main clades: α1 (designated atp1a1a and atp1a1b), α2 (designated atp1a2) and α3 (designated atp1a3a and atp1a3b) isoforms. Transcriptional analysis in larvae showed distinct expression profiles during development. In juvenile tissues, the atp1a1a gene was highly expressed in osmoregulatory organs, atp1a2 in skeletal muscle, atp1a1b in brain and heart and atp1a3a and atp1a3b mainly in brain. Quantification of mRNA abundance after a salinity challenge showed that atp1a1a transcript levels increased significantly in the gill of soles transferred to high salinity water (60 ppt). In contrast, atp1a3a transcripts increased at low salinity (5 ppt). In situ hybridization (ISH) analysis revealed that the number of ionocytes expressing atp1a1a transcripts in the primary gill filaments was higher at 35 and 60 ppt than at 5 ppt and remained undetectable or at very low levels in the lamellae at 5 and 35 ppt but increased at 60 ppt. Immunohistochemistry showed a higher number of positive cells in the lamellae. Whole-mount analysis of atp1a1a mRNA in young sole larvae revealed that it was localized in gut, pronephric tubule, gill, otic vesicle, yolk sac ionocytes and chordacentrum. Moreover, atp1a1a mRNAs increased at mouth opening (3 DPH) in larvae incubated at 36 ppt with a greater signal in gills.