Early ontogenesis from embryo to juvenile in Senegalese sole Solea senegalensis under laboratory conditions.

Senegalese sole, Solea senegalensis, is a flatfish of high commercial value in the world. It has been identified as an interesting and promising species for marine commercial aquaculture diversification in Europe for at least four decades and was introduced to China in 2003. Early ontogenesis from embryo to juvenile stages in S. senegalensis was analysed under controlled laboratory conditions to provide morphological information for aquaculture. From 0 to 59 days post hatching (dph), 10-20 larvae were sampled and measured each day (0-17 dph) or every 2-6 days (17-59 dph). Morphological characteristics from the egg to the juvenile stage were described. The eggs were separate and spherical with multiple oil globules. After 3 dph, the yolk sac was completely absorbed, mouth and anus were open, a swim bladder appeared, and larvae began feeding on rotifers (Brachionus plicatilis). The larvae began metamorphosis as the notochord flexed upward and the left eye migrated upward after 10 dph. The left eye migrated to the dorsal midline at 15 dph. At 19 dph, the left eye was translocated to the right-ocular side, and the juveniles adopted a benthic lifestyle. The swim bladder degenerated, and the juveniles completed metamorphosis at 23 dph. The growth patterns of some parameters (TL, SL, BH, BW) during larval and juvenile development stages were identified. The inflection points, which are slopes of growth changes, were calculated in growth curves. Three inflection points occurring in the growth curves of larvae and juveniles were found to be associated with metamorphosis, weaning, and transitions in feeding habits. The basic information of embryo development and ontogenesis in this study represents a valuable contribution to the S. senegalensis industry, especially in artificial breeding and rearing techniques.

Cloning, expression and functional analysis of the desert hedgehog (dhh) gene in Chinese tongue sole (Cynoglossus semilaevis).

Desert hedgehog (dhh) is a gene that is crucial for spermatogenesis and Leydig cell differentiation, but little is known regarding its influence on gonadal differentiation and development in fish. To understand its function, we cloned and characterized the dhh gene from Cynoglossus semilaevis (csdhh). The full length csdhh cDNA was 2473 bp, including a 1386 bp open reading frame (ORF), a 475 bp 5'-UTR, and a 612 bp 3'-UTR, encoding a predicted protein of 461 amino acid residues. Phylogenetic analysis showed that the putative protein belongs to the hedgehog (HH) family, and contains typical HH-N and HH-C domains. Amino acid sequence analysis revealed that CsDhh shares many features with Dhh analogues in other teleost species. Real-time quantitative PCR showed that csdhh was detected in eight different tissues in male and female tongue sole. During early embryonic development, the relative expression of the csdhh was significantly higher in the neural stage than in other embryonic developmental stages (P < 0.05). csdhh was detected at 20 days after hatching (dah) and at the critical period of male gonadal differentiation (80-95 dah), the relative expression of the csdhh was significantly higher in the male gonads than the female gonads. In 5, 8, and 12 month old gonads, the relative expression of the csdhh was significantly higher in male and pseudo-male than in female fish. The in situ hybridization (ISH) results showed that the hybridization signal was strongly expressed in primary and secondary spermatocytes, spermatids, and sertoli cells of the 1-year-old fish testis, with only weak signal expression in the corresponding ovarian tissue. These results suggest that csdhh is highly conserved in evolution and plays an important role in spermatogenesis in males and pseudo-males.

Sole head transcriptomics reveals a coordinated developmental program during metamorphosis.

Most teleosts undergo a thyroid hormone (TH) regulated larval to juvenile transition known as metamorphosis. In Pleuronectiformes (flatfish), metamorphosis is most dramatic, and one eye of the symmetric pelagic larvae migrates to the opposite side of the head, giving rise to an asymmetric benthic juvenile with both eyes on the same side of the body. Asymmetric development occurs mostly in the head. To understand the genetic mechanisms underlying this developmental change we have generated a Solea senegalensis metamorphosing flatfish head transcriptome. Our results reveal that THs acting as integrative factors direct a stepwise genetic program that initiates a specific organismal level response followed by cell specific responses that lead to the long-term changes that characterise the post-metamorphic identity and physiology of the head. Flatfish head asymmetric development during metamorphosis and its TH dependency is conserved thus we consider the findings in sole most likely representative of all flatfish species.

Effects of triclosan on early development of Solea senegalensis: from biochemical to individual level.

Harmful effects of triclosan (TCS) have been reported on several organisms; however, effects on early life stages of marine vertebrates are limited. Therefore, the objective of this work was to assess the effects of TCS during early development of the flatfish Solea senegalensis after initial characterization of cholinesterases (ChEs) and determination of selected biochemical markers baseline levels. Characterization of ChEs and determination of biochemical markers baseline levels of cholinergic activity, energy metabolism and oxidative stress were analysed in sole at 3 days after hatching (dah) and at the onset and end of metamorphosis. To assess TCS effects, fish were exposed during 96h to 30-500 µg L-1 TCS until 3 dah. Fish at 13 dah were exposed during 48h to 200-1,500 µg L-1 TCS and maintained until complete metamorphosis. Effects on survival, malformations, length, metamorphosis progression and biochemical markers were evaluated. The main ChE active form present in sole early life stages is acetylcholinesterase and baseline levels of oxidative stress and energy metabolism biomarkers changed according to fish developmental stage. Triclosan induced malformations (EC50 = 180 µg L-1 at 3 dah), decreased growth (95 µg L-1 at 3 dah; 548 µg L-1 at 24 dah) and affected metamorphosis progression (391 µg L-1 at 17 dah). Impairment of antioxidant system was observed, with TCS affecting catalase at the end of metamorphosis test, however, no oxidative damage on lipids was detected. Glutathione S-transferase was the most sensitive endpoint during early larval test (LOEC = 30 µg L-1). Exposure to TCS affected S. senegalensis at individual and sub-individual levels, both at early larval stage and during the critical period of metamorphosis.

Expression and localization study of pIgR in the late stage of embryo development in turbot (Scophthalmus maximus).

The receptor responsible for maternofetal transmission of immunoglobulin (Igs) in the teleosts is not clear. Polymeric immunoglobulin receptor (pIgR) specifically binds with IgA and IgM and mediates the transcytosis of intracellular polymeric immunoglobulins (pIgs) at the mucosal surface to protect against pathogens. Hence there is a possibility that it may be involved in the transmission of maternal Igs. The aim of the present study was to detect the expression and localization of pIgR during embryonal development in turbot (Scophthalmus maximus). pIgR gene was first cloned from eggs and embryos of turbot with or without parent immunization. The expression and distribution of pIgR in unfertilized egg and in embryos ranging from day 1 to day 5 after fertilization were analyzed using reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. pIgR gene was detected in all eggs and embryos at different stages of development, with the highest level detected on the 5th day. pIgR mRNA was observed to be first located in the whole blastoderm and enveloped the yolk sac. Later, it was located around entoderm including primary digestive tract and pronephric tubule tract, and finally it was located at the joint of abdomen and vitelline membrane. Then, Eukaryotic expression plasmid carrying pIgR gene was constructed and transfected into HEK293T cells. Results showed mature pIgR protein located on the cellular membrane, and could bound IgM in vitro. Our findings provide information for studying the involvement of pIgR in maternal Igs transportation in turbot.

Functional characterization of Cynoglossus semilaevis R-spondin2 and its role in muscle development during embryogenesis.

R-spondin2 (Rspo2) is a member of the R-spondin family, which plays important roles in cell proliferation, cell fate determination and organogenesis. Rspo2 exhibits important functions during embryonic development and muscle maintenance in adult human, mouse and Xenopus. In the present study, the tongue sole Cynoglossus semilaevis Rspo2 (CsRspo2) gene was isolated and characterized, and its role in muscle development during embryogenesis was studied. Our results showed that CsRspo2 expression was abundant during gastrulation and significantly high during somite formation, but then decreased markedly after hatching. CsRspo2 expression was high in brain and gill, moderate in heart, ovary and testis, and almost undetectable in muscle and other tissues. Moreover, the potential involvement of Rspo2 in muscle development was investigated. We found that overexpression of CsRspo2 mRNA in zebrafish embryos resulted in slow development and abnormal muscle formation at the embryonic stage. Our work provides a fundamental understanding of the structure and potential functions of CsRspo2 during muscle development.

Phylogeny and expression patterns of two apolipoprotein E genes in the flatfish Senegalese sole.

The apolipoprotein E (ApoE) is a key component of several lipoproteins involved in lipid homeostasis. In this study, two cDNA sequences encoding ApoE (referred to as apoEa and apoEb) were characterized in the flatfish Solea senegalensis. The predicted peptides contained conserved structural blocks related with their capacity for lipid binding and lipoprotein receptor interaction. At genomic level, both genes contained five exons and four introns and they were organized into two tandem arrays with apoA-IV gene copies. The phylogenetic analysis clearly separated them into two well-supported clusters that matched with their organization in the genome of teleosts. Whole-mount in situ hybridization located the apoEa signal in the yolk syncytial layer (YSL) of lecitothrophic larval stages (0dph) and in the anterior intestine of exotrophic larvae and benthic fish. In the case of apoEb, hybridization signals were located in the YSL, tail bud, eyes and mouth at 0dph and in the otic vesicle, hindbrain, eyes, pharynx, mouth, heart and intestine at 1dph. In exotrophic larvae, apoEb was ubiquitously expressed in several tissues such as taste buds, brain, mouth, nostril, gills, intestine, liver and around the neuromasts and eyes. Quantification of mRNA levels in pools of whole larvae confirmed distinct expression patterns with a significant reduction of apoEa and an increase of apoEb mRNA levels throughout larval development. Moreover, only apoEa transcripts increased in response to food supply suggesting that this paralog mostly participates in the absorption and transport of dietary lipids and the apoEb in the redistribution of endogenous lipids as well as in neural tissue regeneration.

Effect of Initiation Time of Hydrostatic Pressure Shock on Chromosome Set Doubling of Tetraploidization in Turbot Scophthalmus maximus.

The objective of the study was to clarify the effects of initiation time on chromosome set doubling induced by hydrostatic pressure shock through nuclear phase fluorescent microscopy in turbot Scophthalmus maximus. The ratio of developmentally delayed embryo and chromosome counting was used to assess induction efficiency. For the embryos subjected to a pressure of 67.5 MPa for 6 min at prometaphase (A group), chromosomes recovered to the pre-treatment condition after 11-min recovering. The first nuclear division and cytokinesis proceeded normally. During the second cell cycle, chromosomes did not enter into metaphase after prometaphase, but spread around for about 13 min, then assembled together and formed a large nucleus without anaphase separation; the second nuclear division and cytokinesis was inhibited. The ratio of developmentally delayed embryo showed that the second mitosis of 78% A group embryo was inhibited. The result of chromosome counting showed that the tetraploidization rate of A group was 72%. For the embryos subjected to a pressure of 67.5 MPa for 6 min at anaphase (B group), chromosomes recovered to the pre-treatment condition after about 31-min recovering. Afterwards, one telophase nucleus formed without anaphase separation; the first nuclear division was inhibited. The time of the first cleavage furrow occurrence of B group embryos delayed 27 min compared with that of A group embryos. With the first cytokinesis proceeding normally, 81.3% B group embryos were at two-cell stage around the middle of the second cell cycle after treatment. Those embryos were one of the two blastomeres containing DNA and the other without DNA. The first nuclear division of those embryos was inhibited. During the third cell cycle after treatment, 65.2% of those abovementioned embryos were at four-cell stage, cytokinesis occurred in both blastomeres, and nuclear division only occurred in the blastomere containing DNA. Of those abovementioned embryos, 14.0% were at three-cell stage and cytokinesis only occurred in the blastomere containing DNA. The result of chromosome counting showed that the tetraploidization rate of B group was only 7%. To summarize what had been mentioned above, mechanisms on chromosome set doubling of tetraploid induction would be different with different initiation time of hydrostatic pressure treatment. Chromosome set doubling was mainly due to inhibition of the second mitosis when hydrostatic pressure treatment was performed at prometaphase. Otherwise, chromosome set doubling was mainly due to inhibition of the first nuclear division when hydrostatic pressure treatment was performed at anaphase. Induction efficiency of tetraploidization resulted from inhibition of the second cleavage was higher than which resulted from inhibition of the first nuclear division. This study was the first to reveal biological mechanisms on the two viewpoints of chromosome set doubling through effect of initiation time of hydrostatic pressure treatment on chromosome set doubling in tetraploid induction.

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.

Cloning, expression profiling and promoter functional analysis of Bone morphogenetic protein 6 and 7 in tongue sole (Cynoglossus semilaevis).

Bone morphogenetic proteins (BMPs) play crucial roles in vertebrate developmental process and are associated with the mechanisms which drive early skeletal development. As a first approach to elucidating the role of BMPs in regulating fish bone formation and growth, we describe the cloning, expression profiling and promoter functional analysis of bmp6 and bmp7 in tongue sole (Cynoglossus semilaevis). The full length of bmp6 and bmp7 cDNA sequences is 1939 and 1836 bp, which encodes a protein of 428 and 427 amino acids, respectively. Tissue expression distribution of bmp6 and bmp7 was examined in 14 tissues of mature individuals by quantitative real-time PCR (qRT-PCR). The results revealed that bmp6 was predominantly expressed in the gonad, and bmp7 exhibited the highest expression level in the dorsal fin. Further comparison of bmp6 expression levels between female and male gonads showed that the expression in the ovary was significantly higher than in the testis. Moreover, bmp6 and bmp7 expression levels were detected at 15 sampling time points of early developmental stages (egg, larva, juvenile and fingerling stages). The highest expression level of bmp6 was observed in the egg stage (multi-cell and gastrula stage); while bmp7 exhibited the highest expression in the larva stage (1-4 days old). The high expression levels of BMP6 in the ovary as well as at early embryonic stages indicated that the maternally stored transcripts of bmp6 might play a role in early embryonic development. Whole-mount in situ hybridization showed that bmp6 and bmp7 exhibited similar spatial expression patterns. Both bmp6 and bmp7 signals were first detected in the head and anterior regions in newly hatched larvae, and then, the mRNAs appeared in the crown-like larval fin, jaw, operculum and fins (pectoral, dorsal, pelvic and anal) along with early development. Subsequently, we characterized the 5'-flanking regions of bmp6 and bmp7 by testing the promoter activity by luciferase reporter assays. Positive regulatory regions were, respectively, detected at the location of -272 to +28 and -740 to -396 in bmp6 and bmp7 gene. The predicted transcription factor binding sites (CREB, AP1 and methyl-CpG-binding protein) in the regions might participate in the transcriptional regulation of these two genes.

Molecular characterization and expression profiles of GATA6 in tongue sole (Cynoglossus semilaevis).

GATA-binding protein 6 (GATA6), a transcription factor of the GATA family, plays an important role in gonadal cell proliferation, differentiation, and endoderm development. In this study, the full-length coding sequence of tongue sole (Cynoglossus semilaevis) GATA6 was identified. The sequence consisted of 1494 nucleotides encoding a peptide of 497 amino acids, which included two conserved zinc finger domains. Phylogenetic, gene structure, and synteny analysis showed that C. semilaevis GATA6 was homologous to teleost and tetrapod GATA6. C. semilaevis GATA6 mRNA exhibited high expression in heart, intestine, liver, kidney, and gonad. Embryonic development expression profiles revealed that GATA6 is involved in morphogenesis because its expression increased at the blastula stage. The in situ hybridization results showed strong GATA6 signals in spermatogonia, spermatocytes, and Sertoli cells of the testis. The signals were also detected in the oogonia and oocytes of the ovary. The expression of C. semilaevis GATA6 was sexually dimorphic, and the methylation pattern in the promoter region varied among males, females, and pseudomales. These results suggested that GATA6 might influence the gonad development and reproduction of C. semilaevis. This study provides the groundwork for further development of breeding techniques in C. semilaevis.

Molecular characterization and functional analysis of the GATA4 in tongue sole (Cynoglossus semilaevis).

The GATA family of transcription factors is characterized by two zinc finger domains and is involved in different cellular processes. GATA4 is a highly conserved transcription factor that regulates embryonic morphogenesis and cellular differentiation. GATA4 in vertebrates regulates its target genes to influence genital ridge differentiation. In this study, the GATA4 from tongue sole (Cynoglossus semilaevis) was characterized to understand the function of this transcription factor in sex differentiation. The full-length cDNA of C. semilaevis GATA4 comprised 2031bp, encoding a predicted polypeptide consisting of 402 amino acids with two conserved zinc finger domains. Phylogenetic, gene structure, and synteny analyses showed that C. semilaevis GATA4 was homologous to tetrapod GATA4. The mRNA transcript of C. semilaevis GATA4 exhibited high expression in the heart, liver, and gonad. GATA4 expression is dimorphic in the male and female gonads. Embryonic development expression profiles revealed the possible involvement of C. semilaevis GATA4 in morphogenesis. In situ hybridization results showed strong GATA4 signals in the spermatogonia and spermatocytes of the testis and in the oogonia, primary oocytes, and secondary oocytes of the ovary. The expression of C. semilaevis GATA4 in the male, pseudomale, and female gonads showed significantly different methylation levels of the two CpG sites (-2738 and -2647) among the three genders. Basing on these results, we speculated that GATA4 plays a potential role in sex differentiation. This study lays the groundwork for further sex control breeding techniques in C. semilaevis.

Maternally derived trypsin may have multiple functions in the early development of turbot (Scopthalmus maximus).

Trypsin is an important serine protease that is considered to be involved in digestion of protein in teleost fish. Nevertheless, studies on trypsin/trypsinogen in fish embryos are very limited. In this study, the trypsinogen of turbot (Scophthalmus maximus) (tTG) was identified and the expression patterns and activity of trypsinogen/trypsin were investigated. The results showed that the tTG mRNA was evenly distributed in the oocytes and was also expressed along the yolk periphery in early embryos. At later embryo stages and 1 days after hatching (dph), the tTG mRNA concentrated at the alimentary tract and head. Quantitative expression analysis showed that the tTG transcripts decreased after fertilization until the gastrula stage, then increased with the embryo and larvae development. This result was also confirmed by the specific activity analysis of trypsin and in-situ-hybridization (ISH). All of the results indicated that tTG in early embryo stages was maternally derived and expressed by itself after gastrula stages. Additionally, location of tTG mRNA in embryos and larvae was investigated; we considered that trypsin may have multiple functions during the embryo development process. Based on our results regarding trypsinogen in embryos and early development, we concluded that the trypsin/trypsinogen in turbot embryos was inherited from a maternal source and we suggested that trypsin in early development has multiple functions in the process of development.

Morphogenesis of the saccus vasculosus of turbot Scophthalmus maximus: assessment of cell proliferation and distribution of parvalbumin and calretinin during ontogeny.

The ontogenesis of the saccus vasculosus (SV) of turbot Scophthalmus maximus is described using histological and immunohistochemical methods to assess the general morphology, as well as the distribution of proliferative cells and several calcium-binding proteins (CaBP). The results reveal that the SV begins to differentiate on hatching, when immature coronet cells are morphologically distinguishable. Further morphogenesis involves the formation of a tubular avascular SV, which remains until premetamorphic larval stages. Folding and vascularization of the SV occurs mostly during metamorphosis, when S. maximus settle down on the bottom. Proliferative cells were placed within the SV itself and in the neighbouring infundibular hypothalamus. Their putative relationship with the growth of the SV is discussed. The CaBPs analysed are expressed in coronet cells. Parvalbumin is expressed in these cells from the beginning of their differentiation, while calretinin expression arises in the tubular SV and becomes more widespread over time. These data emphasize the importance of calcium buffering in the function of coronet cells.

Distinct expression profiles of three melatonin receptors during early development and metamorphosis in the flatfish Solea senegalensis.

Melatonin actions are mediated through G protein-coupled transmembrane receptors. Recently, mt1, mt2, and mel1c melatonin receptors were cloned in the Senegalese sole. Here, their day-night and developmental expressions were analyzed by quantitative PCR. These results revealed distinct expression patterns of each receptor through development. mel1c transcripts were more abundant in unfertilized ovulated oocytes and declined during embryonic development. mt1 and mt2 expression was higher at the earliest stages (2-6 days post-fertilization), decreasing before (mt2) or during (mt1) metamorphosis. Only mt1 and mel1c expression exhibited day-night variations, with higher nocturnal mRNA levels. These results suggest different roles and transcriptional regulation of these melatonin receptors during flatfish development and metamorphosis.

Sexually dimorphic expression of vasa isoforms in the tongue sole (Cynoglossus semilaevis).

The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding.

Circadian rhythms of embryonic development and hatching in fish: a comparative study of zebrafish (diurnal), Senegalese sole (nocturnal), and Somalian cavefish (blind).

During early development, most organisms display rhythmic physiological processes that are shaped by daily changes in their surrounding environment (i.e., light and temperature cycles). In fish, the effects of daily photocycles and their interaction with temperature during early developmental stages remain largely unexplored. We investigated the existence of circadian rhythms in embryonic development and hatching of three teleost species with different daily patterns of behavior: diurnal (zebrafish), nocturnal (Senegalese sole), and blind, not entrained by light (Somalian cavefish). To this end, fertilized eggs were exposed to three light regimes: 12 h of light: 12 h of darkness cycle (LD), continuous light (LL), or continuous darkness (DD); and three species-appropriate temperature treatments: 24°C, 28°C, or 32°C for zebrafish and cavefish and 18°C, 21°C, or 24°C for sole. The results pointed to the existence of daily rhythms of embryonic development and hatching synchronized to the LD cycle, with different acrophases, depending on the species: zebrafish embryos advanced their developmental stage during the light phase, whereas sole did so during the dark phase. In cavefish, embryogenesis occurred within 24 h post fertilization (hpf) at the same pace during day or night. The hatching rhythms appeared to be controlled by a clock mechanism that restricted or "gated" hatching to a particular time of day/night (window), so that embryos that reached a certain developmental state by that time hatch, whereas those that have not wait until the next available window. Under LL and DD conditions, hatching rhythms and the gating phenomenon persisted in cavefish, in zebrafish they split into ultradian bouts of hatching occurring at 12-18-h intervals, whereas in sole DD and LL produced a 24-h delay and advance, respectively. Hatching rates were best under the LD cycle and the reported optimal temperature for each species (95.2±2.7% of the zebrafish and 83.3±0.1% of the cavefish embryos hatched at 28°C, and 93.1±2.9% of the sole embryos hatched at 21°C). In summary, these results revealed that hatching rhythms in fish are endogenously driven by a time-keeping mechanism, so that the day and time of hatching are determined by the interplay between the developmental state (temperature-sensitive) and the circadian clock (temperature-compensated), with the particular phasing being determined by the diurnal/nocturnal behavior of the species.

Analysis of the morphogenesis and cell proliferation in the retina of a pleuronectiform fish, the turbot Psetta maxima (Pleuronectiformes: Teleostei).

The morphogenesis and cell proliferation in the retina of turbot (Psetta maxima, Pleuronectiformes: Teleostei) from embryo through metamorphosis have been examined by using proliferating cell nuclear antigen (PCNA)-immunohistochemistry and general histological procedures. In the embryonic retina, cell proliferation and spatial cell reorganization form the anlage of the pigment epithelium and neural retina. Neurogenesis begins around hatching in the temporal retina, dorsal to the optic nerve exit, and then a wave of cell differentiation spreads to the nasal retina to yield a laminated retina by the end of the prolarval turbot stage. Germinal zones in the differentiated retina persist as a rim at the retinal margin, as well as surrounding the optic fissure in premetamorphic and metamorphic turbot larvae. In these zones, progenitor cells with different morphologies show a similar spatial arrangement, which suggests that they have a similar retinogenic potential. During metamorphosis, asymmetric proliferative activity in turbot germinal zones is associated with a marked expansion of the retinal tissue. Scattered stem cells in the laminated retina, related to the lineage of rod photoreceptors, were also observed both in large premetamorphic larvae and metamorphic turbots. The proliferative activity of these cells increases considerably during metamorphosis, when rod photoreceptors become morphologically differentiated.

Cloning, genomic structure and expression analysis of ubc9 in the course of development in the half-smooth tongue sole (Cynoglossus semilaevis).

The small ubiquitin-like modifier (SUMO) pathway is an essential biological process in eukaryote, and Ubc9 is an important E2 conjugating enzyme (UBE2) for SUMO pathway and plays a critical role in cellular differentiation, development and sex modification in various species. However, the relationship between Ubc9 and sex modification and development in fish remains elusive. To elucidate the impact of Ubc9 on sex modification and development, the full length of the cDNA and genomic sequence was cloned from half-smooth tongue sole, Cynoglossus semilaevis. Real-time quantitative RT-PCR demonstrated that ubc9 was ubiquitously expressed in different tissues, and the expression levels varied in the different stages of embryonic and gonadal development. In addition, the expression level was significantly higher in the temperature-treated females than the normal females and males. Moreover, the PET-32-Ubc9 plasmid was constructed and the recombinant protein was expressed in Escherichia coli. Follistatin gene expression was initially up-regulated and FSE genes (cyp19a1a, ctnnb1, foxl2) were initially down-regulated after the injection of Ubc9 protein, prior to 96 h eventually recovered to normal levels. Taken together, the results show that Ubc9 is involved in embryogenesis, gametogenesis and sex modification, and exerts an effect on gene expression.

Incubation temperature induces changes in muscle cellularity and gene expression in Senegalese sole (Solea senegalensis).

Fertilised eggs of Senegalese sole were incubated at 15, 18 or 21 °C, and after hatching all larvae were reared at 21 °C until 30 days post-hatch. By this point larvae from the 18 or 21 °C temperature groups had 11 and 9% more muscle fibres than those from 15 °C, respectively. Hyperplastic growth during metamorphosis was higher in larvae from 18 °C. Embryonic temperature induced gene expression changes, albeit with a variable pattern throughout development. Myf5, myod2, myHC and fst mRNA levels were significantly higher at several stages prior to hatching in embryos incubated at 21 °C, whereas hsp90AB and hsp70 transcripts were present at higher levels in the 15 °C group. Myf5, myod1, myod2, pax7, myog, fst, igf-II, igf1r, hsp90AA and hsp90AB were expressed at higher levels during early development, particularly during somitogenesis. In contrast, mrf4, myHC, mylc2, igf-I, mstn1 and hsp70 were up-regulated at later stages of larval development, namely during and after metamorphosis. This study is the first example of thermal plasticity of myogenesis with prolonged effect in a flatfish.