Embryonic development of the bullseye puffer Sphoeroides annulatus (Tetraodontidae): A morphofunctional approach to ontogenetic steps.

The embryonic development of the bullseye puffer, Sphoeroides annulatus, was characterized on the basis of the theory of saltatory ontogeny. This theory predicts a correlative relationship between the ontogeny-type in an altricial-precocial spectrum and the habitat that a species occupies within an unstable-stable environmental spectrum. Because S. annulatus inhabits a variety of unstable environments along a wide latitudinal range, the hypothesis that this species presents one of the most altricial embryonic developments among tetraodontids was tested. Based on major developmental events that marked the ontogenetic thresholds nine embryonic steps were identified. Developmental features such as small adhesives eggs, lack of vitelline circulation, small free embryos swimming up at hatching guided by positive phototaxis, and small first-feeding larvae actively swam in the water column, suggest that S. annulatus belongs to the reproductive guild of the nonguarders-lithopelagophils. Moreover, a comparative analysis of the developmental sequences, egg size, and first-feeding larvae size between tetraodontids confirms the hypothesis of this study and supports the evolutionary principle of the altricial-precocial spectrum postulated in the theory of saltatory ontogeny.

Differential expression of two Piwil orthologs during embryonic and gonadal development in pufferfish, Takifugu fasciatus.

Piwil was an important regulator gene in germ cell division during gonadal development. Two Piwi-like genes, Piwil1 and Piwil2, were first cloned from T. fasciatus. The full-length cDNAs of Piwil1 and Piwil2 were of 2933 and 3394 bp, respectively. Piwil1 and Piwil2 possessed an open reading frame (ORF) of 2565 and 3138 bp, encoding 854 and 1045 amino acids, respectively. The tissue distribution analysis demonstrated that Piwil1 and Piwil2 were expressed at higher levels in gonad compared to other tissues (brain, liver, gill, etc.). The time-course dynamic expressions of Piwils during embryonic indicated that Piwil1 and Piwil2 were mainly enriched in the early embryonic development. In testis, the expression of Piwil1 and Piwil2 increased at first but then decreased at mRNA and protein levels. However, the expression of Piwil1 and Piwil2 in the ovary showed a downward trend from the beginning. In addition, the expression levels of Piwil1 and Piwil2 were weak in mature testes or ovaries. The immunohistochemistry analysis revealed that Piwil1 and Piwil2 were abundantly expressed in cytoplasm of spermatogonia, spermatocytes, oocyte I and oocytes II, which were mainly presented in the early stages of gonadal development. Our results suggested that Piwil was related to the differentiation of germ cells, and might play an important role in embryonic development. Therefore, our findings provided valuable information of Piwils in the reproductive cycle of T. fasciatus.

Anterior migration of lateral plate mesodermal cells during embryogenesis of the pufferfish Takifugu niphobles: insight into the rostral positioning of pelvic fins.

In vertebrates, paired appendages (limbs and fins) are derived from the somatic mesoderm subsequent to the separation of the lateral plate mesoderm into somatic and splanchnic layers. This is less clear for teleosts, however, because the developmental processes of separation into two layers and of extension over the yolk have rarely been studied. During teleost evolution, the position of pelvic fins has generally shifted rostrally (Rosen; Nelson, 1982, 1994), although at the early embryonic stage the presumptive pelvic fin cells are initially located near the future anus region - the anterior border of hoxc10a expression in the spinal cord - regardless of their final destination. Our previous studies in zebrafish (abdominal pelvic fins) and Nile tilapia (thoracic pelvic fins) showed that the presumptive pelvic fin cells shift their position with respect to the body trunk after its protrusion from the yolk surface. Furthermore, in Nile tilapia, presumptive pelvic fin cells migrate anteriorly on the yolk surface. Here, we examined the embryonic development of the lateral plate mesoderm at histological levels in the pufferfish Takifugu niphobles, which belongs to the highly derived teleost order Tetraodontiformes, and lacks pelvic fins. Our results show that, in T. niphobles, the lateral plate mesoderm bulges out as two separate layers of cells alongside the body trunk prior to its further extension to cover the yolk sphere. Once the lateral plate mesoderm extends laterally, it rapidly covers the surface of the yolk. Furthermore, cells located near the anterior border of hoxc10a expression in the spinal cord reach the anterior-most region of the yolk surface. In light of our previous and current studies, we propose that anterior migration of presumptive pelvic fin cells might be required for them to reach the thoracic or more anterior positions as is seen in other highly derived teleost groups.

Description of embryonic development of spotted green pufferfish (Tetraodon nigroviridis).

Pufferfish species of the Tetraodontidae family carry the smallest genomes among vertebrates. Their compressed genomes are thought to be enriched for functional DNA compared to larger vertebrate genomes, and they are important models for comparative genomics. The significance of pufferfish as model organisms in comparative genomics is due to the availability of two sequenced genomes, that of spotted green pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes). However, there is only a very limited utilization of pufferfish as an experimental model organism, due to the lack of established husbandry and developmental genetics protocols. In this study, we provide the first description of the normal embryonic development of Tetraodon nigroviridis. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored by brightfield microscopy at constant temperature. Tetraodon development was divided into distinct stages based on diagnostic morphological features, which were adopted from published literature on normal development of other fish species like medaka (Oryzias latipes), zebrafish (Danio rerio), and fugu. Tetraodon embryos show more similar morphologies to medaka than to zebrafish, reflecting its phylogenetic position. The early developmental stage series described in this study forms the foundation for the utilization of tetraodon as an experimental model organism for comparative developmental studies.

Replacing the first-generation dentition in pufferfish with a unique beak.

Teleost fishes comprise approximately half of all living vertebrates. The extreme range of diversity in teleosts is remarkable, especially, extensive morphological variation in their jaws and dentition. Some of the most unusual dentitions are found among members of the highly derived teleost order Tetraodontiformes, which includes triggerfishes, boxfishes, ocean sunfishes, and pufferfishes. Adult pufferfishes (Tetraodontidae) exhibit a distinctive parrot-like beaked jaw, forming a cutting edge, unlike in any other group of teleosts. Here we show that despite novelty in the structure and development of this "beak," it is initiated by formation of separate first-generation teeth that line the embryonic pufferfish jaw, with timing of development and gene expression patterns conserved from the last common ancestor of osteichthyans. Most of these first-generation larval teeth are lost in development. Continuous tooth replacement proceeds in only four parasymphyseal teeth, as sequentially stacked, multigenerational, jaw-length dentine bands, before development of the functional beak. These data suggest that dental novelties, such as the pufferfish beak, can develop later in ontogeny through modified continuous tooth addition and replacement. We conclude that even highly derived morphological structures like the pufferfish beak form via a conserved developmental bauplan capable of modification during ontogeny by subtle respecification of the developmental module.

Toxicity of cultured bullseye puffer fish Sphoeroides annulatus.

The toxin content in various life cycle stages of tank-cultivated bullseye puffer (Sphoeroides annulatus) were analyzed by mouse bioassay and ESI-MS spectrometry analysis. The presence of toxin content was determined in extracts of sperm, eggs, embryo, larvae, post-larvae, juvenile, pre-adult, and adult fish, as well as in food items used during the cultivation of the species. Our findings show that only the muscle of juveniles, the viscera of pre-adults, and muscle, liver, and gonad of adult specimens were slightly toxic (<1 mouse unit). Thus, cultivated S. annulatus, as occurs with other cultivated puffer fish species, does not represent a food safety risk to consumers. This is the first report of toxin analysis covering the complete life stages of a puffer fish under controlled conditions.

The calcium channel beta2 (CACNB2) subunit repertoire in teleosts.

BACKGROUND: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts. RESULTS: Cloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult. CONCLUSION: These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

Characterization of hey bHLH genes in teleost fish.

Hairy-related basic helix-loop-helix (bHLH) transcription factors are targets of Delta-Notch signaling and represent essential components for a number of cell fate decisions during vertebrate embryogenesis. Hey genes encode a subfamily of hairy-related proteins that have been implicated in processes like somitogenesis, blood vessel and heart development. We have identified and characterized hey genes in three teleost fish lineages using degenerate PCR and database searches. Phylogenetic analysis of Hey proteins suggests a complex pattern of evolution with high divergence of hey2 in Takifugu rubripes (Fugu, Japanese pufferfish) and possibly loss in the related Tetraodon nigroviridis (the freshwater pufferfish). In addition, duplication of hey1 in both pufferfishes, Fugu and Tetraodon, was observed. Conversely, zebrafish (Danio rerio) has the same complement of three hey genes as known from mammals. All three hey genes show much more restricted gene expression profiles in zebrafish when compared to mouse. Importantly, while all three murine Hey genes are expressed in overlapping patterns in the presomitic mesoderm (PSM) and somites, in zebrafish only hey1 shows PSM and somite expression in a highly dynamic fashion. Therefore, while overlapping expression might account for redundancy of hey function in higher vertebrates, this is unlikely to be the case in zebrafish. In deltaD (dlD) deficient after-eight zebrafish mutants, the dynamic expression of hey1 in the PSM is impaired and completely lost in newly formed somitomeres. Overexpression of dlD on the other hand results in the ectopic expression of hey1 in the axial mesoderm. Hence, hey1 represents a target of Delta-Notch signaling dynamically expressed during somite formation in zebrafish.