Pituitary multi-hormone cells in mammals and fish: history, origin, and roles.

The vertebrate pituitary is a dynamic organ, capable of adapting its hormone secretion to different physiological demands. In this context, endocrinologists have debated for the past 40 years if endocrine cells are mono- or multi-hormonal. Since its establishment, the dominant "one cell, one hormone" model has been continuously challenged. In mammals, the use of advanced multi-staining approaches, sensitive gene expression techniques, and the analysis of tumor tissues have helped to quickly demonstrate the existence of pituitary multi-hormone cells. In fishes however, only recent advances in imaging and transcriptomics have enabled the identification of such cells. In this review, we first describe the history of the discovery of cells producing multiple hormones in mammals and fishes. We discuss the technical limitations that have led to uncertainties and debates. Then, we present the current knowledge and hypotheses regarding their origin and biological role, which provides a comprehensive review of pituitary plasticity.

Functional and developmental heterogeneity of pituitary lactotropes in medaka.

In fish, prolactin-producing cells (lactotropes) are located in the anterior part of the pituitary and play an essential role in osmoregulation. However, small satellite lactotrope clusters have been described in other parts of the pituitary in several species. The functional and developmental backgrounds of these satellite clusters are not known. We recently discovered two distinct prolactin-expressing cell types in Japanese medaka (Oryzias latipes), a euryhaline species, using single cell transcriptomics. In the present study, we characterize these two transcriptomically distinct lactotrope cell types and explore the hypothesis that they represent spatially distinct cell clusters, as found in other species. Single cell RNA sequencing shows that one of the two lactotrope cell types exhibits an expression profile similar to that of stem cell-like folliculo-stellate cell populations. Using in situ hybridization, we show that the medaka pituitary often develops additional small satellite lactotrope cell clusters, like in other teleost species. These satellite clusters arise early during development and grow in cell number throughout life regardless of the animal's sex. Surprisingly, our data do not show a correspondence between the stem cell-like lactotropes and these satellite lactotrope clusters. Instead, our data support a scenario in which the stem cell-like lactotropes are an intrinsic stage in the development of every spatially distinct lactotrope cluster. In addition, lactotrope activity in both spatially distinct lactotrope clusters decreases when environmental salinity increases, supporting their role in osmoregulation. However, this decrease appears weaker in the satellite lactotrope cell clusters, suggesting that these lactotropes are regulated differently.

The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process.

Changes in ovarian gene expression profiles and plasma hormone levels in maturing European eel (Anguilla anguilla); Biomarkers for broodstock selection.

Complete sexual maturation of European eels (Anguilla anguilla) in captivity can only be achieved via injections with gonadotropins. For female eels this procedure takes 4-6months and the response ranges from "unresponsive" to final maturation and ovulation. Reproductive success could be significantly increased via early selection of responders based on predictive markers and minimally invasive sampling methods. To get a better understanding of the genetic background of ovarian maturation of the European eel we performed a pilot deep-sequencing transcriptome analysis of ovarian tissue derived from a yellow eel, a prepubertal silver eel and a post-spawning matured eel. Two key players in steroidogenesis were strongly correlated with advanced sexual maturation, namely P450c17 and liver receptor homolog-1, suggesting that blood plasma steroids might qualify as minimally invasive markers for early detection of responders. Since the predictive value of plasma sex steroid levels for final maturation of the European eel had not yet been carefully examined, we performed an extensive artificial maturation trial. Farmed silver eels were treated with pituitary extracts and sampled at multiple time intervals. Expression of steroidogenesis-related genes in ovarian tissue of responding and non-responding eels after four weekly injections with pituitary extract was compared using a custom-built microarray and RNAseq. Increased expression of 17ß-hsd1 was strongly linked to sexual maturation. Blood plasma levels of sex steroids were measured using ELISAs. We show that a 2.5-fold increase in blood-plasma estradiol level after 4 weekly pituitary extract injections is a strong predictor of final sexual maturation of female European eel.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

The Burmese python genome reveals the molecular basis for extreme adaptation in snakes.

Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

Using normalization to resolve RNA-Seq biases caused by amplification from minimal input.

RNA-Seq has become a widely used method to study transcriptomes, and it is now possible to perform RNA-Seq on almost any sample. Nevertheless, samples obtained from small cell populations are particularly challenging, as biases associated with low amounts of input RNA can have strong and detrimental effects on downstream analyses. Here we compare different methods to normalize RNA-Seq data obtained from minimal input material. Using RNA from isolated medaka pituitary cells, we have amplified material from six samples before sequencing. Both synthetic and real data are used to evaluate different normalization methods to obtain a robust and reliable pipeline for analysis of RNA-Seq data from samples with very limited input material. The analysis outlined here shows that quantile normalization outperforms other more commonly used normalization procedures when using amplified RNA as input and will benefit researchers employing low amounts of RNA in similar experiments.

Day length regulates gonadotrope proliferation and reproduction via an intra-pituitary pathway in the model vertebrate Oryzias latipes.

In seasonally breeding mammals and birds, the production of the hormones that regulate reproduction (gonadotropins) is controlled by a complex pituitary-brain-pituitary pathway. Indeed, the pituitary thyroid-stimulating hormone (TSH) regulates gonadotropin expression in pituitary gonadotropes, via dio2-expressing tanycytes, hypothalamic Kisspeptin, RFamide-related peptide, and gonadotropin-releasing hormone neurons. However, in fish, how seasonal environmental signals influence gonadotropins remains unclear. In addition, the seasonal regulation of gonadotrope (gonadotropin-producing cell) proliferation in the pituitary is, to the best of our knowledge, not elucidated in any vertebrate group. Here, we show that in the vertebrate model Japanese medaka (Oryzias latipes), a long day seasonally breeding fish, photoperiod (daylength) not only regulates hormone production by the gonadotropes but also their proliferation. We also reveal an intra-pituitary pathway that regulates gonadotrope cell number and hormone production. In this pathway, Tsh regulates gonadotropes via folliculostellate cells within the pituitary. This study suggests the existence of an alternative regulatory mechanism of seasonal gonadotropin production in fish.

An RNA-seq time series of the medaka pituitary gland during sexual maturation.

Directing both organismal homeostasis and physiological adaptation, the pituitary is a key endocrine gland in all vertebrates. One of its major tasks is to coordinate sexual maturation through the production and release of hormones stimulating gonad development. In order to study its developmental dynamics in the model fish medaka (Oryzias latipes), we sampled both the pituitary and the ovaries of 68 female fish. Of these, 55 spanned the entire course of sexual maturation from prepubertal juveniles to spawning adults. An additional 13 showed either considerably faster or slower growth and development than the majority of fish. We used histological examination of the ovaries to determine a histological maturation stage, and analyzed the pituitary glands using RNA-seq optimized for low input. Taken together, these data reveal the timing of hormone production priorities, and form a comprehensive resource for the study of their regulation.

Genome structures resolve the early diversification of teleost fishes.

Accurate species phylogenies are a prerequisite for all evolutionary research. Teleosts are the largest and most diversified group of extant vertebrates, but relationships among their three oldest extant lineages remain unresolved. On the basis of seven high-quality new genome assemblies in Elopomorpha (tarpons, eels), we revisited the topology of the deepest branches of the teleost phylogeny using independent gene sequence and chromosomal rearrangement phylogenomic approaches. These analyses converged to a single scenario that unambiguously places the Elopomorpha and Osteoglossomorpha (arapaima, elephantnose fish) in a monophyletic sister group to all other teleosts, i.e., the Clupeocephala lineage (zebrafish, medaka). This finding resolves more than 50 years of controversy on the evolutionary relationships of these lineages and highlights the power of combining different levels of genome-wide information to solve complex phylogenies.

Scaffolding pre-assembled contigs using SSPACE.

SUMMARY: De novo assembly tools play a main role in reconstructing genomes from next-generation sequencing (NGS) data and usually yield a number of contigs. Using paired-read sequencing data it is possible to assess the order, distance and orientation of contigs and combine them into so-called scaffolds. Although the latter process is a crucial step in finishing genomes, scaffolding algorithms are often built-in functions in de novo assembly tools and cannot be independently controlled. We here present a new tool, called SSPACE, which is a stand-alone scaffolder of pre-assembled contigs using paired-read data. Main features are: a short runtime, multiple library input of paired-end and/or mate pair datasets and possible contig extension with unmapped sequence reads. SSPACE shows promising results on both prokaryote and eukaryote genomic testsets where the amount of initial contigs was reduced by at least 75%.

Deep sequencing of the innate immune transcriptomic response of zebrafish embryos to Salmonella infection.

Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethal infection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptome level, we have extended and validated previous microarray data by Illumina next-generation sequencing analysis. We obtained 10 million sequence reads from control and Salmonella-infected zebrafish embryos using a tag-based sequencing method (DGE or Tag-Seq) and 15 million reads using whole transcript sequencing (RNA-Seq), which respectively mapped to circa 65% and 85% of 28,716 known Ensembl transcripts. Both sequencing methods showed a strong correlation of sequence read counts per transcript and an overlap of 241 transcripts differentially expressed in response to infection. A lower overlap of 165 transcripts was observed with previous microarray data. Based on the combined sequencing-based and microarray-based transcriptome data we compiled an annotated reference set of infection-responsive genes in zebrafish embryos, encoding transcription factors, signal transduction proteins, cytokines and chemokines, complement factors, proteins involved in apoptosis and proteolysis, proteins with anti-microbial activities, as well as many known or novel proteins not previously linked to the immune response. Furthermore, by comparison of the deep sequencing data of S. typhimurium infection in zebrafish embryos with previous deep sequencing data of Mycobacterium marinum infection in adult zebrafish we derived a common set of infection-responsive genes. This gene set consists of known and putative innate host defense genes that are expressed both in the absence and presence of a fully developed adaptive immune system and that provide a valuable reference for future studies of host-pathogen interactions using zebrafish infection models.

Characterization of hormone-producing cell types in the teleost pituitary gland using single-cell RNA-seq.

The pituitary is the vertebrate endocrine gland responsible for the production and secretion of several essential peptide hormones. These, in turn, control many aspects of an animal's physiology and development, including growth, reproduction, homeostasis, metabolism, and stress responses. In teleost fish, each hormone is presumably produced by a specific cell type. However, key details on the regulation of, and communication between these cell types remain to be resolved. We have therefore used single-cell sequencing to generate gene expression profiles for 2592 and 3804 individual cells from the pituitaries of female and male adult medaka (Oryzias latipes), respectively. Based on expression profile clustering, we define 15 and 16 distinct cell types in the female and male pituitary, respectively, of which ten are involved in the production of a single peptide hormone. Collectively, our data provide a high-quality reference for studies on pituitary biology and the regulation of hormone production, both in fish and in vertebrates in general.

Eye-Transcriptome and Genome-Wide Sequencing for Scolecophidia: Implications for Inferring the Visual System of the Ancestral Snake.

Molecular genetic data have recently been incorporated in attempts to reconstruct the ecology of the ancestral snake, though this has been limited by a paucity of data for one of the two main extant snake taxa, the highly fossorial Scolecophidia. Here we present and analyze vision genes from the first eye-transcriptomic and genome-wide data for Scolecophidia, for Anilios bicolor, and A. bituberculatus, respectively. We also present immunohistochemistry data for retinal anatomy and visual opsin-gene expression in Anilios. Analyzed in the context of 19 lepidosaurian genomes and 12 eye transcriptomes, the new genome-wide and transcriptomic data provide evidence for a much more reduced visual system in Anilios than in non-scolecophidian (=alethinophidian) snakes and in lizards. In Anilios, there is no evidence of the presence of 7 of the 12 genes associated with alethinophidian photopic (cone) phototransduction. This indicates extensive gene loss and many of these candidate gene losses occur also in highly fossorial mammals with reduced vision. Although recent phylogenetic studies have found evidence for scolecophidian paraphyly, the loss in Anilios of visual genes that are present in alethinophidians implies that the ancestral snake had a better-developed visual system than is known for any extant scolecophidian.

3D Atlas of the Pituitary Gland of the Model Fish Medaka (Oryzias latipes).

In vertebrates, the anterior pituitary plays a crucial role in regulating several essential physiological processes via the secretion of at least seven peptide hormones by different endocrine cell types. Comparative and comprehensive knowledge of the spatial distribution of those endocrine cell types is required to better understand their physiological functions. Using medaka as a model and several combinations of multi-color fluorescence in situ hybridization, we present the first 3D atlas revealing the gland-wide distribution of seven endocrine cell populations: lactotropes, thyrotropes, Lh and Fsh gonadotropes, somatotropes, and pomca-expressing cells (corticotropes and melanotropes) in the anterior pituitary of a teleost fish. By combining in situ hybridization and immunofluorescence techniques, we deciphered the location of corticotropes and melanotropes within the pomca-expressing cell population. The 3D localization approach reveals sexual dimorphism of tshba-, pomca-, and lhb-expressing cells in the adult medaka pituitary. Finally, we show the existence of bi-hormonal cells co-expressing lhb-fshb, fshb-tshba and lhb-sl using single-cell transcriptomics analysis and in situ hybridization. This study offers a solid basis for future comparative studies of the teleost pituitary and its functional plasticity.

Unravelling the changes during induced vitellogenesis in female European eel through RNA-Seq: What happens to the liver?

The life cycle of European eel (Anguilla anguilla), a catadromous species, is complex and enigmatic. In nature, during the silvering process prior to their long spawning migration, reproductive development is arrested, and they cease feeding. In studies of reproduction using hormonal induction, eels are equivalently not feed. Therefore, in female eels that undergo vitellogenesis, the liver plays different, essential roles being involved both in vitellogenins synthesis and in reallocating resources for the maintenance of vital functions, performing the transoceanic reproductive migration and completing reproductive development. The present work aimed at unravelling the major transcriptomic changes that occur in the liver during induced vitellogenesis in female eels. mRNA-Seq data from 16 animals (eight before induced vitellogenesis and eight after nine weeks of hormonal treatment) were generated and differential expression analysis was performed comparing the two groups. This analysis detected 1,328 upregulated and 1,490 downregulated transcripts. Overrepresentation analysis of the upregulated genes included biological processes related to biosynthesis, response to estrogens, mitochondrial activity and localization, while downregulated genes were enriched in processes related to morphogenesis and development of several organs and tissues, including liver and immune system. Among key genes, the upregulated ones included vitellogenin genes (VTG1 and VTG2) that are central in vitellogenesis, together with ESR1 and two novel genes not previously investigated in European eel (LMAN1 and NUPR1), which have been linked with reproduction in other species. Moreover, several upregulated genes, such as CYC1, ELOVL5, KARS and ACSS1, are involved in the management of the effect of fasting and NOTCH, VEGFA and NCOR are linked with development, autophagy and liver maintenance in other species. These results increase the understanding of the molecular changes that occur in the liver during vitellogenesis in this complex and distinctive fish species, bringing new insights on European eel reproduction and broodstock management.

Swimming exercise enhances brain plasticity in fish.

It is well-established that sustained exercise training can enhance brain plasticity and boost cognitive performance in mammals, but this phenomenon has not received much attention in fish. The aim of this study was to determine whether sustained swimming exercise can enhance brain plasticity in juvenile Atlantic salmon. Brain plasticity was assessed by both mapping the whole telencephalon transcriptome and conducting telencephalic region-specific microdissections on target genes. We found that 1772 transcripts were differentially expressed between the exercise and control groups. Gene ontology (GO) analysis identified 195 and 272 GO categories with a significant overrepresentation of up- or downregulated transcripts, respectively. A multitude of these GO categories was associated with neuronal excitability, neuronal signalling, cell proliferation and neurite outgrowth (i.e. cognition-related neuronal markers). Additionally, we found an increase in proliferating cell nuclear antigen (pcna) after both three and eight weeks of exercise in the equivalent to the hippocampus in fish. Furthermore, the expression of the neural plasticity markers synaptotagmin (syt) and brain-derived neurotrophic factor (bdnf) were also increased due to exercise in the equivalent to the lateral septum in fish. In conclusion, this is the first time that swimming exercise has been directly linked to increased telencephalic neurogenesis and neural plasticity in a teleost, and our results pave the way for future studies on exercise-induced neuroplasticity in fish.

Author Correction: Rapid de novo assembly of the European eel genome from nanopore sequencing reads.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

DNA computing of solutions to knapsack problems.

One line of DNA computing research focuses on parallel search algorithms, which can be used to solve many optimization problems. DNA in solution can provide an enormous molecular library, which can be searched by molecular biological techniques. We have implemented such a parallel search for solutions to knapsack problems, which ask for the best way to pack a knapsack of limited volume. Several instances of knapsack problems were solved using DNA. We demonstrate how the computations can be extended by in vivo translation of the DNA library into protein. This combination of DNA and protein allows for multi-criterion optimization. The knapsack computations performed can then be seen as protein optimizations, one of the most complex computations performed by natural systems.

Enhancement of Arabidopsis growth characteristics using genome interrogation with artificial transcription factors.

The rapidly growing world population has a greatly increasing demand for plant biomass, thus creating a great interest in the development of methods to enhance the growth and biomass accumulation of crop species. In this study, we used zinc finger artificial transcription factor (ZF-ATF)-mediated genome interrogation to manipulate the growth characteristics and biomass of Arabidopsis plants. We describe the construction of two collections of Arabidopsis lines expressing fusions of three zinc fingers (3F) to the transcriptional repressor motif EAR (3F-EAR) or the transcriptional activator VP16 (3F-VP16), and the characterization of their growth characteristics. In total, six different 3F-ATF lines with a consistent increase in rosette surface area (RSA) of up to 55% were isolated. For two lines we demonstrated that 3F-ATF constructs function as dominant in trans acting causative agents for an increase in RSA and biomass, and for five larger plant lines we have investigated 3F-ATF induced transcriptomic changes. Our results indicate that genome interrogation can be used as a powerful tool for the manipulation of plant growth and biomass and that it might supply novel cues for the discovery of genes and pathways involved in these properties.