Deep transcriptome analysis of the heat shock response in an Atlantic sturgeon (Acipenser oxyrinchus) cell line.

Despite efforts to restore Atlantic sturgeon in European rivers, aquaculture techniques result in animals with high post-release mortality due to, among other reasons, their low tolerance to increasing water temperature. Marker genes to monitor heat stress are needed in order to identify heat-resistant fish. Therefore, an Atlantic sturgeon cell line was exposed to different heat shock protocols (30 °C and 35 °C) and differences in gene expression were investigated. In total 3020 contigs (∼1.5%) were differentially expressed. As the core of the upregulated contigs corresponded to heat shock proteins (HSP), the heat shock factor (HSF) and the HSP gene families were annotated in Atlantic sturgeon and mapped via Illumina RNA sequencing to identify heat-inducible family members. Up to 6 hsf and 76 hsp genes were identified in the Atlantic sturgeon transcriptome resources, 16 of which were significantly responsive to the applied heat shock. The previously studied hspa1 (hsp70) gene was only significantly upregulated at the highest heat shock (35 °C), while a set of 5 genes (hspc1, hsph3a, hspb1b, hspb11a, and hspb11b) was upregulated at all conditions. Although the hspc1 (hsp90a) gene was previously used as heat shock-marker in sturgeons, we found that hspb11a is the most heat-inducible gene, with up to 3296-fold higher expression in the treated cells, constituting the candidate gene markers for in vivo trials.

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.

Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen.

The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

Robotic injection of zebrafish embryos for high-throughput screening in disease models.

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines.

The Madagascar palm genome provides new insights on the evolution of Apocynaceae specialized metabolism.

Specialized metabolites possess diverse interesting biological activities and some cardenolides- and monoterpene indole alkaloids- (MIAs) derived pharmaceuticals are currently used to treat human diseases such as cancers or hypertension. While these two families of biocompounds are produced by specific subfamilies of Apocynaceae, one member of this medicinal plant family, the succulent tree Pachypodium lamerei Drake (also known as Madagascar palm), does not produce such specialized metabolites. To explore the evolutionary paths that have led to the emergence and loss of cardenolide and MIA biosynthesis in Apocynaceae, we sequenced and assembled the P. lamerei genome by combining Oxford Nanopore Technologies long-reads and Illumina short-reads. Phylogenomics revealed that, among the Apocynaceae whose genomes have been sequenced, the Madagascar palm is so far the species closest to the common ancestor between MIA producers/non-MIA producers. Transposable elements, constituting 72.48% of the genome, emerge as potential key players in shaping genomic architecture and influencing specialized metabolic pathways. The absence of crucial MIA biosynthetic genes such as strictosidine synthase in P. lamerei and non-Rauvolfioideae species hints at a transposon-mediated mechanism behind gene loss. Phylogenetic analysis not only showcases the evolutionary divergence of specialized metabolite biosynthesis within Apocynaceae but also underscores the role of transposable elements in this intricate process. Moreover, we shed light on the low conservation of enzymes involved in the final stages of MIA biosynthesis in the distinct MIA-producing plant families, inferring independent gains of these specialized enzymes along the evolution of these medicinal plant clades. Overall, this study marks a leap forward in understanding the genomic dynamics underpinning the evolution of specialized metabolites biosynthesis in the Apocynaceae family, with transposons emerging as potential architects of genomics restructuring and gene loss.

The Rauvolfia tetraphylla genome suggests multiple distinct biosynthetic routes for yohimbane monoterpene indole alkaloids.

Monoterpene indole alkaloids (MIAs) are a structurally diverse family of specialized metabolites mainly produced in Gentianales to cope with environmental challenges. Due to their pharmacological properties, the biosynthetic modalities of several MIA types have been elucidated but not that of the yohimbanes. Here, we combine metabolomics, proteomics, transcriptomics and genome sequencing of Rauvolfia tetraphylla with machine learning to discover the unexpected multiple actors of this natural product synthesis. We identify a medium chain dehydrogenase/reductase (MDR) that produces a mixture of four diastereomers of yohimbanes including the well-known yohimbine and rauwolscine. In addition to this multifunctional yohimbane synthase (YOS), an MDR synthesizing mainly heteroyohimbanes and the short chain dehydrogenase vitrosamine synthase also display a yohimbane synthase side activity. Lastly, we establish that the combination of geissoschizine synthase with at least three other MDRs also produces a yohimbane mixture thus shedding light on the complex mechanisms evolved for the synthesis of these plant bioactives.

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%.

Genome Assembly of the Medicinal Plant Voacanga thouarsii.

The Apocynaceae tree Voacanga thouarsii, native to southern Africa and Madagascar, produces monoterpene indole alkaloids (MIA), which are specialized metabolites with a wide range of bioactive properties. Voacanga species mainly accumulates tabersonine in seeds making these species valuable medicinal plants currently used for industrial MIA production. Despite their importance, the MIA biosynthesis in Voacanga species remains poorly studied. Here, we report the first genome assembly and annotation of a Voacanga species. The combined assembly of Oxford Nanopore Technologies long-reads and Illumina short-reads resulted in 3,406 scaffolds with a total length of 1,354.26 Mb and an N50 of 3.04 Mb. A total of 33,300 protein-coding genes were predicted and functionally annotated. These genes were then used to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. A transposable element (TE) analysis showed the highest proportion of TE in Voacanga thouarsii compared with all other MIA-producing plants. In a nutshell, this first reference genome of V. thouarsii will thus contribute to strengthen future comparative and evolutionary studies in MIA-producing plants leading to a better understanding of MIA pathway evolution. This will also allow the potential identification of new MIA biosynthetic genes for metabolic engineering purposes.

The Vinca minor genome highlights conserved evolutionary traits in monoterpene indole alkaloid synthesis.

Vinca minor, also known as the lesser periwinkle, is a well-known species from the Apocynaceae, native to central and southern Europe. This plant synthesizes monoterpene indole alkaloids, which are a class of specialized metabolites displaying a wide range of bioactive- and pharmacologically important properties. Within the almost 50 monoterpene indole alkaloids it produces, V. minor mainly accumulates vincamine, which is commercially used as a nootropic. Using a combination of Oxford Nanopore Technologies long read- and Illumina short-read sequencing, a 679,098 Mb V. minor genome was assembled into 296 scaffolds with an N50 scaffold length of 6 Mb, and encoding 29,624 genes. These genes were functionally annotated and used in a comparative genomic analysis to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. Furthermore, homology-based monoterpene indole alkaloid gene predictions together with a metabolic analysis across 4 different V. minor tissue types guided the identification of candidate monoterpene indole alkaloid genes. These candidates were finally used to identify monoterpene indole alkaloid gene clusters, which combined with synteny analysis allowed for the discovery of a functionally validated vincadifformine-16-hydroxylase, reinforcing the potential of this dataset for monoterpene indole alkaloids gene discovery. It is expected that access to these resources will facilitate the elucidation of unknown monoterpene indole alkaloid biosynthetic routes with the potential of transferring these pathways to heterologous expression systems for large-scale monoterpene indole alkaloid production.

An updated version of the Madagascar periwinkle genome.

The Madagascar periwinkle, Catharanthus roseus, belongs to the Apocynaceae family. This medicinal plant, endemic to Madagascar, produces many important drugs including the monoterpene indole alkaloids (MIA) vincristine and vinblastine used to treat cancer worldwide. Here, we provide a new version of the C. roseus genome sequence obtained through the combination of Oxford Nanopore Technologies long-reads and Illumina short-reads. This more contiguous assembly consists of 173 scaffolds with a total length of 581.128 Mb and an N50 of 12.241 Mb. Using publicly available RNAseq data, 21,061 protein coding genes were predicted and functionally annotated. A total of 42.87% of the genome was annotated as transposable elements, most of them being long-terminal repeats. Together with the increasing access to MIA-producing plant genomes, this updated version should ease evolutionary studies leading to a better understanding of MIA biosynthetic pathway evolution.

Analyzing the function of a hox gene: an evolutionary approach.

We present an evolutionary approach to dissecting conserved developmental mechanisms. We reason that important mechanisms for making the bodyplan will act early, to generate the major features of the body and that they will be conserved in evolution across many metazoa, and thus, that they will be available in very different animals. This led to our specific approach of microarrays to screen for very early conserved developmental regulators in parallel in an insect, Drosophila and a vertebrate, Xenopus. We screened for the earliest conserved targets of the ectopically expressed hox gene Hoxc6/Antennapedia in both species and followed these targets up, using in situ hybridization, in the Xenopus system. The results indicate that relatively few of the early Hox target genes are conserved: these are mainly involved in the specification of the antero-posterior body axis and in gastrulation.

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.

Genome and transcriptome analysis of the beet armyworm Spodoptera exigua reveals targets for pest control.

The genus Spodoptera (Lepidoptera: Noctuidae) includes some of the most infamous insect pests of cultivated plants including Spodoptera frugiperda, Spodoptera litura, and Spodoptera exigua. To effectively develop targeted pest control strategies for diverse Spodoptera species, genomic resources are highly desired. To this aim, we provide the genome assembly and developmental transcriptome comprising all major life stages of S. exigua, the beet armyworm. Spodoptera exigua is a polyphagous herbivore that can feed on > 130 host plants, including several economically important crops. The 419 Mb beet armyworm genome was sequenced from a female S. exigua pupa. Using a hybrid genome sequencing approach (Nanopore long-read data and Illumina short read), a high-quality genome assembly was achieved (N50 = 1.1 Mb). An official gene set (18,477 transcripts) was generated by automatic annotation and by using transcriptomic RNA-seq datasets of 18 S. exigua samples as supporting evidence. In-depth analyses of developmental stage-specific expression combined with gene tree analyses of identified homologous genes across Lepidoptera genomes revealed four potential genes of interest (three of them Spodoptera-specific) upregulated during first- and third-instar larval stages for targeted pest-outbreak management. The beet armyworm genome sequence and developmental transcriptome covering all major developmental stages provide critical insights into the biology of this devastating polyphagous insect pest species worldwide. In addition, comparative genomic analyses across Lepidoptera significantly advance our knowledge to further control other invasive Spodoptera species and reveals potential lineage-specific target genes for pest control strategies.

Corrigendum: RNAseq Profiling of Leukocyte Populations in Zebrafish Larvae Reveals a cxcl11 Chemokine Gene as a Marker of Macrophage Polarization During Mycobacterial Infection.

[This corrects the article DOI: 10.3389/fimmu.2019.00832.].

RNAseq Profiling of Leukocyte Populations in Zebrafish Larvae Reveals a cxcl11 Chemokine Gene as a Marker of Macrophage Polarization During Mycobacterial Infection.

Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polarization during inflammation has been visualized by real time imaging in larvae of the zebrafish. This model organism has also become widely used to study macrophage responses to microbial pathogens. To support the increasing use of zebrafish in macrophage biology, we set out to determine the complete transcriptome of zebrafish larval macrophages. We studied the specificity of the macrophage signature compared with other larval immune cells and the macrophage-specific expression changes upon infection. We made use of the well-established mpeg1, mpx, and lck fluorescent reporter lines to sort and sequence the transcriptome of larval macrophages, neutrophils, and lymphoid progenitor cells, respectively. Our results provide a complete dataset of genes expressed in these different immune cell types and highlight their similarities and differences. Major differences between the macrophage and neutrophil signatures were found within the families of proteinases. Furthermore, expression of genes involved in antigen presentation and processing was specifically detected in macrophages, while lymphoid progenitors showed expression of genes involved in macrophage activation. Comparison with datasets of in vitro polarized human macrophages revealed that zebrafish macrophages express a strongly homologous gene set, comprising both M1 and M2 markers. Furthermore, transcriptome analysis of low numbers of macrophages infected by the intracellular pathogen Mycobacterium marinum revealed that infected macrophages change their transcriptomic response by downregulation of M2-associated genes and overexpression of specific M1-associated genes. Among the infection-induced genes, a homolog of the human CXCL11 chemokine gene, cxcl11aa, stood out as the most strongly overexpressed M1 marker. Upregulation of cxcl11aa in Mycobacterium-infected macrophages was found to require the function of Myd88, a critical adaptor molecule in the Toll-like and interleukin 1 receptor pathways that are central to pathogen recognition and activation of the innate immune response. Altogether, our data provide a valuable data mining resource to support infection and inflammation research in the zebrafish model.

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.

The role of the Spemann organizer in anterior-posterior patterning of the trunk.

The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tail organizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk.

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

We have sequenced the genome of the endangered European eel using the MinION by Oxford Nanopore, and assembled these data using a novel algorithm specifically designed for large eukaryotic genomes. For this 860 Mbp genome, the entire computational process takes two days on a single CPU. The resulting genome assembly significantly improves on a previous draft based on short reads only, both in terms of contiguity (N50 1.2 Mbp) and structural quality. This combination of affordable nanopore sequencing and light weight assembly promises to make high-quality genomic resources accessible for many non-model plants and animals.