A universal molecular control for DNA, mRNA and protein expression.

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Growth dynamics and molecular bases of evolutionary novel jaw extensions in halfbeaks and needlefishes (Beloniformes).

Evolutionary novelties-derived traits without clear homology found in the ancestors of a lineage-may promote ecological specialization and facilitate adaptive radiations. Examples for such novelties include the wings of bats, pharyngeal jaws of cichlids and flowers of angiosperms. Belonoid fishes (flying fishes, halfbeaks and needlefishes) feature an astonishing diversity of extremely elongated jaw phenotypes with undetermined evolutionary origins. We investigate the development of elongated jaws in a halfbeak (Dermogenys pusilla) and a needlefish (Xenentodon cancila) using morphometrics, transcriptomics and in situ hybridization. We confirm that these fishes' elongated jaws are composed of distinct base and novel 'extension' portions. These extensions are morphologically unique to belonoids, and we describe the growth dynamics of both bases and extensions throughout early development in both studied species. From transcriptomic profiling, we deduce that jaw extension outgrowth is guided by populations of multipotent cells originating from the anterior tip of the dentary. These cells are shielded from differentiation, but proliferate and migrate anteriorly during the extension's allometric growth phase. Cells left behind at the tip leave the shielded zone and undergo differentiation into osteoblast-like cells, which deposit extracellular matrix with both bone and cartilage characteristics that mineralizes and thereby provides rigidity. Such bone has characteristics akin to histological observations on the elongated 'kype' process on lower jaws of male salmon, which may hint at common conserved regulatory underpinnings. Future studies will evaluate the molecular pathways that govern the anterior migration and proliferation of these multipotent cells underlying the belonoids' evolutionary novel jaw extensions.

mRNA vaccine quality analysis using RNA sequencing.

The success of mRNA vaccines has been realised, in part, by advances in manufacturing that enabled billions of doses to be produced at sufficient quality and safety. However, mRNA vaccines must be rigorously analysed to measure their integrity and detect contaminants that reduce their effectiveness and induce side-effects. Currently, mRNA vaccines and therapies are analysed using a range of time-consuming and costly methods. Here we describe a streamlined method to analyse mRNA vaccines and therapies using long-read nanopore sequencing. Compared to other industry-standard techniques, VAX-seq can comprehensively measure key mRNA vaccine quality attributes, including sequence, length, integrity, and purity. We also show how direct RNA sequencing can analyse mRNA chemistry, including the detection of nucleoside modifications. To support this approach, we provide supporting software to automatically report on mRNA and plasmid template quality and integrity. Given these advantages, we anticipate that RNA sequencing methods, such as VAX-seq, will become central to the development and manufacture of mRNA drugs.

Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

Library adaptors are short oligonucleotides that are attached to RNA and DNA samples in preparation for next-generation sequencing (NGS). Adaptors can also include additional functional elements, such as sample indexes and unique molecular identifiers, to improve library analysis. Here, we describe Control Library Adaptors, termed CAPTORs, that measure the accuracy and reliability of NGS. CAPTORs can be integrated within the library preparation of RNA and DNA samples, and their encoded information is retrieved during sequencing. We show how CAPTORs can measure the accuracy of nanopore sequencing, evaluate the quantitative performance of metagenomic and RNA sequencing, and improve normalisation between samples. CAPTORs can also be customised for clinical diagnoses, correcting systematic sequencing errors and improving the diagnosis of pathogenic BRCA1/2 variants in breast cancer. CAPTORs are a simple and effective method to increase the accuracy and reliability of NGS, enabling comparisons between samples, reagents and laboratories, and supporting the use of nanopore sequencing for clinical diagnosis.

Functional colour genes and signals of selection in colour-polymorphic salamanders.

Coloration has been associated with multiple biologically relevant traits that drive adaptation and diversification in many taxa. However, despite the great diversity of colour patterns present in amphibians the underlying molecular basis is largely unknown. Here, we use insight from a highly colour-variable lineage of the European fire salamander (Salamandra salamandra bernardezi) to identify functional associations with striking variation in colour morph and pattern. The three focal colour morphs-ancestral black-yellow striped, fully yellow and fully brown-differed in pattern, visible coloration and cellular composition. From population genomic analyses of up to 4,702 loci, we found no correlations of neutral population genetic structure with colour morph. However, we identified 21 loci with genotype-phenotype associations, several of which relate to known colour genes. Furthermore, we inferred response to selection at up to 142 loci between the colour morphs, again including several that relate to coloration genes. By transcriptomic analysis across all different combinations, we found 196 differentially expressed genes between yellow, brown and black skin, 63 of which are candidate genes involved in animal coloration. The concordance across different statistical approaches and 'omic data sets provide several lines of evidence for loci linked to functional differences between colour morphs, including TYR, CAMK1 and PMEL. We found little association between colour morph and the metabolomic profile of its toxic compounds from the skin secretions. Our research suggests that current ecological and evolutionary hypotheses for the origins and maintenance of these striking colour morphs may need to be revisited.

Molecular investigation of genetic assimilation during the rapid adaptive radiations of East African cichlid fishes.

Adaptive radiations are characterized by adaptive diversification intertwined with rapid speciation within a lineage resulting in many ecologically specialized, phenotypically diverse species. It has been proposed that adaptive radiations can originate from ancestral lineages with pronounced phenotypic plasticity in adaptive traits, facilitating ecologically driven phenotypic diversification that is ultimately fixed through genetic assimilation of gene regulatory regions. This study aimed to investigate how phenotypic plasticity is reflected in gene expression patterns in the trophic apparatus of several lineages of East African cichlid fishes, and whether the observed patterns support genetic assimilation. This investigation used a split brood experimental design to compare adaptive plasticity in species from within and outside of adaptive radiations. The plastic response was induced in the crushing pharyngeal jaws through feeding individuals either a hard or soft diet. We find that nonradiating, basal lineages show higher levels of adaptive morphological plasticity than the derived, radiated lineages, suggesting that these differences have become partially genetically fixed during the formation of the adaptive radiations. Two candidate genes that may have undergone genetic assimilation, gif and alas1, were identified, in addition to alterations in the wiring of LPJ patterning networks. Taken together, our results suggest that genetic assimilation may have dampened the inducibility of plasticity related genes during the adaptive radiations of East African cichlids, flattening the reaction norms and canalizing their feeding phenotypes, driving adaptation to progressively more narrow ecological niches.

The seahorse genome and the evolution of its specialized morphology.

Seahorses have a specialized morphology that includes a toothless tubular mouth, a body covered with bony plates, a male brood pouch, and the absence of caudal and pelvic fins. Here we report the sequencing and de novo assembly of the genome of the tiger tail seahorse, Hippocampus comes. Comparative genomic analysis identifies higher protein and nucleotide evolutionary rates in H. comes compared with other teleost fish genomes. We identified an astacin metalloprotease gene family that has undergone expansion and is highly expressed in the male brood pouch. We also find that the H. comes genome lacks enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes, which might have led to the loss of mineralized teeth. tbx4, a regulator of hindlimb development, is also not found in H. comes genome. Knockout of tbx4 in zebrafish showed a 'pelvic fin-loss' phenotype similar to that of seahorses.

Regulatory gene networks that shape the development of adaptive phenotypic plasticity in a cichlid fish.

Phenotypic plasticity is the ability of organisms with a given genotype to develop different phenotypes according to environmental stimuli, resulting in individuals that are better adapted to local conditions. In spite of their ecological importance, the developmental regulatory networks underlying plastic phenotypes often remain uncharacterized. We examined the regulatory basis of diet-induced plasticity in the lower pharyngeal jaw (LPJ) of the cichlid fish Astatoreochromis alluaudi, a model species in the study of adaptive plasticity. Through raising juvenile A. alluaudi on either a hard or soft diet (hard-shelled or pulverized snails) for between 1 and 8 months, we gained insight into the temporal regulation of 19 previously identified candidate genes during the early stages of plasticity development. Plasticity in LPJ morphology was first detected between 3 and 5 months of diet treatment. The candidate genes, belonging to various functional categories, displayed dynamic expression patterns that consistently preceded the onset of morphological divergence and putatively contribute to the initiation of the plastic phenotypes. Within functional categories, we observed striking co-expression, and transcription factor binding site analysis was used to examine the prospective basis of their coregulation. We propose a regulatory network of LPJ plasticity in cichlids, presenting evidence for regulatory crosstalk between bone and muscle tissues, which putatively facilitates the development of this highly integrated trait. Through incorporating a developmental time-course into a phenotypic plasticity study, we have identified an interconnected, environmentally responsive regulatory network that shapes the development of plasticity in a key innovation of East African cichlids.

The search for peripheral biomarkers for major depression: benefiting from successes in the biology of smoking.

The search for robust, clinically useful markers for major depression (MD) has been relatively unproductive. This is unfortunate because MD is one of the largest socio-economic challenges for much of the world and the development of reliable biomarkers for MD could aid in the prevention or treatment of this common syndrome. In this editorial, we compare the approaches taken in the search for biomarkers for MD to that of the more successful searches for biomarkers for tobacco use, and identify several substantive barriers. We suggest that many of the existing clinical repositories used in these biomarkers searches for MD may be of limited value. We conclude that in the future greater attention should be given to the clinical definitions, characterization of confounding environmental factors and age of subjects included in studies. © 2014 Wiley Periodicals, Inc.

Revisiting de Beer's textbook example of heterochrony and jaw elongation in fish: calmodulin expression reflects heterochronic growth, and underlies morphological innovation in the jaws of belonoid fishes.

BACKGROUND: Heterochronic shifts during ontogeny can result in adaptively important innovations and might be initiated by simple developmental switches. Understanding the nature of these developmental events can provide insights into fundamental molecular mechanisms of evolutionary change. Fishes from the Suborder Belonoidei display a vast array of extreme craniofacial morphologies that appear to have arisen through a series of heterochronic shifts. We performed a molecular heterochrony study, comparing postembryonic jaw development in representatives of the Suborder Belonoidei, the halfbeak Dermogenys pusilla (where the lower jaw is considerably elongated compared to the upper jaw) and the needlefish Belone belone (where both jaws are elongated), to a representative of their sister group the Suborder Adrianichthyoidei, the medaka Oryzias latipes, which has retained the ancestral morphology. RESULTS: Early in development, the lower jaw displays accelerated growth both in needlefish and halfbeak compared to medaka, and secondary acceleration of the upper jaw is seen in needlefish later in their development, representing a case of mosaic heterochrony. We identified toothless extensions of the dentaries as innovations of Belonoid fishes and the source of heterochronic growth. The molecular basis of growth heterochronies in the Belonoidei was examined through comparing expression of skeletogenic genes during development of halfbeak and medaka. The calmodulin paralogue calm1 was identified as a potential regulator of jaw length in halfbeak as its expression gradually increases in the lower jaw, but not the upper jaw, in a pattern that matches its outgrowth. Moreover, medaka displays equal expression of calm1 in the upper and lower jaws, consistent with the lack of jaw outgrowth in this species. CONCLUSIONS: Heterochronic shifts in jaw growth have occurred repeatedly during the evolution of Belonoid fishes and we identify toothless extensions of the dentaries as an important innovation of this group. Our results suggest that calm1 contributes to jaw heterochrony in halfbeak, potentially driving further heterochronic shifts in jaw growth across the Suborder Belonoidei, such as the upper jaw acceleration observed in needlefish.

Shaping development through mechanical strain: the transcriptional basis of diet-induced phenotypic plasticity in a cichlid fish.

Adaptive phenotypic plasticity, the ability of an organism to change its phenotype to match local environments, is increasingly recognized for its contribution to evolution. However, few empirical studies have explored the molecular basis of plastic traits. The East African cichlid fish Astatoreochromis alluaudi displays adaptive phenotypic plasticity in its pharyngeal jaw apparatus, a structure that is widely seen as an evolutionary key innovation that has contributed to the remarkable diversity of cichlid fishes. It has previously been shown that in response to different diets, the pharyngeal jaws change their size, shape and dentition: hard diets induce an adaptive robust molariform tooth phenotype with short jaws and strong internal bone structures, while soft diets induce a gracile papilliform tooth phenotype with elongated jaws and slender internal bone structures. To gain insight into the molecular underpinnings of these adaptations and enable future investigations of the role that phenotypic plasticity plays during the formation of adaptive radiations, the transcriptomes of the two divergent jaw phenotypes were examined. Our study identified a total of 187 genes whose expression differs in response to hard and soft diets, including immediate early genes, extracellular matrix genes and inflammatory factors. Transcriptome results are interpreted in light of expression of candidate genes-markers for tooth size and shape, bone cells and mechanically sensitive pathways. This study opens up new avenues of research at new levels of biological organization into the roles of phenotypic plasticity during speciation and radiation of cichlid fishes.

Identification and characterization of gene expression involved in the coloration of cichlid fish using microarray and qRT-PCR approaches.

It has been suggested that speciation on the basis of sexual selection is an important mechanism for the generation of new species for East African cichlids, where male body coloration is one of the major discriminatory factors used by females in mate choice. To gain insight into the molecular basis of cichlid coloration, we studied the Lake Malawi cichlid Pseudotropheus saulosi, comparing transcription in the bright blue skin of males to the yellow skin of females. Our cDNA microarray experiments identified 46 clones that exhibited expression differences between the two sexes, of which five were confirmed to be differentially expressed by relative quantitative real-time PCR (qRT-PCR). This gene list includes a representative from the endosomal-to-Golgi vesicle trafficking pathway, Coatomer protein complex, subunit zeta-1 (Copz-1), which is known to be a critical determinant of pigmentation in humans and zebrafish. With the support of microscopic images of the skin of these specimens, we interpret the transcriptional differences between the blue males and yellow females. Here, we provide insight into the putative functional diversification of genes involved in the coloration of cichlids and by extension, on the evolution of coloration in teleost fish.

Ancestral and derived attributes of the dlx gene repertoire, cluster structure and expression patterns in an African cichlid fish.

BACKGROUND: Cichlid fishes have undergone rapid, expansive evolutionary radiations that are manifested in the diversification of their trophic morphologies, tooth patterning and coloration. Understanding the molecular mechanisms that underlie the cichlids' unique patterns of evolution requires a thorough examination of genes that pattern the neural crest, from which these diverse phenotypes are derived. Among those genes, the homeobox-containing Dlx gene family is of particular interest since it is involved in the patterning of the brain, jaws and teeth. RESULTS: In this study, we characterized the dlx genes of an African cichlid fish, Astatotilapia burtoni, to provide a baseline to later allow cross-species comparison within Cichlidae. We identified seven dlx paralogs (dlx1a, -2a, -4a, -3b, -4b, -5a and -6a), whose orthologies were validated with molecular phylogenetic trees. The intergenic regions of three dlx gene clusters (dlx1a-2a, dlx3b-4b, and dlx5a-6a) were amplified with long PCR. Intensive cross-species comparison revealed a number of conserved non-coding elements (CNEs) that are shared with other percomorph fishes. This analysis highlighted additional lineage-specific gains/losses of CNEs in different teleost fish lineages and a novel CNE that had previously not been identified. Our gene expression analyses revealed overlapping but distinct expression of dlx orthologs in the developing brain and pharyngeal arches. Notably, four of the seven A. burtoni dlx genes, dlx2a, dlx3b, dlx4a and dlx5a, were expressed in the developing pharyngeal teeth. CONCLUSION: This comparative study of the dlx genes of A. burtoni has deepened our knowledge of the diversity of the Dlx gene family, in terms of gene repertoire, expression patterns and non-coding elements. We have identified possible cichlid lineage-specific changes, including losses of a subset of dlx expression domains in the pharyngeal teeth, which will be the targets of future functional studies.

Will he still look good with the lights on? Spectral tuning of visual pigments in fish.

Visual perception is a key element in evolution, as it is required for many life processes. Two recent studies in BMC Biology and BMC Evolutionary Biology shed light on the genetic determinants of color detection in strikingly colored fish.

Impact of ecologically relevant heat shocks on Hsp developmental function in the vetigastropod Haliotis asinina.

Heat shock proteins (Hsps) are essential for cellular maintenance, normal differentiation and morphogenesis, and protection against a range of environmental stresses. It is unknown which of these roles takes precedence when they are required simultaneously. Here we examined the impact of thermal stress on the complex developmental expression patterns of HasHsp70 and HasHsp90A in the vetigastropod Haliotis asinina. We find that near-lethal heat shocks do not alter the spatial demarcation of Hsp expression despite such treatments impacting on the external character of the embryos. Using a suite of molecular markers that are both coexpressed with the Hsps (i.e. in ventrolateral ectoderm and prototroch) and expressed in tissues that have lower (basal) Hsp expression (e.g. serotonergic nervous system and shell gland), we determined that Hsp-expressing tissues do not incur markedly less thermal damage than adjacent tissues. To explore the relationship of Hsp expression with sensitivity of specific cell territories to heat shock, we focused on the formation of the prototroch, a tissue where HasHsp70 and HasHsp90A are coexpressed. By heat shocking at specific developmental stages, we determined that the most sensitive period of prototroch development is during its early specification and differentiation, which overlaps with the time the Hsps are expressed at their highest levels in these cells. This correlation is consistent with heat shock impairing the function of Hsps in regions of the H. asinina embryo undergoing morphogenesis.

The role of MAPK signaling in patterning and establishing axial symmetry in the gastropod Haliotis asinina.

Gastropods are members of the Spiralia, a diverse group of invertebrates that share a common early developmental program, which includes spiral cleavage and a larval trochophore stage. The spiral cleavage program results in the division of the embryo into four quadrants. Specification of the dorsal (D) quadrant is intimately linked with body plan organization and in equally cleaving gastropods occurs when one of the vegetal macromeres makes contact with overlying micromeres and receives an inductive signal that activates a MAPK signaling cascade. Following the induction of the 3D macromere, the embryo begins to gastrulate and assumes a bilateral cleavage pattern. Here we inhibit MAPK activation in 3D with U0126 and examine its effect on the formation and patterning of the trochophore, using a suite of territory-specific markers. The head (pretrochal) region appears to maintain quadri-radial symmetry in U0126-treated embryos, supporting a role for MAPK signaling in 3D in establishing dorsoventral polarity in this region. Posterior (posttrochal) structures - larval musculature, shell and foot--fail to develop in MAPK inhibited trochophores. Inhibition of 3D specification by an alternative method--monensin treatment--yields similar abnormal trochophores. However, genes that are normally expressed in the ectodermal structures (shell and foot) are detected in U0126- and monensin-perturbed larvae in patterns that suggest that this region has latent dorsoventral polarity that is manifested even in the absence of D quadrant specification.

Developmental expression of Hsp90, Hsp70 and HSF during morphogenesis in the vetigastropod Haliotis asinina.

Heat shock proteins (Hsps) have dual functions, participating in both the stress response and a broad range of developmental processes. At physiological temperatures, it has been demonstrated in deuterostomes (vertebrates) and ecdysozoans (insects) that Hsps are expressed in tissues that are undergoing differentiation and morphogenesis. Here we investigate the developmental expression of Hsp70, Hsp90 and their regulatory transcription factor heat shock transcription factor (HSF) in the marine gastropod Haliotis asinina, a representative of the 3rd major lineage of bilaterian animals, the Lophotrochozoa. HasHsp70, HasHsp90 and HasHSF are maternally expressed in H. asinina and are progressively restricted to the micromere lineage during cleavage. During larval morphogenesis, they are expressed in unique and overlapping patterns in the prototroch, foot, and mantle. Hsp expression peaked in these tissues during periods of cell differentiation and morphogenesis, returning to lower levels after morphogenesis was complete. These patterns of Hsp and HSF expression in H. asinina are akin to those observed in ecdysozoans and deuterostomes, with Hsps being activated in cells and tissues undergoing morphogenesis.