Transcriptomic data support phylogenetic congruence and reveal genomic changes associated with the repeated evolution of annualism in aplocheiloid killifishes (Cyprinodontiformes).

Repeated evolution of novel life histories that are correlated with ecological variables offers opportunities to study convergence in genetic, developmental, and metabolic features. Nearly half of the 800 species of Aplocheiloid killifishes, a clade of teleost fishes with a circumtropical distribution, are "annual" or seasonal species that survive in ephemeral bodies of water that desiccate and are unfeasible for growth, reproduction, or survival for weeks to months every year. But the repeated evolution of adaptations that are key features of the annual life history among these fishes remains poorly known without a robust phylogenetic framework. We present a large-scale phylogenomic reconstruction of aplocheiloid killifishes evolution using newly sequenced transcriptomes obtained from a diversity of killifish lineages representing putative independent origins of annualism. Ancestral state estimation shows that developmental dormancy (diapause), a key trait of the killifish annual life cycle, may have originated up to seven times independently among African and South American lineages. To further explore the genetic basis of this unique trait, we measure changes in evolutionary rates among orthologous genes across the killifish tree of life by quantifying codon evolution using dN/dS ratios. We show that some genes have higher dN/dS ratios in lineages leading to species with annual life history. Many of them constitute key developmental genes or nuclear-encoded metabolic genes that control oxidative phosphorylation. Lastly, we compare these genes with higher ω to genes previously associated to developmental dormancy and metabolic shifts in killifishes and other vertebrates, and thereby identify molecular evolutionary signatures of repeated transitions to extreme environments.

Suppressors of cGAS-STING are downregulated during fin-limb regeneration and aging in aquatic vertebrates.

During the early stages of limb and fin regeneration in aquatic vertebrates (i.e., fishes and amphibians), blastema undergo transcriptional rewiring of innate immune signaling pathways to promote immune cell recruitment. In mammals, a fundamental component of innate immune signaling is the cytosolic DNA sensing pathway, cGAS-STING. However, to what extent the cGAS-STING pathway influences regeneration in aquatic anamniotes is unknown. In jawed vertebrates, negative regulation of cGAS-STING activity is accomplished by suppressors of cytosolic DNA such as Trex1, Pml, and PML-like exon 9 (Plex9) exonucleases. Here, we examine the expression of these suppressors of cGAS-STING, as well as inflammatory genes and cGAS activity during caudal fin and limb regeneration using the spotted gar (Lepisosteus oculatus) and axolotl (Ambystoma mexicanum) model species, and during age-related senescence in zebrafish (Danio rerio). In the regenerative blastema of wounded gar and axolotl, we observe increased inflammatory gene expression, including interferon genes and interleukins 6 and 8. We also observed a decrease in axolotl Trex1 and gar pml expression during the early phases of wound healing which correlates with a dramatic increase in cGAS activity. In contrast, the plex9.1 gene does not change in expression during wound healing in gar. However, we observed decreased expression of plex9.1 in the senescing cardiac tissue of aged zebrafish, where 2'3'-cGAMP levels are elevated. Finally, we demonstrate a similar pattern of Trex1, pml, and plex9.1 gene regulation across species in response to exogenous 2'3'-cGAMP. Thus, during the early stages of limb-fin regeneration, Pml, Trex1, and Plex9.1 exonucleases are downregulated, presumably to allow an evolutionarily ancient cGAS-STING activity to promote inflammation and the recruitment of immune cells.

Deep evolutionary origin of limb and fin regeneration.

Salamanders and lungfishes are the only sarcopterygians (lobe-finned vertebrates) capable of paired appendage regeneration, regardless of the amputation level. Among actinopterygians (ray-finned fishes), regeneration after amputation at the fin endoskeleton has only been demonstrated in polypterid fishes (Cladistia). Whether this ability evolved independently in sarcopterygians and actinopterygians or has a common origin remains unknown. Here we combine fin regeneration assays and comparative RNA-sequencing (RNA-seq) analysis of Polypterus and axolotl blastemas to provide support for a common origin of paired appendage regeneration in Osteichthyes (bony vertebrates). We show that, in addition to polypterids, regeneration after fin endoskeleton amputation occurs in extant representatives of 2 other nonteleost actinopterygians: the American paddlefish (Chondrostei) and the spotted gar (Holostei). Furthermore, we assessed regeneration in 4 teleost species and show that, with the exception of the blue gourami (Anabantidae), 3 species were capable of regenerating fins after endoskeleton amputation: the white convict and the oscar (Cichlidae), and the goldfish (Cyprinidae). Our comparative RNA-seq analysis of regenerating blastemas of axolotl and Polypterus reveals the activation of common genetic pathways and expression profiles, consistent with a shared genetic program of appendage regeneration. Comparison of RNA-seq data from early Polypterus blastema to single-cell RNA-seq data from axolotl limb bud and limb regeneration stages shows that Polypterus and axolotl share a regeneration-specific genetic program. Collectively, our findings support a deep evolutionary origin of paired appendage regeneration in Osteichthyes and provide an evolutionary framework for studies on the genetic basis of appendage regeneration.

Cellular mechanisms of frontal bone development in spotted gar (Lepisosteus oculatus).

BACKGROUND: The cellular and molecular mechanisms initiating vertebrate cranial dermal bone formation is a conundrum in evolutionary and developmental biology. Decades of studies have determined the developmental processes of cranial dermal bones in various vertebrates and identified possible inducers of dermal bone. However, evolutionarily derived characters of current experimental model organisms, such as non-homologous frontal bones between teleosts and sarcopterygians, hinder investigations of ancestral and conserved mechanisms of vertebrate cranial dermal bone induction. Thus, investigating such mechanisms with animals diverging at evolutionarily informative phylogenetic nodes is imperative. RESULTS: We investigated the cellular foundations of skull frontal bone formation in the spotted gar Lepisosteus oculatus, a basally branching non-teleost actinopterygian. Whole-mount bone and cartilage staining and hematoxylin-eosin section staining revealed that mesenchymal cell condensations in the frontal bone of spotted gar develop in close association with the underlying cartilage. We also identified novel aspects of frontal bone formation: enrichment of F-actin, cellular membranes, and E-cadherin in condensing cells, and extension of podia-like structures from osteoblasts to the frontal bone, which may be responsible for bone mineral transport. CONCLUSION: This study highlights the process of frontal bone formation with dynamic architectural changes of mesenchymal cells in spotted gar, an emerging non-teleost fish model system, illuminating supposedly ancestral and likely conserved developmental mechanisms of skull bone formation among vertebrates.

Holosteans contextualize the role of the teleost genome duplication in promoting the rise of evolutionary novelties in the ray-finned fish innate immune system.

Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrate species that have diversified across virtually all fresh and saltwater ecosystems. This ecological breadth raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.

Faster, reduced cost calibration method development methods for the analysis of fermentation product using near-infrared spectroscopy (NIRS).

Recent innovations in synthetic biology, fermentation, and process development have decreased time to market by reducing strain construction cycle time and effort. Faster analytical methods are required to keep pace with these innovations, but current methods of measuring fermentation titers often involve manual intervention and are slow, time-consuming, and difficult to scale. Spectroscopic methods like near-infrared (NIR) spectroscopy address this shortcoming; however, NIR methods require calibration model development that is often costly and time-consuming. Here, we introduce two approaches that speed up calibration model development. First, generalized calibration modeling (GCM) or sibling modeling, which reduces calibration modeling time and cost by up to 50% by reducing the number of samples required. Instead of constructing analyte-specific models, GCM combines a reduced number of spectra from several individual analytes to produce a large pool of spectra for a generalized model predicting all analyte levels. Second, randomized multicomponent multivariate modeling (RMMM) reduces modeling time by mixing multiple analytes into one sample matrix and then taking the spectral measurements. Afterward, individual calibration methods are developed for the various components in the mixture. Time saved from the use of RMMM is proportional to the number of components or analytes in the mixture. When combined, the two methods effectively reduce the associated cost and time for calibration model development by a factor of 10.

Comprehensive phylogeny of ray-finned fishes (Actinopterygii) based on transcriptomic and genomic data.

Our understanding of phylogenetic relationships among bony fishes has been transformed by analysis of a small number of genes, but uncertainty remains around critical nodes. Genome-scale inferences so far have sampled a limited number of taxa and genes. Here we leveraged 144 genomes and 159 transcriptomes to investigate fish evolution with an unparalleled scale of data: >0.5 Mb from 1,105 orthologous exon sequences from 303 species, representing 66 out of 72 ray-finned fish orders. We apply phylogenetic tests designed to trace the effect of whole-genome duplication events on gene trees and find paralogy-free loci using a bioinformatics approach. Genome-wide data support the structure of the fish phylogeny, and hypothesis-testing procedures appropriate for phylogenomic datasets using explicit gene genealogy interrogation settle some long-standing uncertainties, such as the branching order at the base of the teleosts and among early euteleosts, and the sister lineage to the acanthomorph and percomorph radiations. Comprehensive fossil calibrations date the origin of all major fish lineages before the end of the Cretaceous.

Annual Killifish Transcriptomics and Candidate Genes for Metazoan Diapause.

Dormancy has evolved in all major metazoan lineages. It is critical for survival when environmental stresses are not conducive to growth, maturation, or reproduction. Embryonic diapause is a form of dormancy where development is reversibly delayed and metabolism is depressed. We report the diapause transcriptome of the annual killifish Nematolebias whitei, and compare gene expression between diapause embryos and free-living larvae to identify a candidate set of 945 differentially expressed "diapause" genes for this species. Similarity of transcriptional patterns among N. whitei and other diapausing animals is striking for a small set of genes associated with stress resistance, circadian rhythm, and metabolism, while other genes show discordant patterns. Although convergent evolution of diapause may require shared molecular mechanisms for fundamental processes, similar physiological phenotypes also may arise through modification of alternative pathways. Annual killifishes are a tractable model system for comparative transcriptomic studies on the evolution of diapause.

Structural basis for ion permeation mechanism in pentameric ligand-gated ion channels.

To understand the molecular mechanism of ion permeation in pentameric ligand-gated ion channels (pLGIC), we solved the structure of an open form of GLIC, a prokaryotic pLGIC, at 2.4 Å. Anomalous diffraction data were used to place bound anions and cations. This reveals ordered water molecules at the level of two rings of hydroxylated residues (named Ser6' and Thr2') that contribute to the ion selectivity filter. Two water pentagons are observed, a self-stabilized ice-like water pentagon and a second wider water pentagon, with one sodium ion between them. Single-channel electrophysiology shows that the side-chain hydroxyl of Ser6' is crucial for ion translocation. Simulations and electrostatics calculations complemented the description of hydration in the pore and suggest that the water pentagons observed in the crystal are important for the ion to cross hydrophobic constriction barriers. Simulations that pull a cation through the pore reveal that residue Ser6' actively contributes to ion translocation by reorienting its side chain when the ion is going through the pore. Generalization of these findings to the pLGIC family is proposed.

The dynamics of growth cone morphology.

BACKGROUND: Normal brain function depends on the development of appropriate patterns of neural connections. A critical role in guiding axons to their targets during neural development is played by neuronal growth cones. These have a complex and rapidly changing morphology; however, a quantitative understanding of this morphology, its dynamics and how these are related to growth cone movement, is lacking. RESULTS: Here we use eigenshape analysis (principal components analysis in shape space) to uncover the set of five to six basic shape modes that capture the most variance in growth cone form. By analysing how the projections of growth cones onto these principal modes evolve in time, we found that growth cone shape oscillates with a mean period of 30 min. The variability of oscillation periods and strengths between different growth cones was correlated with their forward movement, such that growth cones with strong, fast shape oscillations tended to extend faster. A simple computational model of growth cone shape dynamics based on dynamic microtubule instability was able to reproduce quantitatively both the mean and variance of oscillation periods seen experimentally, suggesting that the principal driver of growth cone shape oscillations may be intrinsic periodicity in cytoskeletal rearrangements. CONCLUSIONS: Intrinsically driven shape oscillations are an important component of growth cone shape dynamics. More generally, eigenshape analysis has the potential to provide new quantitative information about differences in growth cone behaviour in different conditions.

A time-calibrated, multi-locus phylogeny of piranhas and pacus (Characiformes: Serrasalmidae) and a comparison of species tree methods.

The phylogeny of piranhas, pacus, and relatives (family Serrasalmidae) was inferred on the basis of DNA sequences from eleven gene fragments that include the mitochondrial control region plus 10 nuclear genes (two exons and eight introns). The new data were obtained for a representative sampling of 53 specimens, collected from all major South American rivers, accounting for over 40% of the valid species and all genera excluding Utiaritichthys. Two fossil calibration points and relaxed-clock Bayesian analyses were used to estimate the timing of diversification. The new multilocus dataset also is used to compare several species-tree approaches against the results obtained using the concatenated alignment analyzed under maximum likelihood and Bayesian inference. Individual gene trees showed substantial topological discordance, but analyses based on concatenation and Bayesian and maximum likelihood-based species trees approaches converged onto a single phylogeny. The resulting phylogenetic hypothesis is robust and supports a division of the family into three major clades, consistent with previous results based on mitochondrial DNA alone. The earliest branching event separated a "pacu" clade (Colossoma, Mylossoma and Piaractus) from the rest of the family in the Late Cretaceous (over 68 Ma). The other two clades, that contain most of the diversity, are formed by the "true piranhas" (Metynnis, Pygopristis, Pygocentrus, Pristobrycon, Catoprion, and Serrasalmus) and the Myleus-like pacus (the Myleus clade). The "true" piranha clade originated during the Eocene (∼53 Ma) but the most recent diversification of flesh-eating piranhas within the genera Serrasalmus and Pygocentrus did not start until the Miocene (∼17 Ma). A comparison of species tree approaches indicates that most methods tested are consistent with results obtained by concatenation, suggesting that the gene-tree incongruence observed is mild and will not produce misleading results under simple concatenation analysis. Non-monophyly of several genera (Pristobrycon, Tometes, Myloplus, Mylesinus) and putative species (Serrasalmus rhombeus) was obtained, suggesting that further study of this family is necessary.

Axon guidance by growth-rate modulation.

Guidance of axons by molecular gradients is crucial for wiring up the developing nervous system. It often is assumed that the unique signature of such guidance is immediate and biased turning of the axon tip toward or away from the gradient. However, here we show that such turning is not required for guidance. Rather, by a combination of experimental and computational analyses, we demonstrate that growth-rate modulation is an alternative mechanism for guidance. Furthermore we show that, although both mechanisms may operate simultaneously, biased turning dominates in steep gradients, whereas growth-rate modulation may dominate in shallow gradients. These results suggest that biased axon turning is not the only method by which guidance can occur.

The Complete Genome Sequences of three species from the killifish genus Kryptolebias (Rivulidae, Cyprinodontiformes).

The killifish genus Kryptolebias currently contains seven recognized species found in freshwater and mangrove microhabitats in South and Central America, the Caribbean, and Florida. Kryptolebias species have several unique features. Beyond its amphibious nature, two of the Kryptolebias species (K. marmoratus and K. hermaphroditus sensu Costa 2011) are the only known vertebrates capable of self-fertilization. Although reference genomes for self-fertilizing species are readily available, a complete understanding of the genomic basis leading to the transition in mating systems in the genus requires more genomic resources at a broader taxonomic level. Here, we present the complete genome sequences for three Kryptolebias species from Brazil: the endangered freshwater species Kryptolebias brasiliensis and Kryptolebias gracilis and the androdiecious but obligate outcrossing Kryptolebias ocellatus (sensu Costa 2011). The raw data and assembled genomes are available in GenBank.

Kilo-Scale GMP Synthesis of Renewable Semisynthetic Vaccine-Grade Squalene.

Emulsions of the triterpene squalene ((6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, CAS 111-02-4) have been used as adjuvants in influenza vaccines since the 1990s. Traditionally sourced from shark liver oil, the overfishing of sharks and concomitant reduction in the oceanic shark population raises sustainability issues for vaccine adjuvant grade squalene. We report a semisynthetic route to squalene meeting current pharmacopeial specifications for use in vaccines that leverages the ready availability of trans-ß-farnesene ((6E)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene, CAS 18794-84-8), manufactured from sustainable sugarcane via a yeast fermentation process. The scalability of the proposed route was verified by a kilo-scale GMP synthesis. We also report data demonstrating the synthesized semi-synthetic squalene's physical stability and biological activity when used in a vaccine adjuvant formulation.

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.

Expression, purification and preliminary structural analysis of Escherichia coli MatP in complex with the matS DNA site.

The Escherichia coli chromosome is organized into four macrodomains which are found in the replication-origin region (Ori), at the terminus (Ter) and on both its sides (Right and Left). The localization of the macrodomains is subject to programmed changes during the cell cycle. The compaction of the 800 kb Ter macrodomain relies on the binding of the MatP protein to a 13 bp matS motif repeated 23 times. MatP is a small DNA-binding protein of about 18 kDa that shares homology in its C-terminal region with the ribbon-helix-helix (RHH) motifs present in regulatory DNA-binding proteins such as CopG. In order to understand the DNA-compaction mechanism of MatP at an atomic level, it was decided to study the structure of apo MatP and of the nucleoprotein complex MatP-matS by both X-ray diffraction and SAXS analysis. It was demonstrated that MatP forms dimers that bind a single matS motif. Complete native X-ray data sets were collected and phasing of the diffraction data is under way.

Cyclic nucleotide-dependent switching of mammalian axon guidance depends on gradient steepness.

Correct wiring of the nervous system during development requires axons to respond appropriately to gradients of attractive and repulsive guidance cues. However, the steepness and concentration of these gradients vary in vivo, for instance, with distance from the target. Understanding how these changing conditions affect the navigation strategies used by developing axons is important for understanding how they are guided over long distances. Previous work has shown that cyclic nucleotide levels determine whether axons are attracted or repelled by steep gradients of the same guidance cue, but it is unknown whether this is also true for shallow gradients. We therefore investigated the guidance responses of rat superior cervical ganglion (SCG) axons in both steep and shallow gradients of nerve growth factor (NGF). In steep gradients we found that cyclic nucleotide-dependent switching occurred, consistent with previous reports. Surprisingly however, we found that in shallow NGF gradients, cyclic nucleotide-dependent switching did not occur. These results suggest that there may be substantial differences in the way axons respond to gradient-based guidance cues depending on where they are within the gradient.

Genome of the Rio Pearlfish (Nematolebias whitei), a bi-annual killifish model for Eco-Evo-Devo in extreme environments.

The Rio Pearlfish, Nematolebias whitei, is a bi-annual killifish species inhabiting seasonal pools in the Rio de Janeiro region of Brazil that dry twice per year. Embryos enter dormant diapause stages in the soil, waiting for the inundation of the habitat which triggers hatching and commencement of a new life cycle. Rio Pearlfish represents a convergent, independent origin of annualism from other emerging killifish model species. While some transcriptomic datasets are available for Rio Pearlfish, thus far, a sequenced genome has been unavailable. Here, we present a high quality, 1.2 Gb chromosome-level genome assembly, genome annotations, and a comparative genomic investigation of the Rio Pearlfish as representative of a vertebrate clade that evolved environmentally cued hatching. We show conservation of 3D genome structure across teleost fish evolution, developmental stages, tissues, and cell types. Our analysis of mobile DNA shows that Rio Pearlfish, like other annual killifishes, possesses an expanded transposable element profile with implications for rapid aging and adaptation to harsh conditions. We use the Rio Pearlfish genome to identify its hatching enzyme gene repertoire and the location of the hatching gland, a key first step in understanding the developmental genetic control of hatching. The Rio Pearlfish genome expands the comparative genomic toolkit available to study convergent origins of seasonal life histories, diapause, and rapid aging phenotypes. We present the first set of genomic resources for this emerging model organism, critical for future functional genetic, and multiomic explorations of "Eco-Evo-Devo" phenotypes of resilience and adaptation to extreme environments.

Convergent losses of SCPP genes and ganoid scales among non-teleost actinopterygians.

Various secretory calcium-binding phosphoprotein (SCPP) genes are expressed in the skin and jaw during the formation of bone, teeth, and scales in osteichthyans (bony vertebrates). Among these mineralized skeletal units is the ganoid scale, found in many fossil actinopterygians (ray-finned fish) but confirmed only in Polypteriformes (bichirs, reedfish) and Lepisosteiformes (gars) among extant clades. Here, we examined SCPP genes in the genome of seven non-teleost actinopterygian species that possess or do not possess ganoid scales. As a result, 39-43 SCPP genes were identified in Polypteriformes and Lepisosteiformes, whereas 22-24 SCPP genes were found in Acipenseriformes (sturgeons, paddlefish) and Amiiformes (bowfin). Most of these genes form two clusters in the genome of Polypteriformes, Lepisosteiformes, and Amiiformes, and these two clusters are duplicated in Acipenseriformes. Despite their distant phylogenetic relationship, Polypteriformes and Lepisosteiformes retain many orthologous SCPP genes. These results imply that common ancestors of extant actinopterygians possessed a large repertoire of SCPP genes, and that many SCPP genes were lost independently in Acipenseriformes and Amiiformes. Notably, most SCPP genes originally located in one of the two SCPP gene clusters are retained in Polypteriformes and Lepisosteiformes but were secondarily lost in Acipenseriformes and Amiiformes. In Lepisosteiformes, orthologs of these lost genes show high or detectable expression levels in the skin but not in the jaw. We thus hypothesize that many SCPP genes located in this cluster are involved in the formation of ganoid scales in Polypteriformes and Lepisosteiformes, and that their orthologs and ganoid scales were convergently lost in Acipenseriformes and Amiiformes.

Stereoselective Synthesis of (4S,5S)-5-Vinyloxazolidin-2-one-4-carboxylate as a ß-Vinylserine Synthetic Equivalent by Vinyl Grignard Addition to an N-Tosyl Version of Garner's Aldehyde.

A highly efficient synthesis of a ß-vinylserine synthetic equivalent is reported that exploits the stereodirecting effect of the N-toluenesulfonamide in an anti-diastereoselective (8.5:1) vinyl Grignard addition to an analogue of Garner's aldehyde. Both aryl and alkyl Grignards are shown to give increased anti-selectivity compared with N-Boc Garner's aldehyde.