Non-teleost Ray-finned Fishes Research Articles

Ancient vertebrate dermal armor evolved from trunk neural crest

Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of nonteleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analyses of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin, and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis.

Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates.

In the last decade, the CRISPR/Cas9 bacterial virus defense system has been adapted as a user-friendly, efficient, and precise method for targeted mutagenesis in eukaryotes. Though CRISPR/Cas9 has proven effective in a diverse range of organisms, it is still most often used to create mutant lines in lab-reared genetic model systems. However, one major advantage of CRISPR/Cas9 mutagenesis over previous gene targeting approaches is that its high efficiency allows the immediate generation of near-null mosaic mutants. This feature could potentially allow genotype to be linked to phenotype in organisms with life histories that preclude the establishment of purebred genetic lines; a group that includes the vast majority of vertebrate species. Of particular interest to scholars of early vertebrate evolution are several long-lived and slow-maturing fishes that diverged from two dominant modern lineages, teleosts and tetrapods, in the Ordovician, or before. These early-diverging or “basal” vertebrates include the jawless cyclostomes, cartilaginous fishes, and various non-teleost ray-finned fishes. In addition to occupying critical phylogenetic positions, these groups possess combinations of derived and ancestral features not seen in conventional model vertebrates, and thus provide an opportunity for understanding the genetic bases of such traits. Here we report successful use of CRISPR/Cas9 mutagenesis in one such non-teleost fish, sterlet Acipenser ruthenus, a small species of sturgeon. We introduced mutations into the genes Tyrosinase, which is needed for melanin production, and Sonic hedgehog, a pleiotropic developmental regulator with diverse roles in early embryonic patterning and organogenesis. We observed disruption of both loci and the production of consistent phenotypes, including both near-null mutants’ various hypomorphs. Based on these results, and previous work in lamprey and amphibians, we discuss how CRISPR/Cas9 F0 mutagenesis may be successfully adapted to other long-lived, slow-maturing aquatic vertebrates and identify the ease of obtaining and injecting eggs and/or zygotes as the main challenges.

Whole-Genome Duplications and the Diversification of the Globin-X Genes of Vertebrates.

Globin-X (GbX) is an enigmatic member of the vertebrate globin gene family with a wide phyletic distribution that spans protostomes and deuterostomes. Unlike canonical globins such as hemoglobins and myoglobins, functional data suggest that GbX does not have a primary respiratory function. Instead, evidence suggests that the monomeric, membrane-bound GbX may play a role in cellular signaling or protection against the oxidation of membrane lipids. Recently released genomes from key vertebrates provide an excellent opportunity to address questions about the early stages of the evolution of GbX in vertebrates. We integrate bioinformatics, synteny, and phylogenetic analyses to characterize the diversity of GbX genes in nonteleost ray-finned fishes, resolve relationships between the GbX genes of cartilaginous fish and bony vertebrates, and demonstrate that the GbX genes of cyclostomes and gnathostomes derive from independent duplications. Our study highlights the role that whole-genome duplications (WGDs) have played in expanding the repertoire of genes in vertebrate genomes. Our results indicate that GbX paralogs have a remarkably high rate of retention following WGDs relative to other globin genes and provide an evolutionary framework for interpreting results of experiments that examine functional properties of GbX and patterns of tissue-specific expression. By identifying GbX paralogs that are products of different WGDs, our results can guide the design of experimental work to explore whether gene duplicates that originate via WGDs have evolved novel functional properties or expression profiles relative to singleton or tandemly duplicated copies of GbX.

Molecular Evolution of clock Genes in Vertebrates.

Circadian rhythms not only influence the overall daily routine of organisms but also directly affect life activities to varying degrees. Circadian locomotor output cycle kaput (Clock), the most critical gene in the circadian rhythm feedback system, plays an important role in the regulation of biological rhythms. Here, we aimed to elucidate the evolutionary history of the clock gene family in a taxonomically diverse set of vertebrates, providing novel insights into the evolution of the clock gene family based on 102 vertebrate genomes. Using genome-wide analysis, we extracted 264 clock sequences. In lobe-finned fishes and some basal non-teleost ray-finned fishes, only two clock isotypes were found (clock1 and clock2). However, the majority of teleosts possess three clock genes (two clock1 genes and one clock2 gene) owing to extra whole-genome duplication. The following syntenic analysis confirmed that clock1a, clock1b, and clock2 are conserved in teleost species. Interestingly, we discovered that osteoglossomorph fishes possess two clock2 genes. Moreover, protein sequence comparisons indicate that CLOCK protein changes among vertebrates were concentrated at the N-terminal and poly Q regions. We also performed a dN/dS analysis, and the results suggest that clock1 and clock2 may show distinct fates for duplicated genes between the lobe-finned and ray-finned fish clades. Collectively, these results provide a genome-wide insight into clock gene evolution in vertebrates.

Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned fishes

Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.

The evolutionary origin of developmental enhancers in vertebrates: Insights from non-model species.

How random DNA mutations have established the diverse morphology of extant vertebrates is one of the major challenges in evolutionary biology. Thanks to the recent advancement in DNA sequencing technologies, the genome sequences of many non-model species have been determined, which allows us to address previously inaccessible questions about gene regulatory evolution in vertebrates. In particular, the genome sequences of non-teleost ray-finned fishes and cartilaginous fishes offer clues about when and how vertebrates gained developmental enhancers related to morphological traits that were required for the water-to-land transition. In this review, I examine the evolutionary origin of conserved non-coding elements (CNEs), which often function as tissue-specific developmental enhancers, and discuss how CNEs are related to gene regulatory changes that caused the major morphological transitions of vertebrates.

Cerebrovascular β-dystroglycan immunoreactivity in vertebrates: not detected in anurans and in the teleosts Ostariophysi and Euteleostei.

The aim of the present paper was to check for the presence of cerebrovascular dystroglycan in vertebrates, because dystroglycan, which is localized in the vascular astroglial end-feet, has a pivotal function in glio-vascular connections. In mammalian brains, the immunoreactivity of β-dystroglycan subunit delineates the vessels. The results of the present study demonstrate similar patterns in other vertebrates, except for anurans and the teleost groups Ostariophysi and Euteleostei. In this study, we investigated 1 or 2 representative species of the main groups of Chondrichthyes, teleost and non-teleost ray-finned fishes, urodeles, anurans, and reptiles. We also investigated 5 mammalian and 3 bird species. Animals were obtained from breeders or fishermen. The presence of β-dystroglycan was investigated immunohistochemically in free-floating sections. Pre-embedding electron microscopical immunohistochemistry on Heterodontus japonicus shark brains demonstrated that in Elasmobranchii, β-dystroglycan is also localized in the perivascular glial end-feet despite the different construction of their blood-brain barrier. The results indicated that the cerebrovascular β-dystroglycan immunoreactivity disappeared separately in anurans, and in teleosts, in the latter group before its division to Ostariophysi and Euteleostei. Immunohistochemistry in muscles and western blots from brain homogenates, however, detected the presence of β-dystroglycan, even in anurans and all teleosts. A possible explanation is that in the glial end-feet, β-dystroglycan is masked in these animals, or disappeared during adaptation to the freshwater habitat.

The Molecular Evolution of Circadian Clock Genes in Spotted Gar (Lepisosteus oculatus)

Circadian rhythms are biological rhythms with a period of approximately 24 h. While canonical circadian clock genes and their regulatory mechanisms appear highly conserved, the evolution of clock gene families is still unclear due to several rounds of whole genome duplication in vertebrates. The spotted gar (Lepisosteus oculatus), as a non-teleost ray-finned fish, represents a fish lineage that diverged before the teleost genome duplication (TGD), providing an outgroup for exploring the evolutionary mechanisms of circadian clocks after whole-genome duplication. In this study, we interrogated the spotted gar draft genome sequences and found that spotted gar contains 26 circadian clock genes from 11 families. Phylogenetic analysis showed that 9 of these 11 spotted gar circadian clock gene families have the same number of genes as humans, while the members of the nfil3 and cry families are different between spotted gar and humans. Using phylogenetic and syntenic analyses, we found that nfil3-1 is conserved in vertebrates, while nfil3-2 and nfil3-3 are maintained in spotted gar, teleost fish, amphibians, and reptiles, but not in mammals. Following the two-round vertebrate genome duplication (VGD), spotted gar retained cry1a, cry1b, and cry2, and cry3 is retained in spotted gar, teleost fish, turtles, and birds, but not in mammals. We hypothesize that duplication of core clock genes, such as (nfil3 and cry), likely facilitated diversification of circadian regulatory mechanisms in teleost fish. We also found that the transcription factor binding element (Ahr::Arnt) is retained only in one of the per1 or per2 duplicated paralogs derived from the TGD in the teleost fish, implicating possible subfuctionalization cases. Together, these findings help decipher the repertoires of the spotted gar’s circadian system and shed light on how the vertebrate circadian clock systems have evolved.

Molecular Evolution of Tryptophan Hydroxylases in Vertebrates: A Comparative Genomic Survey.

Serotonin is a neurotransmitter involved in various physiological processes in the central and peripheral nervous systems. Serotonin is also a precursor for melatonin biosynthesis, which mainly occurs in the pineal gland of vertebrates. Tryptophan hydroxylase (TPH) acts as the rate-limiting enzyme in serotonin biosynthesis and is the initial enzyme involved in the synthesis of melatonin. Recently, two enzymes—TPH1 and TPH2—were reported to form the TPH family in vertebrates and to play divergent roles in serotonergic systems. Here, we examined the evolution of the TPH family from 70 vertebrate genomes. Based on the sequence similarity, we extracted 184 predicted tph homologs in the examined vertebrates. A phylogenetic tree, constructed on the basis of these protein sequences, indicated that tph genes could be divided into two main clades (tph1 and tph2), and that the two clades were further split into two subgroups of tetrapods and Actinopterygii. In tetrapods, and some basal non-teleost ray-finned fishes, only two tph isotypes exist. Notably, tph1 in most teleosts that had undergone the teleost-specific genome duplication could be further divided into tph1a and tph1b. Moreover, protein sequence comparisons indicated that TPH protein changes among vertebrates were concentrated at the NH2-terminal. The tertiary structures of TPH1 and TPH2 revealed obvious differences in the structural elements. Five positively selected sites were characterized in TPH2 compared with TPH1; these sites may reflect the functional divergence in enzyme activity and substrate specificity. In summary, our current work provides novel insights into the evolution of tph genes in vertebrates from a comprehensive genomic perspective.

Diversity of Immunoglobulin Light Chain Genes in Non-Teleost Ray-Finned Fish Uncovers IgL Subdivision into Five Ancient Isotypes.

The aim of this study was to fill important gaps in the evolutionary history of immunoglobulins by examining the structure and diversity of IgL genes in non-teleost ray-finned fish. First, based on the bioinformatic analysis of recent transcriptomic and genomic resources, we experimentally characterized the IgL genes in the chondrostean fish, Acipenser ruthenus (sterlet). We show that this species has three loci encoding IgL kappa-like chains with a translocon-type gene organization and a single VJC cluster, encoding homogeneous lambda-like light chain. In addition, sterlet possesses sigma-like VL and J-CL genes, which are transcribed separately and both encode protein products with cleavable leader peptides. The Acipenseriformes IgL dataset was extended by the sequences mined in the databases of species belonging to other non-teleost lineages of ray-finned fish: Holostei and Polypteriformes. Inclusion of these new data into phylogenetic analysis showed a clear subdivision of IgL chains into five groups. The isotype described previously as the teleostean IgL lambda turned out to be a kappa and lambda chain paralog that emerged before the radiation of ray-finned fish. We designate this isotype as lambda-2. The phylogeny also showed that sigma-2 IgL chains initially regarded as specific for cartilaginous fish are present in holosteans, polypterids, and even in turtles. We conclude that there were five ancient IgL isotypes, which evolved differentially in various lineages of jawed vertebrates.

The Divergent Genomes of Teleosts.

Boasting nearly 30,000 species, teleosts account for half of all extant vertebrates and approximately 98% of all ray-finned fish species (Actinopterygii). Teleosts are also the largest and most diverse group of vertebrates, exhibiting an astonishing level of morphological, physiological, and behavioral diversity. Previous studies had indicated that the teleost lineage has experienced an additional whole-genome duplication event. Recent comparative genomic analyses of teleosts and other bony vertebrates using spotted gar (a nonteleost ray-finned fish) and elephant shark (a cartilaginous fish) as outgroups have revealed several divergent features of teleost genomes. These include an accelerated evolutionary rate of protein-coding and nucleotide sequences, a higher rate of intron turnover, loss of many potential cis-regulatory elements and shorter conserved syntenic blocks. A combination of these divergent genomic features might have contributed to the evolution of the amazing phenotypic diversity and morphological innovations of teleosts.

Notch and Fgf signaling during electrosensory versus mechanosensory lateral line organ development in a non-teleost ray-finned fish

The lateral line system is a useful model for studying the embryonic and evolutionary diversification of different organs and cell types. In jawed vertebrates, this ancestrally comprises lines of mechanosensory neuromasts over the head and trunk, flanked on the head by fields of electrosensory ampullary organs, all innervated by lateral line neurons in cranial lateral line ganglia. Both types of sense organs, and their afferent neurons, develop from cranial lateral line placodes. Current research primarily focuses on the posterior lateral line primordium in zebrafish, which migrates as a cell collective along the trunk; epithelial rosettes form in the trailing zone and are deposited as a line of neuromasts, within which hair cells and supporting cells differentiate. However, in at least some other teleosts (e.g. catfishes) and all non-teleosts, lines of cranial neuromasts are formed by placodes that elongate to form a sensory ridge, which subsequently fragments, with neuromasts differentiating in a line along the crest of the ridge. Furthermore, in many non-teleost species, electrosensory ampullary organs develop from the flanks of the sensory ridge. It is unknown to what extent the molecular mechanisms underlying neuromast formation from the zebrafish migrating posterior lateral line primordium are conserved with the as-yet unexplored molecular mechanisms underlying neuromast and ampullary organ formation from elongating lateral line placodes. Here, we report experiments in an electroreceptive non-teleost ray-finned fish, the Mississippi paddlefish Polyodon spathula, that suggest a conserved role for Notch signaling in regulating lateral line organ receptor cell number, but potentially divergent roles for the fibroblast growth factor signaling pathway, both between neuromasts and ampullary organs, and between paddlefish and zebrafish.

"Holostei versus Halecostomi" Problem: Insight from Cytogenetics of Ancient Nonteleost Actinopterygian Fish, Bowfin Amia calva.

Bowfin belongs to an ancient lineage of nonteleost ray-finned fishes (actinopterygians) and is the only extant survivor of a once diverged group, the Halecomorphi or Amiiformes. Owing to the scarcity of extant nonteleost ray-finned lineages, also referred as "living fossils," their phylogenetic interrelationships have been the target of multiple hypotheses concerning their sister group relationships. Molecular and morphological data sets have produced controversial results; bowfin is considered as either the sister group to genome-duplicated teleosts (together forming the group of Halecostomi) or to gars (Lepisosteiformes; together forming the group of Holostei). However, any detailed cytogenetic analysis of bowfin chromosomes has never been performed to address this issue. Here we examined bowfin chromosomes by conventional (Giemsa-staining, C-banding, base-specific fluorescence and silver staining) and molecular (FISH with rDNA probes) cytogenetic protocols. We identified diploid chromosome number 2n = 46 with a middle-sized submetacentric chromosome pair as the major ribosomal DNA-bearing (45S rDNA), GC-positive and silver-positive element. The minor rDNA (5S rDNA) sites were localized in the pericentromeric region of one middle-sized acrocentric chromosome pair. Comparison with available cytogenetic data of other nonteleost actinopterygians (bichirs, sturgeons, gars) and teleost species including representative of basally branching lineages showed bowfin chromosomal characteristics more similar to the teleost type than to any other nonteleosts. Particularly striking differences were identified between bowfin and gars, the latter of which were found to mimic mammalian AT/GC genomic organisation. Such conclusion however contradicts the most recent phylogenomic results and raises the question what states are ancestral and what are derived.

Frequency tuning and directional preferences of eighth nerve afferents in the non-teleost bony fish, Acipenser fulvescens

We investigated the coding mechanisms for spectral analysis and sound source location in Acipenser fulvescens, the lake sturgeon. A. fulvescens belongs to one of the few extant non-teleost ray-finned fishes, with a phylogenetic history that dates back about 200 million years. A shaker system was used to simulate the particle motion component of sound during electrophysiological recordings of isolated single units from the eighth nerve. Data were compared to teleosts and land vertebrates. Peripheral coding strategies for spectral analysis and sound source location in A. fulvescens resembled those found in teleosts. Frequency characteristics generally resembled that of low-frequency afferents of other fishes and land vertebrates and the auditory periphery in A. fulvescens appears to be well suited to encode the intensity of sound. Eighth nerve afferents responded to directional stimuli in a cosine-like manner (as in teleosts). However, differences from teleosts were found that may have implications for the mechanisms for sound source location in azimuth. The common physiological characteristics among A. fulvescens, teleosts, and land vertebrates may reflect important functions of the auditory system that have been conserved throughout the evolution of vertebrates.

Coding of sound direction in the auditory periphery of the lake sturgeon,Acipenser fulvescens

The lake sturgeon, Acipenser fulvescens, belongs to one of the few extant nonteleost ray-finned fishes and diverged from the main vertebrate lineage about 250 million years ago. The aim of this study was to use this species to explore the peripheral neural coding strategies for sound direction and compare these results to modern bony fishes (teleosts). Extracellular recordings were made from afferent neurons innervating the saccule and lagena of the inner ear while the fish was stimulated using a shaker system. Afferents were highly directional and strongly phase locked to the stimulus. Directional response profiles resembled cosine functions, and directional preferences occurred at a wide range of stimulus intensities (spanning at least 60 dB re 1 nm displacement). Seventy-six percent of afferents were directionally selective for stimuli in the vertical plane near 90° (up down) and did not respond to horizontal stimulation. Sixty-two percent of afferents responsive to horizontal stimulation had their best axis in azimuths near 0° (front back). These findings suggest that in the lake sturgeon, in contrast to teleosts, the saccule and lagena may convey more limited information about the direction of a sound source, raising the possibility that this species uses a different mechanism for localizing sound. For azimuth, a mechanism could involve the utricle or perhaps the computation of arrival time differences. For elevation, behavioral strategies such as directing the head to maximize input to the area of best sensitivity may be used. Alternatively, the lake sturgeon may have a more limited ability for sound source localization compared with teleosts.

Phylogeny of nucleus medianus of the posterior tubercle in rayfinned fishes

The brains of ray-finned fishes form a morphocline of increasing complexity, from cladistians through teleosts. This is particularly apparent in the posterior tubercle of the diencephalon. In cladistians, the posterior tubercle consists of a periventricular nucleus and a migrated nucleus medianus that is fused across the midline. In more advanced ray-finned fishes, such as gars and bowfins, the posterior tubercle comprises numerous additional migrated nuclei, termed the preglomerular complex, in addition to a more well developed nucleus medianus. In teleosts, the most derived ray-finned fishes, there is an even more elaborate preglomerular complex, but there is no recognizable nucleus medianus. In an attempt to explain the variation in the posterior tubercle of the diencephalon in ray-finned fishes, the immunohistochemistry and connections of nucleus medianus were examined in cladistians, gars and bowfins. In each of these taxa, nucleus medianus exhibits large numbers of calretinin-positive neurons and has ascending projections that terminate in several divisions of the pallium. Although teleosts, such as goldfish, also exhibit numerous cell groups in the posterior tubercle that are rich in calretinin, none of these cell groups has connections that are comparable to those of nucleus medianus in non-teleost ray-finned fishes. It is possible, therefore, that nucleus medianus was lost with the origin of teleosts.

Visual Thalamotelencephalic Pathways in the Sturgeon Acipenser, a Non-Teleost Actinopterygian Fish

Terrestrial vertebrates (amphibians, reptiles, birds, and mammals) possess two visual systems, the geniculate and extrageniculate pathways to the telencephalon. In cartilaginous fishes (e.g. sharks) both retinal and tectal neurons project to neurons in the thalamus, which themselves project to a single area in the telencephalon. The condition in ray-finned fishes (Actinopterygii) is ambiguous. In many teleosts there is a well developed extrageniculate pathway but no obvious geniculate system. This study reports on the thalamotelencephalic projections of a sturgeon, a non-teleost ray-finned fish. Several tract tracing methods (e.g., HRP, WGA-HRP, biocytin, BDA, DiI) were employed in conjunction with normal techniques for identifying neural structures (e.g., Nissl, Golgi). After injections of tracer into retinal and tectal recipient areas of the thalamus, labeled terminals were observed in the ventrolateral region of the caudal telencephalon, an area referred to as the thalamic projection area. After injections of tracer into the telencephalon, populations of retrogradely filled neurons were located in both the dorsal and ventral thalamus. These data demonstrate that thalamic neurons in both retinal and tectal pathways project directly to the telencephalon. These results support the view that two visual pathways are a primitive feature of vertebrate brain organization. These results are also consistent with the hypothesis that the ancestor of Acipenser and Teleostei (Actinopteri) acquired a novel visual pathway to the telencephalon through the ventral portion of the thalamus.