Soluble adenylyl cyclase (sAC) is a HCO3 -stimulated enzyme that produces the ubiquitous signalling molecule cAMP, and deemed an evolutionarily conserved acid–base sensor. However, its presence is not yet confirmed in bony fishes, the most abundant and diverse of vertebrates. Here, we identified sAC genes in various cartilaginous, ray-finned and lobe-finned fish species. Next, we focused on rainbow trout sAC (rtsAC) and identified 20 potential alternative spliced mRNAs coding for protein isoforms ranging in size from 28 to 186 kDa. Biochemical and kinetic analyses on purified recombinant rtsAC protein determined stimulation by HCO3 at physiologically relevant levels for fish internal fluids (EC50∼ 7 mM). rtsAC activity was sensitive to KH7, LRE1, and DIDS (established inhibitors of sAC from other organisms), and insensitive to forskolin and 2,5-dideoxyadenosine (modulators of transmembrane adenylyl cyclases). Western blot and immunocytochemistry revealed high rtsAC expression in gill ion-transporting cells, hepatocytes, red blood cells, myocytes and cardiomyocytes. Analyses in the cell line RTgill-W1 suggested that some of the longer rtsAC isoforms may be preferentially localized in the nucleus, the Golgi apparatus and podosomes. These results indicate that sAC is poised to mediate multiple acid–base homeostatic responses in bony fishes, and provide cues about potential novel functions in mammals.
more »
« less
Evolution of vertebrate gill covers via shifts in an ancient Pou3f3 enhancer
Whereas the gill chambers of jawless vertebrates open directly into the environment, jawed vertebrates evolved skeletal appendages that drive oxygenated water unidirectionally over the gills. A major anatomical difference between the two jawed vertebrate lineages is the presence of a single large gill cover in bony fishes versus separate covers for each gill chamber in cartilaginous fishes. Here, we find that these divergent patterns correlate with the pharyngeal arch expression of Pou3f3 orthologs. We identify a deeply conserved Pou3f3 arch enhancer present in humans through sharks but undetectable in jawless fish. Minor differences between the bony and cartilaginous fish enhancers account for their restricted versus pan-arch expression patterns. In zebrafish, mutation of Pou3f3 or the conserved enhancer disrupts gill cover formation, whereas ectopic pan-arch Pou3f3b expression generates ectopic skeletal elements resembling the multimeric covers of cartilaginous fishes. Emergence of this Pou3f3 arch enhancer >430 Mya and subsequent modifications may thus have contributed to the acquisition and diversification of gill covers and respiratory strategies during gnathostome evolution.
more » « less- Award ID(s):
- 1656843
- NSF-PAR ID:
- 10193145
- Publisher / Repository:
- Proceedings of the National Academy of Sciences
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 117
- Issue:
- 40
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- p. 24876-24884
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract The bowfin (
Amia calva ) is a ray-finned fish that possesses a unique suite of ancestral and derived phenotypes, which are key to understanding vertebrate evolution. The phylogenetic position of bowfin as a representative of neopterygian fishes, its archetypical body plan and its unduplicated and slowly evolving genome make bowfin a central species for the genomic exploration of ray-finned fishes. Here we present a chromosome-level genome assembly for bowfin that enables gene-order analyses, settling long-debated neopterygian phylogenetic relationships. We examine chromatin accessibility and gene expression through bowfin development to investigate the evolution of immune, scale, respiratory and fin skeletal systems and identify hundreds of gene-regulatory loci conserved across vertebrates. These resources connect developmental evolution among bony fishes, further highlighting the bowfin’s importance for illuminating vertebrate biology and diversity in the genomic era. -
Jawed vertebrates (gnathostomes) have been the dominant lineage of deuterostomes for nearly three hundred fifty million years. Only a few lineages of jawless vertebrates remain in comparison. Composed of lampreys and hagfishes (cyclostomes), these jawless survivors are important systems for understanding the evolution of vertebrates. One focus of cyclostome research has been head skeleton development, as its evolution has been a driver of vertebrate morphological diversification. Recent work has identified hyaline-like cartilage in the oral cirri of the invertebrate chordate amphioxus, making cyclostomes critical for understanding the stepwise acquisition of vertebrate chondroid tissues. Our knowledge of cyclostome skeletogenesis, however, has lagged behind gnathostomes due to the difficulty of manipulating lamprey and hagfish embryos. In this review, we discuss and compare the regulation and histogenesis of cyclostome and gnathostome skeletal tissues. We also survey differences in skeletal morphology that we see amongst cyclostomes, as few elements can be confidently homologized between them. A recurring theme is the heterogeneity of skeletal morphology amongst living vertebrates, despite conserved genetic regulation. Based on these comparisons, we suggest a model through which these mesenchymal connective tissues acquired distinct histologies and that histological flexibility in cartilage existed in the last common ancestor of modern vertebrates.more » « less
-
Vertebrates have distinct tissues which are not present in invertebrate chordates nor other metazoans. The rise of these tissues also coincided with at least one round of whole-genome duplication as well as a suite of lineage-specific segmental duplications. Understanding whether novel genes lead to the origin and diversification of novel cell types, therefore, is of great importance in vertebrate evolution. Here we were particularly interested in the evolution of the vertebrate musculoskeletal system, the muscles and connective tissues that support a diversity of body plans. A major component of the musculoskeletal extracellular matrix (ECM) is fibrillar collagens, a gene family which has been greatly expanded upon in vertebrates. We thus asked whether the repertoire of fibrillar collagens in vertebrates reflects differences in the musculoskeletal system. To test this, we explored the diversity of fibrillar collagens in lamprey, a jawless vertebrate which diverged from jawed vertebrates (gnathostomes) more than five hundred million years ago and has undergone its own gene duplications. Some of the principal components of vertebrate hyaline cartilage are the fibrillar collagens type II and XI, but their presence in cartilage development across all vertebrate taxa has been disputed. We particularly emphasized the characterization of genes in the lamprey hyaline cartilage, testing if its collagen repertoire was similar to that in gnathostomes. Overall, we discovered thirteen fibrillar collagens from all known gene subfamilies in lamprey and were able to identify several lineage-specific duplications. We found that, while the collagen loci have undergone rearrangement, the Clade A genes have remained linked with the hox clusters, a phenomenon also seen in gnathostomes. While the lamprey muscular tissue was largely similar to that seen in gnathostomes, we saw considerable differences in the larval lamprey skeletal tissue, with distinct collagen combinations pertaining to different cartilage types. Our gene expression analyses were unable to identify type II collagen in the sea lamprey hyaline cartilage nor any other fibrillar collagen during chondrogenesis at the stages observed, meaning that sea lamprey likely no longer require these genes during early cartilage development. Our findings suggest that fibrillar collagens were multifunctional across the musculoskeletal system in the last common ancestor of vertebrates and have been largely conserved, but these genes alone cannot explain the origin of novel cell types.more » « less
-
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.more » « less
