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

BackgroundThe 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 vertebrate species, finding possible inducers of dermal bone. However, the evolutionarily derived characters of current experimental model organisms hinder investigations of the ancestral and conserved mechanisms of vertebrate cranial dermal bone induction. Thus, investigating such mechanisms with animals diverging at evolutionarily crucial phylogenetic nodes is imperative.ResultsWe investigated the cellular and molecular foundations of skull frontal bone formation in the spotted gar Lepisosteus oculatus, a basally branching actinopterygian. Whole-mount bone and cartilage stainings and hematoxylin-eosin section stainings 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: Upregulation of F-actin and plasma membrane in condensing cells, and extension of podia from osteoblasts to the frontal bone, which may be responsible for bone mineral transport.ConclusionThis study highlights the process of frontal bone formation with dynamic architectural changes of mesenchymal cells in spotted gar, illuminating supposedly ancestral and likely conserved developmental mechanisms of skull bone formation among vertebrates.

Major histocompatibility complex in Osteichthyes

Based on analysis of available genome sequences, five gene lineages of MHC class I molecules (MHC I-U, -Z, -S, -L and -P) and one gene lineage of MHC class II molecules (MHC II-D) have been identified in Osteichthyes. In the latter lineage, three MHC II molecule sublineages have been identified (MHC II-A, -B and -E). As regards MHC class I molecules in Osteichthyes, it is important to take note of the fact that the lineages U and Z in MHC I genes have been identified in almost all fish species examined so far. Phylogenetic studies into MHC II molecule genes of sublineages A and B suggest that they may be descended from the genes of the sublineage named A/B that have been identified in spotted gar (Lepisosteus oculatus). The sublineage E genes of MHC II molecules, which represent the group of non-polymorphic genes with poor expression in the tissues connected with the immune system, are present in primitive fish, i.e. in paddlefish, sturgeons and spotted gar (Lepisosteus oculatus), as well as in cyprinids (Cyprinidae), Atlantic salmon (Salmo salar), and rainbow trout (Oncorhynchus mykiss). Full elucidation of the details relating to the organisation and functioning of the particular components of the major histocompatibility complex in Osteichthyes can advance the understanding of the evolution of the MHC molecule genes and the immune mechanism.

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.

Assexon: Assembling Exon Using Gene Capture Data

Exon capture across species has been one of the most broadly applied approaches to acquire multi-locus data in phylogenomic studies of non-model organisms. Methods for assembling loci from short-read sequences (eg, Illumina platforms) that rely on mapping reads to a reference genome may not be suitable for studies comprising species across a wide phylogenetic spectrum; thus, de novo assembling methods are more generally applied. Current approaches for assembling targeted exons from short reads are not particularly optimized as they cannot (1) assemble loci with low read depth, (2) handle large files efficiently, and (3) reliably address issues with paralogs. Thus, we present Assexon: a streamlined pipeline that de novo assembles targeted exons and their flanking sequences from raw reads. We tested our method using reads from Lepisosteus osseus (4.37 Gb) and Boleophthalmus pectinirostris (2.43 Gb), which are captured using baits that were designed based on genome sequence of Lepisosteus oculatus and Oreochromis niloticus, respectively. We compared performance of Assexon to PHYLUCE and HybPiper, which are commonly used pipelines to assemble ultra-conserved element (UCE) and Hyb-seq data. A custom exon capture analysis pipeline (CP) developed by Yuan et al was compared as well. Assexon accurately assembled more than 3400 to 3800 (20%-28%) loci than PHYLUCE and more than 1900 to 2300 (8%-14%) loci than HybPiper across different levels of phylogenetic divergence. Assexon ran at least twice as fast as PHYLUCE and HybPiper. Number of loci assembled using CP was comparable with Assexon in both tests, while Assexon ran at least 7 times faster than CP. In addition, some steps of CP require the user’s interaction and are not fully automated, and this user time was not counted in our calculation. Both Assexon and CP retrieved no paralogs in the testing runs, but PHYLUCE and Hybpiper did. In conclusion, Assexon is a tool for accurate and efficient assembling of large read sets from exon capture experiments. Furthermore, Assexon includes scripts to filter poorly aligned coding regions and flanking regions, calculate summary statistics of loci, and select loci with reliable phylogenetic signal. Assexon is available at https://github.com/yhadevol/Assexon .