Evolution of the endothelin pathway drove neural crest cell diversification.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.

Mutations in KIF7 implicated in idiopathic scoliosis in humans and axial curvatures in zebrafish.

Idiopathic scoliosis (IS) is a spinal disorder affecting up to 3% of otherwise healthy children. IS has a strong familial genetic component and is believed to be genetically complex due to significant variability in phenotype and heritability. Previous studies identified putative loci and variants possibly contributing to IS susceptibility, including within extracellular matrix, cilia, and actin networks, but the genetic architecture and underlying mechanisms remain unresolved. Here, we used whole-exome sequencing from three affected individuals in a multigenerational family with IS and identified 19 uncommon variants (minor allele frequency < 0.05). Genotyping of additional family members identified a candidate heterozygous variant (H1115Q, G>C, rs142032413) within the ciliary gene KIF7, a regulator within the hedgehog (Hh) signaling pathway. Resequencing of the second cohort of unrelated IS individuals and controls identified several severe mutations in KIF7 in affected individuals only. Subsequently, we generated a mutant zebrafish model of kif7 using CRISPR-Cas9. kif7co63/co63 zebrafish displayed severe scoliosis, presenting in juveniles and progressing through adulthood. We observed no deformities in the brain, Reissner fiber, or central canal cilia in kif7co63/co63 embryos, although alterations were seen in Hh pathway gene expression. This study suggests defects in KIF7-dependent Hh signaling, which may drive pathogenesis in a subset of individuals with IS.

Evolution of the new vertebrate head by co-option of an ancient chordate skeletal tissue.

A defining feature of vertebrates (craniates) is a pronounced head that is supported and protected by a robust cellular endoskeleton. In the first vertebrates, this skeleton probably consisted of collagenous cellular cartilage, which forms the embryonic skeleton of all vertebrates and the adult skeleton of modern jawless and cartilaginous fish. In the head, most cellular cartilage is derived from a migratory cell population called the neural crest, which arises from the edges of the central nervous system. Because collagenous cellular cartilage and neural crest cells have not been described in invertebrates, the appearance of cellular cartilage derived from neural crest cells is considered a turning point in vertebrate evolution. Here we show that a tissue with many of the defining features of vertebrate cellular cartilage transiently forms in the larvae of the invertebrate chordate Branchiostoma floridae (Florida amphioxus). We also present evidence that during evolution, a key regulator of vertebrate cartilage development, SoxE, gained new cis-regulatory sequences that subsequently directed its novel expression in neural crest cells. Together, these results suggest that the origin of the vertebrate head skeleton did not depend on the evolution of a new skeletal tissue, as is commonly thought, but on the spread of this tissue throughout the head. We further propose that the evolution of cis-regulatory elements near an ancient regulator of cartilage differentiation was a major factor in the evolution of the vertebrate head skeleton.

CRISPR/Cas9-mediated mutagenesis in the sea lamprey Petromyzon marinus: a powerful tool for understanding ancestral gene functions in vertebrates.

Lamprey is one of only two living jawless vertebrates, a group that includes the first vertebrates. Comparisons between lamprey and jawed vertebrates have yielded important insights into the origin and evolution of vertebrate physiology, morphology and development. Despite its key phylogenetic position, studies of lamprey have been limited by their complex life history, which makes traditional genetic approaches impossible. The CRISPR/Cas9 system is a bacterial defense mechanism that was recently adapted to achieve high-efficiency targeted mutagenesis in eukaryotes. Here we report CRISPR/Cas9-mediated disruption of the genes Tyrosinase and FGF8/17/18 in the sea lamprey Petromyzon marinus, and detail optimized parameters for producing mutant F0 embryos. Using phenotype and genotype analyses, we show that CRISPR/Cas9 is highly effective in the sea lamprey, with a majority of injected embryos developing into complete or partial mutants. The ability to create large numbers of mutant embryos without inbred lines opens exciting new possibilities for studying development in lamprey and other non-traditional model organisms with life histories that prohibit the generation of mutant lines.

Roles for FGF in lamprey pharyngeal pouch formation and skeletogenesis highlight ancestral functions in the vertebrate head.

A defining feature of vertebrates (craniates) is a pronounced head supported and protected by a cellularized endoskeleton. In jawed vertebrates (gnathostomes), the head skeleton is made of rigid three-dimensional elements connected by joints. By contrast, the head skeleton of modern jawless vertebrates (agnathans) consists of thin rods of flexible cellular cartilage, a condition thought to reflect the ancestral vertebrate state. To better understand the origin and evolution of the gnathostome head skeleton, we have been analyzing head skeleton development in the agnathan, lamprey. The fibroblast growth factors FGF3 and FGF8 have various roles during head development in jawed vertebrates, including pharyngeal pouch morphogenesis, patterning of the oral skeleton and chondrogenesis. We isolated lamprey homologs of FGF3, FGF8 and FGF receptors and asked whether these functions are ancestral features of vertebrate development or gnathostome novelties. Using gene expression and pharmacological agents, we found that proper formation of the lamprey head skeleton requires two phases of FGF signaling: an early phase during which FGFs drive pharyngeal pouch formation, and a later phase when they directly regulate skeletal differentiation and patterning. In the context of gene expression and functional studies in gnathostomes, our results suggest that these roles for FGFs arose in the first vertebrates and that the evolution of the jaw and gnathostome cellular cartilage was driven by changes developmentally downstream from pharyngeal FGF signaling.

A maternally established SoxB1/SoxF axis is a conserved feature of chordate germ layer patterning.

Despite deep evolutionary roots in the metazoa, the gene regulatory network driving germ layer specification is surprisingly labile both between and within phyla. In Xenopus laevis, SoxB1- and SoxF-type transcription factors are intimately involved in germ-layer specification, in part through their regulation of Nodal signaling. However, it is unclear if X. laevis is representative of the ancestral vertebrate condition, as the precise roles of SoxF and SoxB1 in germ-layer specification vary among vertebrates, and there is no evidence that SoxF mediates germ-layer specification in any invertebrate. To better understand the evolution of germ-layer specification in the vertebrate lineage, we analyzed the expression of soxB1 and soxF genes in embryos and larvae of the basal vertebrate lamprey, and the basal chordate amphioxus. We find that both species maternally deposit soxB1 mRNA in the animal pole, soxF mRNA in the vegetal hemisphere, and zygotically express soxB1 and soxF throughout nascent ectoderm and mesendoderm, respectively. We also find that soxF is excluded from the vegetalmost blastomeres in lamprey and that, in contrast to vertebrates, amphioxus does not express soxF in the oral epithelium. In the context of recent work, our results suggest that a maternally established animal/vegetal Sox axis is a deeply conserved feature of chordate development that predates the role of Nodal in vertebrate germ-layer specification. Furthermore, exclusion of this axis from the vegetal pole in lamprey is consistent with the presence of an extraembryonic yolk mass, as has been previously proposed. Finally, conserved expression of SoxF in the forming mouth across the vertebrates, but not in amphioxus, lends support to the idea that the larval amphioxus mouth is nonhomologous to the vertebrate mouth.

Evolution of retinoic acid receptors in chordates: insights from three lamprey species, Lampetra fluviatilis, Petromyzon marinus, and Lethenteron japonicum.

BACKGROUND: Retinoic acid (RA) signaling controls many developmental processes in chordates, from early axis specification to late organogenesis. The functions of RA are chiefly mediated by a subfamily of nuclear hormone receptors, the retinoic acid receptors (RARs), that act as ligand-activated transcription factors. While RARs have been extensively studied in jawed vertebrates (that is, gnathostomes) and invertebrate chordates, very little is known about the repertoire and developmental roles of RARs in cyclostomes, which are extant jawless vertebrates. Here, we present the first extensive study of cyclostome RARs focusing on three different lamprey species: the European freshwater lamprey, Lampetra fluviatilis, the sea lamprey, Petromyzon marinus, and the Japanese lamprey, Lethenteron japonicum. RESULTS: We identified four rar paralogs (rar1, rar2, rar3, and rar4) in each of the three lamprey species, and phylogenetic analyses indicate a complex evolutionary history of lamprey rar genes including the origin of rar1 and rar4 by lineage-specific duplication after the lamprey-hagfish split. We further assessed their expression patterns during embryonic development by in situ hybridization. The results show that lamprey rar genes are generally characterized by dynamic and highly specific expression domains in different embryonic tissues. In particular, lamprey rar genes exhibit combinatorial expression domains in the anterior central nervous system (CNS) and the pharyngeal region. CONCLUSIONS: Our results indicate that the genome of lampreys encodes at least four rar genes and suggest that the lamprey rar complement arose from vertebrate-specific whole genome duplications followed by a lamprey-specific duplication event. Moreover, we describe a combinatorial code of lamprey rar expression in both anterior CNS and pharynx resulting from dynamic and highly specific expression patterns during embryonic development. This 'RAR code' might function in regionalization and patterning of these two tissues by differentially modulating the expression of downstream effector genes during development.

Experimental tests of trophic dynamics: taking a closer look.

The relative importance of bottom-up versus top-down forces, and the effect of productivity on community dynamics continue to be of much interest to ecologists. Trophic dynamic theories are difficult to test, as they require explicit knowledge of the many organisms involved, as well as the nature of the interactions between them. The Oksanen-Fretwell (OF) theory, which suggests that the relative roles of top-down and bottom-up factors vary with primary productivity, is well known in the literature, but is difficult to test rigorously. Recently, two experimental studies have tried to test OF theory. In this paper we discuss methodological problems associated with these studies that may weaken the conclusions drawn by the authors.

Genetics and morphology in a Borrichia frutescens and B. arborescens (Asteraceae) hybrid zone.

Interspecific plant hybridization is a common and evolutionarily important phenomenon. Here, the results of a study of hybridization in the Florida Keys between two species of sea oxeye daisy, Borrichia frutescens and B. arborescens, are reported. Nuclear and chloroplast genetic loci, log-likelihood assignment tests, and maximum likelihood estimates of genealogical class frequencies were used to identify hybrid and parent genotypes, to investigate the utility of leaf and flower morphology for hybrid identification, and to study symmetry and degree of introgression between the species. Genetic analyses confirmed the identity of the hybrid and parent plants that were used for the morphological studies. Together, leaf and flower morphology can be used to identify hybrid and parental types with moderate accuracy (4% error rate). Population genetic analyses indicate that, in spite of a significant level of hybridization, pure B. frutescens and B. arborescens are persisting in the hybrid zone. Of the nonparentals, about 18% appear to be F(1) hybrids, over 50% F(2) hybrids, and the remainder backcrossed individuals but only with the B. frutescens parent. It is postulated that the hybrid zone in the Florida Keys is being maintained by a combination of positive assortative mating and clonal reproduction.

Induced defensive response of myrtle oak to foliar insect herbivory in ambient and elevated CO2.

The rising level of atmospheric CO2 has stimulated several recent studies attempting to predict the effects of increased CO2 on ecological communities. However, most of these studies have been conducted in the benign conditions of the laboratory and in the absence of herbivores. In the current study, we utilized large octagonal chambers, which enclosed portions of an intact scrub-oak community to investigate the interactive effects of CO2 and insect herbivory on myrtle oak, Quercus myrtifolia. Specifically, we assessed the effects of ambient and elevated CO2 (2x current concentrations) on percent foliar nitrogen, C:N ratio, total relative foliar tannin content, and the presence of leaf damage caused by leaf mining and leaf chewing insects that feed on myrtle oak. Total foliar N declined and C:N ratios increased significantly in oaks in elevated CO2 chambers. The percentages of leaves damaged by either leafminers or leaf chewers tended to be lower in elevated compared to ambient chambers, but they co-occurred on leaves less than expected, regardless of CO2 treatment. Leaves that had been either mined or chewed exhibited a similar wounding or defensive response; they had an average of 25 and 21% higher protein binding ability, which is correlated with tannin concentration, compared to nondamaged control leaves, respectively. While the protein-binding ability (expressed as total percent tannin) of leaves from elevated CO2 was slightly higher than from leaves grown in ambient chambers, this difference was not significant.