Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates-a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

In this review, we outline the gene-regulatory interactions driving neural crest development and compare these to a hypothetical network operating in the embryonic ectoderm of the cephalochordate amphioxus. While the early stages of ectodermal patterning appear conserved between amphioxus and vertebrates, later activation of neural crest-specific factors at the neural plate border appears to be a vertebrate novelty. This difference may reflect co-option of genetic pathways which conferred novel properties upon the evolving vertebrate neural plate border, potentiating the evolution of definitive neural crest.

The vertebrate neural crest migrates from its origin, the neural plate border, to form diverse derivatives. We previously hypothesized that a neural crest gene regulatory network (NC-GRN) guides neural crest formation. Here, we investigate when during evolution this hypothetical network emerged by analyzing neural crest formation in lamprey, a basal extant vertebrate. We identify 50 NC-GRN homologs and use morpholinos to demonstrate a critical role for eight transcriptional regulators. The results reveal conservation in deployment of upstream factors, suggesting that proximal portions of the network arose early in vertebrate evolution and have been conserved for >500 million years. We found biphasic expression of neural crest specifiers and differences in deployment of some specifiers and effectors expected to confer species-specific properties. By testing the collective expression and function of neural crest genes in a single, basal vertebrate, we reveal the ground state of the NC-GRN and resolve ambiguities between model organisms.

The emergence of the neural crest has been proposed to play a key role in early vertebrate evolution by remodeling the chordate head into a “new head” that enabled early vertebrates to shift from filter feeding to active predation. Here we show that the genome of the basal chordate, amphioxus, contains homologs of most vertebrate genes implicated in a putative neural crest gene regulatory network (NC-GRN) for neural crest development. Our survey of gene expression shows that early inducing signals, neural plate border patterning genes, and melanocyte differentiation genes appear conserved. Furthermore, exogenous BMP affects expression of amphioxus neural plate border genes as in vertebrates, suggesting that conserved signals specify the neural plate border throughout chordates. In contrast to this core conservation, many neural crest specifier genes are not expressed at the amphioxus neural plate/tube border, raising the intriguing possibility that this level of the network was co-opted during vertebrate evolution. Consistent with this, the regulatory region of AmphiFoxD, homologous to the vertebrate neural crest specifier FoxD3, drives tissue-specific reporter expression in chick mesoderm, but not neural crest. Thus, evolution of a new regulatory element may have allowed co-option of this gene to the NC-GRN.

Central to the story of vertebrate evolution is the origin of the vertebrate head, a problem difficult to approach using paleontology and comparative morphology due to a lack of unambiguous intermediate forms. Embryologically, much of the vertebrate head is derived from two ectodermal tissues, the neural crest and cranial placodes. Recent work in protochordates suggests the first chordates possessed migratory neural tube cells with some features of neural crest cells. However, it is unclear how and when these cells acquired the ability to form cellular cartilage, a cell type unique to vertebrates. It has been variously proposed that the neural crest acquired chondrogenic ability by recruiting proto-chondrogenic gene programs deployed in the neural tube, pharynx, and notochord. To test these hypotheses we examined the expression of 11 amphioxus orthologs of genes involved in neural crest chondrogenesis. Consistent with cellular cartilage as a vertebrate novelty, we find that no single amphioxus tissue co-expresses all or most of these genes. However, most are variously co-expressed in mesodermal derivatives. Our results suggest that neural crest-derived cartilage evolved by serial cooption of genes which functioned primitively in mesoderm.