Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Bilaterian animals are notably characterized by complex endocrine systems. The receptors for many steroids, retinoids, and other hormones belong to the superfamily of nuclear receptors, which are transcription factors regulating many aspects of development and homeostasis. Despite a diversity of regulatory mechanisms and physiological roles, nuclear receptors share a common protein organization. To obtain the broad picture of bilaterian nuclear hormone receptor evolution, we have characterized the complete set of nuclear receptor genes from nine animal genome sequences and analyzed it in a phylogenetic framework. In addition, expressed sequence tags from key lineages with no available genome sequence were also searched. This allows us to date the evolutionary events that led from an ancestral nuclear receptor gene, in an early metazoan, to present day diversity. We show that there were ∼25 nuclear receptor genes in Urbilateria, the ancestor of bilaterians, at which point the fundamental diversity of the subfamily was already established. Surprisingly, differential gene loss played an important role in the evolution of different nuclear receptor sets in bilaterian lineages. The nuclear receptor distribution was also shaped by periods of gene duplication, essentially in vertebrates, as well as a lineage-specific duplication burst in nematodes. Our results imply that the genes for major receptors such as steroid receptors or thyroid hormone receptors were present in Urbilateria.

Nuclear receptors (NRs) are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes) using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily. NRs are key molecules controlling development, metabolism, and reproduction in metazoans. Since NRs are implicated in many human diseases such as cancer, metabolic syndrome, and hormone resistance, they are important pharmaceutical targets and are under intense scrutiny to better understand their biological functions. In the present study, we determined the expression patterns of all NR genes as well as their main transcriptional coregulators during zebrafish development. We used zebrafish because the transparency of its embryo allows us to perform whole-mount in situ hybridization from early development to late organogenesis. This complete developmental profiling offers an unprecedented view of NR expression during embryogenesis, uncovering their potential function during central nervous system and retina formation. We observed that in contrast to NR genes, only a few coregulators exhibit a restricted expression pattern, suggesting that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Lastly, by evolutionary analysis of expression pattern divergence of duplicated genes, we observed that neofunctionalization occurs at the levels of both protein sequence and mRNA expression patterns. Taken together, our data provide the starting point for functional analysis of an entire gene family during development and call for the study of the intersection between metabolism and development.

Nuclear receptors form a superfamily of ligand-activated transcription factors implicated in various physiological functions from development to homoeostasis. Nuclear receptors share a common evolutionary history revealed by their conserved structure and by their high degree of sequence conservation. Here we review the latest advances on the evolution of nuclear receptors by addressing the following questions. What is known about the appearance and diversification of nuclear hormone receptors? How did their different functional characteristics evolve? What can we infer from the analysis of complete genomes? In summary, the study of the evolution of nuclear receptors will be very important not only for understanding their functions in vivo but also for obtaining insights into the evolution of animal genomes as a whole.

FGF signaling is one of the few cell–cell signaling pathways conserved among all metazoans. The diversity of FGF gene content among different phyla suggests that evolution of FGF signaling may have participated in generating the current variety of animal forms. Vertebrates possess the greatest number of FGF genes, the functional evolution of which may have been implicated in the acquisition of vertebrate-specific morphological traits. In this study, we have investigated the roles of the FGF signal during embryogenesis of the cephalochordate amphioxus, the best proxy for the chordate ancestor. We first isolate the full FGF gene complement and determine the evolutionary relationships between amphioxus and vertebrate FGFs via phylogenetic and synteny conservation analysis. Using pharmacological treatments, we inhibit the FGF signaling pathway in amphioxus embryos in different time windows. Our results show that the requirement for FGF signaling during gastrulation is a conserved character among chordates, whereas this signal is not necessary for neural induction in amphioxus, in contrast to what is known in vertebrates. We also show that FGF signal, acting through the MAPK pathway, is necessary for the formation of the most anterior somites in amphioxus, whereas more posterior somite formation is not FGF-dependent. This result leads us to propose that modification of the FGF signal function in the anterior paraxial mesoderm in an amphioxus-like vertebrate ancestor might have contributed to the loss of segmentation in the preotic paraxial mesoderm of the vertebrate head.

The phylogenetic position of amphioxus, together with its relatively simple and evolutionarily conserved morphology and genome structure, has led to its use as a model for studies of vertebrate evolution. In particular, the recent development of technical approaches, as well as access to the complete amphioxus genome sequence, has provided the community with tools with which to study the invertebrate-chordate to vertebrate transition. Here, we present this animal model, discussing its life cycle, the model species studied and the experimental techniques that it is amenable to. We also summarize the major findings made using amphioxus that have informed us about the evolution of vertebrate traits.