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.

Toll-like receptors (TLRs) are important for raising innate immune responses in both invertebrates and vertebrates. Amphioxus belongs to an ancient chordate lineage which shares key features with vertebrates. The genomic research on TLR genes in Branchiostoma floridae and Branchiostoma belcheri reveals the expansion of TLRs in amphioxus. However, the repertoire of TLRs in Branchiostoma lanceolatum has not been studied and the functionality of amphioxus TLRs has not been reported. We have identified from transcriptomic data 30 new putative TLRs in B. lanceolatum and all of them are transcribed in adult amphioxus. Phylogenetic analysis showed that the repertoire of TLRs consists of both non-vertebrate and vertebrate-like TLRs. It also indicated a lineage-specific expansion in orthologous clusters of the vertebrate TLR11 family. We did not detect any representatives of the vertebrate TLR1, TLR3, TLR4, TLR5 and TLR7 families. To gain insight into these TLRs, we studied in depth a particular TLR highly similar to a B. belcheri gene annotated as bbtTLR1. The phylogenetic analysis of this novel BlTLR showed that it clusters with the vertebrate TLR11 family and it might be more related to TLR13 subfamily according to similar domain architecture. Transient and stable expression in HEK293 cells showed that the BlTLR localizes on the plasma membrane, but it did not respond to the most common mammalian TLR ligands. However, when the ectodomain of BlTLR is fused to the TIR domain of human TLR2, the chimeric protein could indeed induce NF-κB transactivation in response to the viral ligand Poly I:C, also indicating that in amphioxus, specific accessory proteins are needed for downstream activation. Based on the phylogenetic, subcellular localization and functional analysis, we propose that the novel BlTLR might be classified as an antiviral receptor sharing at least partly the functions performed by vertebrate TLR22. TLR22 is thought to be viral teleost-specific TLR but here we demonstrate that teleosts and amphioxus TLR22-like probably shared a common ancestor. Additional functional studies with other lancelet TLR genes will enrich our understanding of the immune response in amphioxus and will provide a unique perspective on the evolution of the immune system.

Homologous long non-coding RNAs (lncRNAs) are elusive to identify by sequence similarity due to their fast-evolutionary rate. Here we develop LincOFinder, a pipeline that finds conserved intergenic lncRNAs (lincRNAs) between distant related species by means of microsynteny analyses. Using this tool, we have identified 16 bona fide homologous lincRNAs between the amphioxus and human genomes. We characterized and compared in amphioxus and Xenopus the expression domain of one of them, Hotairm1, located in the anterior part of the Hox cluster. In addition, we analyzed the function of this lincRNA in Xenopus, showing that its disruption produces a severe headless phenotype, most probably by interfering with the regulation of the Hox cluster. Our results strongly suggest that this lincRNA has probably been regulating the Hox cluster since the early origin of chordates. Our work pioneers the use of syntenic searches to identify non-coding genes over long evolutionary distances and helps to further understand lncRNA evolution.

Control of cell number is crucial to define body size during animal development and to restrict tumoral transformation. The cell number is determined by the balance between cell proliferation and cell death. Although many genes are known to regulate those processes, the molecular mechanisms underlying the relationship between cell number and body size remain poorly understood. This relationship can be better understood by studying planarians, flatworms that continuously change their body size according to nutrient availability. We identified a novel gene family, blitzschnell (bls), which consists of de novo and taxonomically restricted genes that control cell proliferation:cell death ratio. Their silencing promotes faster regeneration and increases cell number during homeostasis. Importantly, this increase in cell number only leads to an increase in body size in a nutrient-rich environment; in starved planarians silencing results in a decrease in cell size and cell accumulation that ultimately produces overgrowths. bls expression is down-regulated after feeding and related with the Insulin/Akt/mTOR network activity, suggesting that the bls family evolved in planarians as an additional mechanism by which to restrict cell number in nutrient-fluctuating environments.

The vertebrate brain is arguably the most complex anatomical and functional structure in nature. During embryonic development, the central nervous system (CNS) undergoes a series of morphogenetic processes that eventually obscure the major axes of the early neural plate to our perception. Notwithstanding this complexity, the “genoarchitecture” of the developing neural tube brings into light homologous regions between brains of different vertebrate species, acting as a molecular barcode of each particular domain. Those homologous regions and their topological inter-relations constitute the ancestral, deeply conserved, bauplan of the vertebrate brain. Remarkably, although simpler, the cephalochordate amphioxus shares multiple features of this bauplan, serving as a privileged reference point to understand the origins of the vertebrate brain. Here, we review the development of the chordate CNS in view of the latest morphological and genoarchitectonic data from amphioxus. This comparison reveals that the amphioxus CNS is far from simple and provides unique insights into the structure of the vertebrate CNS and its evolutionary origins. In particular, we summarize recent research in amphioxus and vertebrates that has challenged views on the major partitions of the vertebrate brain, proposing a novel organization of the chordate CNS bauplan that better reflects developmental and evolutionary data.

RNA editing is a relatively unexplored process in which transcribed RNA is modified at specific nucleotides before translation, adding another level of regulation of gene expression. Cephalopods use it extensively to increase the regulatory complexity of their nervous systems, and mammals use it too, but less prominently. Nevertheless, little is known about the specifics of RNA editing in most of the other clades and the relevance of RNA editing from an evolutionary perspective remains unknown. Here we analyze a key element of the editing machinery, the ADAR (adenosine deaminase acting on RNA) gene family, in an animal with a key phylogenetic position at the root of chordates: the cephalochordate amphioxus. We show, that as in cephalopods, ADAR genes in amphioxus are predominantly expressed in the nervous system; we identify a number of RNA editing events in amphioxus; and we provide a newly developed method to identify RNA editing events in highly polymorphic genomes using orthology as a guide. Overall, our work lays the foundations for future comparative analysis of RNA-editing events across the metazoan tree.