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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.

Multiple invertebrates possess enzymes enabling de novo biosynthesis of essential omega-3 fatty acids.

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Oct4 is considered a master transcription factor for pluripotent cell self-renewal, but its biology remains poorly understood. Here, we investigated the role of Oct4 using the process of induced pluripotency. We found that a defined embryonic stem cell (ESC) level of Oct4 is required for pluripotency entry. However, once pluripotency is established, the Oct4 level can be decreased up to sevenfold without loss of self-renewal. Unexpectedly, cells constitutively expressing Oct4 at an ESC level robustly differentiated into all embryonic lineages and germline. In contrast, cells with low Oct4 levels were deficient in differentiation, exhibiting expression of naive pluripotency genes in the absence of pluripotency culture requisites. The restoration of Oct4 expression to an ESC level rescued the ability of these to restrict naive pluripotent gene expression and to differentiate. In conclusion, a defined Oct4 level controls the establishment of naive pluripotency as well as commitment to all embryonic lineages.

Long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids are essential components of biomembranes, particularly in neural tissues. Endogenous synthesis of ARA, EPA and DHA occurs from precursor dietary essential fatty acids such as linoleic and α-linolenic acid through elongation and Δ5 and Δ6 desaturations. With respect to desaturation activities some noteworthy differences have been noted in vertebrate classes. In mammals, the Δ5 activity is allocated to the Fads1 gene, while Fads2 is a Δ6 desaturase. In contrast, teleosts show distinct combinations of desaturase activities (e.g. bifunctional or separate Δ5 and Δ6 desaturases) apparently allocated to Fads2-type genes. To determine the timing of Fads1-Δ5 and Fads2-Δ6 evolution in vertebrates we used a combination of comparative and functional genomics with the analysis of key phylogenetic species. Our data show that Fads1 and Fads2 genes with Δ5 and Δ6 activities respectively, evolved before gnathostome radiation, since the catshark Scyliorhinus canicula has functional orthologues of both gene families. Consequently, the loss of Fads1 in teleosts is a secondary episode, while the existence of Δ5 activities in the same group most likely occurred through independent mutations into Fads2 type genes. Unexpectedly, we also establish that events of Fads1 gene expansion have taken place in birds and reptiles. Finally, a fourth Fads gene (Fads4) was found with an exclusive occurrence in mammalian genomes. Our findings enlighten the history of a crucially important gene family in vertebrate fatty acid metabolism and physiology and provide an explanation of how observed lineage-specific gene duplications, losses and diversifications might be linked to habitat-specific food web structures in different environments and over geological timescales.

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The Drosophila melanogaster genome has six physically clustered NK-related homeobox genes in just 180 kb. Here we show that the NK homeobox gene cluster was an ancient feature of bilaterian animal genomes, but has been secondarily split in chordate ancestry. The NK homeobox gene clusters of amphioxus and vertebrates are each split and dispersed at two equivalent intergenic positions. From the ancestral NK gene cluster, only the Tlx-Lbx and NK3-NK4 linkages have been retained in chordates. This evolutionary pattern is in marked contrast to the Hox and ParaHox gene clusters, which are compact in amphioxus and vertebrates, but have been disrupted in Drosophila.T he Drosophila melanogaster 93D͞E or NK gene cluster contains six homeobox genes: tinman (tin, NK4), bagpipe (bap, NK3), ladybird late (lbl), ladybird early (lbe), C15 (93Bal), and slouch (slou, NK1) in a gene cluster spanning just 180 kb (1, 2). All six genes possess homeobox sequences of the ANTP class (3), forming a distinct clade within this class along with several NK-related genes (4). The similarity of the genes and their clustered arrangement indicates that they arose through a series of tandem gene duplications, in an analogous way to the Hox gene cluster. We deduce that the NK homeobox gene cluster is ancient, dating at least to the base of the Bilateria. The reasoning is based on the phylogenetic distribution of these genes. Animals from very divergent evolutionary lineages have orthologues of each Drosophila NK gene (except for lbl and lbe, which are recent tandem duplicates). For example, human NKX3.1 and NKX3.2 (BAPX1) genes are orthologues of Drosophila bap, LBX1 and LBX2 genes are orthologues of the ancestral ladybird gene, two human genes equivalent to mouse Nkx1.1 and Nkx1.2 are orthologues of slou, and TLX1, TLX2, and TLX3 (the HOX11 family) are orthologues of C15 (4). It is also likely that human NKX2. 3, NKX2.5, and NKX2.6 are orthologues of Drosophila tin, although sequence similarity is less clear (4, 5). Existence of these human genes implies that the tandem duplications that produced these various NK-related genes had occurred before the divergence of humans and Drosophila and, by implication, before the separation of the Deuterostomia and Ecdysozoa. Therefore, Drosophila NK (and related) genes have remained a tight gene cluster since the origin of these genes Ͼ500 million years (Myr) ago, which implies the existence of a selective reason for the gene clustering in Drosophila.