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.

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.

All vertebrate brains develop following a common Bauplan defined by anteroposterior (AP) and dorsoventral (DV) subdivisions, characterized by largely conserved differential expression of gene markers. However, it is still unclear how this Bauplan originated during evolution. We studied the relative expression of 48 genes with key roles in vertebrate neural patterning in a representative amphioxus embryonic stage. Unlike nonchordates, amphioxus develops its central nervous system (CNS) from a neural plate that is homologous to that of vertebrates, allowing direct topological comparisons. The resulting genoarchitectonic model revealed that the amphioxus incipient neural tube is unexpectedly complex, consisting of several AP and DV molecular partitions. Strikingly, comparison with vertebrates indicates that the vertebrate thalamus, pretectum, and midbrain domains jointly correspond to a single amphioxus region, which we termed Di-Mesencephalic primordium (DiMes). This suggests that these domains have a common developmental and evolutionary origin, as supported by functional experiments manipulating secondary organizers in zebrafish and mice. According to textbooks, vertebrate brains develop from a neural tube that rapidly becomes regionalized into the forebrain (which includes the secondary prosencephalon and diencephalon), midbrain, and hindbrain. These regions are then further subdivided; in particular, the diencephalon gives rise to the prethalamus, thalamus, and pretectum. However, embryological manipulations of brain signaling centers showed that the prethalamus behaves very differently than the thalamus and pretectum, which largely share their developmental potential with the midbrain. Therefore, this classic partition scheme might not be fully consistent from a developmental perspective. To better understand the origin and evolution of the regionalization of the vertebrate brain, we built a comprehensive molecular model of the incipient neural tube of amphioxus, an invertebrate chordate that shares multiple features with its vertebrate relatives. This model shows that the amphioxus nervous system is unexpectedly complex, sharing its basic blueprint with that of vertebrates. However, a single undivided region in amphioxus, which we termed Di-Mesencephalic primordium (DiMes), unambiguously corresponds to the region encompassing the thalamus, pretectum, and midbrain in vertebrates, indicating that these regions are also more closely related evolutionarily. Therefore, the diencephalon as a neuroanatomical compartment as well as the classic separation between forebrain and midbrain in vertebrates seem inconsistent from both an evolutionary and developmental perspective.

D‐Aspartic acid (D‐Asp) is present in invertebrate and vertebrate neuroendocrine tissues’ where it carries out important physiological functions and is implicated in nervous system development. We show here that D‐Asp is a novel endogenous neurotransmitter in two distantly related animals’ a mammal ( Rattus norvégiens ) and a mollusk ( Loligo vulgaris ). Our main findings demonstrate that D‐Asp is present in high concentrations in the synaptic vesicles of axon terminals;synthesis for this amino acid occurs in neurons by conversion of L‐Asp to D‐Asp via D‐aspartate racemase;depolarization of nerve endings with K ⁺ ions evokes an immediate release of D‐Asp in a Ca ²⁺ dependent manner;specific receptors for D‐Asp occur at the postsynaptic membrane’ as demonstrated by binding assays and by the expansion of squid skin chromatophores;D‐aspartate oxidase’ the specific enzyme that oxidizes D‐Asp’ is present in the postsynaptic membranes;and stimulation of nerve endings with D‐Asp triggers signal transduction by increasing the second messenger cAMP. Taken together’ these data demonstrate that D‐Asp fulfills all criteria necessary to be considered a novel endogenous neurotransmitter. Given its known role inneurogenesis’ learning’ and neuropathologies’ our results have important implications for biomedical and clinical research.—D'Aniello, S., Somorjai, I., Garcia‐Fernàndez, J., Topo, E., D'Aniello, A. D‐Aspartic acid is a novel endogenous neurotransmitter. FASEB J. 25’ 1014–1027 (2011). www.fasebj.org

Brain function requires neuronal activity-dependent energy consumption. Neuronal energy supply is controlled by molecular mechanisms that regulate mitochondrial dynamics, including Kinesin motors and Mitofusins, Miro1-2 and Trak2 proteins. Here we show a new protein family that localizes to the mitochondria and controls mitochondrial dynamics. This family of proteins is encoded by an array of armadillo (Arm) repeat-containing genes located on the X chromosome. The Armcx cluster is unique to Eutherian mammals and evolved from a single ancestor gene (Armc10). We show that these genes are highly expressed in the developing and adult nervous system. Furthermore, we demonstrate that Armcx3 expression levels regulate mitochondrial dynamics and trafficking in neurons, and that Alex3 interacts with the Kinesin/Miro/Trak2 complex in a Ca²⁺-dependent manner. Our data provide evidence of a new Eutherian-specific family of mitochondrial proteins that controls mitochondrial dynamics and indicate that this key process is differentially regulated in the brain of higher vertebrates.

An important question in biology is why some animals are able to regenerate, whereas others are not. The basal chordate amphioxus is uniquely positioned to address the evolution of regeneration. We report here the high regeneration potential of the European amphioxus Branchiostoma lanceolatum . Adults regenerate both anterior and posterior structures, including neural tube, notochord, fin, and muscle. Development of a classifier based on tail regeneration profiles predicts the assignment of young and old adults to their own class with >94% accuracy. The process involves loss of differentiated characteristics, formation of an msx -expressing blastema, and neurogenesis. Moreover, regeneration is linked to the activation of satellite-like Pax3/7 progenitor cells, the extent of which declines with size and age. Our results provide a framework for understanding the evolution and diversity of regeneration mechanisms in vertebrates.