Stéphanie Bertrand

Neofunctionalization in Vertebrates: The Example of Retinoic Acid Receptors

Understanding the role of gene duplications in establishing vertebrate innovations is one of the main challenges of Evo-Devo (evolution of development) studies. Data on evolutionary changes in gene expression (i.e., evolution of transcription factor-cis-regulatory elements relationships) tell only part of the story; protein function, best studied by biochemical and functional assays, can also change. In this study, we have investigated how gene duplication has affected both the expression and the ligand-binding specificity of retinoic acid receptors (RARs), which play a major role in chordate embryonic development. Mammals have three paralogous RAR genes—RARα, β, and γ—which resulted from genome duplications at the origin of vertebrates. By using pharmacological ligands selective for specific paralogues, we have studied the ligand-binding capacities of RARs from diverse chordates species. We have found that RARβ-like binding selectivity is a synapomorphy of all chordate RARs, including a reconstructed synthetic RAR representing the receptor present in the ancestor of chordates. Moreover, comparison of expression patterns of the cephalochordate amphioxus and the vertebrates suggests that, of all the RARs, RARβ expression has remained most similar to that of the ancestral RAR. On the basis of these results together, we suggest that while RARβ kept the ancestral RAR role, RARα and RARγ diverged both in ligand-binding capacity and in expression patterns. We thus suggest that neofunctionalization occurred at both the expression and the functional levels to shape RAR roles during development in vertebrates.

Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits

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.

Evolutionary crossroads in developmental biology: amphioxus

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.

Nuclear hormone receptor signaling in amphioxus

The nuclear hormone receptors (NRs) form a superfamily of transcription factors unified by conserved protein structure and mode of function. While most members of this superfamily are activated by ligands, such as thyroid hormones, steroids, vitamin D or retinoic acid, other NRs are called orphan receptors because they have no known ligand. NR-dependent signaling is crucial for vertebrate development with the majority of receptors being expressed in the developing embryo. Due to massive gene duplications during vertebrate diversification, there are usually more NRs in vertebrates than in invertebrates. In this study, we examine the evolutionary diversification of the NR superfamily and of NR-dependent signaling in chordates (vertebrates, tunicates, and amphioxus). We take advantage of the unique features of the genome of the invertebrate amphioxus, which is characterized by a vertebrate-like gene content without having undergone massive duplications, to assess the NR signaling complement (NRs and NR coregulators) of the ancestral chordate. We find 33 NRs in amphioxus, which are more NRs than originally anticipated. This increase is mainly due to an amphioxus-specific duplication of genes encoding receptors of the NR1H group. In addition, there are three heterologous NRs in amphioxus that could not be placed within the framework of the NR superfamily. Apart from these exceptions, there is usually one amphioxus NR or NR signaling coregulator for each paralogous group of two, three, or four human receptors suggesting that the ancestral chordate had a set of 22 different NRs plus one copy of each NR coregulator.

A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation

The HoxA and HoxD gene clusters of jawed vertebrates are organized into bipartite three-dimensional chromatin structures that separate long-range regulatory inputs coming from the anterior and posterior Hox-neighboring regions. This architecture is instrumental in allowing vertebrate Hox genes to pattern disparate parts of the body, including limbs. Almost nothing is known about how these three-dimensional topologies originated. Here we perform extensive 4C-seq profiling of the Hox cluster in embryos of amphioxus, an invertebrate chordate. We find that, in contrast to the architecture in vertebrates, the amphioxus Hox cluster is organized into a single chromatin interaction domain that includes long-range contacts mostly from the anterior side, bringing distant cis-regulatory elements into contact with Hox genes. We infer that the vertebrate Hox bipartite regulatory system is an evolutionary novelty generated by combining ancient long-range regulatory contacts from DNA in the anterior Hox neighborhood with new regulatory inputs from the posterior side.

Evolution of the FGF Gene Family

Fibroblast Growth Factors (FGFs) are small proteins generally secreted, acting through binding to transmembrane tyrosine kinase receptors (FGFRs). Activation of FGFRs triggers several cytoplasmic cascades leading to the modification of cell behavior. FGFs play critical roles in a variety of developmental and physiological processes. Since their discovery in mammals, FGFs have been found in many metazoans and some arthropod viruses. Efforts have been previously made to decipher the evolutionary history of this family but conclusions were limited due to a poor taxonomic coverage. We took advantage of the availability of many new sequences from diverse metazoan lineages to further explore the possible evolutionary scenarios explaining the diversity of the FGF gene family. Our analyses, based on phylogenetics and synteny conservation approaches, allow us to propose a new classification of FGF genes into eight subfamilies, and to draw hypotheses for the evolutionary events leading to the present diversity of this gene family.

Erratum to “Evolution of the FGF Gene Family”

The authors would like to make the following correction. In Figure  4 in the original paper shows two blue stars indicating genome duplications in the wrong place of the phylogenetic tree. This figure should be replaced by the following figure. The previous legend of Figure  4 is correct.

Developmental cell-cell communication pathways in the cephalochordate amphioxus: actors and functions

During embryonic development, cells of metazoan embryos need to communicate in order to construct the correct bodyplan. To do so, they use several signals that usually act through interactions between ligands and receptors. Interestingly, only a few pathways are known to be fundamental during animal development, and they are usually found in all the major metazoan clades, raising the following question: how have evolution of the actors and of the functions of these pathways participated in the appearance of the current diversity of animal morphologies? The chordate lineage comprises vertebrates, their sister group the urochordates, and the cephalochordates (i.e. amphioxus). Urochordates are quite derived relative to the chordate ancestor, whereas cephalochordates and vertebrates share many morphological traits. Thus, comparing embryonic development between vertebrates and cephalochordates should give us some insight into the ancestral characters present in chordates and into the morphological evolution in this clade. However, while much is known about the function of different signalling pathways in vertebrates, data are still scarce in the literature for cephalochordates. In this review, we summarize the current state of the field concerning the expression of actors and the function of the major cell-cell communication pathways, including Hedgehog (Hh), Notch, Nuclear Receptor (NR), Receptor Tyrosine Kinase (RTK), Transforming Growth Factor-β (TGF-β) and Wingless/Int (Wnt), in amphioxus.