Asymmetric dimerization in a transcription factor superfamily is promoted by allosteric interactions with DNA.

Transcription factors, such as nuclear receptors achieve precise transcriptional regulation by means of a tight and reciprocal communication with DNA, where cooperativity gained by receptor dimerization is added to binding site sequence specificity to expand the range of DNA target gene sequences. To unravel the evolutionary steps in the emergence of DNA selection by steroid receptors (SRs) from monomeric to dimeric palindromic binding sites, we carried out crystallographic, biophysical and phylogenetic studies, focusing on the estrogen-related receptors (ERRs, NR3B) that represent closest relatives of SRs. Our results, showing the structure of the ERR DNA-binding domain bound to a palindromic response element (RE), unveil the molecular mechanisms of ERR dimerization which are imprinted in the protein itself with DNA acting as an allosteric driver by allowing the formation of a novel extended asymmetric dimerization region (KR-box). Phylogenetic analyses suggest that this dimerization asymmetry is an ancestral feature necessary for establishing a strong overall dimerization interface, which was progressively modified in other SRs in the course of evolution.

Henri de Lacaze-Duthiers and the ascidian hypothesis.

In 1830, Cuvier and Geoffroy Saint-Hilaire confronted each other in a famous debate on the unity of the animal kingdom, which permeated the zoology of the 19th century. From that time, a growing number of naturalists attempted to understand the large-scale relationships among animals. And among all the questions, that of the origin of vertebrates was one of the most controversial. Analytical methods based on comparative anatomy, embryology and paleontology were developed to identify convincing homologies that would reveal a logical sequence of events for the evolution of an invertebrate into the first vertebrate. Within this context, several theories have clashed on the question of the identity of the ancestor of vertebrates. Among the proposals, a group of rather discrete organisms, the ascidians, played a central role. Because he had discovered an ascidian with a particularly atypical larval development, the Molgula, Henri de Lacaze-Duthiers, a rigorous and meticulous naturalist, became involved in the ascidian hypothesis. While the visionary mind of Lacaze-Duthiers led him to establish a particularly innovative methodology and the first marine biology station in Europe, at Roscoff, the tailless tadpole of the Molgula prevented him from recognizing the ancestor of vertebrates. This old 19th century story echoes the ever-present questions driving the field of Eco-Evo-Devo.

Counting Nemo: anemonefish Amphiprion ocellaris identify species by number of white bars.

The brilliant colors of coral reef fish have received much research attention. This is well exemplified by anemonefish, which have distinct white bar patterns and inhabit host anemones and defend them as a territory. The 28 described species have between 0 and 3 white bars present, which has been suggested to be important for species recognition. In the present study, we found that Amphiprion ocellaris (a species that displays three white bars) hatched and reared in aquaria, when faced with an intruder fish, attacked their own species more frequently than other species of intruding anemonefish. Additionally, we explicitly tested whether this species could distinguish models with different numbers of bars. For this, 120 individuals of A. ocellaris were presented with four different models (no bars, and 1, 2 and 3 bars) and we compared whether the frequency of aggressive behavior towards the model differed according to the number of bars. The frequency of aggressive behavior toward the 3-bar model was the same as against living A. ocellaris, and was higher than towards any of the other models. We conclude that A. ocellaris use the number of white bars as a cue to identify and attack only competitors that might use the same host. We considered this as an important behavior for efficient host defense.

Amphioxus functional genomics and the origins of vertebrate gene regulation.

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.

Thyroid hormones regulate the formation and environmental plasticity of white bars in clownfishes.

Determining how plasticity of developmental traits responds to environmental conditions is a challenge that must combine evolutionary sciences, ecology, and developmental biology. During metamorphosis, fish alter their morphology and color pattern according to environmental cues. We observed that juvenile clownfish (Amphiprion percula) modulate the developmental timing of their adult white bar formation during metamorphosis depending on the sea anemone species in which they are recruited. We observed an earlier formation of white bars when clownfish developed with Stichodactyla gigantea (Sg) than with Heteractis magnifica (Hm). As these bars, composed of iridophores, form during metamorphosis, we hypothesized that timing of their development may be thyroid hormone (TH) dependent. We treated clownfish larvae with TH and found that white bars developed earlier than in control fish. We further observed higher TH levels, associated with rapid white bar formation, in juveniles recruited in Sg than in Hm, explaining the faster white bar formation. Transcriptomic analysis of Sg recruits revealed higher expression of duox, a dual oxidase implicated in TH production as compared to Hm recruits. Finally, we showed that duox is an essential regulator of iridophore pattern timing in zebrafish. Taken together, our results suggest that TH controls the timing of adult color pattern formation and that shifts in duox expression and TH levels are associated with ecological differences resulting in divergent ontogenetic trajectories in color pattern development.

A structural signature motif enlightens the origin and diversification of nuclear receptors.

Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homodimers or as heterodimers. The dimerization is crucial for gene regulation by nuclear receptors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual structural variation of the α-helix, called π-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of the π-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of the π-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analyses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that the π-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for the π-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that the π-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors.

A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Direct development of the catfish pectoral fin: An alternative pectoral fin pattern of teleosts.

BACKGROUND: The study of the teleosts' pectoral fin development touches on many crucial issues of evolutionary biology, from the formation of local adaptations to the tetrapod limbs' origin. Teleosts' pectoral fin is considered a rather developmentally and anatomically conservative structure. It displays larval and adult stages differing in the skeletal and soft tissues' composition. Larva-adult transition proceeds under the thyroid hormone (TH) control that defines pectoral fin ontogeny as an indirect development. However, the outstanding diversity of teleosts allows suggesting the existence of lineage specific developmental patterns. RESULTS: We present a description of the North African catfish, Clarias gariepinus, pectoral fin development. It lacks a clear larval stage and directly develops the adult skeleton with the associated musculature and innervation. Interestingly, the development of catfish pectoral fin appears not to be under the TH dependence. CONCLUSION: This catfish displays a direct pectoral fin developmental trajectory differing from the stereotyped teleost pattern. In the absence of the larval endoskeletal disk and TH control, the catfish's proximal radials arise in a manner somewhat similar to the metapterygial radials in basal actinopterygians and humerus in sarcopterygians. Thus, the catfish fin pattern seems homoplastic, arising by convergence with, or reversion to the ancestral developmental mechanisms.

Magic Traits in Magic Fish: Understanding Color Pattern Evolution Using Reef Fish.

Color patterns provide easy access to phenotypic diversity and allow the questioning of the adaptive value of traits or the constraints acting on phenotypic evolution. Reef fish offer a unique opportunity to address such questions because they are ecologically and phylogenetically diverse and have the largest variety of pigment cell types known in vertebrates. In addition to recent development of their genetic resources, reef fish also constitute experimental models that allow the discrimination of ecological, developmental, and evolutionary processes at work. Here, we emphasize how the study of color patterns in reef fish can be integrated in an Eco/Evo/Devo (ecological evolutionary developmental) perspective and we illustrate that such an approach can bring new insights on the evolution of complex phenotypes.

Colour patterns influence symbiosis and competition in the anemonefish-host anemone symbiosis system.

Colour patterns in fish are often used as an important medium for communication. Anemonefish, characterized by specific patterns of white bars, inhabit host anemones and defend the area around an anemone as their territory. The host anemone is used not only by the anemonefish, but also by other fish species that use anemones as temporary shelters. Anemonefish may be able to identify potential competitors by their colour patterns. We first examined the colour patterns of fish using host anemones inhabited by Amphiprion ocellaris as shelter and compared them with the patterns of fish using surrounding scleractinian corals. There were no fish with bars sheltering in host anemones, although many fish with bars were found in surrounding corals. Next, two fish models, one with white bars and the other with white stripes on a black background, were presented to an A. ocellaris colony. The duration of aggressive behaviour towards the bar model was significantly longer than that towards the stripe model. We conclude that differences in aggressive behaviour by the anemonefish possibly select the colour patterns of cohabiting fish. This study indicates that colour patterns may influence not only intraspecific interactions but also interspecific interactions in coral reef ecosystems.

Modeling Edar expression reveals the hidden dynamics of tooth signaling center patterning.

When patterns are set during embryogenesis, it is expected that they are straightly established rather than subsequently modified. The patterning of the three mouse molars is, however, far from straight, likely as a result of mouse evolutionary history. The first-formed tooth signaling centers, called MS and R2, disappear before driving tooth formation and are thought to be vestiges of the premolars found in mouse ancestors. Moreover, the mature signaling center of the first molar (M1) is formed from the fusion of two signaling centers (R2 and early M1). Here, we report that broad activation of Edar expression precedes its spatial restriction to tooth signaling centers. This reveals a hidden two-step patterning process for tooth signaling centers, which was modeled with a single activator-inhibitor pair subject to reaction-diffusion (RD). The study of Edar expression also unveiled successive phases of signaling center formation, erasing, recovering, and fusion. Our model, in which R2 signaling center is not intrinsically defective but erased by the broad activation preceding M1 signaling center formation, predicted the surprising rescue of R2 in Edar mutant mice, where activation is reduced. The importance of this R2-M1 interaction was confirmed by ex vivo cultures showing that R2 is capable of forming a tooth. Finally, by introducing chemotaxis as a secondary process to RD, we recapitulated in silico different conditions in which R2 and M1 centers fuse or not. In conclusion, pattern formation in the mouse molar field relies on basic mechanisms whose dynamics produce embryonic patterns that are plastic objects rather than fixed end points.

Transcriptomes of Giant Sea Anemones from Okinawa as a Tool for Understanding Their Phylogeny and Symbiotic Relationships with Anemonefish.

The relationship between anemonefish and sea anemones is one of the most emblematic examples of mutualistic symbiosis in coral reefs. Although this is a textbook example, the major aspects of this symbiosis are still not fully understood in mechanistic terms. Moreover, since studies of this relationship have usually been focused on anemonefish, much less is known about giant sea anemones, their similarities, their phylogenetic relationships, and their differences at the molecular level. Since both partners of the symbiotic relationship are important, we decided to explore this well-known phenomenon from the perspective of giant sea anemones. Here, we report reference transcriptomes for all seven species of giant sea anemones that inhabit fringing reefs of Okinawa (Japan) and serve as hosts for six species of local anemonefish. Transcriptomes were used to investigate their phylogenetic relations, genetic differences and repertoires of nematocyte-specific proteins. Our data support the presence of three distinct groups corresponding to three genera: Entacmaea, Heteractis, and Stichodactyla. The basal position among the three groups belongs to Entacmaea, which was the first to diverge from a common ancestor. While the magnitude of genetic difference between the representatives of Entacmaea and Stichodactyla is large, intra-specific variation within Stichodactyla is much smaller and seems to result from recent speciation events. Our data reconfirms that Heteractis magnifica belongs to the genus Stichodactyla, despite an overall morphological similarity with representatives of the genus Heteractis. The availability of reference transcriptomes will facilitate further research into the fascinating relationship between sea anemones and anemonefish.

The Vertebrate Tooth Row: Is It Initiated by a Single Organizing Tooth?

Teeth are one of the most fascinating innovations of vertebrates. Their diversity of shape, size, location, and number in vertebrates is astonishing. If the molecular mechanisms underlying the morphogenesis of individual teeth are now relatively well understood, thanks to the detailed experimental work that has been performed in model organisms (mainly mouse and zebrafish), the mechanisms that control the organization of the dentition are still a mystery. Mammals display simplified dentitions when compared to other vertebrates with only a single tooth row positioned in the anterior part of the mouth, whereas other vertebrates exhibit tooth rows in many locations. As proposed 60 years ago, tooth rows can be formed sequentially from an initiator tooth. Recent results in zebrafish have now largely confirmed this hypothesis. Here this observation is generalized upon and it is suggested that in most vertebrates tooth rows could form sequentially from a single initiator tooth.

The real Nemo movie: Description of embryonic development in Amphiprion ocellaris from first division to hatching.

BACKGROUND: Amphiprion ocellaris is one of the rare reef fish species that can be reared in aquaria. It is increasingly used as a model species for Eco-Evo-Devo. Therefore, it is important to have an embryonic development table based on high quality images that will allow for standardized sampling by the scientific community. RESULTS: Here we provide high-resolution time-lapse videos to accompany a detailed description of embryonic development in A ocellaris. We describe a series of developmental stages and we define six broad periods of embryogenesis: zygote, cleavage, blastula, gastrula, segmentation, and organogenesis that we further subdivide into 32 stages. These periods highlight the changing spectrum of major developmental processes that occur during embryonic development. CONCLUSIONS: We provide an easy system for the determination of embryonic stages, enabling the development of A ocellaris as a coral reef fish model species. This work will facilitate evolutionary development studies, in particular studies of the relationship between climate change and developmental trajectories in the context of coral reefs. Thanks to its lifestyle, complex behavior, and ecology, A ocellaris will undoubtedly become a very attractive model in a wide range of biological fields.

A star is born again: Methods for larval rearing of an emerging model organism, the False clownfish Amphiprion ocellaris.

As interest increases in ecological, evolutionary, and developmental biology (Eco-Evo-Devo), wild species are increasingly used as experimental models. However, we are still lacking a suitable model for marine fish species, as well as coral reef fishes that can be reared at laboratory scales. Extensive knowledge of the life cycle of anemonefishes, and the peculiarities of their biology, make them relevant marine fish models for developmental biology, ecology, and evolutionary sciences. Here, we present standard methods to maintain breeding pairs of the anemonefish Amphiprion ocellaris in captivity, obtain regular good quality spawning, and protocols to ensure larval survival throughout rearing. We provide a detailed description of the anemonefish husbandry system and life prey culturing protocols. Finally, a "low-volume" rearing protocol useful for the pharmacological treatment of larvae is presented. Such methods are important as strict requirements for large volumes in rearing tanks often inhibit continuous treatments with expensive or rare compounds.

Staging and normal table of postembryonic development of the clownfish (Amphiprion ocellaris).

BACKGROUND: The clownfish Amphiprion ocellaris is one of the rare coral reef fish species that can be reared in aquaria. With relatively short embryonic and larval development, it could be used as a model species to study the impact of global changes such as temperature rise or anthropogenic threats (eg, pollution) on the postembryonic development at molecular and endocrinological levels. Establishing a developmental table allows us to standardize sampling for the scientific community willing to conduct experiments on this species on different areas: ecology, evolution, and developmental biology. RESULTS: Here, we describe the postembryonic developmental stages for the clownfish A. ocellaris from hatching to juvenile stages (30 days posthatching). We quantitatively followed the postembryonic growth and described qualitative traits: head, paired and unpaired fins, notochord flexion, and pigmentation changes. The occurrence of these changes over time allowed us to define seven stages, for which we provide precise descriptions. CONCLUSIONS: Our work gives an easy system to determine A. ocellaris postembryonic stages allowing, thus, to develop this species as a model species for coral reef fishes. In light of global warming, the access to the full postembryonic development stages of coral reef fish is important to determine stressors that can affect such processes.

The first formed tooth serves as a signalling centre to induce the formation of the dental row in zebrafish.

The diversity of teeth patterns in actinopterygians is impressive with tooth rows in many locations in the oral and pharyngeal regions. The first-formed tooth has been hypothesized to serve as an initiator controlling the formation of the subsequent teeth. In zebrafish, the existence of the first tooth (named 4 V1) is puzzling as its replacement is induced before the opening of the mouth. Functionally, it has been shown that 4 V1 formation requires fibroblast growth factor (FGF) and retinoic acid (RA) signalling. Here, we show that the ablation of 4 V1 prevents the development of the dental row demonstrating its dependency over it. If endogenous levels of FGF and RA are restored after 4 V1 ablation, embryonic dentition starts again by de novo formation of a first tooth, followed by the dental row. Similarly, induction of anterior ectopic teeth induces subsequent tooth formation, demonstrating that the initiator tooth is necessary and sufficient for dental row formation, probably via FGF ligands released by 4 V1 to induce the formation of subsequent teeth. Our results show that by modifying the formation of the initiator tooth it is possible to control the formation of a dental row. This could help to explain the diversity of tooth patterns observed in actinopterygians and more broadly, how diverse traits evolved through molecular fine-tuning.

Ontogenetic and phylogenetic simplification during white stripe evolution in clownfishes.

BACKGROUND: Biologists have long been fascinated by the striking diversity of complex color patterns in tropical reef fishes. However, the origins and evolution of this diversity are still poorly understood. Disentangling the evolution of simple color patterns offers the opportunity to dissect both ultimate and proximate causes underlying color diversity. RESULTS: Here, we study clownfishes, a tribe of 30 species within the Pomacentridae that displays a relatively simple color pattern made of zero to three vertical white stripes on a dark body background. Mapping the number of white stripes on the evolutionary tree of clownfishes reveals that their color pattern diversification results from successive caudal to rostral losses of stripes. Moreover, we demonstrate that stripes always appear with a rostral to caudal stereotyped sequence during larval to juvenile transition. Drug treatments (TAE 684) during this period leads to a dose-dependent loss of stripes, demonstrating that white stripes are made of iridophores and that these cells initiate the stripe formation. Surprisingly, juveniles of several species (e.g., Amphiprion frenatus) have supplementary stripes when compared to their respective adults. These stripes disappear caudo-rostrally during the juvenile phase leading to the definitive color pattern. Remarkably, the reduction of stripe number over ontogeny matches the sequences of stripe losses during evolution, showing that color pattern diversification among clownfish lineages results from changes in developmental processes. Finally, we reveal that the diversity of striped patterns plays a key role for species recognition. CONCLUSIONS: Overall, our findings illustrate how developmental, ecological, and social processes have shaped the diversification of color patterns during the radiation of an emblematic coral reef fish lineage.

The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase.

The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65ß of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65ß and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation.