Vincent Laudet

Bgee: Integrating and Comparing Heterogeneous Transcriptome Data Among Species

Gene expression patterns are a key feature in understanding gene function, notably in development. Comparing gene expression patterns between animals is a major step in the study of gene function as well as of animal evolution. It also provides a link between genes and phenotypes. Thus we have developed Bgee, a database designed to compare expression patterns between animals, by implementing ontologies describing anatomies and developmental stages of species, and then designing homology relationships between anatomies and comparison criteria between developmental stages. To define homology relationships between anatomical features we have developed the software Homolonto, which uses a modified ontology alignment approach to propose homology relationships between ontologies. Bgee then uses these aligned ontologies, onto which heterogeneous expression data types are mapped. These already include microarrays and ESTs. Bgee is available at http://bgee.unil.ch/

Structural and Evolutionary Innovation of the Heterodimerization Interface between USP and the Ecdysone Receptor ECR in Insects

Understanding how the variability of protein structure arises during evolution and leads to new structure-function relationships ultimately promoting evolutionary novelties is a major goal of molecular evolution and is critical for interpreting genome sequences. We addressed this issue using the ecdysone receptor (ECR), a major developmental factor that controls development and reproduction of arthropods. The functional ECR is a heterodimer of two nuclear receptors: ECR, which binds ecdysteroids, and its obligatory partner ultraspirade (USP), which is orthologous to the retinoid X receptor of vertebrates. Both genes underwent a dramatic increase of evolutionary rate in Mecopterida, the major insect terminal group containing Dipteras and Lepidopteras. We therefore questioned the implication of this event in terms of coevolution of their dimerization interface. A structural comparison revealed a 30% larger ligand-binding domain (LBD) heterodimerization surface in the Lepidoptera Heliothis when compared with basal insects, associated with a symmetrization of the interface, which is exceptional for nuclear receptors. Reconstruction of ancestral sequences and homology modeling of the ancestral Mecopterida ECR-USP reveal that this enlarged dimerization surface is a synapomorphy for Mecopterida. Furthermore, we show that the residues implicated in the new dimerization surface underwent specific evolutionary constraints in Mecopterida indicative of their new and conserved role in the dimerization interface. Most of all, the novel surface originates from a 15 degrees torsion of a subdomain of USP LBD toward its partner ECR, which is a long-range consequence of the peculiar position of a Mecopterida-specific insertion in loop L1-3, located outside of the interaction surface, in a less crucial domain of the partner protein. These results indicate that the coevolution between ECR and USP occurred through a novel mechanism of intramolecular epistasis that will undoubtedly be generalized for other molecules because it uses flexibility of a less-constrained region of a protein to modify the structure of another, critical part of the molecule.

Evolution of nuclear retinoic acid receptor alpha (RARα) phosphorylation sites. Serine gain provides fine-tuned regulation

The human nuclear retinoic acid (RA) receptor alpha (hRARα) is a ligand-dependent transcriptional regulator, which is controlled by a phosphorylation cascade. The cascade starts with the RA-induced phosphorylation of a serine residue located in the ligand-binding domain, S(LBD), allowing the recruitment of the cdk7/cyclin H/MAT1 subcomplex of TFIIH through the docking of cyclin H. It ends by the subsequent phosphorylation by cdk7 of an other serine located in the N-terminal domain, S(NTD). Here, we show that this cascade relies on an increase in the flexibility of the domain involved in cyclin H binding, subsequently to the phosphorylation of S(LBD). Owing to the functional importance of RARα in several vertebrate species, we investigated whether the phosphorylation cascade was conserved in zebrafish (Danio rerio), which expresses two RARα genes: RARα-A and RARα-B. We found that in zebrafish RARαs, S(LBD) is absent, whereas S(NTD) is conserved and phosphorylated. Therefore, we analyzed the pattern of conservation of the phosphorylation sites and traced back their evolution. We found that S(LBD) is most often absent outside mammalian RARα and appears late during vertebrate evolution. In contrast, S(NTD) is conserved, indicating that the phosphorylation of this functional site has been under ancient high selection constraint. This suggests that, during evolution, different regulatory circuits control RARα activity.

Retinoic acid expands the evolutionarily reduced dentition of zebrafish

Zebrafish lost anterior teeth during evolution but retain a posterior pharyngeal dentition that requires retinoic acid (RA) cell‐cell signaling for its development. The purposes of this study were to test the sufficiency of RA to induce tooth development and to assess its role in evolution. We found that exposure of embryos to exogenous RA induces a dramatic anterior expansion of the number of pharyngeal teeth that later form and shifts anteriorly the expression patterns of genes normally expressed in the posterior tooth‐forming region, such as pitx2 and dlx2b. After RA exposure, we also observed a correlation between cartilage malformations and ectopic tooth induction, as well as abnormal cranial neural crest marker gene expression. Additionally, we observed that the RA‐induced zebrafish anterior teeth resemble in pattern and number the dentition of fish species that retain anterior pharyngeal teeth such as medaka but that medaka do not express the aldh1a2 RA‐synthesizing enzyme in tooth‐forming regions. We conclude that RA is sufficient to induce anterior ectopic tooth development in zebrafish where teeth were lost in evolution, potentially by altering neural crest cell development, and that changes in the location of RA synthesis correlate with evolutionary changes in vertebrate dentitions.—Seritrakul, P., Samarut, E., Lama, T. T. S., Gibert, Y., Laudet, V., Jackman, W. R. Retinoic acid expands the evolutionarily reduced dentition of zebrafish. FASEB J. 26, 5014–5024 (2012). www.fasebj.org

Altered retinoic acid signalling underpins dentition evolution

Small variations in signalling pathways have been linked to phenotypic diversity and speciation. In vertebrates, teeth represent a reservoir of adaptive morphological structures that are prone to evolutionary change. Cyprinid fish display an impressive diversity in tooth number, but the signals that generate such diversity are unknown. Here, we show that retinoic acid (RA) availability influences tooth number size in Cyprinids. Heterozygous adult zebrafish heterozygous for the cyp26b1 mutant that encodes an enzyme able to degrade RA possess an extra tooth in the ventral row. Expression analysis of pharyngeal mesenchyme markers such as dlx2a and lhx6 shows lateral, anterior and dorsal expansion of these markers in RA-treated embryos, whereas the expression of the dental epithelium markers dlx2b and dlx3b is unchanged. Our analysis suggests that changes in RA signalling play an important role in the diversification of teeth in Cyprinids. Our work illustrates that through subtle changes in the expression of rate-limiting enzymes, the RA pathway is an active player of tooth evolution in fish.

Origin of an ancient hormone/receptor couple revealed by resurrection of an ancestral estrogen

Cladistic analysis allows inference of the structure of an ancestral steroid that acts as a ligand for the ancestral receptor. The origin of ancient ligand/receptor couples is often analyzed via reconstruction of ancient receptors and, when ligands are products of metabolic pathways, they are not supposed to evolve. However, because metabolic pathways are inherited by descent with modification, their structure can be compared using cladistic analysis. Using this approach, we studied the evolution of steroid hormones. We show that side-chain cleavage is common to most vertebrate steroids, whereas aromatization was co-opted for estrogen synthesis from a more ancient pathway. The ancestral products of aromatic activity were aromatized steroids with a side chain, which we named “paraestrols.” We synthesized paraestrol A and show that it effectively binds and activates the ancestral steroid receptor. Our study opens the way to comparative studies of biologically active small molecules.

6 – Evolution of Nuclear Receptors in Insects

Publisher Summary This chapter discusses the evolution of nuclear receptors in insects. Nuclear receptors are metazoan transcription factors activated by small lipophilic ligands, such as steroid hormones, thyroid hormones, retinoids, vitamin D, or fatty acids. The availability of the ligand controls the transcriptional activity of nuclear receptors. Nuclear receptors are involved in a considerable diversity of developmental and physiological processes. Nuclear receptors are present in all metazoans, and only in metazoans. They are absent from the genome of the unicellular Monosiga brevicollis, which belongs to choanoflagellates, the sister group of animals. The set of nuclear receptors is strongly conserved in pterygote insects, ranging from 19 to 21 genes that are distributed into 22 groups. Unlike nematodes and chordates, insects did not experience any lineage-specific expansions within the nuclear receptors family.

Rev-erbα2 mRNA Encodes a Stable Protein with a Potential Role in Circadian Clock Regulation

Circadian rhythms are observed in nearly all aspects of physiology and behavior. In mammals, such biological rhythms are supported by a complex network of self-sustained transcriptional loops and posttranslational modifications, which regulate timely controlled production and degradation of critical factors on a 24-h basis. Among these factors, the orphan nuclear receptor rev-erbalpha plays an essential role by linking together positive and negative regulatory loops. As an essential part of the circadian core clock mechanism, REV-ERBalpha expression shows a precisely scheduled oscillation reflecting the tight control of its production and degradation. In previous studies, we identified two alternative transcripts encoding two protein variants referred to as REV-ERBalpha1 and -alpha2. Interestingly, recent work identified structural elements present only in REV-ERBalpha1 that controls its turnover and thereby influences circadian oscillations. In the present work, we comparatively analyze the two variants and show that REV-ERBalpha2 exhibits a half-life incompatible with a circadian function, suggesting that this variant exerts different biological functions. However, our comparative study clearly indicates undistinguishable DNA-binding properties and transcriptional repression activity as well as a similar regulation mechanism. The only consistent difference appears to be the relative expression level of the two transcripts, rev-erbalpha1 being one to 100 times more expressed than alpha2 depending on tissue and circadian time. Taking this finding into consideration, we reassessed REV-ERBalpha2 turnover and were able to show that this variant exhibits a reduced half-life when coexpressed with REV-ERBalpha1. We propose that the relative expression levels of the two REV-ERBalpha variants fine-tune the circadian period length by regulating REV-ERBalpha half-life.

Evolution of retinoic acid receptors in chordates: insights from three lamprey species, Lampetra fluviatilis, Petromyzon marinus, and Lethenteron japonicum.

BackgroundRetinoic acid (RA) signaling controls many developmental processes in chordates, from early axis specification to late organogenesis. The functions of RA are chiefly mediated by a subfamily of nuclear hormone receptors, the retinoic acid receptors (RARs), that act as ligand-activated transcription factors. While RARs have been extensively studied in jawed vertebrates (that is, gnathostomes) and invertebrate chordates, very little is known about the repertoire and developmental roles of RARs in cyclostomes, which are extant jawless vertebrates. Here, we present the first extensive study of cyclostome RARs focusing on three different lamprey species: the European freshwater lamprey, Lampetra fluviatilis, the sea lamprey, Petromyzon marinus, and the Japanese lamprey, Lethenteron japonicum.ResultsWe identified four rar paralogs (rar1, rar2, rar3, and rar4) in each of the three lamprey species, and phylogenetic analyses indicate a complex evolutionary history of lamprey rar genes including the origin of rar1 and rar4 by lineage-specific duplication after the lamprey-hagfish split. We further assessed their expression patterns during embryonic development by in situ hybridization. The results show that lamprey rar genes are generally characterized by dynamic and highly specific expression domains in different embryonic tissues. In particular, lamprey rar genes exhibit combinatorial expression domains in the anterior central nervous system (CNS) and the pharyngeal region.ConclusionsOur results indicate that the genome of lampreys encodes at least four rar genes and suggest that the lamprey rar complement arose from vertebrate-specific whole genome duplications followed by a lamprey-specific duplication event. Moreover, we describe a combinatorial code of lamprey rar expression in both anterior CNS and pharynx resulting from dynamic and highly specific expression patterns during embryonic development. This ‘RAR code’ might function in regionalization and patterning of these two tissues by differentially modulating the expression of downstream effector genes during development.

The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation

Vitamin A–dependent intercellular signaling was originally regulated by a low-affinity sensor and acted in neural development. Retinoic acid (RA) is an important intercellular signaling molecule in vertebrate development, with a well-established role in the regulation of hox genes during hindbrain patterning and in neurogenesis. However, the evolutionary origin of the RA signaling pathway remains elusive. To elucidate the evolution of the RA signaling system, we characterized RA metabolism and signaling in the marine annelid Platynereis dumerilii, a powerful model for evolution, development, and neurobiology. Binding assays and crystal structure analyses show that the annelid retinoic acid receptor (RAR) binds RA and activates transcription just as vertebrate RARs, yet with a different ligand-binding pocket and lower binding affinity, suggesting a permissive rather than instructive role of RA signaling. RAR knockdown and RA treatment of swimming annelid larvae further reveal that the RA signal is locally received in the medial neuroectoderm, where it controls neurogenesis and axon outgrowth, whereas the spatial colinear hox gene expression in the neuroectoderm remains unaffected. These findings suggest that one early role of the new RAR in bilaterian evolution was to control the spatially restricted onset of motor and interneuron differentiation in the developing ventral nerve cord and to indicate that the regulation of hox-controlled anterior-posterior patterning arose only at the base of the chordates, concomitant with a high-affinity RAR needed for the interpretation of a complex RA gradient.