Estradiol-17ß and bisphenol A affect growth and mineralization in early life stages of seabass.

Natural and synthetic estrogens are contaminants present in aquatic ecosystems. They can have significant consequences on the estrogen-sensitive functions of organisms, including skeletal development and growth of vertebrate larvae. Synthetic polyphenols represent a group of environmental xenoestrogens capable of binding the receptors for the natural hormone estradiol-17ß (E2). To better understand how (xeno-)estrogens can affect the skeleton in fish species with high ecological and commercial interest, 16 days post-hatch larvae of the seabass were experimentally exposed for 7 days to E2 and Bisphenol A (BPA), both used at the regulatory concentration of surface water quality (E2: 0.4 ng.L-1, BPA: 1.6 µg.L-1) or at a concentration 100 times higher. Skeletal mineralization levels were evaluated using Alizarin red staining, and expression of several genes playing key roles in growth, skeletogenesis and estrogen signaling pathways was assessed by qPCR. Our results show that E2 exerts an overall negative effect on skeletal mineralization at the environmental concentration of 0.4 ng.L-1, correlated with an increase in the expression of genes associated only with osteoblast bone cells. Both BPA exposures inhibited mineralization with less severe effects and modified bone homeostasis by regulating the expression of gene encoding osteoblasts and osteoclasts markers. Our results demonstrate that environmental E2 exposure inhibits larval growth and has an additional inhibitory effect on skeleton mineralization while both BPA exposures have marginal inhibitory effect on skeletal mineralization. All exposures have significant effects on transcriptional levels of genes involved in the skeletal development of seabass larvae.

Experimental evolution of environmental tolerance, acclimation, and physiological plasticity in a randomly fluctuating environment.

Environmental tolerance curves, representing absolute fitness against the environment, are an empirical assessment of the fundamental niche, and emerge from the phenotypic plasticity of underlying phenotypic traits. Dynamic plastic responses of these traits can lead to acclimation effects, whereby recent past environments impact current fitness. Theory predicts that higher levels of phenotypic plasticity should evolve in environments that fluctuate more predictably, but there have been few experimental tests of these predictions. Specifically, we still lack experimental evidence for the evolution of acclimation effects in response to environmental predictability. Here, we exposed 25 genetically diverse populations of the halotolerant microalgae Dunaliella salina to different constant salinities, or to randomly fluctuating salinities, for over 200 generations. The fluctuating treatments differed in their autocorrelation, which determines the similarity of subsequent values, and thus environmental predictability. We then measured acclimated tolerance surfaces, mapping population growth rate against past (acclimation) and current (assay) environments. We found that experimental mean and variance in salinity caused the evolution of niche position (optimal salinity) and breadth, with respect to not only current but also past (acclimation) salinity. We also detected weak but significant evidence for evolutionary changes in response to environmental predictability, with higher predictability leading notably to lower optimal salinities and stronger acclimation effect of past environment on current fitness. We further showed that these responses are related to the evolution of plasticity for intracellular glycerol, the major osmoregulatory mechanism in this species. However, the direction of plasticity evolution did not match simple theoretical predictions. Our results underline the need for a more explicit consideration of the dynamics of environmental tolerance and its underlying plastic traits to reach a better understanding of ecology and evolution in fluctuating environments.

Parallel Evolution of Ameloblastic scpp Genes in Bony and Cartilaginous Vertebrates.

In bony vertebrates, skeletal mineralization relies on the secretory calcium-binding phosphoproteins (Scpp) family whose members are acidic extracellular proteins posttranslationally regulated by the Fam20°C kinase. As scpp genes are absent from the elephant shark genome, they are currently thought to be specific to bony fishes (osteichthyans). Here, we report a scpp gene present in elasmobranchs (sharks and rays) that evolved from local tandem duplication of sparc-L 5' exons and show that both genes experienced recent gene conversion in sharks. The elasmobranch scpp is remarkably similar to the osteichthyan scpp members as they share syntenic and gene structure features, code for a conserved signal peptide, tyrosine-rich and aspartate/glutamate-rich regions, and harbor putative Fam20°C phosphorylation sites. In addition, the catshark scpp is coexpressed with sparc-L and fam20°C in tooth and scale ameloblasts, similarly to some osteichthyan scpp genes. Despite these strong similarities, molecular clock and phylogenetic data demonstrate that the elasmobranch scpp gene originated independently from the osteichthyan scpp gene family. Our study reveals convergent events at the sparc-L locus in the two sister clades of jawed vertebrates, leading to parallel diversification of the skeletal biomineralization toolkit. The molecular evolution of sparc-L and its coexpression with fam20°C in catshark ameloblasts provides a unifying genetic basis that suggests that all convergent scpp duplicates inherited similar features from their sparc-L precursor. This conclusion supports a single origin for the hypermineralized outer odontode layer as produced by an ancestral developmental process performed by Sparc-L, implying the homology of the enamel and enameloid tissues in all vertebrates.

Divergent Expression of SPARC, SPARC-L, and SCPP Genes During Jawed Vertebrate Cartilage Mineralization.

While cartilage is an ancient tissue found both in protostomes and deuterostomes, its mineralization evolved more recently, within the vertebrate lineage. SPARC, SPARC-L, and the SCPP members (Secretory Calcium-binding PhosphoProtein genes which evolved from SPARC-L) are major players of dentine and bone mineralization, but their involvement in the emergence of the vertebrate mineralized cartilage remains unclear. We performed in situ hybridization on mineralizing cartilaginous skeletal elements of the frog Xenopus tropicalis (Xt) and the shark Scyliorhinus canicula (Sc) to examine the expression of SPARC (present in both species), SPARC-L (present in Sc only) and the SCPP members (present in Xt only). We show that while mineralizing cartilage expresses SPARC (but not SPARC-L) in Sc, it expresses the SCPP genes (but not SPARC) in Xt, and propose two possible evolutionary scenarios to explain these opposite expression patterns. In spite of these genetic divergences, our data draw the attention on an overlooked and evolutionarily conserved peripheral cartilage subdomain expressing SPARC or the SCPP genes and exhibiting a high propensity to mineralize.

Evolution of Matrix Gla and Bone Gla Protein Genes in Jawed Vertebrates.

Matrix Gla protein (Mgp) and bone Gla protein (Bgp) are vitamin-K dependent proteins that bind calcium in their γ-carboxylated versions in mammals. They are recognized as positive (Bgp) or negative (Mgp and Bgp) regulators of biomineralization in a number of tissues, including skeletal tissues of bony vertebrates. The Mgp/Bgp gene family is poorly known in cartilaginous fishes, which precludes the understanding of the evolution of the biomineralization toolkit at the emergence of jawed vertebrates. Here we took advantage of recently released genomic and transcriptomic data in cartilaginous fishes and described the genomic loci and gene expression patterns of the Mgp/Bgp gene family. We identified three genes, Mgp1, Mgp2, and Bgp, in cartilaginous fishes instead of the single previously reported Mgp gene. We describe their genomic loci, resulting in a dynamic evolutionary scenario for this gene family including several events of local (tandem) duplications, but also of translocation events, along jawed vertebrate evolution. We describe the expression patterns of Mgp1, Mgp2, and Bgp in embryonic stages covering organogenesis in the small-spotted catshark Scyliorhinus canicula and present a comparative analysis with Mgp/Bgp family members previously described in bony vertebrates, highlighting ancestral features such as early embryonic, soft tissues, and neuronal expressions, but also derived features of cartilaginous fishes such as expression in fin supporting fibers. Our results support an ancestral function of Mgp in skeletal mineralization and a later derived function of Bgp in skeletal development that may be related to the divergence of bony vertebrates.