Modular Evolution and Population Variability of Oikopleura dioica Metallothioneins

Chordate Oikopleura dioica probably is the fastest evolving metazoan reported so far, and thereby, a suitable system in which to explore the limits of evolutionary processes. For this reason, and in order to gain new insights on the evolution of protein modularity, we have investigated the organization, function and evolution of multi-modular metallothionein (MT) proteins in O. dioica. MTs are a heterogeneous group of modular proteins defined by their cysteine (C)-rich domains, which confer the capacity of coordinating different transition metal ions. O. dioica has two MTs, a bi-modular OdiMT1 consisting of two domains (t-12C and 12C), and a multi-modular OdiMT2 with six t-12C/12C repeats. By means of mass spectrometry and spectroscopy of metal-protein complexes, we have shown that the 12C domain is able to autonomously bind four divalent metal ions, although the t-12C/12C pair -as it is found in OdiMT1- is the optimized unit for divalent metal binding. We have also shown a direct relationship between the number of the t-12C/12C repeats and the metal-binding capacity of the MTs, which means a stepwise mode of functional and structural evolution for OdiMT2. Finally, after analyzing four different O. dioica populations worldwide distributed, we have detected several OdiMT2 variants with changes in their number of t-12C/12C domain repeats. This finding reveals that the number of repeats fluctuates between current O. dioica populations, which provides a new perspective on the evolution of domain repeat proteins

Pth reloaded: a new evolutionary perspective

The parathyroid hormone (PTH) family is a group of structurally-related secreted peptides involved in bone mineral homeostasis and multitude of developmental processes in vertebrates. These peptides mediate actions through PTH receptors (PTHRs), which belong to the transmembrane G protein-coupled receptor group. To date, genes encoding for PTH and PTHR have only been identified in chordates, suggesting that this signaling pathway may be an evolutionary innovation of our phylum. In vertebrates, we found up to six PTH and three PTHR different paralogs, varying in number between mammals and teleost fishes due to the different rounds of whole-genome duplication and specific gene losses suffered between the two groups of animals. The diversification of the PTH gene family has been accompanied by both functional divergence and convergence, making sometimes difficult the comparison between PTH peptides of teleosts and mammals. Here, we review the roles of all Pth peptides in fishes, and based on the evolutionary history of PTH paralogs, we propose a new and simple nomenclature from PTH1 to PTH4. Moreover, the recent characterization of the Pth4 in zebrafish allows us to consider the prominent role of the brain-to-bone signaling pathway in the regulation of bone development and homeostasis. Finally, comparison between PTH peptides of fish and mammals allows us to discuss an evolutionary model for PTH functions related to bone mineral balance during the vertebrate transition from an aquatic to a terrestrial environment

Retinoic Acid metabolic genes, meiosis, and gonadal sex differentiation in zebrafish.

To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female pathway in zebrafish

Sex Reversal in Zebrafish fancl Mutants is Caused by Tp53-Mediated Germ Cell Apoptosis

The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination

Massive Gene Loss and Function Shuffling in Appendicularians Stretch the Boundaries of Chordate Wnt Family Evolution

Gene loss is a pervasive source of genetic variation that influences species evolvability, biodiversity and the innovation of evolutionary adaptations. To better understand the evolutionary patterns and impact of gene loss, here we investigate as a case study the evolution of the wingless (Wnt) family in the appendicularian tunicate Oikopleura dioica, an emergent EvoDevo model characterized by its proneness to lose genes among chordates. Genome survey and phylogenetic analyses reveal that only four of the thirteen Wnt subfamilies have survived in O. dioica¿Wnt5, Wnt10, Wnt11, and Wnt16,¿representing the minimal Wnt repertoire described in chordates. While the loss of Wnt4 and Wnt8 likely occurred in the last common ancestor of tunicates, representing therefore a synapomorphy of this subphylum, the rest of losses occurred during the evolution of appendicularians. This work provides the first complete Wnt developmental expression atlas in a tunicate and the first insights into the evolution of Wnt developmental functions in appendicularians. Our work highlights three main evolutionary patterns of gene loss: (1) conservation of ancestral Wnt expression domains not affected by gene losses; (2) function shuffling among Wnt paralogs accompanied by gene losses; and (3) extinction of Wnt expression in certain embryonic directly correlated with gene losses. Overall our work reveals that in contrast to 'conservative' pattern of evolution of cephalochordates and vertebrates, O. dioica shows an even more radical 'liberal' evolutionary pattern than that described ascidian tunicates, stretching the boundaries of the malleability of Wnt family evolution in chordates

Metallothioneins of the urochordate Oikopleura dioica have Cys-rich tandem repeats, large size and cadmium-binding preference

The increasing levels of heavy metals derived from human activity are poisoning marine environments, threating zooplankton and ocean food webs. To protect themselves from the harmful effects of heavy metals, living beings have different physiological mechanisms, one of which is based on metallothioneins (MTs), a group of small cysteine-rich proteins that can bind heavy metals counteracting their toxicity. The MT system of urochordate appendicularians, an ecologically relevant component of the zooplankton,remained, however, unknown. In this work, we have characterized the MTs of the appendicularian species Oikopleura dioica, revealing that O. dioica has two MT genes, named OdMT1 and OdMT2, which encode for Cys-rich proteins, the former with 72 amino acids comparable with the small size MTs of other organisms, but the second with 399 amino acids representing the longest MT reported to date for any living being. Sequence analysis revealed that OdMT2 gene arose from a duplication of an ancestral OdMT1 gene followed by up to six tandem duplications of an ancestral repeat unit (RU) in the current OdMT2 gene. Interestingly, each RU contained, in turn, an internal repeat of a 7-Cys subunit (X3CX3CX2CX2CX3-6CX2CXCX), which is repeated up to 12 times in OdMT2. Finally, ICP-AES analyses of heterologously expressed OdMT proteins showed that both MTs were capable to form metalcomplexes, with preference for cadmium ions. Collectively, our results provide the first characterization of the MT system in an appendicularian species as an initial step to understand the zooplankton response to metal toxicity and other environmental stress situations

Diatom bloom-derived biotoxins cause aberrant development and gene expression in the appendicularian chordate Oikopleura dioica.

Investigating environmental hazards than could affect appendicularians is of prime ecological interest because they are among the most abundant components of the mesozooplankton. This work shows that embryo development of the appendicularian Oikopleura dioica is compromised by diatom bloom-derived biotoxins, even at concentrations in the same range as those measured after blooms. Developmental gene expression analysis of biotoxin-treated embryos uncovers an aberrant golf ball-like phenotype affecting morphogenesis, midline convergence, and tail elongation. Biotoxins induce a rapid upregulation of defensome genes, and considerable delay and silencing of zygotic transcription of developmental genes. Upon a possible future intensification of blooms associated with ocean warming and acidification, our work puts an alert on the potential impact that an increase of biotoxins may have on marine food webs, and points to defensome genes as molecular biosensors that marine ecologists could use to monitor the genetic stress of natural populations exposed to microalgal blooms

Roles of brca2 (fancd1) in Oocyte Nuclear Architecture, Gametogenesis, Gonad Tumors, and Genome Stability in Zebrafish

Mild mutations in BRCA2 (FANCD1) cause Fanconi anemia (FA) when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd)-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53) rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture

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

Caracterització molecular i evolutiva de la família de les alcohol deshidrogenases de cadena mitjana als cordats.

En la present tesi doctoral, hem caracterizat el gen Adh3 a dos cefalocordats ("B. floridae" i "B. lanceolatum") i un urocordat ("C. intestinalis"), com l'únic membre de la família de les alcohol deshidrogenases (ADHs) de cadena mitjana en organismes procordats. Les nostres dades suggereixen fermament que l'expansió d'aquesta família gènica als vertebrats va tenir lloc fa uns 500 milions d'anys, després de la separació entre cefalocordats i vertebrats