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The urochordate Oikopleura dioica is emerging as a nonclassical animal model in the field of evolutionary developmental biology (a.k.a. evo-devo) especially attractive for investigating the impact of gene loss on the evolution of mechanisms of development. This is because this organism fulfills the requirements of an animal model (i.e., has a simple and accessible morphology, a short generation time and life span, and affordable culture in the laboratory and amenable experimental manipulation), but also because O. dioica occupies a key phylogenetic position to understand the diversification and origin of our own phylum, the chordates. During its evolution, O. dioica genome has suffered a drastic process of compaction, becoming the smallest known chordate genome, a process that has been accompanied by exacerbating amount of gene losses. Interestingly, however, despite the extensive gene losses, including entire regulatory pathways essential for the embryonic development of other chordates, O. dioica retains the typical chordate body plan. This unexpected situation led to the formulation of the so-called inverse paradox of evo-devo, that is, when a genetic diversity is able to maintain a phenotypic unity. This chapter reviews the biological features of O. dioica as a model animal, along with the current data on the evolution of its genes and genome. We pay special attention to the numerous examples of gene losses that have taken place during the evolution of this unique animal model, which is helping us to understand to which the limits of evo-devo can be pushed off.

Reporter analyses of Hox1 and Brachyury (Bra) genes have revealed examples of redundant enhancers that provide regulatory robustness. Retinoic acid (RA) activates through an RA-response element the transcription of Hox1 in the nerve cord of the ascidian Ciona intestinalis. We also found a weak RA-independent neural enhancer within the second intron of Hox1. The Hox1 gene in the larvacean Oikopleura dioica is also expressed in the nerve cord. The O. dioica genome, however, does not contain the RA receptor-encoding gene, and the expression of Hox1 has become independent of RA. We have found that the upstream sequence of the O. dioica Hox1 was able to activate reporter gene expression in the nerve cord of the C. intestinalis embryo, suggesting that an RA-independent regulatory system in the nerve cord might be common in larvaceans and ascidians. This RA-independent redundant regulatory system may have facilitated the Oikopleura ancestor losing RA signaling without an apparent impact on Hox1 expression domains. On the other hand, vertebrate Bra is expressed in the ventral mesoderm and notochord, whereas its ascidian ortholog is exclusively expressed in the notochord. Fibroblast growth factor (FGF) induces Bra in the ventral mesoderm in vertebrates, whereas it induces Bra in the notochord in ascidians. Disruption of the FGF signal does not completely silence Bra expression in ascidians, suggesting that FGF-dependent and independent enhancers might comprise a redundant regulatory system in ascidians. The existence of redundant enhancers, therefore, provides regulatory robustness that may facilitate the acquisition of new expression domains.

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.

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.

[eng] Proteins are composed of domains, a well-defined region within a protein that constitutes a stable, independently folding, compact structural unit, and that usually performs a specific function. During protein evolution, domains have been used as a ‘modules’, and the vast majority of the current proteins show a modular organization in which two or more domains are combined. While the creation of multi-domain proteins through shuffling different domains has been studied extensively, the evolution of proteins made of tandem repeats of a same domain has been less investigated. It is known, for instance, that the fraction of proteins with domain repeats is higher in multicellular organisms than in unicellular organisms, or that proteins with domain repeats seem to be related with stress response functions or with the acquisition of high yielding capacity, but the origin, the genetic mechanisms and the evolutionary forces controlling the expansion of domains repeats in modular proteins are still poorly understood. To investigate the functional and structural evolution of modular proteins with domain repeats, we have chosen the Metallothioneins (MTs) as a case study. MTs are present across all the tree of life and due to their metal-binding ability have been involved in metal homeostasis and detoxification of many organisms. They are modular cysteine-rich proteins that bind metal ions through functionally and structurally independent domains that form metal-thiolate clusters. Many MTs are bi-modular proteins made of a tandem repetition of two similar (although not identical) metal-binding domains. There also are large multi-modular MTs that have expanded the number of their domain repeats, and therefore, their metal-binding capacity. Studying the evolution of metallothionein domains in several phyla can help us to understand, at some level, the evolvability of organisms to adapt to new environmental conditions. In our studies we have identified, analysed, characterized and compared many MTs from different groups of selected organisms (chordates and molluscs) in order to study their domain composition and arrangement, and to untangle the evolution of their modular organization., [spa] Las proteínas están compuestas por dominios, una región bien definida dentro de una proteína que constituye una unidad estructural compacta, estable y que se pliega independientemente, y que normalmente realiza una función específica. Durante la evolución de las proteínas, los dominios se han utilizado como "módulos", y la gran mayoría de las proteínas actuales muestran una organización modular en la que se combinan dos o más dominios. Si bien la creación de proteínas de múltiples dominios mediante la combinación aleatoria de diferentes dominios se ha estudiado ampliamente, la evolución de proteínas compuestas de repeticiones en tándem de un mismo dominio se ha investigado menos. Se sabe, por ejemplo, que la fracción de proteínas con dominios repetidos es mayor en organismos multicelulares que en organismos unicelulares, o que las proteínas con dominios repetidos parecen estar relacionadas con funciones de respuesta al estrés o con la adquisición de una alta capacidad productiva, pero el origen, los mecanismos genéticos y las fuerzas evolutivas que controlan la expansión de las repeticiones de dominios en las proteínas modulares aún son poco conocidos. Para investigar la evolución funcional y estructural de las proteínas modulares con dominio repetido, hemos elegido las metalotioneínas (MT) como caso de estudio. Los MT están presentes en todo el árbol de la vida y, debido a su capacidad de unión a metales, han estado involucrados en la homeostasis de metales y la desintoxicación de muchos organismos. Son proteínas modulares ricas en cisteína que se unen a iones metálicos a través de dominios funcional y estructuralmente independientes que forman grupos de tiolatos metálicos. Muchos MT son proteínas bimodulares hechas de una repetición en tándem de dos dominios de unión a metales similares (aunque no idénticos). También hay grandes MT multimodulares que han ampliado el número de repeticiones de su dominio y, por lo tanto, su capacidad de unión a metales. El estudio de la evolución de los dominios de las metalotioneínas en varios filos puede ayudarnos a comprender, hasta cierto punto, la capacidad de evolución de los organismos para adaptarse a nuevas condiciones ambientales. En nuestros estudios hemos identificado, analizado, caracterizado y comparado muchos MT de diferentes grupos de organismos seleccionados (cordados y moluscos) para estudiar la composición y disposición de sus dominios y desentrañar la evolución de su organización modular.

[eng] The bloom of genomic data has revealed a vast amount of gene losses across all life kingdoms. However, the impact of gene loss on the evolution of the mechanisms of embryo development remains an important challenge. In this work, we have used the successful gene loser Oikopleura dioica, to study the impact of gene loss on the evolution of the cardiopharyngeal gene regulatory network (GRN), and we have extrapolated our results to decipher the ancestral condition of tunicates as free-living or sessile, a hot topic of discussion. To address this question, we have searched for gene losses by combining best reciprocal blast hit (BRBH) with exhaustive phylogenetic reconstructions of the gene family of interest. We have also performed expression analyses of the present orthologs to test for their cardiac function as well as, in the case of lost orthologs, with paralogs trying to detect potential events of function shuffling. Finally, we performed functional analyses by inhibiting the FGF and BMP signaling pathways and started the implementation of a microinjection facility for future functional analyses by gene targeting. Our results show a clear deconstruction of the cardiopharyngeal GRN with the loss of many genes (Mesp, Ets1/2a, Gata4/5/6, Mek1/2, Tbx1/10, and RA- and FGF-signaling related genes) and cardiac subfunctions (FoxF, Islet, Ebf, Mrf, Dach, and Bmp signaling) crucial for cardiopharyngeal development in ascidians and vertebrates. All these losses have led to the dismantling of two genetic modules related to the maintenance of multipotency in the cardiopharyngeal precursors. This has been accompanied by the loss of the second heart field and pharyngeal muscles in appendicularians, which has been phenotypically translated into an open bilaminar heart with an accelerated development compared to the tubular heart present in the rest of chordates. The deconstruction of the cardiopharyngeal GRN in appendicularians can therefore be interpreted as an evolutionary adaptation to the transition from a sessile to a free-living lifestyle based on the innovation of the filter-feeding house. Therefore, our results show O. dioica as a paradigmatic example of the advantages of using species that along their evolution has lost many genes (evolutionary knockout models, eKO) to better understand the evolution of GRNs, mechanisms of embryo development, or any physiological adaptation in the absence of any given gene of interest.

[cat] El creixent nombre de genomes seqüenciats està posant de manifest que la pèrdua gènica és un fenomen recurrent que pot haver generat diversitat al llarg de la història evolutiva dels diferents grups d’organismes. En aquest sentit, un dels grans reptes en el camp de la EvoDevo és entendre l’impacte de la pèrdua gènica en l’evolució dels mecanismes del desenvolupament animal considerant diferents escenaris evolutius. En escenaris de selecció positiva, la pèrdua hauria comportat canvis morfològics o fisiològics que haurien permès l’adaptació de les espècies a noves condicions ambientals. En escenaris de selecció neutre (o quasi neutre), la pèrdua hauria afectat a gens que haurien esdevingut prescindibles per l’existència de robustesa mutacional o perquè les condicions ambientals haurien canviat. Saber si una pèrdua gènica ha estat adaptativa o neutre és generalment molt complex ja que molts cops es difícil establir relacions directes de causalitat entre la pèrdua d’un gen i l’aparició d’una nova característica evolutivament avantatjosa. Malgrat aquesta dificultat, en aquesta tesi doctoral vàrem voler estudiar l’impacte de la pèrdua gènica en l’EvoDevo a partir d’analitzar l’evolució de les vies de senyalització de l’àcid retinoic (RA) i de Wnt -vies essencials pel desenvolupament de tots els cordats- a l’urocordat Oikopleura. dioica com a sistema model de referència. Per establir O. dioica com a model animal, però, a més de les característiques biològiques que la fan atractiva per aquests tipus estudis –desenvolupament embrionari i cicle de vida extremadament ràpid, simplicitat i transparència corporal, genoma reduït i totalment seqüenciat, possibilitat de manipulació gènica per estudis funcionals– ha estat fonamental desenvolupar unes instal·lacions per cultivar-la en el laboratori, així com protocols de manteniment assequibles (low-cost) per qualsevol grup d’investigació. Gràcies a aquest sistema de cultiu, em pogut analitzar els components de la xarxa gènica del metabolisme de l’RA (RA-MGN), així com els lligands de la via de senyalització per Wnt. En referència a la RA-MGN, el nostre treball ens permet concloure que les pèrdues gèniques que han afectat a aquesta via durant l’evolució de O. dioica han tingut lloc en un sistema genètic no robust, on el desballestament de la via no han tingut un impacte dramàtic en el pla corporal típic de cordats que preserva aquest organisme. Per altre banda, l’anàlisi de la família Wnt a cordats, ens ha permès concloure que a més de la duplicació gènica o de la redundància funcional per processos d’evolució convergent, els fenòmens de reassignació de funcions (function shuffling) augmenten també la robustesa mutacional i faciliten les pèrdues gèniques en les famílies de gens. En resum, els resultats d’aquesta tesi doctoral posen de manifest que O. dioica és un model animal atractiu per estudiar tant aspectes bàsics de l’impacte de les pèrdues gèniques en l’evolució dels mecanismes del desenvolupament, com en aspectes aplicats en que certes pèrdues confereixen a O. dioica la condició de knockout evolutiu que pot ser interessant per l’estudi de mecanismes moleculars concrets com la toxicitat de les PUAs en el desenvolupament embrionari d’organismes marins., [eng] The bloom of genomics is revealing gene loss as a pervasive evolutionary force generating genetic diversity that shapes the evolution of species. In this sense, one of the great challenges in the field of EvoDevo is to understand the impact of gene loss on the evolution of animal developmental mechanisms. In this doctoral thesis we wanted to study the impact of gene loss in EvoDevo, using the analysis of the evolution of retinoic acid (RA) and Wnt signaling pathways - essential pathways in the development of all the chordates- in the urochordate Oikopleura. dioica as a model reference system. To establish O. dioica as an animal model, in addition to the biological characteristics that make it attractive for his type of studies-embryonic development and extremely fast life cycle, simplicity and body transparency, reduced and fully sequenced genome, possibility of gene manipulation for functional studies - It has been essential to develop a facility to grow O. dioica in the laboratory, as well as low-cost maintenance protocols affordable for any research group. Thanks to this culture system, we were able to analyze the components of the RA metabolic gene network (RA-MGN), as well as the ligands of the Wnt signaling pathway. In reference to the RA-MGN, our work allows us to conclude that the gene losses that have affected this pathway during the evolution of O. dioica have taken place in a non-robust genetic system, where the dismantling of the network has not had a dramatic impact on the typical body plan of chordates that preserves this organism. On the other hand, the analysis of the Wnt family in chordates, has allowed us to conclude that in addition to gene duplication or functional redundancy due to processes of convergent evolution, function shuffling also increase the mutational robustness and facilitate gene losses in gene families. In summary, the results of this doctoral thesis show that O. dioica is an attractive animal model for studying both basic aspects of the impact of gene losses in the evolution of development mechanisms, and in applied aspects. For instance, certain losses give to O. dioica an evolutionary knockout condition that may be interesting for the study of concrete molecular mechanisms such as the toxicity of PUAs in the embryonic development of marine organisms.