ODETE MARINHO GONÇALVES THE ORIGIN OF STOMACH VERTEBRATE AND THE PARADOX OF LOSS Tese de Candidatura ao grau de Doutor em Ciências Biomédicas submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Doutor Jonathan M. Wilson Categoria – Professor auxiliar/Investigador Afiliação – Universidade de Wilfrid Laurier, Waterloo, Canadá/ CIIMAR- Centro Interdisciplinar de Investigação marinha e ambiental. Coorientador – Doutor L. Filipe C. Castro Categoria – Investigador auxiliar Afiliação – CIIMAR- Centro Interdisciplinar de Investigação do mar e ambiente. Coorientador – Professor João Coimbra Categoria – Professor emérito/ Investigador Afiliação – ICBAS- Instituto de Ciências Biomédicas Abel Salazar/ CIIMAR- Centro Interdisciplinar de Investigação marinha e ambiental. This work was conducted in laboratory of Molecular Physiology (MP) at CIIMAR- Centro Interdisciplinar de Investigação Marinha e Ambiental. The animals were maintained at BOGA- Biotério de Organismos Aquáticos. The Chapter 5 of this thesis was partially developed at Station Biologique de Roscoff (France) in laboratory of Dr. Sylvie Mazan (2013). This Chapter was done in collaboration with Dr. Renata Freitas from IBMC. Some work was also developed in Laboratory of Professor Eduardo Rocha at ICBAS- Instituto de Ciências Biomédicas Abel Salazar. This work was funded by FCT scholarship with reference: SFRH/ BD/ 79821/ 2011 “The theories exposed in this work are the exclusive responsibility of the author” AKNOWLEDGMENTS I am grateful to my supervisor Dr. Jonathan Wilson and my co-supervisor Dr. Filipe Castro. This fascinating work begun almost one decade ago (2007) with my master that allow we work together until now. Thanks to Professor João Coimbra to be so kind and to show interested in my work. Thanks to him and Professor Eduardo Rocha, I had the privilege to be Biomedical Sciences phD student at ICBAS. I am deeply grateful to Dr. Renata Freitas that helps me with the developmental chapter 5 and that supported my ideas when everything was going down. Thank you to Patrícia Ferreira to be always available to help and to Dr. Ana Isabel Teixeira that was so helpful in this last weeks. Thanks to all my friends, since the “ancient” of CIIMAR as Filipa Gonçalves, Inês Coelho, Claudia Escórcio, Marisa Passos, Daniela Lima and Joana Moreira da Silva (lucky me!) until the new friends that accompanied me in these last years as Diogo Martins, Salman Malakpoor, Claudia Moreira. A special thanks to Joana Saiote, Catarina Cruzeiro, Mónica Lopes Marques, Alice Ramos and Ivone Pinheiro that for a reason or another are precious friends. Por muito que eu tente, não encontro palavras para agradecer aos meus pais, Elvira and António, e à minha irmã, Estelle. Tenho muito orgulho na minha família. Este doutoramento foi um desafio constante, não apenas por causa do trabalho laboratorial mas também por diversos motivos que foram aparecendo ao longo dos anos. O vosso apoio constante é inestimável. Obrigada Mãe por me ajudares a apanhar cada pedra que me aparece pelo caminho que sigo usando para construir o belo castelo que é a minha vida. Não tenho palavras para expressar a gratidão que tenho para com o meu marido, Vítor Ferreira. Sem ti, esta tese não existiria. Ao longo desta jornada, tive a sorte de divider a minha vida contigo e de ter o nosso adorado filho, Tiago. Nada disto seria possível sem o teu amor, carinho, apoio e amizade (e algum mindfulness…). Independentemente do que vier a seguir, tenho-vos aos dois e estou profundamente grata por isso. O Tiago e tu são o sol da minha vida! A special thanks to Horatio, Hera, Hercules, small Calisto and his brothers and all the animals that made my days much happier on the lab. v ABSTRACT The stomach represents a well conserved gnathostome innovation that presumably evolved in an ancestor 450 MYA. It is characterized by the secretion of hydrochloric acid (HCl) by the gastric proton pump (H+/K+-ATPase), a heterodimer with an α-subunit and β-subunit encoded by ATP4A and ATP4B respectively and by the aspartic protease pepsin that are both produced in gastric glands. However, functional loss of the stomach in jawed vertebrates’ evolution has occurred multiple times, in a pattern indicating secondary loss. The principal groups known as agastric are the Holocephali (Chimera), Lipidosireniformes (Lungfish) and members of several Actinopterygii orders as Cypriniformes (loaches), Beloniformes (needlefishes) and Tetraodontiformes (pufferfishes). The Perciformes (gobies) and Gasterosteiformes (seahorses and pipfishes) also are known for the several agastric families. Thus, this thesis focus on the central question of the frequent loss of the stomach in vertebrate evolution focusing mostly on H+/K+-ATPase genes. An understanding of the underlying relationship between stomach-loss with gastric genes as the basis of loss is fundamental. Phylogenetics and synteny analyses of H+/K+-ATPase genes, and the investigation and identification of gastric proton pump genes in key species were undertaken. The results clearly demonstrate that the loss of gastric glands correlates with the persistent and complete absence of the gastric gene kit in the analysed species [H+/K+-ATPase (Atp4a and Atp4a) and pepsinogens (Pga, Pgc, Cym)]. The patterns of stomach loss in Tetraodontiform and Cypriniform orders were also studied using a combination of immunohistochemistry and RT-PCR based approaches. To clarify the pattern of stomach loss in the Tetraodontiformes, specimens from the more basal families were examined: Balistidae, Monacanthidae and Ostraciidae in addition to the crown family Tetraodontidae. An absent gastric phenotype is visible in Tetraodontidae as well as in basal Ostraciidae but gastric glands and Atp4a are present in the foregut of the intermediate sister families Balistidae and Monacanthidae. These results indicate that stomach loss occurred at least twice in the Tetraodontiformes. In Cypriniformes, the foregut regions of several Cobitoidea superfamily members were examined for the presence of gastric glands and gastric proton pump (atp4a) α subunit expression since multiple loss events occurred making questionable a reinvention of the stomach. A functional stomach was detected in representative species from Nemacheilidae/ Balitoridae and Botiidae, but not Cobitidae (Misgurnus anguillicaudatus). Thus, an uninterrupted gastric evolutionary lineage in the Cobitoidea is evident being vi improbable that the stomach was reinvented in the Nemacheilidae/Balitoridae clade consistent with Dollo’s principle. The developmental origin of stomach is associated with important molecular interactions between the embryonic gut endoderm and the underlying mesenchyme. The molecular mechanisms behind these interactions involve expression of the homeobox gene BARX1 and the genes SOX2, SFRP2 and FGF10 (generally associated with stomach development) and homeobox genes CDX1 and CDX2 (associated with intestine development). In spite to some reported variation in gnathostomes and in fishes, the available data is scarce regarding the molecular cascade leading to gut differentiation. Thus, the conservation of gut developmental program in a basal gnathostome (Scyliorhinus canicula) was investigated by qPCR-real time, immunohistochemistry and in situ hybridization. Results suggest that the gastric glands first appear in the last developmental stage and the gastric proton pump H+/K+-ATPase expression is also visible. Both Barx1 and Sox2 have a higher expression in the anterior portion of the digestive tract. In contrast, Cdx2 has a higher expression on the posterior portion of the gastrointestinal tract (GIT). These findings suggest that the GIT developmental program is largely conserved in the gnathostome lineage. Then, the several results presented on this work are extremely useful to elucidate some questions related with the curious case of the agastric condition in so many gnathostomes. vii RESUMO O estômago representa uma inovação bem conservada nos Gnathostomata que provavelmente evoluiu num ancestral comum há 450 milhões de anos. Este órgão é caracterizado pelas glândulas gástricas que secretam ácido clorídrico (HCl) através da bomba de protões gástrica (H+/K+-ATPase), um heterodímero com duas subunidades α e β, codificadas pelos genes ATP4A e ATP4B respectivamente, e pepsina, uma protease aspártica. No entanto, a perda do estômago funcional na evolução dos vertebrados com mandíbulas ocorreu múltiplas vezes num padrão que indica perda secundária. Os principais grupos conhecidos por serem agástricos são Holocephali (Quimeras), Lepidosireniformes (Peixes pulmonados) e membros de diversas ordens de actinopterígeos como os Cypriniformes, Beloniformes e Tetraodontiformes. Os Perciformes (alcabozes) e os Gasterosteiformes (cavalos-marinhos) tambéms são conhecidos pelas numerosas famílias agástricas. Assim, esta tese centra-se particularmente na questão relativa à perda frequente de estômago na evolução dos vertebrados focando-se sobretudo nos genes da H+/K+-ATPase. É fundamental entender a relação entre a perda de estômago e os genes gástricos. Para isso foram realizadas análises filogenéticas e de sintenia aos genes da H+/K+-ATPase. Adicionalmente, os genes da bomba gástrica de protões foram investigados e identificados em algumas espécies chave. Os resultados mostram de forma clara que a perda das glândulas gástricas está relacionada com a persistente e completa ausência do grupo de genes gástricos nas espécies analisadas [H+/K+-ATPase (Atp4a