ojoplano-mediated basal constriction is essential for optic cup morphogenesis

11 páginas, 7 figuras. To the memory of Dr José-Santiago Martínez-Vinjoy. Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/13/2165/DC1Although the vertebrate retina is a well-studied paradigm for organogenesis, the morphogenetic mechanisms that carve the architecture of the vertebrate optic cup remain largely unknown. Understanding how the hemispheric shape of an eye is formed requires addressing the fundamental problem of how individual cell behaviour is coordinated to direct epithelial morphogenesis. Here, we analyze the role of ojoplano (opo), an uncharacterized gene whose human ortholog is associated with orofacial clefting syndrome, in the morphogenesis of epithelial tissues. Most notably, when opo is mutated in medaka fish, optic cup folding is impaired. We characterize optic cup morphogenesis in vivo and determine at the cellular level how opo affects this process. opo encodes a developmentally regulated transmembrane protein that localizes to compartments of the secretory pathway and to basal end-feet of the neuroepithelial precursors. We show that Opo regulates the polarized localization of focal adhesion components to the basal cell surface. Furthermore, tissue-specific interference with integrin-adhesive function impairs optic cup folding, resembling the ocular phenotype observed in opo mutants. We propose a model of retinal morphogenesis whereby opo-mediated formation of focal contacts is required to transmit the mechanical tensions that drive the macroscopic folding of the vertebrate optic cup.This work was supported by grants from the Deutsche Forschungsgemeinschaft, Collaborative Research Centre 488, the EU and HFSPO to J.W.; and MEC:BFU2008-04362/BMC to J.R.M.-M.Peer reviewe

Combining in vivo imaging and mechanistic approaches to investigate Wnt regulation of retinal stem cells

Adult postembryonic stem cells reside in tissues throughout the body of most vertebrates. Little is known, however, about the growth mode and regulation of single stem and progenitor cells in vivo. The continuous life-long growth and the accompanying presence of stem cells in all adult organs renders medaka a perfect model organism to address these unknowns. In particular, medaka’s retinal stem cells are an ideal model for stem cell biology. Their position in surface proximity, their exclusive contribution to one of both retinal layers (neural retina or retinal pigmented epithelium) and their multipotency render retinal stem cells a great experimental system. Furthermore, medaka retinal stem cells can be investigated by in vivo assays in the context of the whole organism. In combination with the Cre/loxP system it is possible to mark and/or alter the signaling state of single cells. Subsequently, these cells and their progeny can be followed and clonally examined. Taken together, single cell spatial resolution and long-term observation of medaka retinal stem cells is possible. This thesis focused on the in vivo behavior and Wnt signaling regulation of retinal stem and progenitor cells by in vivo imaging and clonal analysis. To address this aim, three experimental lines were followed. First, in vivo imaging of medaka was enhanced to perform in vivo investigation of retinal stem cells. I optimized the choice of fluorescent proteins, anesthesia and presence of interfering pigmentation. Second, long-term in vivo microscopy of retinal stem and progenitor cells was performed, followed by tracking and track analysis. Finally, the Wnt signaling state of single retinal stem and progenitor cells was altered and the change in proliferative capacity and differentiation potential was investigated. In conclusion, using the established in vivo imaging toolset, I unraveled fundamental mechanisms of the regulation of in vivo stem cells by Wnt, while being embedded in their organismal context. I showed that high Wnt stimulation in all cell types of the retina led to a high incidence of apoptosis. In contrast, low Wnt stimulation in retinal stem and progenitor cells restricts their proliferative capacity without altering their differentiation potential

Doctor of Philosophy

dissertationHeparan sulfate proteoglycans (HSPG) are a family of cell surface and extracellular matrix proteins with glycosaminoglycan (GAG) chains covalently attached to a protein core. O-sulfotransferases modify these GAG chains by catalyzing the transfer of a sulfate to a specific position on HSPG GAG chains. Although the role of specific HSPG modifications have been described in cell culture and invertebrates, little is known about their functions or abilities to modulate specific cell signaling pathways in vertebrate development. My thesis research focused on the many roles a particular GAG chain modifying enzyme, 2-O-sulfotransferase (2-OST), plays in early development. To characterize the function of 2-OST in early zebrafish development we knocked down this gene via morpholino and found that 2-O-sulfation controls epiboly. Epiboly is the process by which the cells of the blastodisc move towards the vegetal pole from the animal pole to envelop the yolk cell. Knocking down 2-OST results in a failure to initiate and progress through epiboly due to alterations in assembly of filamentous actin, in microtubule organization, and in yolk cell endocytosis. Syndecans 2 and 4, HSPG core proteins, appear to be targets of the 2-OST activity which contribute to this phenotype. 2-OST activity modulates the activity of multiple signaling cascades. We found that 2-OST is an essential component of canonical Wnt signaling in zebrafish development. 2-OST deficient embryos have decreased β-catenin and E-cadherin protein levels, reduced cell adhesion, and altered cell cycle regulation. The cell cycle and iv adhesion defects in 2-OST-deficient embryos can be rescued by reactivation of intracellular Wnt pathway components, but not by overexpression of Wnt8 ligand. Together these results indicate that 2-OST functions within the Wnt pathway downstream of ligand signaling and upstream of intracellular localization and function. Knocking down 2-OST also affects FGF signaling. While Wnt epistasis experiments show loss of 2-O-sulfation impairs some signaling interactions at the cell surface, epistasis experiments with components of the FGF signaling cascade suggest loss of 2-O-sulfation in some contexts can promote signaling which occurs at lower levels in regular developmental contexts. These results suggest HSPG function is essential for early development

NANOG IN THE TWIN FISH MODELS MEDAKA AND ZEBRAFISH: FUNCTIONAL DIVERGENCE OR PLEIOTROPY OF VERTEBRATE PLURIPOTENCY GENE

Ph.DDOCTOR OF PHILOSOPHY (FOS

The immunome and embryo quality in sea bream and sea bass

Gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) are teleosts belonging to Eupercaria and are the most important aquaculture fish species in the Mediterranean region. These two species are ranked second after the Atlantic salmon (Salmo salar) in production volume and value in the European Union (EU) aquaculture sector. Unpredictable fertilized egg/embryo quality and performance remain a bottleneck that threatens sustainability of sea bream and sea bass aquaculture, impeding the increased productivity of aquaculture that entirely depends on hatchery production. To address this issue, criteria and molecular markers linked to embryo quality that could be used to monitor and manage hatchery production were procured. Comparative molecular approaches using molecular biology, proteomics and transcriptomics were performed to analyze embryo performance and immunity in samples from several European commercial hatcheries. The core achievements were the: a) identification and characterization of lysozyme and complement 5 (C5) gene families and embryo and larval gene expression and enzyme activity from a diversity of hatcheries, b) characterization of the embryo proteome from three Mediterranean fish species [white sea bream (Diplodus sargus), meagre (Argyrosomus regius) and sea bream] 24h before hatch and at hatch and identification of common and species specific molecular patterns linked to biological function and putative quality-related proteins, c) comparative transcriptomics of good and poor quality sea bream embryos from several Mediterranean hatcheries. Qualitylinked transcripts and some elements of the regulatory epitranscriptome (non-coding RNA) were identified as well as the contribution of maternal proteins to embryos. Taken together, the results provide a comprehensive description of the molecular basis of sea bream and sea bass embryo development and reveal that immune-related molecules in fertilized eggs are low abundance. The development (quality)-related candidate markers identified will be of value for management of fish embryos in aquaculture hatcheries.O mar Mediterrâneo cobre apenas 0,7% da área oceânica mundial, mas é um dos principais reservatórios de biodiversidade marinha e costeira com cerca de 28% de espécies endémicas. A dourada (Sparus aurata) e o robalo (Dicentrarchus labrax), são duas espécies de peixe que pertencem à série Eupercaria e das mais comercializadas pela indústria de aquicultura nesta região. No sector da aquicultura da União Europeia (EU), estas duas espécies ocupam o segundo lugar no “ranking” da cadeia de valor, depois do salmão do Atlântico. Contudo, as suas características morfológicas e de crescimento são os parâmetros de qualidade relevantes considerados pela indústria. Esta abordagem deve-se à sua comercialização em formato de peixe inteiro, o que reduz o valor de mercado na cadeia de valor e também à ausência de critérios de qualidade para seleção de ovos e embriões, bem como, de marcadores moleculares de qualidade com maior grau de sensibilidade. Todos estes obstáculos, limitam substancialmente o desenvolvimento das indústrias associadas á comercialização destas duas espécies de peixe, impedindo a expansão da aquicultura e das “hatcheries” (maternidades incubadoras de ovos/embriões controlados artificialmente para fins comerciais). Para mitigar este problema, utilizou-se técnicas de biologia molecular e tecnologias ómicas e estabeleceu-se uma abordagem comparativa direcionada á descoberta de moléculas e vias metabólicas funcionais de importância crítica para o sistema imune dos peixes associada á “performance” de desenvolvimento de ovos e embriões. Esta tese está organizada em seis capítulos. Inicia-se com uma visão geral dos critérios morfológicos, físico-químicos e moleculares existentes para avaliar a qualidade de ovos e embriões para melhorar a gestão da aquicultura de peixes (Capítulo 1). Subsequentemente, caracterizou-se a família de genes do sistema imune: a) a das lisozimas em peixes teleósteos com enfase na sua caracterização molecular e funcional em dourada (Capítulo 2) e análises moleculares estruturais e evolutivas e b) a do complemento C5 (C5) em peixes especialmente em espécies da faamilia Cyprinidae (Capítulo 3). Os capítulos 4 e 5, integram abordagens de proteómica e transcriptómica em espécies de peixes mediterrânicos [pargo (Diplodus sargus), corvina (Argyrosomus regius) e dourada], focando os processos de desenvolvimento e de eclosão e na função da enzima “hatching enzyme” em dourada e robalo. Foi feita uma associação entre os capítulos e a análise integrada dos dados do transcritoma do embrião (Capítulo 5) revelou um padrão de expressão significativamente diferente (p-valor < 0,05) para o C5 (Capítulo 3) em diferentes lotes de embriões de dourada nas comparações entre graus de qualidade (Boa vrs Má) e entre estágios de desenvolvimento (Pré- eclosão vrs Eclosão). A variação do C5 em relação ao lote de embriões não foi afetada pela origem da “hatchery”, indicando que as prática de manejo ou os próprios reprodutores não influenciam a sua expressão. Os resultados sugerem que este gene e o seu produto proteico, são provavelmente importantes na proteção imunológica precoce e também em outras funções ainda não descritas na dourada ou em outras espécies de peixes. Também a integração dos resultados do proteoma (Capítulo 4) e do transcritoma (Capítulo 5) do embrião de dourada nos mesmos estágios de desenvolvimento, identificou um grupo de proteínas que se especula serem de origem materna. No último capítulo, sumarizou-se os resultados e são apresentadas perspetivas baseadas nos avanços e desafios atuais e propostas para o desenvolvimento de uma ferramenta integrada de monitorização da qualidade dos embriões e uma base biológica do desenvolvimento de ovos e embriões de peixes (Capítulo 6). Neste projeto foram: 1) identificadas duas importantes famílias de genes associadas à imunidade inata em peixes, a das lisozimas e a do C5. Caracterizou-se pela primeira vez a função das lisozimas através da sua expressão e atividade enzimática em embriões e em diferentes estágios larvares de uma diversidade de reprodutores de dourada. Estudou-se a função do C5 em peixes, através da construção de redes génicas, modelação por homologia e “docking” molecular entre o C5 e o seu receptor (C5R/CD88); 2) mapeou-se e caracterizou-se o proteoma do embrião de três espécies de peixes mediterrânicos (sargo, corvina e dourada) em duas fases do seu desenvolvimento (24h antes da eclosão e na eclosão) e identificou-se um grupo de proteínas potencialmente relacionadas com a imunidade e a qualidade dos embriões. Avaliou-se a função do gene para enzima “hatching enzyme”, com base na sua expressão em embriões de dourada e robalo nos estágios acima referidos; 3) mapeou-se e caracterizou-se o transcritoma de embriões de dourada com origem em diferentes “hatcheries” na região mediterrânica através de uma abordagem comparativa entre qualidade e estágios de desenvolvimento (qualidade- Boa vrs Má; estágios- Pré- eclosão vrs Eclosão) e identificou-se uma diversidade de transcritos, vias metabólicas e elementos do epitranscriptoma regulatório do RNA-não codificante. Foram identificados em comum 42 candidatos a marcadores de qualidade e enriquecidas duas vias metabólicas relacionadas com o sistema imunológico e associadas às “hatcheries”: a via de infeção por Salmonella (constituída por 7 genes relacionados com o sistema imune) e a via de sinalização MAPK (mitogen-activated protein kinase). Foram identificadas 543 proteínas que são expressas apenas no proteoma, sugerindo que podem ter origem materna e destas, 7 (diferencialmente expressas) estão potencialmente relacionadas com o sistema imune. Globalmente, os resultados forneceram um grupo de marcadores relacionados à imunidade e ao desenvolvimento (qualidade), com potencial de se traduzirem em critérios de qualidade de ovos e embriões para a indústria da aquicultura. Estes resultados, foram amplamente estudados para descrever a base molecular biológica entre os diferentes estágios de desenvolvimento de ovos e embriões de peixes e também, entre diferentes lotes de embriões de diferente qualidade. As ferramentas biológicas e critérios desenvolvidos neste trabalho, oferecem uma orientação para as “hatcheries” de peixes e a sua aplicação contribuirá para melhorar no futuro o sector da aquicultura.O apoio financeiro ao trabalho relatado na presente tese de doutoramento é reconhecido com gratidão, pois foi crucial para o progresso positivo e sucesso do trabalho científico. Uma diversidade de fontes de financiamento apoiou o trabalho desenvolvido nesta tese, quer diretamente através da compra de consumíveis/materiais (PerformFISH a European Union’s Horizon 2020 research and innovation grant, agreement Nº 727610), quer indiretamente através do financiamento do CCMAR de apoio a serviços e equipamentos utilizados durante a execução do trabalho (projetos da Fundação para a Ciência e Tecnologia (FCT) - UIDB/04326/2020, UIDP/04326/2020 e dos programas operacionais CRESC Algarve 2020 e COMPETE 2020 através do projeto EMBRC.PT ALG-01-0145-FEDER-022121)

Combining in vivo imaging and mechanistic approaches to investigate Wnt regulation of retinal stem cells

Adult postembryonic stem cells reside in tissues throughout the body of most vertebrates. Little is known, however, about the growth mode and regulation of single stem and progenitor cells in vivo. The continuous life-long growth and the accompanying presence of stem cells in all adult organs renders medaka a perfect model organism to address these unknowns. In particular, medaka’s retinal stem cells are an ideal model for stem cell biology. Their position in surface proximity, their exclusive contribution to one of both retinal layers (neural retina or retinal pigmented epithelium) and their multipotency render retinal stem cells a great experimental system. Furthermore, medaka retinal stem cells can be investigated by in vivo assays in the context of the whole organism. In combination with the Cre/loxP system it is possible to mark and/or alter the signaling state of single cells. Subsequently, these cells and their progeny can be followed and clonally examined. Taken together, single cell spatial resolution and long-term observation of medaka retinal stem cells is possible. This thesis focused on the in vivo behavior and Wnt signaling regulation of retinal stem and progenitor cells by in vivo imaging and clonal analysis. To address this aim, three experimental lines were followed. First, in vivo imaging of medaka was enhanced to perform in vivo investigation of retinal stem cells. I optimized the choice of fluorescent proteins, anesthesia and presence of interfering pigmentation. Second, long-term in vivo microscopy of retinal stem and progenitor cells was performed, followed by tracking and track analysis. Finally, the Wnt signaling state of single retinal stem and progenitor cells was altered and the change in proliferative capacity and differentiation potential was investigated. In conclusion, using the established in vivo imaging toolset, I unraveled fundamental mechanisms of the regulation of in vivo stem cells by Wnt, while being embedded in their organismal context. I showed that high Wnt stimulation in all cell types of the retina led to a high incidence of apoptosis. In contrast, low Wnt stimulation in retinal stem and progenitor cells restricts their proliferative capacity without altering their differentiation potential

Research towards the effective disruption of reproductive competence in Nile tilapia Oreochromis niloticus

Reproductive containment in farmed fish is highly desired for sustainable aquaculture to prevent genetic introgression with wild conspecifics and enhance productivity by suppressing sexual maturation. A number of strategies have already been implemented or have been tested in commercially important fish (e.g. triploidy, monosexing, hormonal therapies); however, they either do not result in 100% containment, or they cannot be applied to all species. One promising new approach consists in disrupting primordial germ cells (PGCs), at the origin of germline cells, to induce sterility. The work carried out in this doctoral thesis aimed to investigate the genes involved in the survival of germ cells and subsequently conduct a functional analysis of candidate genes using CRISPR/Cas9 gene editing system to ultimately provide the basis for the development of a novel sterilisation technique. Nile tilapia was chosen as the experimental animal as it is a major aquaculture species worldwide and the control of reproduction plays a critical role in the farming productivity in this species. In addition, the species has clear advantages as its whole genome sequence is accessible, the generation time is relatively short and zygotes can be available all year round. Initially, a panel of 11 candidate genes with reported roles in survival of PGCs was investigated during the ontogenic development which led to the selection of piwi-like (piwil) gene as a target for genome editing. Then, high temperature was tested as a means to induce germ cell loss to better understand the mechanism underlying germ cell survival and apoptosis, and this study confirmed the functional importance of piwil genes in relation to germ cell loss and proliferation. In addition, the study suggested potential subfunctionalisation within the Bcl-2 gene family which requires further investigation. The next step aimed to optimise the CRISPR/Cas9 gene editing method by improving the microinjection system and testing different concentrations of sgRNAs. Over 95% of injected embryos showed on-target mutation in piwil2 via zygote injection of CRISPR/Cas9 reagents and complete KO larvae were shown in half of the mutants, producing putative sterile fish. However, there was no clear association between the phenotypes in PGCs and the mutation rate. Further comparative studies of mutant screening methods including T7E1, RGEN, HRMA, fragment analysis and NGS revealed that the genotypes of F0 are highly mosaic, suggesting that deep sequencing is recommended for accurate and high throughput F0 screening and further improvement for predictable genome editing is required for a reliable gene functional analysis in F0. In summary, the current thesis provided new scientific knowledge and supporting evidence for the use of the CRISPR/Cas9 gene editing platform to study gene function associated with sterility, with the ultimate goal to develop an alternative sterilisation method in fish

Genotype by temperature interaction effects on sex determination in zebrafish (Danio rerio)

The mechanism of sex determination and gonad differentiation in zebrafish is one of the more challenging research questions and complex puzzles in biology. The regulation of sexual dimorphism in this species is not yet fully understood. According to current knowledge, the sex of zebrafish is determined genetically on the basis of a polygenic sex determination system. In this system the interaction of genetic and environmental factors contributes to determine the sexual fate of an organism. The main objective of this thesis was to investigate the effects of high ambient temperature, as one of the major environmental factors, in interaction with the genome on sexual plasticity and phenotypic traits of zebrafish. Chapter 1 introduces a general overview of sexual diversity in teleost fish and explains the main features of sex determination and gonad differentiation mechanisms in zebrafish. The sexual selection theory in relation to the reproductive success and the effect of environment on secondary sexual traits is generally described. In chapter 2, the phenotypic plasticity of zebrafish in response to increased water temperature is investigated. The study was designed to test the influence of transient temperature perturbations during the thermosensitive embryonic developmental period (from gastrula to pharyngula) in a high number of families (69 families) on various biological functions. The physiological response to the high ambient temperature revealed a lower hatchability and survival ability during embryonic and post-embryonic development. The results of survival trajectories until adulthood demonstrated that in zebrafish the life stages most sensitive to thermal changes are the first day after fertilization and the first two weeks after hatching. Consideration of the effect of temperature on morphometric traits (weight and length) in order to investigate the temperature × sex interaction effect indicated a higher growth performance in temperature-treated animals at different time points of adulthood development. A heat-induced masculinization was clearly observed across all families, while a wide range of interfamily sex ratio variations was detected. These observations emphasize a genetic × environment (G×E) interaction of sex determination and constitute a strong confirmation for a polygenic sex determination system in zebrafish. In chapter 3, a fully automated phenotypic sex classification in zebrafish was established for the first time using two different machine learning methods: Deep Convolutional Neural Networks (DCNNs) and Support Vector Machine (SVM). Based on phenotypic characteristics, a high accuracy of sex differentiation was obtained using these two methods in non-heat-treated groups. However, in treated animals, some males were misclassified using SVM due to reduced pigmentation intensity, suggesting these animals were probably masculinized females. Investigation of the colour intensity of the caudal fin using SVM shows that males exhibited a higher pigmentation intensity compared to females. Furthermore, a positive association of male caudal fin colouration with the morphometric traits (weight and length) was determined. These results imply the effect of temperature on secondary phenotypic sexual characteristics, which in turn may influence the sexual attractiveness for mating and reproductive success. Phenotypic modifications in response to different environmental conditions indicate the changes in the expression of genes responsible for those traits. Hence chapter 4 examines, whether the sex and colour genes are differentially expressed in the two sexes with respect to the observed sexually dimorphic in the caudal fin colouration. This hypothesis is derived from chapter 3, where the sexually differing colour intensity in the caudal fin of the two sexes was detected. Therefore, a transcriptome analysis of caudal fins and gonads was performed in chapter 4 in order to identify the differentially expressed genes and pathways regulating sexual dimorphism in interaction with high water temperature. In addition, the mechanism of gene expression in the masculinization process, as one of the most important consequences of increased temperature, was studied in this chapter. A significantly differentiated expression of sex determination and colour pattern genes was identified in the gonad. In the caudal fin, a high expression magnitude of a set of colour pattern genes was observed, although they were not differentially expressed in two sexes of adult fish. The enrichment of a subset of pathways containing sex and colour genes provides an evidence of the involvement of those genes in the regulation of phenotypic sexual dimorphism in zebrafish. These results led to additional analyses in chapter 5 examining the validity of this hypothesis. In chapter 5, the topics of previous chapters are first discussed in more detail, and then the hypothesis of chapter 4 is further investigated by pathway analysis, gene interaction networks and transcription factor analysis. All analyses in this context supported the association between sex determination and colour pattern genes in zebrafish, as proposed in chapter 4 of this thesis. The conclusion of this chapter highlights the perspective for future studies answering the open questions derived from this thesis