Proteomics of early embryonic development of zebrafish (Danio rerio)

Doctoral thesis (PhD) – Nord University, 2021publishedVersio

Single-cell profiling for advancing birth defects research and prevention

Cellular analysis of developmental processes and toxicities has traditionally entailed bulk methods (e.g., transcriptomics) that lack single cell resolution or tissue localization methods (e.g., immunostaining) that allow only a few genes to be monitored in each experiment. Recent technological advances have enabled interrogation of genomic function at the single-cell level, providing new opportunities to unravel developmental pathways and processes with unprecedented resolution. Here, we review emerging technologies of single-cell RNA-sequencing (scRNA-seq) to globally characterize the gene expression sets of different cell types and how different cell types emerge from earlier cell states in development. Cell atlases of experimental embryology and human embryogenesis at single-cell resolution will provide an encyclopedia of genes that define key stages from gastrulation to organogenesis. This technology, combined with computational models to discover key organizational principles, was recognized by Science magazine as the “Breakthrough of the year” for 2018 due to transformative potential on the way we study how human cells mature over a lifetime, how tissues regenerate, and how cells change in diseases (e.g., patient-derived organoids to screen disease-specific targets and design precision therapy). Profiling transcriptomes at the single-cell level can fulfill the need for greater detail in the molecular progression of all cell lineages, from pluripotency to adulthood and how cell–cell signaling pathways control progression at every step. Translational opportunities emerge for elucidating pathogenesis of genetic birth defects with cellular precision and improvements for predictive toxicology of chemical teratogenesis

Using single-cell genomics to understand developmental processes and cell fate decisions.

High-throughput -omics techniques have revolutionised biology, allowing for thorough and unbiased characterisation of the molecular states of biological systems. However, cellular decision-making is inherently a unicellular process to which "bulk" -omics techniques are poorly suited, as they capture ensemble averages of cell states. Recently developed single-cell methods bridge this gap, allowing high-throughput molecular surveys of individual cells. In this review, we cover core concepts of analysis of single-cell gene expression data and highlight areas of developmental biology where single-cell techniques have made important contributions. These include understanding of cell-to-cell heterogeneity, the tracing of differentiation pathways, quantification of gene expression from specific alleles, and the future directions of cell lineage tracing and spatial gene expression analysis.J.A.G. was supported by Wellcome Trust Grant “Systematic Identification of Lineage Specification in Murine Gastrulation” (109081/Z/15/A). A.S. was supported by Wellcome Trust Grant “Tracing early mammalian lineage decisions by single cell genomics” (105031/B/14/Z). J.C.M. was supported by core funding from Cancer Research UK (award no. A17197) and EMBL

Classifying Chemical Bioactivity by Coupling High-throughput Phenotypic Anchoring and Transcriptome Profiling in Zebrafish

There are more than 87,000 chemicals in current use with little to no toxicity information available. Assessing such a large number of chemicals using traditional methods would take an unreasonable amount of time and money, and require the use a large number of animals. The incorporation of high-throughput in vivo model systems that can overcome limitations of in vitro screens while providing a rapid genome-wide view of chemical bioactivity are needed to help fully realize the vision of 21st century toxicity testing. In this dissertation, I employed transcriptomics using the high-throughput in vivo developmental zebrafish model to generate hypotheses regarding the mechanism of action of specific chemicals and to identify unique transcriptional signatures for a set of endocrine disrupting chemicals that can be used to classify the activity of known and unknown chemicals. We employed comparative transcriptomics in wild-type and ahr2-null zebrafish exposed to mono-substituted isopropylated triaryl phosphate (mITP), a component of the Firemaster 550 flame retardant mixture, and demonstrated that the cardiotoxic effects of mITP are likely mediated through inhibition of retinoic acid receptor signaling and not the aryl hydrocarbon receptor (AhR). Although mITP induced a transcriptional profile indicative of AhR activation in wild-type zebrafish, these signatures were not related to cardiotoxicity. I found that mITP exposure, independent of ahr2 status, decreased the expression of many Hox genes as well as enzymes responsible for retinoic acid metabolism, which are known to be regulated by retinoic acid receptors. Several of the dysregulated Hox genes are involved in cardiac cell lineage determination in early heart development, and in heart tube elongation and looping via cell recruitment in the second heart field. I also employed transcriptomics to investigate the mechanism of developmental toxicity of the antimicrobial agent, triclosan (TCS). Exposure to TCS resulted in robust transcriptome changes with a large number of transcripts being significantly decreased. Downstream functional analyses showed that many of the transcripts significantly affected by TCS are involved in liver functioning and development, suggesting that TCS is hepatotoxic in embryonic zebrafish. I also compared our transcriptomic analysis with the comprehensive in vitro bioactivity profile of TCS from the Environmental Protection Agency’s Toxicity Forecaster (ToxCast) program, and observed low concordance between these two screening strategies. Lastly, I conducted transcriptome profiling of 25 endocrine disrupting chemicals in order to identify discriminatory transcriptional signatures that would help classify chemicals with estrogen, androgen, or thyroid hormone activity. Clustered correlation analysis of the top 1000 significantly differentially expressed transcripts revealed four chemicals with highly similar and unique transcriptional profiles compared to the other 21 chemicals. These four chemicals included three known thyroid hormone receptor agonists, and one unknown chemical, which was later identified as a failed pharmaceutical with thyroid receptor agonist activity. I identified a panel of 27 transcripts as well as a unique pigmentation phenotype for these four chemicals which can be used in future chemical screens to detect other thyroid receptor agonist chemicals in zebrafish. Overall, the work presented here demonstrates the utility of phenotypically anchored whole genome transcriptomics in zebrafish to identify putative mechanisms of action for many chemicals. Furthermore, the information obtained from these types of analyses can be used to develop predictive models to classify the bioactivity of known and unknown chemicals, or can identify biomarkers that can be used in future high-throughput screens

Reprogrammed Transcriptome in Rhesus-Bovine Interspecies Somatic Cell Nuclear Transfer Embryos

Global activation of the embryonic genome (EGA), one of the most critical steps in early mammalian embryo development, is recognized as the time when interspecies somatic cell nuclear transfer (iSCNT) embryos fail to thrive.In this study, we analyzed the EGA-related transcriptome of rhesus-bovine iSCNT 8- to 16-cell embryos and dissected the reprogramming process in terms of embryonic gene activation, somatic gene silencing, and maternal RNA degradation. Compared with fibroblast donor cells, two thousand and seven genes were activated in iSCNT embryos, one quarter of them reaching expression levels comparable to those found in in vitro fertilized (IVF) rhesus embryos. This suggested that EGA in iSCNT embryos had partially recapitulated rhesus embryonic development. Eight hundred and sixty somatic genes were not silenced properly and continued to be expressed in iSCNT embryos, which indicated incomplete nuclear reprogramming. We compared maternal RNA degradation in bovine oocytes between bovine-bovine SCNT and iSCNT embryos. While maternal RNA degradation occurred in both SCNT and iSCNT embryos, we saw more limited overall degradation of maternal RNA in iSCNT embryos than in SCNT embryos. Several important maternal RNAs, like GPF9, were not properly processed in SCNT embryos.Our data suggested that iSCNT embryos are capable of triggering EGA, while a portion of somatic cell-associated genes maintain their expression. Maternal RNA degradation seems to be impaired in iSCNT embryos. Further understanding of the biological roles of these genes, networks, and pathways revealed by iSCNT may expand our knowledge about cell reprogramming, pluripotency, and differentiation

Gene regulation and cis-regulatory element usage during sea urchin development

Die Grundlage der Entwicklung multizellulärer Lebewesen bildet die Herausbildung stabiler Zelllinien aus einer einzelnen Zygote. Die Expression spezifischer Kombinationen von Transkriptionsfaktoren ist von zentraler Bedeutung für diesen Prozess. Des Weiteren sind epigenetische Veränderungen des Genoms von Bedeutung, über deren Verhältnis zu Änderungen der Zell-Identitäten weniger bekannt ist. In dieser Arbeit nutze ich Daten aus scATAC-seq und scRNA-seq (mit metabolischer Markierung; scSLAM-seq) um die regulierenden Elemente im Seeigel zu charakterisieren – vom 4-Zell-Stadium, über die Aktivierung des zygotischen Genoms, bis hin zu den Strukturen der frühen Pluteus-Larve. Die Schicksale der einzelnen Zellen des Seeigels sind gut erforscht und ab der vierten Zellteilung etabliert (auch wenn einzelne Veränderungen bis zum 128-Zell Stadium induziert werden können), wonach die Keimblätter fest verankert sind. Plastizität innerhalb der Keimblätter ist mindestens bis zum Ende der Gastrulation möglich. Mithilfe dieser Daten, sowie den bekannten Ergebnissen der Erforschung der Zellschicksale vergangener Jahrzehnte, ist eine Diversifizierung von Zelltypen ersichtlich, die beim 128-Zell-Stadium mit der großen Welle der zygotischen Genomaktivierung (ZGA), einer Veränderung des Chromatinzustandes und dem Verlust der Entwicklungsplastizität einhergeht. Ein kleiner Teil von Genen, im Allgemeinen zelltypspezifische Transkriptionsfaktoren, wird schon vor der großen Welle der ZGA exprimiert, während sich gut offene Chromatinstrukturen auf wenige, distinkte regulatorische Sequenzen beschränken. Von diesem Zeitpunkt an, wird die Genexpression zelltypspezifisch, auch wenn viele Chromatinelemente weiterhin zelltypübergreifend offen sind. Insgesamt sind die Chromatinelemente recht kompakt und Elemente innerhalb der Gene häufig. Danach porträtiere ich noch die regulatorischen Veränderungen die mit der neuralen Entwicklung, sowie der Diversität von skelettbildenden Zellen im Seeigel einhergehen. Zuletzt identifiziere ich ~ 100 Gene, die in die Kalzifizierung des Skelettes des Seeigels involviert sind.The emergence of multiple, stable cell lineages from a single-cell zygote is the essence of multicellular development. Combinatorial transcription factor expression is central to this process, as well as epigenetic changes whose relationship to changes in cell-identity are far less well understood. In this thesis, I use data from scATAC-seq and scRNA-seq (with metabolic labeling; scSLAM-seq) to characterize the regulatory landscapes of sea urchin development spanning from the 4-cell embryo through maternal zygotic transition (MZT), gastrulation, and the early pluteus larvae with its ecologically relevant structures (~72h). The early fate-map in sea urchins is well understood, providing an ideal model for this analysis; the basic fate-map is established by the fourth cleavage (though inducible lineage changes are possible up to the 128-cell stage) after which germ-layer identity is locked, though there remains considerable plasticity within lineages at least through gastrulation. Using these data, along-side classic research into cell fate maps in the early embryo, I find that cell-type diversification and a loss of plasticity at 128-cell stage corresponds to a major wave of zygotic genome activation (ZGA) and a clear resolution of the chromatin landscape in the sea urchin. However, a subset of genes, often cell-type-specific transcription factors, shows evidence of pre-MZT zygotic expression with early chromatin accessibility limited to few sites with distinct regulatory sequences. From this time, gene expression profiles become highly cell-type-specific, though many regulatory elements remain ubiquitously accessible, suggesting differential transcription factor occupancy at broadly accessible sites. Overall, the regulatory landscape is fairly compact with accessible intragenic elements being frequent. Subsequently, I profile the regulatory changes underlying neurodevelopment and the diversity of skeletogenic cells in the sea urchin. Finally, I also identify ~ 100 genes that are associated with calcification of the sea urchin skeleton

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)

Genome-wide gene expression surveys and a transcriptome map in chicken

The chicken (Gallus gallus) is an important model organism in genetics, developmental biology, immunology, evolutionary research, and agricultural science. The completeness of the draft chicken genome sequence provided new possibilities to study genomic changes during evolution by comparing the chicken genome to that of other species. The development of long oligonucleotide microarrays based on the genome sequence made it possible to survey genome-wide gene expression in chicken. This thesis describes two gene expression surveys across a range of healthy chicken tissues in both adult and embryonic stages. Specifically, we focus on the mechanisms of regulation of gene transcription and their evolution in the vertebrate genome. Chapter 1 provides a brief history of the chicken as a model organism in biological and genomics research. In particular a brief overview is presented about expression profiling experiments, followed by an introduction to gene transcription regulation in general. Finally, the aim and outline of this thesis is presented. An important aim of this thesis is to generate surveys of genome-wide gene expression data in chicken using microarrays. In chapter 2, we introduce microarray data normalization including background correction, within-array normalization and between-array normalization. Based on these results an analysis approach is recommended for the analysis of two-color microarray data as performed in the experiments described in this thesis. We also briefly explain the relevant methodology for the identification of differentially expressed genes and how to translate resulting gene lists into biological knowledge. Finally, specific issues related to updating microarray probe annotation in farm animals, is discussed. For the analysis of the microarray data in this thesis re-annotation of the probes on the chicken 20K oligoarray was done using the oligoRAP, analysis pipeline. The vast amount of data generated from a single transcriptomics study makes it impossible to extract meaningful biological knowledge by manually going through individual genes from a list with hundreds and thousands of differentially expressed genes. In chapter 3, we present a practical approach using a collection of R/Bioconductor packages to extract biological knowledge from a microarray experiment in farm animals. Furthermore, a locally adaptive statistical procedure (LAP) analysis approach is used to identify differentially expressed chromosomal regions in a microarray experiment. Chapter 4 presents a genome-wide gene expression survey across eight different tissues (brain, bursa of Fabricius, kidney, liver, lung, small intestine, spleen, and thymus from 10-week old chickens) in adult birds using a chicken 20K microarray. To a certain extent, most genes show some tissue-specific pattern of expression. Housekeeping and tissue-specific genes are identified based on gene expression patterns across the eight different tissues. The results show that housekeeping genes are more compact, i.e. are smaller, with shorter, coding sequence length, intron length, and smaller length of the intergenic regions. This observed compactness of housekeeping genes may be a result of selection on economy of transcription during evolution. Furthermore, a comparative analysis of gene expression among mouse, chicken, and frog showed that the expression patterns of orthologous genes are conserved during evolution between mammals, birds, and amphibians. The chicken embryo has been a very popular model for developmental biology. To study the overall gene expression pattern in whole chicken embryos at different developmental stages and/or embryonic tissues, a genome-wide gene expression survey across different developmental and embryonic stages was performed (chapter 5). The study included four different developmental stages (HH stage 3, 10, 15, 22) and eight different embryonic tissues (brain, bursa of Fabricius, heart, kidney, liver, lung, small intestine, and spleen from HH stage 36). We were able to identify several embryonic stage- and tissue-specific genes in our analysis. Genomic features of genes widely expressed under these 12 conditions suggest that widely expressed genes are more compact than tissue-specific genes, confirming the findings described in chapter 4. The analysis of the differentially expressed genes during the different developmental stages of whole embryo indicates a gradual change in gene expression during embryo development. A comparison of the gene expression profiles between the same organs, of adults and embryos reveals both striking similarities as well as differences. The overall goal of this thesis was to improve our understanding of the mechanisms of transcriptional regulation in the chicken. In chapter 6, a transcriptome map for all chicken chromosomes is presented based on the expression data described in chapter 4. The results reveal the presence of two distinct types of chromosomal regions characterized by clusters of highly or lowly expressed genes respectively. Furthermore, these regions show a high correlation with a number of genome characteristics, like gene density, gene length, intron length, and GC content. A comparative analysis between the chicken and human transcriptome maps suggests that the regions with clusters of highly expressed genes are relatively conserved between the two genomes. Our results revealed the presence of a higher order organization of the chicken genome that affects gene expression, confirming similar observations in other species. Finally, in chapter 7 I summarize the main findings and discuss some of the limitations of the analyses described in this thesis. I also discuss the different merits and shortcomings of studying gene expression using either microarrays or next-generation sequencing technology and propose directions for future research. The rapid developments in new-generation sequencing technology will facilitate better coverage and depth of the chicken genome. This will provide a better genome assembly and an improved genome annotation. The sequence-based approaches for studying gene expression will reduce noise levels compared to hybridization-based approaches. Overall, next-generation sequencing is already providing greatly enhance tools to further improve our understanding of the chicken transcriptome and its regulation. <br/

Revealing routes of cellular differentiation by single-cell RNA-seq

Differentiation of multipotent stem cells is controlled by the intricate regulatory interactions of thousands of genes. It remains one of the major challenges to understand how nature has designed such robust and reproducible regulatory mechanisms. Knowing the detailed structure of the underlying lineage trees is the basis for investigating the molecular control of this process. The recent availability of large-scale sensitive single-cell RNAseq protocols has enabled the generation of snapshot data covering the entire spectrum of cell states in a systemof interest. Consequently, a large number of computational methods for the reconstruction of cellular differentiation trajectories have been developed. Here, I will provide a detailed overview of the concepts and ideas behind some of these algorithms and discuss the particular aspects addressed by each method

Identification of biomarkers of the retinoic acid signaling pathway in the zebrafish embryo model to predict human developmental toxicants

This dissertation falls within the context of the paradigm shift in regulatory toxicology testing which promotes using a mechanistic-based approach based on in vitro tests instead of traditional animal testing to predict chemical hazards to human such as developmental toxicity. The novel research expands the understanding of developmental toxicity pathways by studying chemically-induced gene expression changes related to the perturbation of the retinoic acid signaling pathway (RA-SP) in a vertebrate embryo model. By using the zebrafish embryo (ZE) model it was possible to take advantage of the conservation of this biological pathway across vertebrate taxa, to predict potential human developmental toxicity. The ZE model is not new, however it has been primarily used and optimized for its morphology readout due to the transparent eggs enabling morphological observations during chemical exposure. However, improvements and harmonization are necessary to utilize this model with a reliable molecular level readout, to reveal relevant changes in gene expression. In chapter 2, the protocol design was refined to identify gene expression (GE) changes in the ZE. This was done by investigating the optimal exposure duration to study such changes due to the perturbation of the RA-SP. An exposure of ZE to the RA-SP agonist all-trans retinoic acid (ATRA) was performed using 6 different exposure durations, ranging from 2-117 hrs. These results identified that 4h exposure was the optimal exposure duration to study chemically-induced GE regulation specifically related to the RA-SP perturbation, thereby optimizing the ZE protocol for GE analysis. In chapters 3 and 4, the optimized ZE-GE protocol was employed to identify GE biomarker candidates for maldevelopment. After exposing ZE to two teratogenic compounds known to perturb the RA-SP (ATRA and Valproic Acid, VPA) and one non-teratogenic control compound (Folic Acid, FA), the chemically-induced perturbation of the RA-SP was explored using a whole genome scale GE analysis approach (RNAseq). The 3 test compounds each showed a specific mRNA expression profile, with 248 genes commonly regulated by both teratogenic compounds (ATRA and VPA) but not by FA. These 248 genes were implicated in several developmental processes. 62 differentially expressed genes (DEGs) were associated with nervous system development and were further examined in Chapter 3. These 62 genes were identified as potential biomarkers of early neurodevelopmental toxicity. In chapter 4, the perturbation of RA-SP on the GE associated with development of mesoderm derived tissues was investigated using bioinformatics methods. The investigation identified gene ontology (GO)-terms related to 47 DEGs. Literature indicates that these genes were normally expressed among 3 mesodermal sections (paraxial, intermediate, and lateral plate section) and 6 mesodermal tissues (somites, striated muscle, bone, kidney, circulatory system, and blood). These 47 DEGs were identified as potential biomarkers of early mesodermal maldevelopment or novel potential biomarkers for specific mesodermal organs. These proposed biomarker candidates advance the knowledge on the retinoic acid-mediated developmental toxicity mechanism. As their responses become even more broadly characterized by exploring different exposure regimes and the profiles of different chemicals, these biomarkers could contribute to predictive tools in animal-free chemical hazard and risk assessment