Proteomics analysis of early developmental stages of zebrafish embryos

publishedVersio

Analysis of Sublethal Toxicity in Developing Zebrafish Embryos Exposed to a Range of Petroleum Substances using BE-SPME and Whole Transcriptome Microarray

The OECD 236 Fish Embryo Acute Toxicity Test guideline relies on four endpoints to describe exposure related effects (coagulation, lack of somite formation, tail-bud detachment from yolk-sac, and presence of heartbeat). Danio rerio (zebrafish) embryos were used to investigate these endpoints along with a number of additional sublethal effects (i.e. cardiac dysfunction, pericardial edema, yolk sac edema, tail curvature, hatch success, pericardial edema area, craniofacial malformation, swim bladder development, fin development, and heart rate) following five day exposures to one of seven petroleum substances. The substances investigated included two crude oils, three gas oils, a diluted bitumen, and a petrochemical containing a mixture of branched alcohols. Biomimetic extraction via solid phase microextraction (BE-SPME) was used to quantify freely dissolved test substances as the exposure metric. The most prevalent effects observed were pericardial and yolk sac edema, tail curvature, and lack of embryo viability. Whole transcriptome microarray was used to profile gene expression following exposure to two petroleum substances. Meaningful downregulated differential expression was localized to concentrations that already displayed sublethal morphological effects; therefore, whole transcriptome profiling did not provide sufficient data to be able to predict sublethal morphological effects. A BE-SPME threshold was determined to characterize sublethal morphological alterations that preceded embryo mortality. Overall, this work aids in the understanding of aquatic hazards of petroleum substances to developing zebrafish beyond traditional OECD 236 test endpoints and shows applicability of BE-SPME as an analytical tool to predict sublethal embryotoxicity

Comparative analysis of single-cell transcriptomics in human and Zebrafish oocytes

Background: Zebrafish is a popular model organism, which is widely used in developmental biology research. Despite its general use, the direct comparison of the zebrafish and human oocyte transcriptomes has not been well studied. It is significant to see if the similarity observed between the two organisms at the gene sequence level is also observed at the expression level in key cell types such as the oocyte. Results: We performed single-cell RNA-seq of the zebrafish oocyte and compared it with two studies that have performed single-cell RNA-seq of the human oocyte. We carried out a comparative analysis of genes expressed in the oocyte and genes highly expressed in the oocyte across the three studies. Overall, we found high consistency between the human studies and high concordance in expression for the orthologous genes in the two organisms. According to the Ensembl database, about 60% of the human protein coding genes are orthologous to the zebrafish genes. Our results showed that a higher percentage of the genes that are highly expressed in both organisms show orthology compared to the lower expressed genes. Systems biology analysis of the genes highly expressed in the three studies showed significant overlap of the enriched pathways and GO terms. Moreover, orthologous genes that are commonly overexpressed in both organisms were involved in biological mechanisms that are functionally essential to the oocyte. Conclusions: Orthologous genes are concurrently highly expressed in the oocytes of the two organisms and these genes belong to similar functional categories. Our results provide evidence that zebrafish could serve as a valid model organism to study the oocyte with direct implications in human

Building toxicity pathway models and estimating transcriptomic points-of-departure (tPODs) in three phylogenetically distant ray-finned fishes exposed to 17alpha-ethinylestradiol and fluoxetine

The continuous development and production of new chemicals to meet societal needs results in their increasing release and prevalence in the environment. These contaminants of emerging concern (CECs) are continuously discharged from wastewater treatment plants and other sources that are often not equipped to remove these classes of compounds. Thus, the pseudo-persistence of these CECs poses potential toxicological risks to aquatic organisms and, therefore, constitute a significant issue in many aquatic environments. However, current testing frameworks supporting chemical and environmental risk assessment (ERA) are falling short of the global need to rapidly test the increasing numbers of CECs because they rely on the extensive use of live animals, which is time-consuming, costly, and presents significant ethical concerns. Thus, there is an urgent need for the development and implementation of new approach methodologies (NAMs) aimed to replace, reduce, and refine (3Rs) live animal testing while increasing throughput and decreasing costs to improve hazard assessment strategies and support regulatory decision-making. This dissertation aimed to develop and evaluate a NAM system based on short-term embryonic exposure assays to (1) characterize the toxicity pathways of two priority CECs, 17α-ethinylestradiol (EE2) and fluoxetine (FLX) as model chemicals, through cross-organizational level assessments linking molecular mechanistic response patterns with physiological/apical outcomes; and (2) derive transcriptomics points-of-departure (tPODs) that can support quantitative hazard assessment across phylogenetically distant fish. Specifically, fathead minnow (FHM), rainbow trout (RBT), and white sturgeon (WS) were exposed to graded concentrations of each CEC from an early embryonic stage. At 4 days post-hatch (dph), changes across the whole transcriptome and proteome were characterized, and higher organizational-level responses were evaluated at 28 dph (FHM) and 60 dph (RBT and WS). Molecular alterations were then compared to downstream responses to build toxicity pathway models for each chemical. In addition, transcriptomic datasets from 4 dph were used to model dose-response for genes/transcripts and calculate benchmark concentrations (geneBMCs) of toxicant-responsive genes/transcripts. These geneBMCs were used to derive transcriptomics points-of-departure (tPODs) using a number of statistical strategies. These tPODs were then compared to chronic and physiological/apical PODs obtained in this study and from the literature. tPOD estimates were also compared across species. Results demonstrated that when exposed to EE2, transcriptomic and proteomic profiles of FHM at 4 dph were predictive of histological and apical outcomes at 28 dph. Similarly, transcriptomic profiles of RBT at 4 dph were predictive of histological and apical responses at 60 dph, when exposed to EE2, although proteomic profiles at 4 dph did not correlate well with transcriptomics and apical outcomes. On the other hand, FLX-target genes and proteins were not significantly altered in FHM at 4 dph, but integrated functional analyses of transcriptomics and proteomics revealed molecular processes that were predictive of apical outcomes observed in this study and those reported in the literature. Overall, results showed that transcriptomic responses from whole-body early-life stage (ELS) fish were related to downstream and apical outcomes observed at more advanced life stages. Proteomic responses were limited in predicting relevant responses but added a valuable layer of biological information that enhanced the understanding of the connectivity between molecular events and apical outcomes. Hence, proteomics provided an additional line-of-evidence in support of enhancing our understanding of the mode of action of the tested CECs across biological levels. Estimated tPODs in all species closely approximated and were protective of chronic apical PODs observed in this study and in the literature, both for EE2 and FLX. tPODs derived from the median of the 20 most sensitive active genes (omicBMC20) were the most protective among tPOD estimates within a species, with FLX tPOD values of 0.02, 0.02, and 0.56 μg/L, and with EE2 tPOD values of 0.06, 0.12, and 2.39 ng/L for WS, RBT, and FHM, respectively. These values were significantly more sensitive than pathway-level BMCs (pathBMC) and were independent of species annotations, which is one of the main limitations when working with non-model species for which no functionally annotated genomes or transcriptomes are available. In general, RBT appeared to be the most sensitive species in terms of tPOD estimates, closely followed by WS, with FHM being the least sensitive to exposure to both chemicals. Regardless, tPOD estimates from ELS fish were within a tight range of concentrations despite minor differences in exposure conditions and data analysis workflow. Overall, this dissertation demonstrated that the fish embryo assays established in this thesis project represent a promising approach to (1) characterize toxicity pathways for CECs that can inform cross-species and chemical hazard assessment, and (2) derive quantitative BMCs (tPODs) that are protective of apical PODs, and therefore show significant promise as a NAM to support chemical hazard assessment and regulatory decision making. Future studies should be directed towards the optimization and validation of this approach using more chemicals and other species that are relevant to ERA

Regulation of early cell fate specification in the crustacean embryo, Parhyale hawaiensis

Early cell fate specification is important in the development of animal embryos. This specification is reliant on maternally deposited mRNAs and proteins which help activate lineage-specific gene regulatory networks and requires mechanisms that can establish asymmetries. Previous studies have shown that there is germ layer specification at the 8-cell stage and that some mRNAs are asymmetrically distributed amongst the early progenitors of the germ layers of the amphipod crustacean, Parhyale hawaiensis. These early asymmetries must translate into differential regulation of maternal to zygotic transition (MZT) with different germ layers to ensure correct specification and patterning during development. This makes Parhyale an attractive model to study how early cell fate specification occurs and how early lineage restriction subsequently regulates MZT. In this thesis, we establish single blastomere lineage transcriptomic datasets and test various CRISPR/Cas9 strategies to aid in our understanding of early cell fate specification and the role of maternally loaded mRNAs. Firstly, I describe a method to generate single blastomere lineage transcriptomic datasets. I sequenced mRNAs of each blastomere of the 4- and 8-cell Parhyale embryo to describe genes implicated by asymmetric distribution to be potentially involved in early specification. I show that many early patterning genes, germline specific genes, and translational repression RNA-binding proteins that are asymmetrically distributed in Drosophila and C. elegans are ubiquitously expressed in the Parhyale embryo by in situ hybridization, verifying that transcriptomic data correctly reflects expression of these genes. The establishment of these transcriptomic datasets will now allow us to pick candidate genes for functional studies of early cell fate specifiers as well as analysis of lineage specification as cells divide. Using principal component analysis, we show that macromeres of the 8-cell embryo (Er, Ep, El, Mav) are more similar to their parent cells (Emr, Epen, Eml, Mavg) than their sister cells (mr, en, ml, g) are to their parents. Further analysis also shows that many genes while ubiquitously expressed throughout the embryo have higher expression in specific blastomeres. In addition, I tested various CRISPR/Cas9 strategies for knock-out and epitope tag knock-in experiments to improve current CRISPR mutagenesis methods on Parhyale and to combine other imaging and -omic technologies with the CRISPR/Cas9 system. I used the pioneer transcription factor Zelda as the study gene because it is expressed both before and after MZT, and may be a conserved regulator of MZT in Parhyale. I designed guide RNAs (sgRNAs) to target Zelda for CRISPR knock-outs and designed plasmid constructs to tag Zelda with various epitope tags. I tested non-homologous end-joining and microhomology-mediated end-joining methods for efficiency of CRISPR knock-ins and found that both methods fail to increase penetrance. In the future, we hope to further analyze and test candidate genes from the blastomere-specific transcriptomic datasets as well as improve CRISPR/Cas9 mutagenesis strategies so we can better study maternally expressed genes in the Parhyale embryo

Full transcriptome analysis of early dorsoventral patterning in zebrafish

Understanding the molecular interactions that lead to the establishment of the major body axes during embryogenesis is one of the main goals of developmental biology. Although the past two decades have revolutionized our knowledge about the genetic basis of these patterning processes, the list of genes involved in axis formation is unlikely to be complete. In order to identify new genes involved in the establishment of the dorsoventral (DV) axis during early stages of zebrafish embryonic development, we employed next generation sequencing for full transcriptome analysis of normal embryos and embryos lacking overt DV pattern. A combination of different statistical approaches yielded 41 differentially expressed candidate genes and we confirmed by in situ hybridization the early dorsal expression of 32 genes that are transcribed shortly after the onset of zygotic transcription. Although promoter analysis of the validated genes suggests no general enrichment for the binding sites of early acting transcription factors, most of these genes carry "bivalent" epigenetic histone modifications at the time when zygotic transcription is initiated, suggesting a "poised" transcriptional status. Our results reveal some new candidates of the dorsal gene regulatory network and suggest that a plurality of the earliest upregulated genes on the dorsal side have a role in the modulation of the canonical Wnt pathway

An improved zebrafish transcriptome annotation for sensitive and comprehensive detection of cell type-specific genes

The zebrafish is ideal for studying embryogenesis and is increasingly applied to model human disease. In these contexts, RNA-sequencing (RNA-seq) provides mechanistic insights by identifying transcriptome changes between experimental conditions. Application of RNA-seq relies on accurate transcript annotation for a genome of interest. Here, we find discrepancies in analysis from RNA-seq datasets quantified using Ensembl and RefSeq zebrafish annotations. These issues were due, in part, to variably annotated 3\u27 untranslated regions and thousands of gene models missing from each annotation. Since these discrepancies could compromise downstream analyses and biological reproducibility, we built a more comprehensive zebrafish transcriptome annotation that addresses these deficiencies. Our annotation improves detection of cell type-specific genes in both bulk and single cell RNA-seq datasets, where it also improves resolution of cell clustering. Thus, we demonstrate that our new transcriptome annotation can outperform existing annotations, providing an important resource for zebrafish researchers

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

Single cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs

Haematopoietic stem and progenitor cells (HSPCs) develop through distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) is able to recapitulate some of these processes, however, it has proven difficult to generate functional haematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we exploited single cell RNA sequencing (scRNAseq) in combination with single cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors as well as erythroid, megakaryocyte and leukocyte-committed progenitors and we identified CD44, CD326, ICAM2/CD9 and CD18 as markers of these progenitors, respectively. Using an artificial neural network (ANN), that we trained on a scRNAseq derived from human fetal liver, we were able to identify a wide range of hPSCs-derived HPSC phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded and was completely missing in the dataset that had been generated using cells selected on the basis of CD43expression. By comparing the single cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver we identified transcription factors and molecular pathways that can be exploited in the future to improve the in vitro production of HSCs