Comparison of RNA localization during oogenesis within Acipenser ruthenus and Xenopus laevis

The oocyte is a unique cell, from which develops a complex organism comprising of germ layers, tissues and organs. In some vertebrate species it is known that the asymmetrical localization of biomolecules within the oocyte is what drives the spatial differentiation of the daughter cells required for embryogenesis. This asymmetry is first established to produce an animal-vegetal (A-V) axis which reflects the future specification of the ectoderm, mesoderm, and endoderm layers. Several pathways for localization of vegetal maternal transcripts have already been described using a few animal models. However, there is limited information about transcripts that are localized to the animal pole, even though there is accumulating evidence indicating its active establishment. Here, we performed comparative TOMO-Seq analysis on two holoblastic cleavage models: Xenopus laevis and Acipenser ruthenus oocytes during oogenesis. We found that there were many transcripts that have a temporal preference for the establishment of localization. In both models, we observed vegetal transcript gradients that were established during either the early or late oogenesis stages and transcripts that started their localization during the early stages but became more pronounced during the later stages. We found that some animal gradients were already established during the early stages, however the majority were formed during the later stages of oogenesis. Some of these temporally localized transcripts were conserved between the models, while others were species specific. Additionally, temporal de novo transcription and also degradation of transcripts within the oocyte were observed, pointing to an active remodeling of the maternal RNA pool

Bioinformatics analysis of sequences required for localization of RNA during development

The development of a complex organism from the fusion of two cells (oocyte and sperm) has been a fascinating aspect of developmental biology. It is now known that certain spatially and temporally regulated molecules tightly regulate embryogenesis. The asymmetrical gradient of these molecules within a given cell or within groups of cells helps to guide the differentiation of certain parts of the developing embryo. In fishes and frogs, the establishment of the maternal animal-vegetal transcript gradient within the egg produces the first developmental axis and subsequent formation of the ectoderm, mesoderm, and endoderm regions. Despite this important process, most of our knowledge on this initial animal-vegetal distribution in vertebrates has been limited primarily to the Xenopus laevis model, involved the analysis of only few transcripts and also analyzed only polar regions of the oocyte. This thesis aims to address this deficit in knowledge by leveraging high throughput analysis (RNA sequencing) to characterize and compare the maternal transcriptome and its sub-compartmentalization within the egg of four distantly related models. Additionally, we analyzed different stages of oocyte maturation to determine where the observed localization occurs. The models used were the African clawed frog (Xenopus..

Bioinformatická analýza sekvencí pro lokalizaci RNA ve vývoji

Vývoj komplexního organismu po spojení dvou buněk (vajíčka a spermie) je jedním z fascinujících aspektů vývojové biologie. Je známo, že embryogeneze je řízena pomocí prostorové a časové produkce molekul, které tvoří asymetrické gradienty v buňce nebo ve skupině buněk a pomáhají tak regulovat diferenciaci jednotlivých částí vyvíjejícího se embrya. Animálně-vegetativní gradienty transkriptů ve vajíčcích ryb a žab dávají vzniknout první vývojové ose, která je následně využita v tvorbě ektodermu, mezodermu a endodermu. Přes důležitost tohoto procesu je většina vědomostí o distribuci molekul podél animálně-vegetativní osy u obratlovců získána studiem modelu drápatky, založena na několika málo transkriptech a analyzovány jsou zejména póly vajíček. Tato práce doplňuje dosavadní poznatky využitím RNA sekvenování k charakterizaci a srovnání maternálního transkriptomu a jeho rozložení v rámci vajíček čtyř vzdáleně příbuzných modelových organismů - drápatky vodní (Xenopus laevis), axolotla mexického (Ambystoma mexicanum), jesetera malého (Acipenser ruthenus) a dánia pruhovaného (Danio rerio). Nalezli jsme několik animálně-vegetativních gradientů a můžeme je rozdělit do podskupin extrémně animální, animální, centrální, vegetativní a extrémně vegetativní. Existuje velmi nízká shoda u vegetativních transkriptů...The development of a complex organism from the fusion of two cells (oocyte and sperm) has been a fascinating aspect of developmental biology. It is now known that certain spatially and temporally regulated molecules tightly regulate embryogenesis. The asymmetrical gradient of these molecules within a given cell or within groups of cells helps to guide the differentiation of certain parts of the developing embryo. In fishes and frogs, the establishment of the maternal animal-vegetal transcript gradient within the egg produces the first developmental axis and subsequent formation of the ectoderm, mesoderm, and endoderm regions. Despite this important process, most of our knowledge on this initial animal-vegetal distribution in vertebrates has been limited primarily to the Xenopus laevis model, involved the analysis of only few transcripts and also analyzed only polar regions of the oocyte. This thesis aims to address this deficit in knowledge by leveraging high throughput analysis (RNA sequencing) to characterize and compare the maternal transcriptome and its sub-compartmentalization within the egg of four distantly related models. Additionally, we analyzed different stages of oocyte maturation to determine where the observed localization occurs. The models used were the African clawed frog (Xenopus...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyPřírodovědecká fakultaFaculty of Scienc

THE MICROBIAL DIVERSITY PRESENT IN A TROPICAL, CIRCUMNEUTRAL IRON SPRING IN GRENADA

Microbial evolution has allowed for microbial adaptation to many diverse and harsh environments. As a result, microbial diversity varies spatially, depending on the environmental pressures that are limiting. This research focused on uncovering the microbial diversity present in a Tropical Iron Spring in the Caribbean island, Grenada. Any microorganism from this island is expected to be genetically unique due to its spatial isolation and unique chemical composition of the water and gas in the Spring. The diversity was discovered using both culture dependent and culture independent techniques. Microorganisms were visualized and quantified using Florescence In Situ Hybridization (FISH) in conjunction with both florescent and light microscopy. Culture based analysis was used to isolate iron oxidizing autotrophic microbes. Temporal Gradient Gel Electrophoresis (TGGE) and Cloning were used to screen and isolate individual microbial species. Results indicated the possibility of microbes involved in carbon cycling, nitrogen cycling and iron cycling. In addition, the results point to the likelihood of a microbial community that is self-sustained or requires very few external resources. Such microbial ingenuity may help to provide greater knowledge on the aspects of energy conservation and mineral recyclin

Table1_Comparison of RNA localization during oogenesis within Acipenser ruthenus and Xenopus laevis.xlsx

The oocyte is a unique cell, from which develops a complex organism comprising of germ layers, tissues and organs. In some vertebrate species it is known that the asymmetrical localization of biomolecules within the oocyte is what drives the spatial differentiation of the daughter cells required for embryogenesis. This asymmetry is first established to produce an animal-vegetal (A-V) axis which reflects the future specification of the ectoderm, mesoderm, and endoderm layers. Several pathways for localization of vegetal maternal transcripts have already been described using a few animal models. However, there is limited information about transcripts that are localized to the animal pole, even though there is accumulating evidence indicating its active establishment. Here, we performed comparative TOMO-Seq analysis on two holoblastic cleavage models: Xenopus laevis and Acipenser ruthenus oocytes during oogenesis. We found that there were many transcripts that have a temporal preference for the establishment of localization. In both models, we observed vegetal transcript gradients that were established during either the early or late oogenesis stages and transcripts that started their localization during the early stages but became more pronounced during the later stages. We found that some animal gradients were already established during the early stages, however the majority were formed during the later stages of oogenesis. Some of these temporally localized transcripts were conserved between the models, while others were species specific. Additionally, temporal de novo transcription and also degradation of transcripts within the oocyte were observed, pointing to an active remodeling of the maternal RNA pool.</p

Image1_Comparison of RNA localization during oogenesis within Acipenser ruthenus and Xenopus laevis.pdf

The oocyte is a unique cell, from which develops a complex organism comprising of germ layers, tissues and organs. In some vertebrate species it is known that the asymmetrical localization of biomolecules within the oocyte is what drives the spatial differentiation of the daughter cells required for embryogenesis. This asymmetry is first established to produce an animal-vegetal (A-V) axis which reflects the future specification of the ectoderm, mesoderm, and endoderm layers. Several pathways for localization of vegetal maternal transcripts have already been described using a few animal models. However, there is limited information about transcripts that are localized to the animal pole, even though there is accumulating evidence indicating its active establishment. Here, we performed comparative TOMO-Seq analysis on two holoblastic cleavage models: Xenopus laevis and Acipenser ruthenus oocytes during oogenesis. We found that there were many transcripts that have a temporal preference for the establishment of localization. In both models, we observed vegetal transcript gradients that were established during either the early or late oogenesis stages and transcripts that started their localization during the early stages but became more pronounced during the later stages. We found that some animal gradients were already established during the early stages, however the majority were formed during the later stages of oogenesis. Some of these temporally localized transcripts were conserved between the models, while others were species specific. Additionally, temporal de novo transcription and also degradation of transcripts within the oocyte were observed, pointing to an active remodeling of the maternal RNA pool.</p

Transcriptome and Proteome Analyses Reveal Stage-Specific DNA Damage Response in Embryos of Sturgeon (<i>Acipenser ruthenus</i>)

DNA damage during early life stages may have a negative effect on embryo development, inducing mortality and malformations that have long-lasting effects during adult life. Therefore, in the current study, we analyzed the effect of DNA damage induced by genotoxicants (camptothecin (CPT) and olaparib) at different stages of embryo development. The survival, DNA fragmentation, transcriptome, and proteome of the endangered sturgeon Acipenser ruthenus were analyzed. Sturgeons are non-model fish species that can provide new insights into the DNA damage response and embryo development. The transcriptomic and proteomic patterns changed significantly after exposure to genotoxicants in a stage-dependent manner. The results of this study indicate a correlation between phenotype formation and changes in transcriptomic and proteomic profiles. CPT and olaparib downregulated oxidative phosphorylation and metabolic pathways, and upregulated pathways involved in nucleotide excision repair, base excision repair, and homologous recombination. We observed the upregulated expression of zona pellucida sperm-binding proteins in all treatment groups, as well as the upregulation of several glycolytic enzymes. The analysis of gene expression revealed several markers of DNA damage response and adaptive stress response, which could be applied in toxicological studies on fish embryos. This study is the first complex analysis of the DNA damage response in endangered sturgeons

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints

Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CII, serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CII leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CII is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy