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Jeseter (Acipenser) představuje evoluční přechod od holoblastického k meroblastickému rýhování a unikátní způsob vývoje střeva
SHAH, Mujahid Ali
A vertebrate embryo's cleavage pattern is either holoblastic (complete) or meroblastic (partial). Holoblastic cleavage is thought to be ancestral to vertebrates and is most likely to occur in amphibians, mammals, and chondrosteans. Meroblastic cleavage has evolved five times in vertebrate lineages, including hagfish, elasmobranchs, coelacanths, teleosts, and amniotes. In holoblastic cleavage (as in Xenopus laevis embryos), all blastomeres contribute to one of the germ layers. On the contrary, in meroblastic cleavage pattern (as in teleosts and amniotes-including birds and reptiles), only the animal pole contributes the formation of the germ layers. The transition from holoblastic to meroblastic is usually occurred by an increase in egg size in comparison to the lineage's ancestral state. Sturgeons evolved about 200 million years ago (mya). Their eggs are significantly larger than that of X. laevis. Despite the variation in sizes, their embryos retain nearly characteristics the same as that of X. laevis. Nevertheless, vegetal blastomeres of sturgeons are bigger and divide slower than that of X. laevis. It was speculated that vegetal blastomeres of sturgeon are extraembryonic as in yolk of teleost (zebrafish) and Yolk cells of (YCs) of bichir-earliest diverged living group of actinopterygian fishes, agnathan lampreys (Petromyzontidae)-an extant lineage of jawless fishes and an Eleutherodactylus coqui (direct developing frog). Furthermore, the gut development pattern of sturgeon (Acipenser) and its evolutionary conservation was poorly understood so far. First, we developed the robust technique for specific blastomeres inhibition of sturgeon embryos using diatoms-derived polyunsaturated aldehydes, 2, 4-Decadienal (DD; a model aldehyde for experimental studies). The sturgeon's embryos were injected with optimal DD percentage (0.01 v/v) and subsequently irradiating them by visible light (91.15 - 44.86 W m2). Notably, DD plus light, and not DD injection or light irradiation alone can inhibit cleavage. Furthermore, qPCR-tomography revealed that localized pattern of maternal mRNA remained constant through animal-vegetal axis in partially cleaved embryos when compared to normal. Second, fate-mapping of sturgeon vegetal blastomeres revealed that these blastomeres gave rise to primordial germ cells (PGCs), and the rest of the descendants were vegetal yolk cells. Plastic section histology showed that the nuclei of vegetal yolky cells sharply declined as embryos developed. In addition, inhibition of vegetal blastomeres, RT-qPCR and BrdU pulse revealed that yolk cells become transcriptionally inactive after mid-blastula transition. Here, our results suggested that the meroblastic cleavage in actinopterygian lineage had evolved by the fusion of vegetal blastomeres, which is parallel to the closely related group, e.g., gar (Lepisosteidae), that evolved at approximately 57 mya. Lastly, we continued the observation of sturgeon gut development and its comparison with other taxa including holoblastic (X. laevis, bichir, and mice) and meroblastic (chicks, gars, and zebrafish) representatives. For this purpose, we used histology, in-situ hybridization (HCR), and Immunohistochemistry. We found that sturgeon's endodermal cells formed the Archenteron (primitive gut) as frog and bichir. However, these cells continued to proliferate lateroventrally to encompass a massive amount of yolk mass to give rise "yolk inside the gut." Cross-species comparison revealed that sturgeon retained a unique mode of gut developmental pattern during vertebrate evolution. In conclusion, our current findings suggest that sturgeon embryo development represents a distinct transition from holoblastic to meroblastic cleavage, as well as a distinct archaic mode of gut-endoderm development.
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The foundation of maternal factors in sturgeon: from oocyte to embryo
POCHERNIAIEVA, Kseniia Kostyantynivna
The effective application of embryo engineering to endangered sturgeon species requires fundamental knowledge of its embryonic development and information about structure and characteristics of sturgeon oocyte itself. To reveal intracellular geometry, mechanisms of maternal determinants organization and its later reorganization and morphogenetic aspects we used several techniques such as qPCR tomography, inhibition of transcription and visualization of nucleuos. The qPCR tomography was discovered as reliable technique to determine the role of the genes detected in the animal and vegetal hemispheres of the sturgeon oocyte, and to identify profiles of these genes during early developmental stages of sturgeon embryos. The 12 selected maternal genes were investigated. Two groups of transcriptomes categorized as animal or vegetal with evident gradient profile were identified. The primarily germplasm markers such as dnd, vasa, ddx25 were localized toward the extreme vegetal pole. This finding reveals localization of primordial germ cells in the body plan of the sturgeon oocyte. Another aspect of applying such technique was comparative analysis of RNA profiles in the oocyte of distantly-related species Xenopus laevis and Acipenser ruthenus. We found clear similarity in the localization of mRNA molecules in Acipenser ruthenus and Xenopus laevis, which revealed significant aspects of early development that have been conserved during evolution. Such similarities in expression profiles of distantly related species indicate that their ancestors could have arisen from more closely related lineages. The maternal to zygotic transition (MZT) is a separate developmental period that begins with the elimination of maternal transcripts, continues through the production of zygotic transcripts, and concludes with the first major morphological requirement for zygotic transcripts in embryo development.The alpha-Amanitin as transcript inhibition factor was used to determine the zygotic genome switch in sterlet embryos. The transition in sterlet was observed after the tenth cleavage during late blastula, when blastomeres in the animal pole are surpassed 1000 cells. Mid-blastula transition (MBT) in early embryogenesis can be defined as a time point characterized by cell cycle lengthening, loss of synchrony and acquisition of cell motility. We opted to use oocytes of crosses sterlet A. ruthenus and Russian sturgeon Acipenser gueldenstaedtii, since the hybridization results in increased DNA content in their hybrid offspring compared to parental species A. ruthenus making the embryo a useful model for investigation of changes in the timing of early development. Nucleous vizualization by 4'-6-diaminido-2-phenylindole (DAPI) staining showed that cells divided synchronously at a constant rate until MBT at the ninth cell cycle in control sterlet embryos that corresponds to 1000 cell stage (13 hpf). The sterlet x Russian sturgeon hybrid embryos showed transition from synchronous to asynchronous division at the eighth cell cycle which is the 512 cells stage (12 hpf). In both sterlet and hybrid embryos, the transition occurred within 1 h. Thus, our study confirmed hypothesis the MBT in sturgeon is governed by the ratio of nucleus to cytoplasm, which can be controlled using hybridization, induction of polyspermy or injecting plasmid DNA Embryos of sturgeon injected with alpha-Amanitin also showed cell cycle kinetics similar to controls, with no delay or malformation during cleavage, which most likely indicates that MBT in the sturgeon proceeds independently of onset of zygotic transcripts production. The results and observations presented in this study demonstrate the path from an egg to a developed embryo, which are the basis for improving the production methods and preservation of sturgeons listed in the IUCN Red List, and which is equally important, provide the fundamental knowledge about the nature of sturgeons.
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Regulation of translation in mammalian oocytes and early embryos
Jindrová, Anna ; Šušor, Andrej (advisor) ; Flemr, Matyáš (referee) ; Fulková, Helena (referee)
Fully grown oocytes undergo their further development in the absence of transcription. Completion of meiosis and early embryo development rely on the maternal mRNAs synthetized and stored during earlier development. Thus, the regulation of gene expression in oocytes during that period is controlled almost exclusively at the level of mRNA stabilization and translation. In the same vein, any mRNA metabolism could play a critical function at this stage of development. RNA localization followed by a local translation is a mechanism responsible for the control of spatial and temporal gene expression in the cell. We focused on visualization of mRNA and in situ translation in the mammalian oogenesis and embryogenesis. We characterized localization of global RNA population in the oocyte and early embryo nucleus together with RNA binding proteins. Additionally we visualized specific ribosomal proteins that contribute to translation in the oocyte and embryo. We have shown that the key player of cap-dependent translation mTOR becomes highly active post nuclear envelope breakdown (NEBD) and in turn its substrate, translational repressor 4E-BP1 becomes inactive. Precise localization of inactivated 4E-BP1 at the newly forming spindle of the oocyte indicates the ongoing translation in this area. Furthermore, from...
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