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Gene expression in chicken embryo: micromanipulation and visualization methods
Bendová, Michaela ; Dvořák, Michal (advisor) ; Hirsch, Ivan (referee) ; Krylov, Vladimír (referee)
The aim of this work was to obtain better insight into the principles of cell structures and organs in the chicken embryo development. To reach this goal special methods of micromanipulations and visualization in vitro, ex vivo, ex ovo and in ovo were implemented and adjusted. These methods were used to study gene expression in neural crest development and eye development. In the course of long term research in our laboratory we observed that oncoprotein v-Myb influences the development of the neural crest and has the capacity to change natural cell fate. We performed a series of experiments to investigate v-Myb protein influence on neural crest cells differentiation, especially melanocyte lineage development, and its influence on gene expression in the neural crest. Therefore we focused on Gremlin 2 (PRDC), the gene upregulated by v-Myb in the neural crest. The established procedure of electroporation in ovo was adjusted to transfect cells of the developing eye and used to study gene expression during lens induction. The results obtained from chicken embryo experiments endorsed the study performed on mouse embryos. Futhermore, the electroporation technique was slightly modified for manipulations of the neural retina in the developing eye in ovo. Thereafter, the retinas were processed ex vivo and...
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The embryonic development of the pharyngeal region in vertebrates
Píchová, Lenka ; Černý, Robert (advisor) ; Minařík, Martin (referee)
The fully-formed pharynx is for adult vertebrates indeed a vital structure. The pharynx provides two main functions - dealing with food and breathing. During embryonic development, pharynx is visible like a series of bulges on the lateral surface of the head. Embryonic development of the pharyngeal region starts with evagination of the endoderm to form the pouches, opposit to that the ectoderm invaginates to form the ectodermal clefts. Pharyngeal arches are formed after fusion of these epithelial layers, and pharynx is thus bordered by ectoderm from the inner, and endoderm from the outer side. Each pharyngeal arch consists of mesenchymal core of mesodermal and neural crest derived cells. All vertebrates develop through the so called phylotypic stage, being represented by the - pharyngula with the present pharyngeal arches. Accordingly, it was generally believed that development of the pharyngeal region is rather conservative in all vertebrates. My comparative analysis of pharyngeal development in different vertebrates species reveals that - only early embryonic formation of pharyngeal arches seems conserved, however, that later in development pharyngeal arches form various and diverse derivatives. Key words: Vertebrates, ectodermal fleft, endodermal pouch, pharyngeal arches, neural crest
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Migration and skeletogenesis of trunk neural crest cells
Suchánek, Tomáš ; Černý, Robert (advisor) ; Mašek, Jan (referee)
The neural crest is specific cell population of vertebrates embryos. It emerges in dorsal part of the neural tube from which it separate as individual cells. After that, these cells migrate throughout the embryo to their final destination. There they are exposed to a molecular signals and then they differentiate into various types of derivatives. The neural crest is distinguished into two main parts - cranial and trunk. While the cranial neural crest has been in the forefront of interest due its skeletogenic potential and participation in formation of the "new head", the trunk neural crest had been neglected due its "less attractive " derivates. Hence this bachelor thesis is focused on trunk neural crest. Especially it highlights the migratory behavior of trunk neural crest cells and its skeletogenic potential which represents opportunity for prospective research about neural crest.
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Differential potential of neural crest cells
Wróblová, Aneta ; Krylov, Vladimír (advisor) ; Machoň, Ondřej (referee)
The neural crest is a transient structure formed during the neurulation which undergoes change of phenotype in the process of epithelial-mesenchymal transition. Subsequently, neural crest cells delaminate and migrate collectively and individually to their place of destination, where they differentiate into a broad repertoir of mesenchymal and non-mesenchymal cell types. This thesis aims to examine true differential potential of these cells based on crucial in vitro experiments. Neural crest cells show not only high migration potential, but also stem cells characteristics like multipotency and self-renewal capacity. I also provide answers to questions about cellular potency at the level of neural crest population and along anterior-posterior axis. It was necessary to clarify the essence of events leading to induction, specification, epithelial-mesenchymal transition and migration of neural crest cells, since they are crucial for their differentiation potential. A major role in differentiation as well as in previous processes plays the gene regulatory network which is comprised of mutually affecting signalling pathways. Studying the behaviour of migratory and post-migratory neural crest cells is important for the research of regenerative medicine and even cancer and neurocristopathic treatment.
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The trabecular cartilage as a key structure for development and evolution of the anterior vertebrate cranium
Horáčková, Agáta ; Černý, Robert (advisor) ; Němec, Pavel (referee)
Anterior part of the skull is in embryos composed of two rod-like cartilages called trabeculae cranii. The final shape of the skull of jawed vertebrates is affected by the manner of composition of trabeculae. There are two types of arrangement of vertebrate skull: the platybasic, which is probably the original one, and the tropibasic, which has developed independently in birds, bony fishes and mammals. Jawless vertebrates (lampreys and hagfishes) also have cartilaginous elements called trabeculae, but there are doubts about homology of these structures between groups of jawed and jawless vertebrates. The trabeculae initially became interesting elements because of their possible position in premandibular segment as a former premandibular branchial arch. The two theories of origin of jaws, which do not assume there ever were any branchial arches before the mandibular, strictly disagree in the question of the nature of the trabeculae cranii.
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The role of transcriptional factor Tcf7l1 and Wnt/β-catenin signaling pathway during differentiation of the head ectoderm.
Mašek, Jan ; Kozmik, Zbyněk (advisor) ; Černý, Robert (referee) ; Procházka, Jan (referee)
Differentiation of the head ectoderm is crucial for the evolutionary diversification of vertebrates. Expression of the genes responsible for this process is orchestrated troughout complex gene regulatory networks that are induced and modulated by Wnt, FGF and BMP signaling pathways. In addition, Wnt/β-catenin signaling, in combination with expression of the Wnt antagonists from the rostral-most part of the head ectoderm, represent a key source of information for the regionalization of the tissue along the antero-posterior axis. This allows the differentiation of the anterior ectoderm that gives rise to the anterior neural fold (ANF) and anterior part of the presumptive placodal region (PPR), and more posterior ectoderm where higher levels of active Wnt/β-catenin signaling promote differentiation into the neural crest (NC) and posterior PPR. Although the requirement of Wnt/β-catenin signalling for ANF, PPR and NC development has been intensively studied in non-mammalian vertebrate model organisms, we lack a clear picture about the situation in mammals. Furthermore, current knowledge in mammals has been gathered via experiments on the level of β-catenin and very little is known about the individual roles of the Tcf/Lef transcription factors. Thereby, we decided to manipulate the Tcf7l1, member of the...
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Comparison of migration and morphogenesis of neural crest cells in Ray-finned fishes: towards identification of developmental sources of craniofacial diversity
Štundl, Jan ; Černý, Robert (advisor) ; Drobná Krejčí, Eliška (referee)
Extensively migrating population of neural crest cells, which contributes to many tissues and builds up most of craniofacial vertebrate structures, has a crucial role in embryonic development of vertebrate body. The migratory pathways of neural crest cells are thought to be very conserved throughout the vertebrates and cranial neural crest migration is defined by progression of three migratory streams: trigeminal, hyoid and a common branchial stream. In this diploma thesis, migration of cranial neural crest was analysed using embryos of the Senegal bichir (Polypterus senegalus) and of sterlet (Acipenser ruthenus), which represent two basal-most lineages of extant ray-finned fishes. A combination of several techniques was used in both species in order to study cranial neural crest cells from their sites of origin to post- migratory stages and the pattern of migration was compared and discussed in revealed embryonic context. In the Senegal bichir the hyoid neural crest stream was shown to migrate first and it is also the most abundant; this heterochrony shift is apparently related to formation of external gills, which in bichir are situated on the hyoid arch only. In sterlet, neural crest cells migrate in a classic pattern of three progressive streams but their dynamics and patterning is influenced by...
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Developmental origin of cartilage skull elements in axolotl
Kloučková, Lenka ; Černý, Robert (advisor) ; Roček, Zbyněk (referee)
Despite the fact that some aspects of single studies differ, there's a generally accepted view that the whole cartilaginous viscerocranium of vertebrates is neural crest derived. By the series of isotopic transplantation experiments of presumptive neural crest on the model organism Ambystoma mexicanum I partly specify this oppinion and prove that the most ventro-caudal cartilage, the second basibranchial, is of a different origin. Furher I mention the level of the presumptive neural crest where the single parts of cartilaginous viscerocranium arise from. Moreover there is one element, the first basibranchial, which has double origin. I discuss also some other neural crest derivatives such as head and outer gills mesenchyme, the trabeculae cranii, part of the cartilaginous otic capsule or the connective tissue in the head. I have performed 179 transplantations between transgenic and normal axolotl embryos. My final analysis is composed of 65 embryos of stage 40 - 42 and 7 larvae of lenght of 15 - 17 mm.
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Neural crest biology with respect to diversity of vertebrates
Štundl, Jan ; Černý, Robert (advisor) ; Němec, Pavel (referee)
Neural crest is an extensively migrating population of cells that arise during early development of vertebrate embryos. It provides a huge variety of different cell types that generate new tissues which occur only in vertebrates. Neural crest cells together with the mesoderm participate on the formation of the head of vertebrates, which is viewed as one of the most important innovations in the evolution of vertebrates. Thanks to their skeletogenic potencial neural crest cells are percieved as a key factor causing massive craniofacial diversity. The aim of this thesis was to get acquainted with the population of neural crest cells and try to understand its importance for the evolution of vertebrates and especially for generating craniofacial diversity.
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