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The role of transcription factors in mouse eye development
Sunny, Sweetu Susan ; Kozmik, Zbyněk (advisor) ; Pavlínková, Gabriela (referee) ; Fafílek, Bohumil (referee)
Vision is a complex process that begins with the transmission and refraction of light through a highly specialised transparent tissue called the cornea. The cornea acts as a protective barrier and contributes to the focusing power of the eye. The development of mammalian cornea is a multiphase process involving the formation of the corneal epithelium (CE), stroma and endothelium (CEn) during embryogenesis, followed by the postnatal stratification of epithelium and constant renewal of desquamated outermost cells. Paired box protein (Pax) 6 is an evolutionarily conserved transcription factor important for the proper development of the eye. To provide further insights into the role of Pax6 in corneal development, we took the advantage of Cre-loxP system for selectively inactivating Pax6 in two ocular domains, specifically, the postnatal CE and the ocular surface epithelium (OSE) (cornea, limbus, and conjunctiva). We generated a novel postnatal CE-specific Cre-expressing transgenic mouse line, Aldh3-Cre. Inactivation of Pax6 in the postnatal CE using Aldh3-Cre resulted in the abnormal thin cornea with defective cell-cell adhesion, thus providing direct evidence for the function of Pax6 in postnatal corneal development. Subsequently, the OSE-specific depletion of Pax6 using K14-Cre, resulted in the...
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Functional analysis of invertebrate (Branchiostoma floridae) promoters in heterologous systems
Gurská, Daniela ; Kozmik, Zbyněk (advisor) ; Krylov, Vladimír (referee)
Understanding the mechanisms of transcriptional regulation and the constraints that operate in gene promoter sequences is the key step in understanding the evolutionary conservation of transcriptional regulation. It is well known that regulatory regions with the same expression outputs do not have to share the sequence similarity. The most important elements in regulatory sequences are transcription factor binding sites and their position relocation does not usually influence the expression output. The least complex transcriptional regulation is characteristic for housekeeping genes. For their expression they require only basal core promoter elements (sometimes only CpG islands are sufficient) and general transcription factors, so they can be transcribed easily and immediately whenever they are needed. In this study we focused on transcriptional regulation of invertebrate amphioxus (Branchiostoma floridae) housekeeping genes in vertebrate systems. We prepared a set of constructs with amphioxus regulatory regions for testing their activity in different mammalian cell lines and a set of constructs with the same amphioxus regulatory regions for observing their spatial recognition in developing medaka fish embryo. We found that half of investigated amphioxus regulatory regions are recognized by...
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The role of vent genes family in early development and brain development
Fabian, Peter ; Kozmík, Zbyněk (advisor) ; Němec, Pavel (referee) ; Balaštík, Martin (referee)
6 III ABSTRACT (ENGLISH) In chordates, the central nervous system (CNS) is derived from the dorsal part of gastrula. Induced dorsal part of the embryo - the neural plate - gives rise to the neural tube or primordial brain. The developing dorsal part of the embryo is shaped by BMP/Smad signaling from the ventral part. Using the basal chordate amphioxus, we show here the conserved evolutionary role BMP/Smad signaling in axial cell fate determination. Pharmalogical inhibition of BMP/Smad signaling induces dorsalization of Branchiostoma floridae (amphioxus) and Oryzias latipes (medaka) embryos and expansion of neural plate markers. We provide evidence for the presence of the positive regulatory loop within the BMP/Smad signaling network of amphioxus. Thus, our data suggest that early emergence of a positive feedback loop within the BMP/Smad signaling network may represent a crucial molecular event in the evolutionary history of the chordate cell fate determination. The dorso-ventral body axis formation is mediated by genes of the vent family, which are the direct targets of BMP/Smad signaling. The function of vent gene family in early development is relatively well known, however, its role in developing CNS is not yet clear. Therefore, we decided to manipulate vox transcription factor, a vent family member....
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Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii
Žídek, Radim ; Kozmik, Zbyněk (advisor) ; Janečková, Lucie (referee) ; Macůrková, Marie (referee)
Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...
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Evolution of light detection in chordates
Pergner, Jiří ; Kozmik, Zbyněk (advisor) ; Markoš, Anton (referee) ; Musilová, Zuzana (referee)
Light detection is one of the crucial abilities of all animals. The light cues are important e.g. for maintaining of circadian rhythms, regulation of spawning cycles, changes of pigmentation and arguably most importantly for vision. Most animals detect light by opsins, members of the G protein coupled receptors superfamily. Amphioxus belongs to earliest branching chordate clade, cephalochordates. Thanks to their phylogenetic position, physiology and morphology, cephalochordates became the most relevant model organism for understanding the evolutionary origins of vertebrate specific traits. Amphioxus evince various reactions to light throughout its development. In the presented thesis light detecting systems of amphioxus were studied thoroughly. More specifically characterization of the opsin gene repertoire of two amphioxus species Branchiostoma floridae and Branchiostoma lanceolatum and their comparison with opsins from other animals is presented. In addition, remarkable similarity on the gene expression level between one of amphioxus visual organs, so called frontal eye, and neurons and retinal pigmented epithelium in vertebrate retina was shown. These data confirm the long time ago proposed homology between amphioxus frontal eye and vertebrate lateral eyes. Taken together all the presented data...
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Refractive index gradient in the cubozoan eye: gene expression analysis
Jonášová, Kristýna ; Kozmík, Zbyněk (advisor) ; Markoš, Anton (referee)
Lenses are spread through animal kingdom as an improvement of different eye types. Despite conservation of some key regulators and shared use of photopigment opsin, eyes and their lenses develop by variable mechanisms impeaching their monophyletic origin. Tripedalia cystophora (T. c), a cubozoan jellyfish, is an emerging new model for studying eye evolution. The presence of advanced lens-containing eyes (firstly incident within metazoans in this phylum of Cnidaria), the two types of lesser eyes, the use of pax gene and vertebrate type of phototransduction cascade for eye establishment make this jellyfish an useful tool for comparing eye development and different evolutionary strategies. We focused on the lenses of T.c. and studied formation of their refractive index gradient to reveal its mechanism. Using new antibodies raised against J1 and J2 crystallins (proteins of the T. c. lens), TEM and histology we found that graded refractive index is of protein origin and formed by unequal accumulation of proteins (particularly J1 and J2 crystallins) in different layers of the lens. We have shown that J1 crystallin occurs also in the lesser eyes (ocelli) suggesting how the lens mass can evolved. The synthesis of J2 crystallin in lens development has been examined. Furthermore we have prepared J2 crystallin fused...
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Characterization of mice with constitutively active Wnt/beta-catenin signaling pathway in lens
Antošová, Barbora ; Kozmik, Zbyněk (advisor) ; Pavlínková, Gabriela (referee)
Lens development and differentiation are strictly regulated processes. Various disturbances of these processes can lead to vision-limiting pathologies. The vertebrate lens is composed of epithelial cells and terminally differentiated fiber cells. Differentiation of fiber cells is connected with expression of fiber cell specific proteins (such as crystallins), cell cycle exit, and finally with the degradation of cell nucleus and organelles. Wnt/β-catenin signaling plays important roles during early eye development as well as later during the lens differentiation. To investigate the consequences of constitutive activation of Wnt/β-catenin signaling in lens fiber cells transgenic mouse strain, called CLEF, was created. Constitutive activation of Wnt/β-catenin signaling in fiber cells of CLEF mouse is achieved by transgenic protein CLEF that contains C-terminal activation domain of β-catenin fused to the amino terminus of full-length protein Lef1. The expression of CLEF transgene is under the control of αA-crystallin promoter. As a result of constitutive activation of Wnt/β-catenin signaling in fiber cells, adult CLEF mice develop cataracts and microphthalmia, and the morphology of adult mutant lenses is disrupted. Transgenic CLEF mRNA is expressed starting from E13.5 and by E16.5 transgenic CLEF protein is...
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