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Bmp signaling in the evolution of chordate axial patterning
Kozmikova, Iryna ; Kozmík, Zbyněk (advisor) ; Bryja, Vítězslav (referee) ; Markoš, Anton (referee)
Formation of a dorsoventral axis is a key event in the early development of most animal embryos. In vertebrates, early separation of cell fate domains precedes specification of ectoderm to neural and non-neural as well as mesoderm to dorsal and ventral during development. Maintaining such division with the establishment of an exact border between the domains is required for the formation of highly differentiated structures such as neural tube and chorda. In the cephalochordate amphioxus, genes encoding Chordin, Bmps and transcription factors downstream of Bmp signaling such as Vent are expressed in patterns reminiscent of those of their vertebrate orthologues. However, the key question is whether the conservation of expression patterns of network constituents implies conservation of functional network interactions, and if so, how an increased functional complexity can evolve. Here, we therefore investigated the role of Bmp signaling in axial patterning and cell fate determination in amphioxus, the basal chordate possessing a centralized nervous system and dorsal mesoderm. Using heterologous systems, namely by reporter gene assays in mammalian cell lines and by transgenesis in medaka fish, we have compared the gene regulatory network implicated in dorsoventral patterning of the basal chordate...
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The role of Wnt signaling in embryonic development.
Dupačová, Naoko ; Kozmík, Zbyněk (advisor) ; Peterka, Miroslav (referee) ; Pavlínková, Gabriela (referee)
Ph.D. thesis: The role of Wnt signaling in embryonic development Naoko Dupačová (Fujimura) ABSTRACT Wnt signaling plays important roles in multiple developmental processes. The binding of Wnt ligands to their receptors and coreceptors activates three main downstream pathways: canonical Wnt/β-catenin signaling, which results in the activation of β-catenin/Tcf mediated gene expression and noncanonical Wnt/PCP and Wnt/Ca2+ pathways. In this thesis, we aimed at studying the role of Wnt/β-catenin signaling during embryonic development, especially in the telencephalon and the eye. Wnt/β-catenin signaling is essential for the maintenance of proliferation of neuronal progenitor cells and dorso-ventral specification during the telencephalon development. To provide further insights, we studied transcriptional targets of canonical Wnt signaling. We show that the ectopic activation of Wnt/β-catenin signaling results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene contains five Tcf/Lef binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We further provide evidence that Sp5 works as a transcriptional repressor. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and...
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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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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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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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Evolution of photoreceptors: insights from amphioxus
Vopálenský, Pavel ; Kozmik, Zbyněk (advisor) ; Černý, Robert (referee) ; Markoš, Anton (referee)
The topic of eye evolutionwas touchedas early as at the very beginningof the evolutionarytheoryitself.In his book'On theOrign of Species'CharlesDarwinexpresses concemsfor theexplanationof theocclurenceof complexeyesby naturalselection.Eventhe simplest'proto-eye'composedof a singlephotoreceptorcellanda shadingpigmentrequires properfrrnctioningof dozensof genesinvolvedin the phototansductioncascade,signal transmissionand pigmentation.Photoreceptorcells can be classifiedaccordingto their morphology as ciliary or rhabdomeric.Primary observationsrevealedthe rhabdomeric photoreceptorsbeing predominantlypresentin the eyes of invertebrates,whereasthe vertebrateandcnidarianseyesemploytheciliary type.Key componentsof thephotoreceptor cells arevisualpigments(opsins)andassociatedGa subunitsresponsiblefor absorptionof photonandthefirst stepof aphotoffansductioncascade.To properlyorchesftatetheassembly of thesestructuralgenesmediatingbiochemicalprocessesinto a functionalorgan,a tight regulationby a dedicatedsetof tanscriptionfactorsis required.Oneof themostremarkable andbeststudiedexamplesof geneswith anevolutionaryconservedrole in eyeorganogenesis is a paired-homeoboxgenePax6.However,availableliteraturepointsto othertranscription factors participatingin photoreceptordevelopmentacrossanimal phyla, for examplea...
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