|
|
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...
|
|
|
Molecular mechanisms in the development of the cochlea
Procházková, Šárka ; Pavlínková, Gabriela (advisor) ; Novák, Josef (referee)
Molecular mechanisms in the development of the cochlea The cochlea is the primary structure for the perception of sound. A spiraled cochlear duct contains the auditory organ, the organ of Corti, is innervated by sensory neurons of the spiral ganglion. The organ of Corti contains hair cells, the sensory receptors of the inner ear, that are responsible for the mechano-transduction of sound waves into electrical signals. The development of the cochlea is regulated by the cellular and molecular processes, including cochlear outgrowth, cellular differentiation, and patterning. The precise coordination of multiple processes during the transformation of a flat embryonic epithelium into the cellular mosaic of the adult structure requires a multitude of transcription factors and signaling pathways. These regulatory networks dictate cell fate and timing decisions during cochlear development. Interestingly, these mechanisms operate as well during sensory epithelial cell regeneration after damage and during stem cell directed differentiation, making developmental studies instrumental for improving therapies for hearing impairment.
|
|
|
Hif1a role in cardiovascular function and heart disease
Matějková, Kateřina ; Pavlínková, Gabriela (advisor) ; Holzerová, Kristýna (referee)
Hypoxia inducible factor 1 alpha (HIF1A) is a transcriptional factor, which plays a central role in the maintenance of homeostasis under hypoxic conditions. It regulates a wide variety of genes encoding proteins that influence metabolism, extracellular matrix composition, oxidoreductase activity or angiogenesis in response to reduced oxygen levels. When HIF1A protein function is impaired, the organism is unable respond appropriately to hypoxia. Altered HIF1A regulation can result in severe tissue damage and eventually lead to death. The heart, as an organ with a huge oxygen consumption, is susceptible to various pathologies caused by hypoxic stress. The role of HIF1A in the heart is rather ambiguous and remains to be elucidated. It plays a role in cardioprotective mechanisms but also promotes the development of inflammation and apoptosis. This thesis aims to clarify the role of HIF1A in maintaining physiological functions of the heart during adaptation to hypoxic conditions using a mouse model with heterozygous Hif1a+/- deletion. Experiments involving molecular and cell biology methods performed on left ventricular tissue were preceded by bioinformatic analysis of data obtained by RNA sequencing of isolated cardiomyocytes. RNA sequencing data were analyzed using the R scripting language (packages...
|
|
|
Role of NEUROD1 transcriptional network on the development and function of inner ear neurons
Merc, Veronika ; Pavlínková, Gabriela (advisor) ; Mašek, Jan (referee)
Identification of transcription factors involved in a complex network regulating the development of neurosensory cells in the inner ear is a key point for understanding the pathophysiology of hearing loss, development of new therapeutic tools, and for hearing loss prevention. The aim of this thesis was to elucidate the function of the transcription factor NEUROD1 in the development of the inner ear and sensory neurons. Using the Cre-loxP recombination system, a unique mouse model was created with tissue-specific deletion of Neurod1 in NEUROD1-Cre positive cells (Neurod1ST). In the inner ear, Neurod1 was deleted only in neurons permitting to identify the secondary effects of Neurod1 elimination in neurons on sensory cell development. We showed that neither the early development of the inner ear nor the formation of the statoacoustic ganglia was significantly affected by Neurod1 deletion. The primary consequence of the deletion was manifested by increased neuronal death due to apoptosis, which resulted in a reduced number of differentiated neurons in the inner ear. Spiral and vestibular ganglia were smaller in the mutants, and there was a number of neurons misplaced, indicating impaired migration. The cochlear sensory epithelium was shortened probably due to the reduced number of neurons within the...
|
|
|
The effect of maternal diabetes on embryonic cardiovascular development and fetal programing
Čerychová, Radka ; Pavlínková, Gabriela (advisor) ; Nováková, Olga (referee) ; Neckář, Jan (referee)
Maternal diabetes mellitus negatively affects embryonic development and increases the risk for congenital malformations. Besides direct teratogenicity, diabetic intrauterine milieu can predispose an individual to chronic diseases later in life, including cardiovascular diseases, obesity, and diabetes mellitus, in a process termed fetal programing. Molecular mechanisms of embryonic and fetal responses to maternal diabetes are still not fully elucidated. Using mouse model, we show that maternal diabetes induces gene expression changes in the hearts of developing embryos. The most significant changes in the expression of 11 selected genes were detected at the developmental stage associated with completion of cardiac septation, myocardial mass expansion, and increased insulin production in the embryonic pancreas. These affected genes encode products involved in the epithelial-to-mesenchymal transition, a crucial process in heart development. Using immunohistochemistry, we detected increased hypoxia in the diabetes-exposed hearts at the critical stage of cardiac development. Correspondingly to increased hypoxia, the expression of hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor A was increased in the heart of diabetes-exposed embryos. Based on our results indicating the...
|
|
|
Molecular mechanisms in diabetic embryopathy
Čerychová, Radka ; Pavlínková, Gabriela (advisor) ; Kolář, František (referee)
Diabetic embryopathy is one of many serious complications associated with diabetes. It is known that maternal diabetes increases the frequency of congenital defects up to ten times. The most common defects are cardiovascular and neural tube defects. Molecular mechanisms of diabetic embryopathy are still not known. This work contributes to elucidation of molecular processes leading to development of cardiovascular defects in diabetic embryopathy. This study is based on observation that maternal diabetes affects transcriptional regulation of hypoxia-inducible factor 1 (HIF-1) in developing embryo. To study the influence of maternal diabetes on HIF-1 signaling pathway, we used mouse model heterozygous for "knock-out" of Hif1α gene. Our analyses showed the negative combinational effects of maternal diabetes and Hif1α+/- genotype on embryonic development and increased risk of diabetic embryopathy. Histological analysis demonstrated the increased incidence of cardiovascular defects, particularly defects of interventricular septum and hypoplastic compact left ventricular wall in embryonic day (E) 14.5 Hif1α+/- embryos compared to wt littermates from the diabetic pregnancy. Using qPCR, we analyzed gene expression changes in the embryonic hearts at E9.5 and E10.5. We selected genes important for the...
|
|
|
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...
|
|
|
The role of Pax6 transcription factor in mouse eye development
Žílová, Lucie ; Kozmík, Zbyněk (advisor) ; Krylov, Vladimír (referee) ; Pavlínková, Gabriela (referee)
The formation of the eye is a multistep process of complex morphogenetic events. It begins with the formation of the optic vesicle (OV) and its subsequent interaction with the overlying head surface ectoderm (SE). Reciprocal interaction between OV and SE evoke lens placode formation within the SE followed by coordinated invagination of both, the lens placode and OV. These events result in formation of lens, retina and retinal pigmented epithelium (RPE) with lens originating from the SE and retina/RPE originating from the OV. Early after the retinal domain is established, retinal progenitor cells start to differentiate in seven retinal cell types that are further stratified in the structure of the retina. The transcription factor Pax6 plays a pivotal role in eye formation in various animal species. In mammals, it is expressed from very early stages of eye development in OV and SE. As Pax6-/- mice are anopthalamic, with eye development arrested at OV/SE stage, much attention has been paid to elucidate the Pax6 function in different eye structures. However, whether and/or how Pax6 regulates the early signaling events leading to eye formation as well as the mechanism by which Pax6 regulates the differentiation of all retinal cell types is still only poorly understood. Using the mouse as a model, we...
|
|
|
Changes in embryonal programing induced by diabetes mellitus
Landsmann, Lukáš ; Pavlínková, Gabriela (advisor) ; Tlapáková, Tereza (referee)
Embryonic development is sensitive to environmental changes. These changes may lead to changes in the embryonic programming. Changes in programming embryos can occur due to inadequate nutrition, stress, treatment with chemicals and also due to diabetes. Epigenome reacts sensitively to environmental factors regulating gene transcriptional activity. Changes in the epigenome lead to a changes in gene expression, which can have a negative impact on the physiology and metabolism of organism. Maternal diabetes may alter embryonic and fetal development and may result in diabetic embryopathy. Furthermore, maternal diabetic enviromental plays an important role in the predisposition of offspring to a number of chronic diseases later in life. The offspring of diabetic pregnancies demonstrate differences in metabolic, cardiovascular, and inflammatory variables, compared to the offspring of nondiabetic mothers. This thesis summarizes the genetic and epigenetic factors involved in the development of diabetic embryopathy and in the embryonic programming. Key words: Diabetes mellitus, diabetic embryopathy, transcriptional regulation, genetic and epigenetic factors , embryonic programming, genome
|
|
|
Functional role of Islet1 in pancreatic development
Malfatti, Jessica ; Pavlínková, Gabriela (advisor) ; Krausová, Michaela (referee)
1 Abstract Diabetes mellitus is characterized by the dysfunction and reduction of insulin-producing cells, resulting in hyperglycemia, which in long term harms the organism. For future therapy, it is crucial to understand the function of various factors participating in the differentiation and maturation of endocrine pancreatic cells. The aim of this study was to unravel the functional role of ISL1 during the development of the pancreas. ISL1 is expressed in all endocrine cells of the islets of Langerhansbut its function remains unclear, especially during early pancreatogenesis. As the global deletion of this gene is embryonically lethal, we used the tissue specific deletion of Isl1 in Neurod1 possitive cells using the Cre-loxP system. In this work we studied the effect of this deletion on the structure of islets of Langerhans, the formation of endocrine cell types and relative expression of genes during early pancreatic development. A defective achitecture of islets together with postnatal absence of α-cells was found in the Isl1 deletion mutant. Also, the expression of genes important for the specification of α-cell lineage and their subsequent function was decreased. The secondary outcome was the optimalization of a protocol for effective sorting of endocrine cells using fluorescent flow cytometry, which...
|
guest ::
