As estruturas oculares dos vertebrados apresentam diversas adaptações relacionadas aos hábitats e atividades das espécies. A infra-ordem Serpentes possui amplo número de espécies distribuídas em quase todas as regiões da Terra e seu sistema visual apresenta variações que apontam para adaptações ecológicas. O presente estudo teve por objetivo fazer uma análise comparativa do sistema visual de diferentes espécies de serpentes Caenophidia, das famílias Dipsadidae e Colubridae, centrada no potencial de visão de cores, na densidade e topografia celular da retina e na acuidade visual. Para tanto, foram identificados os genes de opsinas expressos nas retinas, e analisadas a densidade e distribuição dos diferentes tipos de fotorreceptores e das células da camada de células ganglionares (CCG). As serpentes obtidas junto ao Laboratório de Herpetologia do Instituto Butantan foram sacrificadas com dose letal do anestésico thiopental. Os olhos foram enucleados e as retinas dissecadas para estudos genéticos e morfológicos, com imunohistoquímica e coloração de Nissl. Para sequenciamento dos genes das opsinas SWS1, Rh1 e LWS, dois olhos de 17 espécies foram utilizados. A amplificação por PCR mostrou que os três genes são expressos nas retinas de todas as espécies analisadas; o pico de sensibilidade espectral (max) de cada opsina foi estimado a partir das sequências de aminoácidos. O max do fotopigmento SWS1 foi estimado em 360 nm (UV), para todas as espécies. O fotopigmento Rh1, apresentou três diferentes combinações de aminoácidos que geram picos de sensibilidade em 500 nm, 494 nm e 484 nm. Todas as espécies de serpentes diurnas apresentaram a combinação de aminoácidos que gerou o max 484 nm. O fotopigmento LWS apresentou 4 diferentes combinações de aminoácidos, com max variando entre 543 nm e 560 nm. Para os estudos morfológicos foram utilizadas 86 retinas de 20 diferentes espécies. Retinas íntegras foram marcadas com anticorpos específicos para quantificação e análise topográfica de fotorreceptores. A coloração de Nissl foi empregada em retinas planas para quantificação de células da CCG e cálculo da acuidade visual. As análises morfológicas em retinas de serpentes noturnas mostraram uma grande densidade média de fotorreceptores (82.042 ± 37.945 células/mm2), com predominância de bastonetes, enquanto espécies diurnas apresentaram baixa densidade média de fotorreceptores (11.290 ± 2.810 células/mm2) e ausência de bastonetes. Serpentes noturnas apresentaram densidade média mais baixa de células da CCG (7.653 ± 1.636 4 células/mm2) comparada às diurnas (9.575 ± 2.301 células/mm2). O poder de resolução espacial também foi maior em espécies diurnas (2,3 ± 0,7 cpg) do que nas noturnas (1,45 ± 0,4 cpg). A distribuição de fotorreceptores e células da CCG nas retinas mostrou a presença de area centralis em diferentes regiões das retinas de espécies noturnas, e faixa horizontal em retinas das espécies diurnas, com exceção da serpente aquática e diurna Helicops modestus, que apresentou area centralis. As diferenças de localização das areae centralis variaram de acordo com hábitat e características comportamentais das espécies. Serpentes fossoriais do gênero Atractus, por exemplo, apresentaram area centralis na região dorsal da retina, que favorece o campo de visão inferior e auxilia no comportamento de escavar. Os resultados obtidos neste abrangente estudo apontam para a complexidade das adaptações do sistema visual deste grupo de vertebrados. As variações do padrão de atividade (diurna ou noturna) e uso de hábitat parecem ser fatores de forte influência sobre as características do sistema visual, como a sensibilidade espectral dos pigmentos visuais, a densidade e distribuição de neurônios nas retinas e o poder de resolução espacial do olho / The structures of vertebrate eyes have many adaptations related to the habitats and activities of the species. The infra-order Serpentes has a large number of species distributed in almost all regions of the Earth and its visual system presents variations that point to ecological adaptations. This study aimed to compare the visual system of different species of Caenophidian snakes, from the Dipsadidae and Colubridae families. To do so, the opsin genes expressed in the retinas were identified and the density and distribution of the different types of photoreceptors and the cells of the ganglion cell layer (GCC) were analyzed. The snakes were colected from Butantan Institute and were sacrificed with a lethal dose of the anesthetic thiopental. The eyes were enucleated and the retinas dissected for genetic and morphological studies, using Nissl stainig technique and immunohistochemistry. For the sequencing the opsins genes SWS1, Rh1 and LWS, two eyes of 17 species were colected. PCR amplification showed that the three opsin genes are expressed in the retinas of all species analyzed; the maximum spectral sensitivity (max) of each opsin was estimated based on the amino acid sequences. The max of the SWS1 photopigment was estimated at 360 nm (UV), for all species. The photopigment Rh1 had three different combinations of amino acids that generate max at 500 nm, 494 nm and 484 nm. All diurnal species had the amino acid combination that generate the max at 484 nm. The photopigment LWS had 4 different combinations of amino acids with max ranging from 543 nm to 560 nm. For morphological studies, 86 retinas of 20 different species were analyzed. Wholemounted retinas were stained with specific antibodies for analysis of the photoreceptors density and topography. The Nissl stainig technique was used for quantification of GCL cells in flatmounted retinas and estimation of the spatial resolving power. Nocturnal snakes had retinas with higher photoreceptor densities (82,042 ± 37,945 cells/mm2), with predominance of rods, compared to diurnal species that had low photoreceptors density (11,290 ± 2,810 cells/mm2) and the absence of rods. On the other hand, diurnal snakes had higher density of GCL cells (9,575 ± 2,301 cells/mm2) and spatial resolving power (2.3 ± 0.7 cpd), compared to nocturnal (7,653 ± 1,636 cells/mm2 and 1.45 ± 0.4 cpg). The distribution of cells in the retinas had variations related to the circadian rhythm of the species, with the presence of area centralis in retinas of nocturnal species and horizontal streak in retinas of diurnal snakes, except for the diurnal and aquatic Helicops 6 modestus, that had an area centralis in the ventral retina. The location of the area centralis varies according the habitat and specific behavior of each species. The fossorial snake Atractus, for example, had an area in the dorsal retina, which improves the resolution of the inferior visual field and benefits the digging habit in this snake. The results of this comprehensive study point to the complexity of adaptations of the visual system of this group of vertebrates. The differences in the activity pattern (diurnal or nocturnal) and habitat seem to be of great influence on the characteristics of the visual system, such as the spectral sensitivity of the visual pigments, the density and distribution of neurons in the retina and the spatial resolving power of eye

The overall objective of this thesis was to understand better the mechanisms that shape the diversity in colour vision of fish, and to explore the adaptive significance of this divergence. Among the vertebrates, teleost fish show the greatest diversity in colour vision systems. The cichlid model system illustrates that the visual system of fish may differ among species, sexes, individuals, and life stages of individuals. The large number of available cone opsin genes, which have resulted from multiple opsin gene duplications, facilitates this high degree of variation in the mechanisms of colour vision. In general, cichlids possessed complements of four to five cone pigments, and these complements varied across species, sexes, and individuals. Additionally, lens transmission, cone pigment expression, post-receptoral sensitivity, and retinal circuitry differed across life stages of individuals. My results suggest that the diversification of colour vision across species and across life stages of individuals contributes to sensory adaptations that enhance both the contrast of zooplanktonic prey, and the detection of optical signals from conspecifics. Therefore, both natural and sexual selection may have worked in concert to shape colour vision in fish. Since light is more complex under water than on land, fish required four to six cone classes to reconstruct the colour signals reflected from aquatic objects. This suggests that the large number of cone pigments in fish have likely evolved to enhance the reconstruction of the complex colour-signals in aquatic environments. Taken together, these findings improve our understanding of the variable nature of fish colour vision, and, more generally, help unravel the evolution of photoreceptors and colour vision. / Thesis (Ph.D, Biology) -- Queen's University, 2012-05-14 13:16:50.276

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Biological Sciences, 2009. / Includes bibliographical references.

Title from first page of PDF document. Includes bibliographical references (p. 26-29).

Les rétinites pigmentaires (RP) sont des pathologies rétiniennes cécitantes d'origine génétique caractérisées par une perte des photorécepteurs. Nous avons ciblé des formes de RP autosomique dominante consécutives à des mutations dans le gène du pigment visuel de la RHODOPSINE, du facteur d'épissage PRPF31 et du facteur de transcription impliqué dans le développement des photorécepteurs NR2E3. Les fibroblastes, issus de biopsies de peau de patients, ont été reprogrammés en cellules iPS par une technique dite non intégrative. Après stabilisation des cellules iPS, nous avons vérifié leur propriété de pluripotence et l'absence d'anomalies caryotypiques.Les cellules iPS porteuses d'une mutation sur le gène RHODOPSINE ont été différenciées dans le lignage des photorécepteurs. Nos résultats montrent que les photorécepteurs porteurs de la mutation P347L du le gène RHODOPSINE récapitulent la dégénérescence observée chez les patients.Nous montrons que les cellules de l'épithélium pigmentaire rétinien (EPR) dérivées de cellules iPS porteuses de la mutation Cys294X du gène PRPF31 présentent des problèmes d'adhésion cellulaire due à l'absence de lame basale. Leur activité de phagocytose est alors perturbée, suggérant qu'un dysfonctionnement de l'EPR pourrait être à la base de la RP causée par la mutation Cys294X du gène PRPF31. Les modèles développés nous ont permis de mieux comprendre les processus à la base de la pathogénèse de certaines RP. Ces modèles associés à des protocoles de criblage, pourraient permettre d'évaluer l'efficacité et la toxicité de nouvelles molécules pharmacologiques, mais également être utilisés pour valider des approches de thérapie génique. / Retinitis pigmentosa (RP) is an inherited retinal diseases characterized by a loss of photoreceptors. We focused specific forms of autosomal dominant RP with mutations in the rod visual pigment RHODOPSIN, the ubiquitous splicing factor PRPF31 and the transcription factor involved in the development of photoreceptors NR2E3. Fibroblasts from skin biopsies of patients were reprogrammed into iPS cells by a non-integrative approach. After stabilization of iPS cell lines, we verified their pluripotency property and the absence of karyotype abnormalities. Based on the retinal differentiation protocol, iPS cells carrying a mutation in the RHODOPSIN gene have been differentiated in the photoreceptor lineage. Our results showed that the photoreceptors expressing the mutated form of RHODOPSIN summarizing the process of degeneration observed in RP patients. We show that retinal pigment epithelium (RPE) cells derived from iPS cells carrying a mutation in the PRPF31 gene lack basal membranes and have cell adhesion disorders. Consequently, their phagocytic activity is disturbed, suggesting that a malfunction of the RPE could be the primary step of the development of RP caused by mutation Cys294X in the PRPF31 gene. The models developed from specific-patient iPS cells have enabled us to better understand the processes underlying the pathogenesis of some RP. These models associated with screening protocols could be used to evaluate the efficacy and toxicity of new pharmacologic compounds but also used to validate new gene therapy approaches.

Human senses are specifically designed to recognize and understand the world that surrounds us. Even though we have five senses, vision alone is responsible for at least 30 % of the sensory input to our brain. The visual process is initiated in a highly specialized cell type, the photoreceptors. These are light-sensitive cells located in the retina, a layered nervous tissue situated at the back of the eye. Retinal degeneration diseases are a highly heterogeneous group of conditions that include mutations affecting the survival, maintenance and proper functioning of photoreceptors or the adjacent retinal pigment epithelium (RPE). Such mutations, alone or in combination with environmental factors, cause the loss of the affected cells, and therefore, impairment of the visual sense. Retinitis Pigmentosa and Age-related Macular Degeneration are typical examples of retinal degenerative diseases eventually leading to blindness. In the first one, rod photoreceptors degenerate and consequently also cone photoreceptors are lost. The second is characterized by malfunction and loss of both, RPE and photoreceptor cells. Many current therapeutic approaches for the treatment of retinal degenerative diseases focus on slowing down the progression of the disease, rather than restoring the visual function. Currently, new therapies with the potential to recover the visual signal are under development. Some of these therapeutic strategies have already reached clinical stages, including gene therapy or retinal prosthesis. However, gene therapy approaches require the presence of remaining photoreceptors and, furthermore, particular targeting of disease-related genes. Retinal prosthesis still require improvement in terms of long-term biocompatibility and relevant visual function recovery. An alternative strategy for vision restoration is cell replacement of the lost photoreceptors, which is potentially suitable for targeting late stages of retinal degeneration diseases, independently of the inherent cause of the disease. Human vision relies primarily on cone photoreceptors, which are the cells responsible for color and high acuity vision under daylight conditions. However, cones represent a minority of the photoreceptors within the retina, and so, due to the low availability of these cells, cone photoreceptor transplantation studies lag behind rod transplantation studies. Consequently, in this study, strategies to increase the numbers of cone photoreceptors within mouse embryonic stem cells (mESC)-derived retinal organoids, which represent a potential cell source for transplantation studies, were explored. In this regard, I manipulated developmental pathways known to be involved in retinal development, such as Notch signaling, through the addition of various compounds in the retinal organoid maturation media. However, early cone markers have not yet been definitively identified, complicating the detection and isolation of cone photoreceptor precursors within the organoids. Therefore, a new early cone-reporter mESC line was generated in the course of this study as a valuable tool with the potential to facilitate the development of novel cone photoreceptor replacement therapies. Equally important in the field of photoreceptor cell replacement is the understanding of how the transplanted donor cells interact with the host retina. Previous studies have shown that visual function improvement is possible after transplanting rod or cone-like photoreceptor precursors into the sub-retinal space of mouse models for retinal degeneration. For many years it has been assumed that the underlying mechanism for the observed vision improvement was the migration and structural integration of donor cells into the host outer nuclear layer, where they mature and establish synaptic connections with the host retinal circuitry. However, experiments performed in this study demonstrate, for the first time, that upon transplantation donor and host photoreceptors exchange cytoplasmic material rather than structurally integrate into the host outer nuclear layer. Furthermore, insights into the transferred cytoplasmic content are given, i.e. that mRNA, but not mitochondria are exchanged by donor and host photoreceptors. This novel way of photoreceptor-photoreceptor communication led to a paradigm change in the field of retinal transplantation, requiring a re-interpretation of former transplantation studies. In addition, the discovery of the material transfer phenomenon might serve as a starting point for the development of novel therapeutic strategies based on cell-cell support for the treatment of retinal degenerative diseases. This study generated new knowledge in two important topics related to the development of cell therapies for retinal degeneration diseases, including the development of tools for cone transplantation studies as well as elucidating the interaction between donor and host cells upon transplantation.

Motile microorganisms navigate through their environment using special molecular machinery in order to sense gradients of various signals: chemotaxis (reactions to chemical compounds) and phototaxis (to light) sensory cascades. Transmembrane receptors play a central role in these cascades as they receive input signals and transmit them inside the cell, where they modulate activity of the kinases CheA, which are tightly bound to their cytoplasmic domains. CheA further phosphorylates the response regulator protein CheY, which regulates the flagella. At the same time, CheA phosphorylates and, by means of this, activates another response regulator, CheB, which, along with the constantly active CheR protein, catalyzes two opposite reactions: methylation and demethylation of the specific glutamic acid residues located at the cytoplasmic domain of the receptors. The latter reactions establish the adaptation mechanism, which allows microbes to sense in a very broad range of the input signal intensities. Many functional, structural and dynamical aspects of the signal propagation through the prokaryotic receptors as well as a mechanism of the signal amplification remain still unclear. In the present thesis we have used various techniques of computational biophysics, chiefly molecular dynamics (MD) simulations, in order to approach these problems. In Chapter 3, we have carried out MD simulations of the isolated linker domain (HAMP) from the E. coli Tsr chemoreceptor. The MD simulations revealed highly dynamical nature of this domain, which allows for interconversion between several metastable states. These metastable states feature a number of structural and dynamical properties, which were previously reported for HAMP domains of various receptors obtained from different organisms. It allowed us to reconcile numerous experimental data and to hypothesize that different HAMP domains share similar mechanism of their action. In Chapter 4, we have performed MD simulations of the whole cytoplasmic domain of the Tsr chemoreceptor. The simulations revealed a mechanism for the inter-domain coupling between the HAMP domain and a part of the cytoplasmic domain adjacent to the HAMP, the adaptation subdomain, by means of the regulated unfolding of a short linker region termed the stutter. Also, we have found that the reversible methylation/demethylation of the cytoplasmic domain affects its flexibility and symmetry. Altogether, these findings suggest a mechanism of signal propagation at the level of an individual chemoreceptor dimer. In Chapter 5, we have built a model of the trimer-of-dimers of the archaeal phototaxis receptor complex (NpSRII:NpHtrII). Subsequent MD simulations revealed an important role of dynamics in signal transduction and, potentially, in the kinase activation. In Chapter 6, we have reconstructed a whole transmembrane lattice formed by the NpSRII:NpHtrII complexes. The concave shape of the obtained lattice naturally explains polar localization of the receptor arrays in prokaryotic cells. At the same time, additional MD simulations of an individual unit of this lattice (a dimer of the photosensor) revealed global motional modes in its transmembrane region, which presumably co-occur with its activation and can spread across the tightly packed transmembrane arrays allowing for the signal amplification.

Les modifications post-traductionnelles (MPTs) permettent un haut degré de régulation de l'expression des gènes en générant une diversité fonctionnelle au niveau du protéome. Dans le système nerveux, les MPTs régulent entre autres des facteurs de transcription permettant une adaptation rapide à un microenvironnement dynamique. Dans ce contexte, je me suis concentrée sur l’étude des Glycogène Synthase Kinases 3 (GSK3s). Elles sont au centre de la régulation de nombreuses voies de signalisation et contrôlent la stabilité de multiples cibles par phosphorylation. Au cours du développement du cerveau, les kinases GSK3 contrôlent la balance entre la prolifération et la différenciation. La dérégulation de l'activité des kinases GSK3 a un rôle clé dans les maladies neurodégénératives du cerveau. En revanche, le rôle important de ces kinases au cours du développement rétinien ainsi que dans les maladies neurodégénératives rétiniennes reste une question ouverte.L'objectif de ma thèse était d'étudier le rôle de ces kinases au cours du développement et de l'homéostasie rétinienne. J’ai montré que l'absence totale de Gsk3α et de Gsk3β très tôt au cours du développement rétinien entraîne une microphtalmie chez l'adulte. Les deux kinases jouent des rôles redondants puisque l'expression d'un seul allèle Gsk3 est suffisante pour prévenir le phénotype de microphtalmie. Cependant, une analyse phénotypique approfondie dans ce contexte génétique (un seul allèle Gsk3) a révélé une forte augmentation du nombre de cellules ganglionnaires déplacées (dRGCs) dans la couche nucléaire interne, associée à une modification des projections axonales des cellules ganglionnaires dans le cerveau par rapport aux contrôles. Dans l’ensemble, ces données suggèrent que les kinases GSK3s sont essentielles au maintien des progéniteurs rétiniens et sont impliquées dans la genèse des dRGCs. Compte tenu du très faible nombre de dRGCs en conditions normales, la fonction de ces cellules a été très peu étudiée à ce jour. Le modèle génétique que j’ai développé offre par conséquent un modèle de choix pour étudier l’ontogenèse et la fonction de ces cellules.Mes travaux de thèse se sont ensuite concentrés sur le rôle de GSK3 dans les photorécepteurs. En effet, des défauts de développement ou leur mort est l’une des principales causes de dégénérescence rétiniennes. Afin de mieux comprendre la fonction de ces kinases dans la maintenance des photorécepteurs, j'ai donc utilisé des souris invalidées de manière conditionnelle pour Gsk3α et Gsk3β spécifiquement dans les précurseurs des photorécepteurs. L’absence de GSK3 conduit à une altération de la maturation et de la fonction des photorécepteurs, suivie de leur dégénérescence. J’ai alors combiné des analyses transcriptomiques et des approches in vitro pour élucider les mécanismes sous-jacents. Mes données m’ont conduit à proposer un modèle selon lequel l’absence de GSK3 dans les photorécepteurs conduit à des défauts de phosphorylation de NRL (facteur de transcription nécessaire au développement des photorécepteurs de type bâtonnet), augmentant sa stabilité. Cette dérégulation post-traductionnelle conduit à la diminution d’expression d'un sous-ensemble de gènes cibles de NRL, co-régulés par CRX, et impliqués dans le développement et l'homéostasie des photorécepteurs. Cette dérégulation conduirait alors à la dégénérescence des photorécepteurs observée dans les mutants GSK3. Ce travail suggère donc que GSK3 joue un rôle essentiel dans la régulation de NRL pour contrôler la maturation et l'homéostasie des photorécepteurs. De telles données suggèrent également que ce mécanisme de régulation pourrait être déficient chez les patients atteints de rétinites pigmentaires dues à des mutations de NRL empêchant sa phosphorylation par GSK3. / Post-translational modifications (PTMs) allow a higher degree of regulation for the control of gene expression by generating functional diversity at the proteome level. In the central nervous system, PTMs regulate stability or activity of transcription factors allowing a rapid response to external signals and a quick adaptation to a dynamic cellular microenvironment. In this context, I focused on the ubiquitously expressed and highly conserved Glycogen Synthase Kinases 3 (GSK3s). They are at the crossroad of multifunctional signalling pathways. During mammalian brain development, GSK3 kinases control the balance between proliferation and differentiation. Deregulation of GSK3 kinases activity has also a key role in neurodegenerative diseases by causing the accumulation/aggregations of proteins causing neuronal cell death. Drugs targeting GSK3s hold a lot of promises to treat such diseases. Whether these kinases are also important during retinal development and involved in retinal diseases remains an open question. Several studies suggest the importance of regulating GSK3 function in photoreceptor under pathological conditions. Therefore, the main objective of my PhD was to investigate the role of these kinases during photoreceptor development and homeostasis. To better understand the role of these two kinases during retinal development and to highlight potential differences with the developing brain, we also investigated their function in the control of the balance between proliferation and differentiation of retinal progenitors. To achieve my work, I used conditional knockout mice for Gsk3α and Gsk3β specifically deleted either in photoreceptor precursors or in retinal progenitors during early development. The lack of GSK3 kinases in photoreceptor precursors led to impaired photoreceptor maturation and function followed by their degeneration. Transcriptomic analysis (RNAseq) 6, 10 and 14 days postnatally prior degeneration revealed several genes downregulated belonging to biological processes involved in eye development and visual functions. Among them, the expression of the transcription factor Nrl that is required for rod photoreceptor development was decreased. Astonishingly, NRL expression was highly increased at protein level. By in vitro approaches, I demonstrated that GSK3-dependent phosphorylation regulates NRL protein stability. Despite such increase, a large number of NRL target genes were downregulated leading to impaired photoreceptor maturation and function. Surprisingly, a vast majority of these downregulated genes were also target genes for CRX, another transcription factor working in synergy with NRL. This work demonstrates that PTMs of NRL play a critical role in fine tuning the expression of a subset of genes involved photoreceptor development and homeostasis. Such findings could allow the development of innovative therapeutic strategies for retinal dystrophies. The functional characterisation of GSK3 in the course of retinal development by invalidating both Gsk3α and Gsk3β in retinal progenitors early during development revealed their requirement for controlling cell cycle exit and neuronal differentiation. Indeed, the complete lack of Gsk3α and Gsk3β led to microphtalmia in adults. Interestingly, the expression of only one Gsk3 allele was enough to rescue the phenotype. However, further analysis revealed a large number of displaced ganglion cells in the inner nuclear layer. The function of these cells remains to be determined, but their timing of production corresponds to other ganglion cells. Strikingly, these displaced ganglion cells project in distinct brain regions than normal ganglion cells. Therefore, our work could provide the first step toward determining the function of the displaced ganglion cells, which appear at low number in wildtype but whose function remains to be clarified.

Photoreceptors are sensory neurons in our eyes’ retinas that convert light into electrochemical signals thus allowing us to see the world around us. Human retinas have two types of photoreceptors: rods important for night vision and cones important for high-acuity daylight vision. In some retinal diseases, photoreceptors degenerate leaving patients visually impaired or even blind. One of the promising therapeutic approaches is cell therapy, which acts by supporting surviving or by substituting lost photoreceptors by transplanting donor photoreceptors produced in vitro. Human induced pluripotent stem cells (hiPSCs) represent a favorable donor cell source for transplantation, as they are patient-specific and have the ability to self-renew. While photoreceptors isolated from human retinal organoids represent bona fide cells for cell therapy, long time needed for their production, which coincides with developmental time, hampers their clinical application. Another approach for hiPSC differentiation is by overexpressing different transcription factors (TFs), the so-called forward programming. Although proved fast and efficient in producing multiple neuronal cell types, efficient forward programming protocols for engineering photoreceptors were so far not established. Therefore, aim of my thesis was to find TFs that drive in vitro photoreceptor differentiation from hiPSCs and to establish a fast forward programming protocol for producing photoreceptors in high yields. To find TFs that drive photoreceptor differentiation, I performed a TF-library-on-library screening in a reporter hiPSC line. The reporter hiPSCs expressed fluorescent markers only if synthetic photoreceptor-specific promoters were activated, i.e. in case of photoreceptor differentiation. The specificity of the reporter was confirmed in human retinal organoids. For the screening, I transduced the reporter cell line with two lentiviral libraries: a biased one consisting of 16 TFs known from in vivo photoreceptor development and an unbiased one consisting of 1756 TFs. After overexpressing TFs, cells that activated photoreceptor promoters were fluorescently sorted and analyzed. As 80 % of the sorted cells were positive for photoreceptor-specific genes, the screening was highly specific. Furthermore, the screening identified TFs that I validated in the reporter cell line as single, double and triple combinations to find the most efficient one in driving photoreceptor differentiation. The double combination of OTX2 and NEUROD1, known players in photoreceptor development, activated the cone reporter in 10 % of the cells, while additional overexpression of GON4L increased the activation to 25 %. GON4L was never before associated with photoreceptor development showing that in vitro differentiation might be uncoupled from its in vivo counterpart. The cone differentiation efficiency was increased to 50% by treating the cells with AraC, a cell cycle inhibitor that removes all proliferating cells from the cultures. Whether the cell will activate the cone reporter depended on expression levels of individual TFs. Higher and unequal levels, with NEUROD1 having the highest expression, were favorable for obtaining cells with activated cone reporter. Thus, by producing monoclonal cell lines, I identified competent clones with differentiation efficiencies higher than that of a polyclonal cell line and going up to 58%. Except activating the cone reporter, the cone precursor-like cells differentiated from hiPSCs by overexpressing the three TFs OTX2, NEUROD1 and GON4L acquired neuronal morphology and expressed photoreceptor precursor markers. As precursors are the optimal developmental time point for transplantation studies, the engineered cells were transplanted into mouse model of cone degeneration to assess their possible therapeutic potential. Some of the transplanted cells survived in vivo in the subretinal space but did not show any maturation or integration into the remaining retinal circuitry. Thus, further maturation of the cells in vitro is needed before the transplantation. So far, cone precursor-like cells showed ability to mature in vitro when co-cultured with retinal pigment epithelial cells derived from hPSCs and when cultured in presence of additional previously published growth factors. Therefore, changing culturing conditions from stem cell to photoreceptor-specific showed beneficial for further in vitro maturation and paved the way for further research. In conclusion, this study advanced TF-mediated cone photoreceptor engineering. It showed that overexpressing the three TFs OTX2, NEUROD1 and GON4L is enough to push differentiation of more than 50% of hiPSCs into cone precursor-like cells in only 10 days. Additional research to improve maturity and homogeneity of engineered cells – overexpressing additional TFs and changing culturing conditions is ongoing. Fast and efficient protocol established in this study is beneficial for bringing in vitro differentiated cone photoreceptors closer to their commercial application. Such engineered cones could be used as biomedical testbeds for drug discovery and research and represent a promising donor material for cell transplantation to treat blindness.

Licht und Pflanzenhormone sind essentiell für das Wachstum und die Entwicklung von Pflanzen. Es ist nur wenig darüber bekannt, wie sie die Transkription organellärer Gene beeinflussen. In Arabidopsis thaliana gibt es drei kernkodierte Phagentyp-RNA-Polymerasen (RpoT), welche für die organelläre Transkription verantwortlich sind. Diese werden in die Plastiden (RpoTp), die Mitochondrien (RpoTm) oder zu beiden Organellen (RpoTmp) transportiert. Neben den beiden kernkodierten RNA-Polymerasen (NEP) existiert in den Plastiden eine plastidärkodierte RNA-Polymerase (PEP), welche zusätzliche Sigmafaktoren zur Promotererkennung benötigt. Um die Lichtabhängigkeit der Expression der RpoT Gene sowie NEP-transkribierter Chloroplastengene zu analysieren, wurde die Akkumulation von RpoT- und rpoB-Transkripten in 7-Tage alten Keimlingen unter verschiedenen Lichtbedingungen mittels quantitativer real-time PCR untersucht. Die Änderungen in der Transkriptakkumulation deuten darauf hin, dass rote, blaue und grüne Wellenlängen die Expression der drei RpoT Gene unterschiedlich stark stimulieren. Untersuchungen an verschiedenen Lichtrezeptor-Mutanten zeigten, dass die Lichtinduktion der RpoT Genexpression überaus komplex ist und ein interagierendes Netzwerk aus multiplen Rezeptoren und Transkriptionsfaktoren an der Signalweiterleitung beteiligt ist. Das Phytohormon Cytokinin wird durch Histidin Kinase Rezeptoren (AHK) detektiert. Es gibt drei unterschiedliche Rezeptoren: AHK2, AHK3 und AHK4. Diese sind Teil eines Zwei-Komponenten-Systems, welches Signale mit Hilfe einer Phosphorylierungskette überträgt. Der Einfluss von Cytokinin auf die plastidäre Transkription wurde mit Hilfe von Cytokininrezeptor-Mutanten untersucht, um die Funktion von AHK2, AHK3 und AHK4 zu analysieren. Um weitere Informationen darüber zu erhalten, wie die plastidäre Transkription durch PEP mittels Cytokinin reguliert wird, wurden die Hormoneffekte auf die plastidäre Transkription in Sigmafaktor-Mutanten untersucht. / Light and plant hormones are essential for plant growth and development. Only little information is available about how these signals influence the transcription of organellar genes. Arabidopsis thaliana possesses three nuclear-encoded phage-type RNA polymerases (RpoT) for organellar transcription. They are imported into plastids (RpoTp), mitochondria (RpoTm), or into both organelles (RpoTmp). Besides the two nuclear-encoded plastid polymerases (NEP), plastids contain an additional plastid-encoded RNA polymerase (PEP), which needs additional sigma factors for promoter recognition. Interested in the expression of RpoT genes and NEP-transcribed plastid genes in response to light we analyzed transcript levels of RpoT and rpoB genes in 7-day-old wild-type plants under different light conditions by quantitative real-time-PCR. The observed changes in transcript accumulation indicated that red, blue, and green light differentially stimulated the expression of all three RpoT genes. Further analyses using different photoreceptor mutants showed that light induction of RpoT gene expression is surprisingly complex based on a network of multiple photoreceptors an d downstream pathways. Cytokinin signals are perceived by the histidine kinase (AHK) receptor family. There exist three different membrane-bound receptors: AHK2, AHK3 and AHK4/CRE1. These receptors are part of a two-component signaling system which transfers signals via phosphorelay mechanisms. Interested in the potential role of AHK2, AHK3 and AHK4/CRE1 in the transduction of cytokinin signals into the chloroplast, we analyzed the influence of cytokinin on plastidial transcription in receptor mutants. To gain more information on how plastid transcription by PEP is regulated by cytokinin, the influence of cytokinin in sigma factor mutants was also studied.