La retina de los vertebrados está compuesta por cinco tipos de neuronas: fotorreceptores (FRs, conos y bastones), bipolares, ganglionares, horizontales y amacrinas, y células no neurales entre las que se destacan las células gliales de Müller. Durante el desarrollo, estas neuronas se originan a partir de células progenitoras que pasan a través de una serie de estados de competencia determinados por factores genéticos, celulares y moleculares, lo que permite la aparición ordenada y secuencial de los distintos tipos celulares (Livesey y Cepko, 2001b). Entre las diversas moléculas y factores tróficos que influencian el desarrollo de los bastones se encuentran el Ácido Retinoico (AR) y el Ácido Docosahexaenoico (ADH). El AR ejerce una amplia variedad de efectos durante el desarrollo de los vertebrados y la diferenciación celular. Juega un rol crucial en la determinación del patrón antero-posterior del cuerpo, en la espermatogénesis, y en la formación y crecimiento de los miembros y de la piel. Además, es crítico para el desarrollo temprano del ojo y diferenciación de los FRs (Stenkamp y col., 1993; Prabhudesai y col., 2005; Hyatt y col., 1996; Khanna y col., 2006). El AR ejerce sus efectos en las células uniéndose y activando a receptores nucleares que funcionan como factores de transcripción y regulan así la transcripción génica. Por otro lado, en nuestro laboratorio se ha establecido que el ADH promueve la supervivencia y diferenciación de los FRs de retina de rata en cultivo, y que sus efectos anti-apoptóticos ocurren a través de la estimulación de la vía de la ERK/MAPK y de la modulación de la expresión de proteínas anti y pro-apoptóticas.El objetivo general de este trabajo fue estudiar los efectos del AR en el desarrollo de neuronas amacrinas y FRs de retina in vitro. Para ello utilizamos cultivos neuronales de retinas de rata postnatal desarrollados en medio químicamente definido, los cuales fueron suplementados con AR y/o ADH. Dado que el AR es un factor de diferenciación celular nuestra hipótesis fue que, al igual que otros factores tróficos, esta molécula promovería además la supervivencia de los FRs. Sin embargo, cuando el AR se agregó al día 0 se incrementó el porcentaje de FRs apoptóticos, lo cual se correspondió con una pérdida de funcionalidad mitocondrial. Esta apoptosis pudo ser bloqueada completamente por el tratamiento con un pan-inhibidor de caspasas previo a la suplementación con AR. Estos resultados sugieren que el AR induciría la muerte de los FRs a través de un mecanismo apoptótico que involucra la pérdida de la actividad mitocondrial y activación de caspasas. Como el AR está ubicuamente presente en la retina y es esencial para su desarrollo, la preservación de FRs viables requeriría que su efecto pro-apoptótico fuera contrarrestado por la presencia simultánea de moléculas de supervivencia, como el ADH. Para poner a prueba esta hipótesis agregamos ADH a los cultivos previo al tratamiento con AR. Este agregado previno la muerte de los FRs inducida por el AR, respaldando la hipótesis de que durante el desarrollo se requeriría la presencia de otros factores de supervivencia para prevenir esta muerte. Notablemente, la inducción de apoptosis por AR afectó selectivamente a los FRs, resultando inalteradas las neuronas amacrinas.Dado que el AR es reconocido por sus efectos promotores de la diferenciación, su efecto inductor de la muerte de los FRs fue un hallazgo inesperado. Esta observación hizo necesario verificar si, en las condiciones experimentales ensayadas, el AR favorecía o no la diferenciación. Comprobamos que el AR promovió marcadamente la diferenciación, en paralelo al aumento en el porcentaje de células apoptóticas. Determinamos, por inmunocitoquímica y Western Blot, que el AR incrementó la cantidad de FRs que expresaron opsina y periferina, proteínas características de FRs maduros, y que desarrollaron procesos apicales, rudimentos de los segmentos externos propios de estas neuronas maduras. Además, el AR aumentó el número de FRs que desarrollaron neuritas y la extensión alcanzada por las mismas.Cabe destacar que a diferencia de los otros parámetros analizados, la estimulación del desarrollo de neuritas no fue selectiva para los FRs: el tratamiento con AR indujo el crecimiento de neuritas también en las neuronas amacrinas. Dado que el AR y el ADH tienen efectos similares sobre la diferenciación, y que se unen a receptores que forman heterodímeros (RAR y RXR respectivamente), decidimos estudiar sus posibles efectos aditivos o sinérgicos. El tratamiento simultáneo con ambos factores aumentó la expresión de opsina y periferina a valores semejan la suma de los dos por separado. Estos resultados implican que el AR y ADH contribuyen a la diferenciación de los FRs en forma aditiva, y sugieren que estimularían vías independientes para promover sus efectos. El hecho de que el AR indujera mayor expresión de proteínas y formación de estructuras de neuronas maduras, nos llevó a proponer que la funcionalidad de los FRs también podría estar estimulada. Sin embargo, observamos que el AR no estimuló la hidrólisis del GMPc, característica indicativa de una cascada de fototransducción activa y por consiguiente de capacidad de respuesta a la luz, ni la capacidad de incorporar neurotransmisores (como glutamato en los FRs y GABA en las neuronas amacrinas) del medio extracelular. Estos resultados indican que, aunque el AR promueve la diferenciación de los FRs y neuronas amacrinas, por sí solo no logra la maduración funcional de estas neuronas en cultivo, sugiriendo que se requeriría la presencia de otros factores. La observación de que el AR inducía simultáneamente la diferenciación y simultáneamente la apoptosis nos hizo suponer que podría tener efectos distintos sobre distintas sub-poblaciones de FRs o sobre sub-poblaciones celulares en distintos estadios de maduración. Para corroborar esta hipótesis, se suplementaron los cultivos con AR al día 0, cuando la proliferación aún era activa, y al día 2, momento en el cual ya no había progenitores en proliferación. Notablemente, al tratar los cultivos al día 2, el AR estimuló la diferenciación de los FRs, aunque ya no se observó un aumento en la apoptosis. Estos resultados indican que el AR actuaría en forma diferencial según el estadio de desarrollo de los FRs, induciendo la apoptosis en una sub-población de aquellos que aun son progenitores indiferenciados y acelerando la diferenciación en los que ya han abandonado el ciclo celular. Diversos trabajos han demostrado que el AR influye en la proliferación y la adquisición de un fenotipo particular en progenitores de retina embrionarios. Esto sugirió que el incremento en el número de células diferenciadas inducido por el AR podría ser resultado de un mayor número total de FRs debido a que el AR podría estar modificando la proliferación o redirigiendo el destino celular. Sin embargo, al analizar distintos parámetros relacionados con estos eventos, como la incorporación de BrdU, la expresión de p27, nestina, Crx y HPC-1 (marcadores de FRs y neuronas amacrinas, respectivamente), observamos que el AR no indujo una salida temprana del ciclo ni modificó la determinación de la identidad celular. Esto implica que al menos en las condiciones experimentales descritas, y en ese momento del desarrollo postnatal temprano, el AR no altera la salida del ciclo ni regula la identidad celular de estas neuronas in vitro. Para comprender mejor los mecanismos de acción del AR sobre los FRs, estudiamos la modulación de las vías de señalización intracelular implicadas en sus efectos. Se ha involucrado al AR en la activación de la quinasa p38, relacionada con la regulación de la apoptosis en varios tipos celulares. Cuando investigamos si el AR activaba la vía de p38 en los FRs, el análisis por Western Blot e inmunocitoquímica demostró que el AR promovió rápidamente la activación de esta vía de señalización, y que el bloqueo de dicha activación con un inhibidor específico de p38 evitó la apoptosis de los FRs. Paralelamente, la inhibición de esta vía redujo significativamente, aunque no por completo, la diferenciación de los FRs. Esto sugiere que la vía de señalización de p38 sería la preferencialmente activada por el AR para activar la apoptosis de los FRs y al menos una de las involucradas en inducir su diferenciación. Trabajos previos han mostrado que en la estimulación de la supervivencia de los FRs promovida por ADH interviene la activación de ERK/MAPK. Por ello, sería posible que el efecto deletéreo del AR implicara una modulación de esta vía. Sin embargo, no observamos cambios en la activación de dicha vía, indicando que no estaría afectada en el proceso de muerte inducido por AR. Por otro lado, teniendo en cuenta que la actividad de p38/MAPK podría ser regulada por interacción con la vía de PI3K/Akt, determinamos si el AR era capaz de modular esta vía en los FRs. El tratamiento con AR redujo la cantidad de P-Akt, respaldando la hipótesis de que el efecto estimulatorio del AR sobre la vía de p38 involucraría también una inhibición de la actividad de PI3K/Akt. En conjunto, estos resultados muestran que el AR es requerido para promover la diferenciación de los FRs y que este proceso de diferenciación no está necesariamente ligado a la supervivencia de estas neuronas. Su presencia prematura podría inducir la muerte de los progenitores al inducirlos a diferenciarse cuando aun están demasiado inmaduros, lo que resalta la importancia de la presencia simultánea de factores tróficos para prevenir dicha muerte. En conclusión, este trabajo remarca la importancia de una adecuada sincronización entre los niveles de diferentes señales moleculares esenciales para el desarrollo de los FRs. El AR podría así ser una de las moléculas cruciales que contribuyen a definir el número final de FRs en la retina. Las principales conclusiones de esta tesis son: a) El AR induce la muerte por apoptosis en los progenitores de FRs mientras se encuentran en el ciclo celular, por una vía que involucra la pérdida de funcionalidad mitocondrial y la activación de caspasas. b) El AR induce la diferenciación de los FRs, estimulando la expresión de opsina, periferina y el crecimiento de neuritas. c) El AR promueve el crecimiento de las neuritas en las neuronas amacrinas. d) La inducción de apoptosis por parte del AR es selectiva para los FRs. e) El AR no altera la proliferación ni modifica el destino de los progenitores. f) La inducción de de la diferenciación es independiente de que las células estén activas o no en el ciclo celular. g) Los procesos de apoptosis y diferenciación en los FRs inducidos por el AR dependen de la activación de la vía de p38/MAPK, que a su vez interacciona con la vía de PI3K/Akt. h) Un factor trófico lipídico, el ADH, protege a los FRs de la muerte inducida por AR. / The vertebrate retina has five neuronal types: photoreceptors (PHRs, rods and cones), bipolar, ganglion, horizontal and amacrine neurons, and non neuronal cells including the Müller glial cells. During development, these neurons are originated from progenitor cells that undergo a series of competence states, determined by genetic, cellular and environmental factors, thus allowing the sequential and organized appearance of the different cell types (Livesey y Cepko, 2001b). Retinoic Acid (RA) and Docosahexaenoic Acid (DHA) are among the different molecules and trophic factors that influence the development of rod PHRs. RA exerts a wide variety of effects during vertebrate development and cell differentiation. It plays a major role in the determination of the antero-posterior body axis, spermatogenesis, the formation and growth of body limbs and skin. Moreover, it is critical for the early development of the eye and PHR differentiation (Stenkamp y col., 1993; Prabhudesai y col., 2005; Hyatt y col., 1996; Khanna y col., 2006). RA binds to and activates nuclear receptors that function as transcription factors, thus regulating gene transcription. On the other hand, in our lab we have established that DHA promotes the survival and differentiation of rat PHRs in culture, and that these anti-apoptotic effects require the activation of the ERK/MAPK signaling pathway and the modulation of anti- and pro-apoptotic protein the expression. The general purpose of this work was to study the effects of RA on the development of amacrine neurons and PHRs in vitro. To that end, we used cultures obtained from postnatal rat retinas, developed in chemically defined media, which were supplemented with RA and/or DHA. Given that RA is a cell differentiation factor; our hypothesis was that, like other trophic factors, this molecule would also promote PHR survival. However, when RA was added at day 0, the percentage of apoptotic PHRs increased, in parallel with a loss of mitochondrial functionality. This apoptosis was completely blocked by incubating the cultures with a caspase inhibitor before RA addition. These results suggest that RA would induce PHR death through an apoptotic mechanism involving a loss of mitochondrial activity and caspase activation. Since RA is ubiquitously present in the retina and it is essential for development, the preservation of viable PHRs would require its pro-apoptotic effects to be counteracted by the simultaneous presence of survival molecules, such as DHA. To test this hypothesis, we added DHA to the cultures prior RA treatment; this addition prevented RA-induced PHR death, supporting the hypothesis of the necessity of other survival factors to prevent death during development. Noteworthy, RA-induced apoptosis was selective for PHRs, since amacrine neurons were not affected. Since RA is well known for its differentiation-promoting effects, the fact that it induced apoptosis was rather unexpected. This observation led us to test whether, under these experimental conditions, RA would promote or not PHR differentiation. RA indeed promoted differentiation, in parallel with an increase in the percentage of apoptotic PHRs. We determined, by immunocytochemistry and Western Blot, that RA increased the amount of PHRs that expressed opsin and peripherin, characteristic proteins of mature PHRs and of PHRs that developed apical processes, structures that resemble the initial steps of outer segment formation. Moreover, RA increased the percentage of PHRs that developed neurites and promoted neurite outgrowth. It is worth to note that, unlike other evaluated features, the stimulation of neurite outgrowth was not exclusive for PHRs; RA treatment also induced also neurite outgrowth in amacrine cells. Since RA and DHA have similar effects on differentiation, and they bind to receptors that form heterodimers (RAR y RXR respectively), we evaluated their possible additive or synergistic effects. The simultaneous treatment with both factors increased opsin and peripherin expression up to a value that resembled the sum of both metabolites alone. These results imply that RA and DHA contribute to PH differentiation in an additive fashion, and suggest that they stimulate independent pathways to that end. The fact that RA induced the expression of proteins and formation of structures of mature neurons, led us to propose that the functionality of these cells could also be stimulated. However, RA neither stimulated cGMP hydrolysis, a characteristic that would indicate an active phototransduction cascade and the ability to respond to light, nor the capacity to take up neurotransmitters (like glutamate in PHRs and GABA in amacrine neurons) from the extracellular medium. These results indicate that, although RA promotes PHR and amacrine cell differentiation, it is not enough of a stimulus to achieve functional maturity of these cells, suggesting that this functionality requires the presence of other factors. The finding that RA simultaneously induced differentiation and apoptosis led us to propose that it might have distinct effects on different PHR sub-populations or on populations at different developmental stages. To test this hypothesis, cultures were supplemented with RA at day 0, when proliferation is still active, and at day 2, when there are no longer proliferating progenitors. Noteworthy, when added at day 2, RA stimulated PHR differentiation, although no increase in apoptosis was evident. These results indicate that RA would act differentially depending on PHRs developmental stages, inducing apoptosis in a sub-population of undifferentiated progenitors and accelerating the differentiation in those which have already abandoned the cell cycle. Several studies have shown that RA influences proliferation and in the acquisition of a particular phenotype in embryonic retina progenitors. For that reason, the increase in the number of differentiated cells induced by RA could be due to a higher total number of PHRs, since RA might be redirecting cell fate or modifying proliferation. However, when we analyzed a number of parameters related to these events, such as BrdU incorporation and the expression of p27, nestin, CRX and HPC-1 (markers of PHRs and amacrine cells, respectively), we found RA neither induced cell cycle exit nor modified cell fate. This implies that, at least under the described experimental conditions, and at this particular time of development, RA would not alter the cell cycle exit or regulate cell identity. To better understand the mechanisms by which RA exerted its effects on PHRs, we studied the modulation of signaling pathways. RA has been involved in the activation of p38/MAPK, which related to the regulation of apoptosis in several cell types. When we evaluated whether RA activated the p38 pathway in PHRs, Western Blot and immunocytochemical analyses showed that it induced a rapid activation of this pathway, and the blockade of such activation with a specific inhibitor prevented PHR apoptosis. Moreover, the inhibition of this pathway led to a significant, though not complete, reduction of PHR differentiation. This suggests that the p38/MAPK would be the preferred signaling pathway activated by RA to induce apoptosis in PHRs, and at least one of the involved in the induction of their differentiation. Previous work has shown that DHA-stimulated survival in PHRs requires the activation of the ERK/MAPK pathway. Hence, the deleterious effect of RA might involve the modulation of this pathway. However, we found no changes in the activation of this pathway, indicating that it would not be related to RA-induced PHR death. On the other hand, given that p38/MAPK activity has been shown to be regulated by interaction with the PI3K/Akt pathway, we determined whether RA was capable of modulating this pathway in PHRs. Treatment with RA reduced the amount of P-Akt, supporting the hypothesis that the stimulatory effect of RA on the p38 pathway would involve the inhibition of PI3K/Akt activity. As a whole, these results show that RA is required for the induction of PHR differentiation, and that this process is not necessarily linked to the survival of these neurons. The premature presence of RA could elicit progenitor death as it might induce them to differentiate at a stage when they are still too immature, highlighting the need of the simultaneous presence of trophic factors to prevent this death. In summary, this work underscores the relevance of an adequate synchronization between the levels of different molecular cues essential for PHR development. RA might thus be one of the crucial molecules that contribute to define the final number of PHRs in the retina. The main conclusions of this thesis are: a) RA induces PHR progenitor apoptosis while they are active in the cell cycle, through a mechanism that involves the loss of mitochondrial activity and caspase activation. b) RA induces PHR differentiation, stimulating opsin and peripherin expression, and neurite outgrowth. c) RA promotes neurite outgrowth in amacrine neurons. d) RA-induced apoptosis is selective for PHRs. e) RA does not alter progenitor proliferation or the acquisition of cell fate. f) The induction of differentiation occurs regardless of the cells being active in the cell cycle or not. g) RA-induced differentiation and apoptosis processes in PHRs depend on the activation of p38/MAPK, which also interacts with PI3K/Akt. h) A lipid trophic factor, DHA, protects PHRs from RA-induced apoptosis.

Note:

published_or_final_version / abstract / Medicine / Master / Doctor of Medicine

No description available.

No description available.

Introduction Retinal vascular reactivity is impaired in patients with diabetes and is thought to be involved in the onset and progression of diabetic retinopathy (DR). Previous studies that have utilized hyperoxia to assess retinal vascular reactivity have been limited due to confounding factors associated with the administration of oxygen and have used a variety of different instruments to measure retinal blood flow. The influence of blood glucose at the time of blood flow assessment has also not been systemically investigated. The specific aims of each Chapter are as follows: Chapter 3: To compare three systems used to administer hyperoxia to human subjects. Chapter 4: To quantify the magnitude and timeline of change of retinal hemodynamic parameters induced by an isocapnic hyperoxic stimulus. Chapters 5, 6 and 7: To quantify the magnitude of change of retinal hemodynamic parameters induced by hyperoxia, hyperglycemia and combined hyperoxia / hyperglycemia, respectively, in groups of diabetic patients with no clinically visible, and mild-to-moderate, DR and in age-matched subjects without diabetes. Methods Chapter 3: Subjects breathed air followed by oxygen, or oxygen plus carbon dioxide using a non-rebreathing system, or air followed by oxygen using a sequential rebreathing system. The magnitude of change and variability of CO2 concentrations was compared between systems. Chapter 4: Baseline retinal blood flow data was acquired while the subjects breathed air using a sequential rebreathing system. An isocapnic hyperoxic stimulus was initiated and maintained for 20 minutes. Air was then re-administered for 10 minutes. Retinal blood flow measurements were acquired every minute over the course of the study. The magnitude of change of each hemodynamic parameter was determined by fitting individual data with a sigmoidal function. For Chapter 5, 6 and 7 diabetic patients with no clinically visible, and mild-to-moderate, DR were stratified into groups based upon their retinopathy status. Age-matched non-diabetic subjects were recruited as controls. Baseline retinal blood flow data was acquired while subjects breathed air. Retinal blood flow measurements were then acquired after exposure to (a) hyperoxia, (b) hyperglycemia and (c) combined hyperoxic / hyperglycemic stimuli. Change in hemodynamic parameters was compared between groups and correlated with objective measures of retinal edema. Results Chapter 3: The difference in group mean end-tidal CO2 levels between baseline and hyperoxia was significant for oxygen administration using a non-rebreathing system. The sequential rebreathing technique resulted in a significantly lower variability of individual CO2 levels than either of the other techniques. Chapter 4: An ~11% decrease of diameter, ~36% decrease of velocity and ~48% decrease of blood flow was observed in response to isocapnic hyperoxia in young, healthy subjects. A response time of 2.30??0.53 minutes and 2.62??0.54 minutes was observed for diameter and velocity, respectively. Chapter 5: Retinal blood velocity, flow, and WSR significantly decreased in response to isocapnic hyperoxia in all groups. The magnitude of the reduction of blood flow was significantly reduced with increasing severity of retinopathy. There was a significant relationship between baseline objective edema index values and retinal vascular reactivity. Chapter 6: A significant change in blood glucose level was observed for all groups. No significant change in any hemodynamic parameter was found in patients with diabetes and in age-matched subjects without diabetes. Chapter 7: Retinal blood velocity and flow significantly decreased in all groups in response to combined hyperoxic / hyperglycemic provocation. The vascular reactivity response was not significantly different across the groups. Conclusions Chapter 3: Control of CO2 is necessary to attain standardized, reproducible hyperoxic stimuli for the assessment of retinal vascular reactivity. Chapter 4: Arteriolar retinal vascular reactivity to isocapnic hyperoxic provocation occurs within a maximum of 4 minutes. Although there was a trend for diameter to respond before velocity, the response characteristics were not significantly different between diameter and velocity. Different response characteristics of the retinal vasculature to transmural pressure mediated autoregulation as opposed to metabolic mediated vascular reactivity are suggested. Chapter 5: The vascular reactivity response in terms of the reduction of blood flow relative to baseline was significant in all groups but the magnitude of the change in flow was significantly reduced with increasing severity of retinopathy. A loss of retinal vascular reactivity is indicated in patients with moderate DR without clinically evident diabetic macular edema (DME), and in patients with DME. Chapter 6: Unaltered retinal arteriolar blood flow was found 1 hour after glucose ingestion in patients with diabetes and in age-matched subjects without diabetes. These results do not support the theory that retinal blood flow is affected by an acute increase of blood glucose in diabetic patients and in subjects without diabetes. Chapter 7: The vascular reactivity response to a combined hyperoxic / hyperglycemic provocation produced a pronounced reduction in blood flow. Unlike the response to hyperoxia alone, the vascular reactivity response was not significantly different across the groups. This suggests that hyperglycemia may influence the retinal vascular reactivity response to hyperoxia.

Inherited retinal and corneal dystrophies represent a group of disorders with great genetic heterogeneity. Over 250 genes are associated with retinal diseases and 16 genes are causative of corneal dystrophies. This thesis is focused on finding the genetic causes of corneal dystrophy, Leber congenital amaurosis (LCA), Stargardt disease and retinitis pigmentosa in families from northern Sweden.  By whole exome sequencing a novel mutation, c.2816C>T, p.Thr939Ile, in Collagen Type XVII, Alpha 1 chain, COL17A1, gene was identified in several families with epithelial recurrent erosion dystrophy (ERED). We showed that the COL17A1 protein is expressed in the basement membrane of the cornea, explaining the mutation involvement in the corneal symptoms. We could link all the families in this study to a couple born in the late 1700s confirming a founder mutation in northern Sweden. Our finding highlights role of COL17A1 in ERED and suggests screening of this gene in patients with similar phenotype worldwide. Furthermore the genetic causes in several retinal degenerations were identified. In one family with two recessive disorders, LCA and Stargardt disease, a novel stop mutation, c.2557C>T, p.Gln853Stop, was detected in all LCA patients. In the Stargardt patients two intronic variants, the novel c.4773+3A>G and c.5461-10T>C, were detected in the ABCA4 gene. One individual was homozygous for the known variant c.5461-10T>C and the other one was compound heterozygote with both variants present. Both variants, c.4773+3A>G and c.5461-10T>C caused exon skipping in HEK293T cells demonstrated by in vitro splice assay, proving their pathogenicity in Stargardt disease. Finally, in recessive retinitis pigmentosa, Bothnia Dystrophy (BD), we identified a second mutation in the RLBP1 gene, c.677T>A, p.Met226Lys. Thus, BD is caused not only by common c.700C>T variant but also by homozygosity of c.677T>A or compound heterozygosity. Notably, known variant, c.40C>T, p.R14W in the CAIV gene associated with a dominant retinal dystrophy RP17 was detected in one of the compound BD heterozygote and his unaffected mother. This variant appears to be a benign variant in the population of northern Sweden. In conclusion, novel genetic causes of retinal dystrophies in northern Sweden were found demonstrating the heterogeneity and complexity of retinal diseases. Identification of the genetic defect in COL17A1 in the corneal dystrophy contributes to understanding ERED pathogenesis and encourages refinement of IC3D classification. Our results provide valuable information for future molecular testing and genetic counselling of the families.

Fibroblast growth factors (FGFs) are critical in many aspects of embryonic development and other cellular functions including apoptosis, cell adhesion, and proliferation. FGF8a, specifically, is known to initiate retinal ganglion cell (RGC) differentiation along with FGF3 early in retinal development (Martinez-Morales et al., 2005b). There has been little research into later roles for FGF8a in eye development. Here we show mRNA expression of fgf8a in the presumptive RGCs of 2 day-old zebrafish, past the time of RGC differentiation (28-48 hours)(Schmitt and Dowling, 1996). In addition, mRNA expression of putative receptor, FGFR1b, was localized outside the retina on the presumptive vasculature. Acerebellar (ace) mutants lacking FGF8a show mispatterned retinal vasculature and a lack of blood flow through the eye at 48 hpf. Further, we looked to see if this lack of blood flow had any effect on the developing neural retina. We found a significant reduction in the size of ace mutant eyes and also a reduction in total cell numbers in the retina starting at 48 hours post fertilization (hpf) suggesting a role for fgf8a in neurovascular signaling. The cause of the small eye phenotype was found to be due to a lack of proliferating cells and not an increase in cell death. We hypothesized if this phenotype was a result of a lack of blood flow to the retina. It has previously been reported that zebrafish survive and develop normally for 7 days without blood flow as the embryo receives nutrients by simple diffusion with its surroundings (Sehnert et al., 2002). To investigate the role that blood flow plays on the developing retina we utilized a silent heart mutant (sih) fish line, which lacks cardiac troponin t resulting in embryos without blood flow, as heart contractility does not initiate. To explore lack of blood flow to the retina as a cause for the observed ace mutant phenotype, sih mutant eye phenotypes were assessed. Retina cell counts from these embryos show a decreased eye diameter and a loss in total retina cell numbers due to lack of proliferation, phenocopying ace mutants. sih mutants also show a mis-patterning of their retinal vasculature with ectopic vessel branches similar to ace mutants. Our data support the small eye phenotype seen in both mutants is a result due to lack of proliferation. After morpholino knock down of the receptor, fgfr1b, we see mispatterend vasculature that phenocopies what we see in ace mutants. These finding led us to hypothesize that FGF8a, secreted by the RGCs, signals through its receptor, FGFR1b, on the retinal vasculature to promote cell growth and development. Further these data suggest that the retinal vasculature subsequently responds by secreting an unknown factor to support the proliferation and maintenance of the RGCs.

by Sun-on Chan. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 132-157.

Late-onset retinal macular degeneration (L-ORMD) is a fully penetrant autosomal dominant macular degeneration resulting from a Ser163Arg substitution in the gene encoding the protein C1QTNF5. Clinically L-ORMD results in dark adaptation delay in the fifth decade, central visual loss in the sixth decade and further progressive visual field loss in successive decades of life. Pathologically the disease results in thick sub-retinal deposits, which have a similar composition to drusen seen in AMD, retinal pigment epithelial (RPE) loss, and neuro-retinal atrophy. The function of C1QTNF5 is incompletely understood however within the eye it is expressed most strongly by the RPE cells. An in vitro model for L-ORMD was developed using human induced pluripotent stem cells (hiPSCs) derived from patients and with stem cells from patient’s unaffected siblings used as controls. The hiPSCs were differentiated to RPE (hiPSC-RPE). L-ORMD hiPSC-RPE shared baseline characteristics with sibling control hiPSC-RPE. In order to model in vivo conditions hiPSC-RPE were grown on permeable supports in human serum enriched media. Case hiPSC-RPE cell lines were found to activate the complement pathway resulting in increased deposition of the terminal complement complex (TCC) C5b-9 when compared to control hiPSC-RPE. Using depleted serum, deposition was not affected by depletion of classical and lectin pathway components but was reduced by depletion of alternative complement pathway components. Depletion of complement components C3 and C5 abolished TCC deposition. The addition of a monoclonal antibody against C5 also reduced TCC deposition. The role of complement dysregulation in L-ORMD pathogenesis was confirmed by immunostaining of L-ORMD and age-matched control human donor retinal sections. L-ORMD retinal sections displayed increased C3d and C5b-9 deposition. Using mutant and wild type-protein generated from a bacterial expression system it was found that the mutant protein was less stable than the wild-type. In addition the wild type protein formed multimers whilst the mutant was mainly monomeric. A surface plasmon resonance (SPR) study showed an increased affinity of wild-type C1QTNF5, especially in multimeric form for complement factor H (CFH), a key regulator of the alternative complement pathway when compared to mutant protein. Taken together these studies implicate dysfunction of the alternative complement pathway in L-ORMD disease mechanism and have suggested a role for C1Q TNF5 in the extracellular matrix. The studies also show that L-ORMD and AMD share a pathogenic and clinical similarities.