LMO4 is a transcription cofactor expressed during retinal development and in amacrine neurons at birth. A previous study in zebrafish reported that morpholino RNA ablation of one of two related genes, LMO4b, increases the size of the eye in embryos. However, the significance of LMO4 in mammalian eye development and function remained unknown since LMO4 null mice die prior to birth. We observed the presence of a smaller eye and/or coloboma in ~40% of LMO4 null mouse embryos. To investigate the postnatal role of LMO4 in retinal development and function, LMO4 was conditionally ablated in retinal progenitor cells using the Pax6 alpha-enhancer Cre/LMO4flox mice. We found that these mice have fewer Bhlhb5-positive GABAergic amacrine and OFF-cone bipolar cells. The deficit appears to affect the postnatal wave of Bhlhb5+ neurons, suggesting a temporal requirement for LMO4 in retinal neuron development. In contrast, cholinergic and dopaminergic amacrine, rod bipolar and photoreceptor cell numbers were not affected. The selective reduction in these interneurons was accompanied by a functional deficit revealed by electroretinography, with reduced amplitude of b-waves, indicating deficits in the inner nuclear layer of the retina. Thus, LMO4 is necessary for normal GABAergic amacrine and OFF-cone bipolar cell development during retina development.

Indiana University-Purdue University Indianapolis (IUPUI) / Human pluripotent stem cells (hPSCs) allow for the unprecedented ability to recapitulate early stages of human development, which are otherwise inaccessible to investigation. This is especially true for one of the earliest events in human development, the establishment of a neuroretinal fate from an unspecified pluripotent population. To test the ability of hPSCs to serve in this capacity, hPSCs were generated using mRNA-reprogamming methods and maintained in xenogeneic-free differentiation conditions. These cells were directed to differentiate using a three-dimensional approach to analyze their ability to successfully recapitulate early events in human development in a temporal and developmentally-appropriate fashion. To do so, hPSCs were first directed to an anterior neural phenotype, which was confirmed by analysis of stage-specific neural transcription factors via immunocytochemistry and quantitative RT-PCR. Next, the cells were directed to an optic vesicle-like stage, where the presumptive retinal cells were identified by the expression of specific transcription factors. Finally, three-dimensional optic vesicle-like retinal organoids were identified, isolated, and further analyzed for the expression of markers associated with some of the differentiated cell types of the neural retina. Upon establishment of hPSC-derived retinal organoids, this system was further utilized to study the neurodegeneration in glaucoma and provide insights into the disease mechanisms. Overall, the results of this study help to demonstrate the suitability of hPSC-differentiation approaches as an effective tool to model retinal development and disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Abnormal blood vessel growth from pre-existing blood vessels, termed pathological angiogenesis, is a common characteristic of vascular diseases like proliferative diabetic retinopathy (PDR) and wet age-related macular degeneration (wet AMD). Retinal detachment, hemorrhage, and loss of vision are only some of the debilitating consequences of advanced pathological angiogenesis. Current therapeutics targeting these blood vessels are ineffective in many patients. We previously identified a novel angiogenesis target, ferrochelatase (FECH), from the heme synthesis pathway, and found that FECH inhibition is antiangiogenic in cell and animal models. Heme synthesis occurs in mitochondria, where FECH inserts Fe2+ into protoporphyrin IX (PPIX) to produce heme. However, the relationship between heme metabolism and angiogenesis is poorly understood. I sought to understand the mechanism of how FECH and thus, heme is involved in endothelial cell function. First, I determined the energetic state of retinal and choroidal endothelial cells, previously uncharacterized. I found that mitochondria in endothelial cells had several functional defects after heme inhibition. FECH loss changed the shape of mitochondria and depleted expression of genes maintaining mitochondrial dynamics. FECH blockade elevated oxidative stress and depolarized mitochondrial membrane potential. Heme depletion had negative effects on cellular metabolism, affecting oxidative phosphorylation and glycolysis. Mitochondrial complex IV of the electron transport chain (cytochrome c oxidase) was decreased in cultured human retinal endothelial cells and in murine retina ex vivo after FECH inhibition. Supplementation with heme partially rescued phenotypes of FECH blockade. Additionally, I discovered that partial loss-of-function Fech mutation in mice caused PPIX accumulation with no change in normal vasculature, as assessed by fundoscopy. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis. My studies identify a novel role of a heme synthesis enzyme in blood vessel formation and provide an opportunity to exploit these findings therapeutically for patients with PDR and wet AMD.

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