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ABSTRACT Background Leber congenital amaurosis (LCA) is a genetically heterogeneous group of retinal diseases that cause congenital blindness in infants and children. Mutations in the GUCY2D gene that encodes retinal [...]

Monocular lid closure (MC) causes a profound shift in the ocular dominance (OD) of neurons in primary visual cortex (V1). Anatomical studies in both cat and mouse V1 suggest that large-scale structural rearrangements of eye-specific thalamocortical (TC) axons in response to MC occur much more slowly than the shift in OD. Consequently, there has been considerable debate as to whether the plasticity of TC synapses, which transmit competing visual information from each eye to V1, contributes to the early functional consequences of MC or is simply a feature of long-term deprivation. Here, we used quantitative immuno-electron microscopy to examine the possibility that alterations of TC synapses occur rapidly enough to impact OD after brief MC. The effect of short-term deprivation on TC synaptic structure was examined in male C57BL/6 mice that underwent 3 and 7 d of MC or monocular retinal inactivation (MI) with tetrodotoxin. The data show that 3 d of MC is sufficient to induce substantial remodeling of TC synapses. In contrast, 3 d of MI, which alters TC activity but does not shift OD, does not significantly affect the structure of TC synapses. Our results support the hypothesis that the rapid plasticity of TC synapses is a key step in the sequence of events that shift OD in visual cortex. [ABSTRACT FROM AUTHOR]

Purpose: Specific mutations of the retinal guanylyl cyclase-1 (retGC1) gene have been linked to Leber congenital amaurosis type 1 (LCA1) and cone-rod dystrophies in humans, diseases that are amenable to treatments using molecular based therapies. As a step towards developing a therapeutic transgene for LCA1, we analyzed the cell specific and developmental activity profiles of fragments of the human retGC1 5' flanking region in vivo., Methods: We generated self inactivating lentiviral vector constructs carrying three different fragments of the human retGC1 promoter fused to a nuclear localized beta-galactosidase reporter gene (nlacZ). The transgenes were packaged into lentiviral vectors, which were then used to transduce retinal progenitor cells of the developing chick. We monitored the expression of nlacZ in the retina over the course of development and in the retina, brain and pineal gland just prior to hatching., Results: A 1.8 kb fragment of the retGC1 5' flanking region upstream of Exon 2 was capable of targeting nlacZ expression to photoreceptor cells in vivo and its activity was augmented by the presence of intron 1. We also demonstrated that the cell specific activity of this fragment arises, at least in part, by silencing expression in non-photoreceptor cells during the final stages of retinal development., Conclusions: We have identified a human retGC1 promoter fragment that exhibits photoreceptor cell specific activity in vivo. Our results suggest that an element located in the proximal promoter may play a role in silencing expression of this gene in non-photoreceptor cells, thereby by shaping the restricted expression pattern of GC1 in the retina.

Purpose: To determine the activity, cell specificity, and developmental expression profiles of fragments of the chicken guanylate cyclase activating protein (GCAP)-1 promoter., Methods: The intrinsic activities of five GCAP1 promoter-luciferase constructs were measured in transiently transfected primary chicken embryonic retinal cultures. Lentivirus vectors carrying GCAP1 promoter-nlacZ transgenes were used to examine the cell specificities and temporal expression characteristics of selected promoter fragments in developing retina., Results: Three of the five GCAP1 promoter fragments exhibited significant activity in vitro. The expression characteristics of the promoter fragments in vivo varied as a function of promoter length. Expression of nlacZ driven by the 0.6- and 1.7-kb GCAP1 promoter fragments was first observed on embryonic day (E)12 and was restricted to the inner nuclear layer (INL). By E16, nlacZ staining was also detected in the outer nuclear layer (ONL). Expression of nlacZ driven by the 4.2-kb GCAP1 promoter fragment was not observed until E16 and was restricted to the ONL., Conclusions: The general organization of regulatory cis elements within the GCAP1 promoter is different from other photoreceptor-specific gene promoters. Elements located within 0.3 kb upstream of the transcription start point are capable of producing efficient gene expression; however, additional elements located within 4.0 kb upstream of the transcription start point are necessary to confer on the fragment the cell specificity and developmental expression characteristics of the native GCAP1 promoter. The results of the current study show that the lentiviral vector system is a useful tool for the characterization of the promoters of genes expressed in neural retina.