Transition to Chronic Fibrosis in an Animal Model of Retinal Detachment With Features of Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of failure of surgically repaired rhegmatogenous retinal detachment (RRD). Chemically induced and cell injection PVR models do not fully simulate the clinical characteristics of PVR in the post-RRD context. There is an unmet need for translational models in which to study mechanisms and treatments specific to RRD-PVR. Methods: RRD was induced in adult Dutch Belted rabbits. Posterior segments were fixed or processed for RNA sequencing at 6 hours and 2, 7, 14, and 35 days after induction. Histochemical staining and immunolabeling for glial fibrillary acidic protein, alpha smooth muscle actin, vascular endothelial growth factor receptor 2, CD68, and RPE 65 kDa protein were performed, and labeling intensity was scored. Single cell RNA sequencing was performed. Results: Acute histopathological changes included intravitreal and intraretinal hemorrhage, leukocytic vitritis, chorioretinitis, and retinal rarefaction. Chronic lesions showed retinal atrophy, gliosis, fibrotic subretinal membranes, and epiretinal fibrovascular proliferation. Fibrillar collagen was present in the fibrocellular and fibrovascular membranes in chronic lesions. Moderate to strong labeling of glia and vasculature was detected in chronic lesions. At day 14, most cells profiled by single cell sequencing were identified as Mϋller glia and microglia, consistent with immunolabeling. Expression of several fibrillar collagen genes was upregulated in chronic lesions. Conclusions: Histological and transcriptional features of this rabbit model simulate important features of human RRD-PVR, including the transition to chronic intraretinal and periretinal fibrosis. This animal model of RRD with features of PVR will enable further research on targeted treatment interventions.

Comparative Analysis of Single-cell and Single-nucleus RNA-sequencing in a Rabbit Model of Retinal Detachment-related Proliferative Vitreoretinopathy.

Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of failure of retinal reattachment surgery, and the molecular changes leading to this aberrant wound healing process are currently unknown. Our ultimate goal is to study PVR pathogenesis by employing single-cell transcriptomics to dissect cellular heterogeneity. Design: Here we aimed to compare single-cell RNA sequencing (scRNA-seq)  and single-nucleus RNA-sequencing (snRNA-seq) of retinal PVR samples in the rabbit model. Participants: Unilateral induction of PVR lesions in rabbit eyes with contralateral eyes serving as controls. Methods: Proliferative vitreoretinopathy was induced unilaterally in Dutch Belted rabbits. At different timepoints after PVR induction, retinas were dissociated into either cells or nuclei suspension and processed for scRNA-seq or snRNA-seq. Main Outcome Measures: Single cell and nuclei transcriptomic profiles of retinas after PVR induction. Results: Single-cell RNA sequencing and snRNA-seq were conducted on retinas at 4 hours and 14 days after disease induction. Although the capture rate of unique molecular identifiers and genes were greater in scRNA-seq samples, overall gene expression profiles of individual cell types were highly correlated between scRNA-seq and snRNA-seq. A major disparity between the 2 sequencing modalities was the cell type capture rate, however, with glial cell types overrepresented in scRNA-seq, and inner retinal neurons were enriched by snRNA-seq. Furthermore, fibrotic Müller glia were overrepresented in snRNA-seq samples, whereas reactive Müller glia were overrepresented in scRNA-seq samples. Trajectory analyses were similar between the 2 methods, allowing for the combined analysis of the scRNA-seq and snRNA-seq data sets. Conclusions: These findings highlight limitations of both scRNA-seq and snRNA-seq analysis and imply that use of both techniques together can more accurately identify transcriptional networks critical for aberrant fibrogenesis in PVR than using either in isolation. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Single-cell transcriptome analysis of xenotransplanted human retinal organoids defines two migratory cell populations of nonretinal origin.

Human retinal organoid transplantation could potentially be a treatment for degenerative retinal diseases. How the recipient retina regulates the survival, maturation, and proliferation of transplanted organoid cells is unknown. We transplanted human retinal organoid-derived cells into photoreceptor-deficient mice and conducted histology and single-cell RNA sequencing alongside time-matched cultured retinal organoids. Unexpectedly, we observed human cells that migrated into all recipient retinal layers and traveled long distances. Using an unbiased approach, we identified these cells as astrocytes and brain/spinal cord-like neural precursors that were absent or rare in stage-matched cultured organoids. In contrast, retinal progenitor-derived rods and cones remained in the subretinal space, maturing more rapidly than those in the cultured controls. These data suggest that recipient microenvironment promotes the maturation of transplanted photoreceptors while inducing or facilitating the survival of migratory cell populations that are not normally derived from retinal progenitors. These findings have important implications for potential cell-based treatments of retinal diseases.

Lhx2 is a progenitor-intrinsic modulator of Sonic Hedgehog signaling during early retinal neurogenesis.

An important question in organogenesis is how tissue-specific transcription factors interact with signaling pathways. In some cases, transcription factors define the context for how signaling pathways elicit tissue- or cell-specific responses, and in others, they influence signaling through transcriptional regulation of signaling components or accessory factors. We previously showed that during optic vesicle patterning, the Lim-homeodomain transcription factor Lhx2 has a contextual role by linking the Sonic Hedgehog (Shh) pathway to downstream targets without regulating the pathway itself. Here, we show that during early retinal neurogenesis in mice, Lhx2 is a multilevel regulator of Shh signaling. Specifically, Lhx2 acts cell autonomously to control the expression of pathway genes required for efficient activation and maintenance of signaling in retinal progenitor cells. The Shh co-receptors Cdon and Gas1 are candidate direct targets of Lhx2 that mediate pathway activation, whereas Lhx2 directly or indirectly promotes the expression of other pathway components important for activation and sustained signaling. We also provide genetic evidence suggesting that Lhx2 has a contextual role by linking the Shh pathway to downstream targets. Through these interactions, Lhx2 establishes the competence for Shh signaling in retinal progenitors and the context for the pathway to promote early retinal neurogenesis. The temporally distinct interactions between Lhx2 and the Shh pathway in retinal development illustrate how transcription factors and signaling pathways adapt to meet stage-dependent requirements of tissue formation.

Cell-specific regulation of gene expression using splicing-dependent frameshifting.

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.

Gene regulatory networks controlling temporal patterning, neurogenesis, and cell-fate specification in mammalian retina.

Gene regulatory networks (GRNs), consisting of transcription factors and their target sites, control neurogenesis and cell-fate specification in the developing central nervous system. In this study, we use integrated single-cell RNA and single-cell ATAC sequencing (scATAC-seq) analysis in developing mouse and human retina to identify multiple interconnected, evolutionarily conserved GRNs composed of cell-type-specific transcription factors that both activate genes within their own network and inhibit genes in other networks. These GRNs control temporal patterning in primary progenitors, regulate transition from primary to neurogenic progenitors, and drive specification of each major retinal cell type. We confirm that NFI transcription factors selectively activate expression of genes promoting late-stage temporal identity in primary retinal progenitors and identify other transcription factors that regulate rod photoreceptor specification in postnatal retina. This study inventories cis- and trans-acting factors that control retinal development and can guide cell-based therapies aimed at replacing retinal neurons lost to disease.

C5a Activates a Pro-Inflammatory Gene Expression Profile in Human Gaucher iPSC-Derived Macrophages.

Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic GBA1 mutations that reduce the activity of the lysosomal enzyme ß-glucocerebrosidase (GCase). GCase catalyzes the conversion of glucosylceramide (GluCer), a ubiquitous glycosphingolipid, to glucose and ceramide. GCase deficiency causes the accumulation of GluCer and its metabolite glucosylsphingosine (GluSph) in a number of tissues and organs. In the immune system, GCase deficiency deregulates signal transduction events, resulting in an inflammatory environment. It is known that the complement system promotes inflammation, and complement inhibitors are currently being considered as a novel therapy for GD; however, the mechanism by which complement drives systemic macrophage-mediated inflammation remains incompletely understood. To help understand the mechanisms involved, we used human GD-induced pluripotent stem cell (iPSC)-derived macrophages. We found that GD macrophages exhibit exacerbated production of inflammatory cytokines via an innate immune response mediated by receptor 1 for complement component C5a (C5aR1). Quantitative RT-PCR and ELISA assays showed that in the presence of recombinant C5a (rC5a), GD macrophages secreted 8-10-fold higher levels of TNF-α compared to rC5a-stimulated control macrophages. PMX53, a C5aR1 blocker, reversed the enhanced GD macrophage TNF-α production, indicating that the observed effect was predominantly C5aR1-mediated. To further analyze the extent of changes induced by rC5a stimulation, we performed gene array analysis of the rC5a-treated macrophage transcriptomes. We found that rC5a-stimulated GD macrophages exhibit increased expression of genes involved in TNF-α inflammatory responses compared to rC5a-stimulated controls. Our results suggest that rC5a-induced inflammation in GD macrophages activates a unique immune response, supporting the potential use of inhibitors of the C5a-C5aR1 receptor axis to mitigate the chronic inflammatory abnormalities associated with GD.

Transcriptomic Profiling of Control and Thyroid-Associated Orbitopathy (TAO) Orbital Fat and TAO Orbital Fibroblasts Undergoing Adipogenesis.

Purpose: Orbital fat hyperplasia commonly occurs in thyroid-associated orbitopathy (TAO). To understand molecular mechanisms underlying orbital adipogenesis, we used transcriptomics to compare gene expression in controls and patients with TAO, as well as in orbital fibroblasts (OFs) undergoing adipogenic differentiation. Methods: We performed bulk RNA sequencing (RNA-Seq) on intraconal orbital fat from controls and patients with TAO. We treated cultured OFs derived from patients with TAO with adipogenic media to induce adipogenesis. We used single nucleus RNA-Seq (snRNA-Seq) to profile treated and control OFs, identifying genes that are dynamically expressed during orbital adipogenesis in vitro, and compared these results to data from control and TAO orbital fat. Results: Gene expression profiles in control and TAO orbital fat are distinct. Signaling pathways including PI3K-Akt signaling, cAMP signaling, AGE-RAGE signaling, regulation of lipolysis, and thyroid hormone signaling are enriched in orbital fat isolated from patients with TAO. SnRNA-Seq of orbital fibroblasts undergoing adipogenesis reveals differential expression of the adipocyte-specific genes FABP4/5, APOE, PPARG, and ADIPOQ during adipogenic differentiation. The insulin-like growth factor-1 receptor and Wnt signaling pathways appear to be enriched early in adipogenesis. Gene modules that are enriched in TAO orbital fat are upregulated in orbital adipocytes during differentiation in vitro, whereas genes that are enriched in control orbital fat are enriched in undifferentiated OFs. Conclusions: We identified pathways enriched in TAO orbital fat, and dynamic changes in gene expression that occur during adipogenic differentiation of orbital fibroblasts. These findings may help guide functional studies of genes and pathways critical for orbital adipogenesis.

Retinal homeostasis and metformin-induced protection are not affected by retina-specific Pparδ knockout.

Peroxisome proliferator-activated receptors (PPARs) are a family of three nuclear hormone receptors (PPARα, PPARδ, and PPARγ) that are known to regulate expression of lipid metabolism and oxidative stress genes. Given their role in reducing oxidative stress in a variety of tissues, these genes are likely important for retinal homeostasis. This hypothesis has been further supported by recent studies suggesting that PPAR-activating drugs are protective against retinal degenerations. The objective of the present study was to determine the role of PPARδ in the neuroretina. RNA-seq data show that Pparα and Pparδ are both expressed in the retina, but that Pparδ is expressed at 4-fold higher levels. Single-cell RNAseq data show that Pparδ is broadly expressed in all retinal cell types. To determine the importance of Pparδ to the retina, we generated retina-specific Pparδ knockout mice. We found that deletion of Pparδ had a minimal effect on retinal function or morphology out to 12 months of age and did not increase retinal sensitivity to oxidative stress induced by exposure to bright light. While data show that PPARδ levels were increased by the drug metformin, PPARδ was not necessary for metformin-induced protection from light damage. These data suggest that Pparδ either has a redundant function with Pparα or is not essential for normal neuroretina function or resistance to oxidative stress.

ASCOT identifies key regulators of neuronal subtype-specific splicing.

Public archives of next-generation sequencing data are growing exponentially, but the difficulty of marshaling this data has led to its underutilization by scientists. Here, we present ASCOT, a resource that uses annotation-free methods to rapidly analyze and visualize splice variants across tens of thousands of bulk and single-cell data sets in the public archive. To demonstrate the utility of ASCOT, we identify novel cell type-specific alternative exons across the nervous system and leverage ENCODE and GTEx data sets to study the unique splicing of photoreceptors. We find that PTBP1 knockdown and MSI1 and PCBP2 overexpression are sufficient to activate many photoreceptor-specific exons in HepG2 liver cancer cells. This work demonstrates how large-scale analysis of public RNA-Seq data sets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events.

Clarin-1 expression in adult mouse and human retina highlights a role of Müller glia in Usher syndrome.

Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Single-Cell RNA-Seq Analysis of Retinal Development Identifies NFI Factors as Regulating Mitotic Exit and Late-Born Cell Specification.

Precise temporal control of gene expression in neuronal progenitors is necessary for correct regulation of neurogenesis and cell fate specification. However, the cellular heterogeneity of the developing CNS has posed a major obstacle to identifying the gene regulatory networks that control these processes. To address this, we used single-cell RNA sequencing to profile ten developmental stages encompassing the full course of retinal neurogenesis. This allowed us to comprehensively characterize changes in gene expression that occur during initiation of neurogenesis, changes in developmental competence, and specification and differentiation of each major retinal cell type. We identify the NFI transcription factors (Nfia, Nfib, and Nfix) as selectively expressed in late retinal progenitor cells and show that they control bipolar interneuron and Müller glia cell fate specification and promote proliferative quiescence.

A Drug-Tunable Gene Therapy for Broad-Spectrum Protection against Retinal Degeneration.

Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of light-sensitive photoreceptors that impairs the vision of 9.1 million people in the US. An attractive treatment option is to use gene therapy to deliver broad-spectrum neuroprotective factors. However, this approach has had limited clinical translation because of the inability to control transgene expression. To address this problem, we generated an adeno-associated virus vector named RPF2 that was engineered to express domains of leukemia inhibitory factor fused to the destabilization domain of bacterial dihydrofolate reductase. Fusion proteins containing the destabilization domain are degraded in mammalian cells but can be stabilized with the binding of the drug trimethoprim. Our data show that expression levels of RPF2 are tightly regulated by the dose of trimethoprim and can be reversed by trimethoprim withdrawal. We further show that stabilized RPF2 can protect photoreceptors and prevent blindness in treated mice.

Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2.

Cloned type III secretion systems have much potential to be used for bacterial engineering purposes involving protein secretion and substrate translocation directly into eukaryotic cells. We have previously cloned the SPI-1 and SPI-2 type III systems from the Salmonella enterica serovar Typhimurium genome using plasmid R995 which can conveniently capture large genomic segments for transfer between bacterial strains. However, though expressed and functional in Salmonella strains, cloned SPI-1 was previously observed to have a serious expression defect in other Gram negative bacteria including Escherichia coli. Here we show that cloned SPI-1 expression and secretion can be detected in the secretion preps from E. coli and Citrobacter indicating the first observation of non-Salmonella SPI-1 expression. We describe a compatible plasmid system to introduce engineered SPI-1 substrates into cloned SPI-1 strains. However, a SPI-1 translocation defect is still observed in E. coli, and we show that this is likely due to a defect in SipB expression/secretion in this species. In addition, we also examined the requirement for the hilA and ssrAB regulators in the expression of cloned SPI-1 and SPI-2, respectively. We found a strict requirement for hilA for full cloned SPI-1 expression and secretion. However, though we found that ssrAB is required for full cloned SPI-2 expression in a range of media across different bacteria, it is not required for cloned SPI-2 expression in MgM8 inducing media in S. Typhimurium. This suggests that under SPI-2 inducing conditions in S. Typhimurium, other factors can substitute for loss of ssrAB in cloned SPI-2 expression. The results provide key foundational information for the future use of these cloned systems in bacteria.

Self-transmissible IncP R995 plasmids with alternative markers and utility for Flp/FRT cloning strategies.

The IncP plasmid R995 has been a useful self-transmissible, broad-host-range vector for a number of applications including the recombinase/conjugation-based cloning of large genomic DNA segments. However, R995 derivatives (or related plasmids) expressing a wide range of different resistance markers and Flp recombinase target sites do not exist in the literature. In addition, documented strategies for applying such plasmids in cloning applications that take advantage of conjugation for the convenient isolation and recovery of constructs are extremely limited. Here, we report a new series of R995 plasmids with alternative markers to increase options for applications in backgrounds already expressing resistance to a particular antibiotic(s). These R995 plasmids have been engineered to contain FRT sites that can be used for recombinase-based cloning. We demonstrate the utility of this approach by cloning 20 kb regions from the Salmonella Typhimurium and Escherichia coli genomes and by cloning DNA from an exogenous plasmid source. To our knowledge, this represents the first systematic engineering of an intact, self-transmissible IncP plasmid with a series of alternative antibiotic markers and FRT sites.