Biology subjects

Santiago, C. P.

Publications and source records attributed to Santiago, C. P..

8 recordsLinked to original sources

bioRxiv2025

Blocking apoptosis promotes survival and alters developmental dynamics of human retinal ganglion cells in retinal organoids

Retinal ganglion cells (RGCs) are the projection neurons that transmit visual information from the retina to the brain. In many species, a substantial proportion of RGCs are eliminated by programmed cell death during development to regulate their final number, but how cell death impacts human RGC development remains poorly understood. Here, we characterized the timing and cell-type-specificity of cell death in human fetal retinas and retinal organoids. Both retinas and organoids exhibited two waves of apoptosis: an early wave targeting neurogenic retinal progenitor cells and neuronal precursors, and a late wave affecting RGCs and other neurons. Additionally, organoids displayed a distinct wave of necrosis. To investigate how the apoptotic waves affect retinal development, we differentiated human BAX/BAK double mutant organoids deficient in apoptosis. In these mutants, RGC lifespan and survival increased, while RGC neurogenesis and maturation were delayed. Thus, developmental apoptosis controls not only the quantity of RGCs but also their developmental dynamics. Together, our results highlight the roles of apoptosis in human RGC development and the challenges in retinal organoid design. Addressing these limitations will improve the utility of organoids for studying human retinal development and modeling optic neuropathies like glaucoma.

developmental biology↗

bioRxiv2022

Cellular and Fibrillar Collagen Analyses in an Animal Model of Retinal Detachment-Related Proliferative Vitreoretinopathy Reveals a Defined Transition to Chronic Fibrosis

PurposeProliferative 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. MethodsRRD-PVR was induced in adult Dutch Belted rabbits. Posterior segments of enucleated globes were fixed or processed for RNA-Seq at 6 hours and 2, 7, 14, and 35 days post-induction. Histochemical staining and immunolabeling for glial fibrillary acidic protein (GFAP), alpha smooth muscle actin (SMA), vascular endothelial growth factor receptor 2 (VEGFR2), CD68, and retinal pigment epithelium 65 kDa protein (RPE65) were performed, and labeling intensity was scored. Single cell RNA sequencing was performed. ResultsAcute histopathologic 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 Muller glia and microglia, consistent with immunolabeling. Expression of several fibrillar collagen genes were upregulated in chronic lesions. ConclusionsHistologic and transcriptional features of this rabbit model simulate important features of human RRD-PVR, including the transition to chronic intra and periretinal fibrosis. This high-fidelity in vivo model of RRD-PVR will enable further research on targeted treatment interventions.

pathology↗

bioRxiv2022

Comparative analysis of single-cell and single-nucleus RNA-sequencing in a rabbit model of retinal detachment-related proliferative vitreoretinopathy.

Structured AbstractO_ST_ABSPurposeC_ST_ABSProliferative vitreoretinopathy (PVR) is the most common cause of failure of retinal reattachment surgery and the molecular changes leading to this aberrant wound healing process is currently unknown. We aimed to study PVR pathogenesis using single-cell transcriptomics to dissect cellular heterogeneity in a rabbit PVR model. MethodsPVR was induced unilaterally in Dutch Belted rabbits. At different timepoints following PVR induction, retinas were dissociated into either cells or nuclei suspension and processed for single-cell or single-nucleus RNA sequencing (scRNA-seq or snRNA-seq). ResultsscRNA-Seq and snRNA-Seq were conducted on retinas at 4 hours and 14 days after disease induction. While the capture rate of unique molecular identifiers (UMI) 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 two sequencing modalities is the cell type capture rate, however, with glial cell types over-represented in scRNA-seq, and inner retinal neurons were enriched by snRNA-seq. Furthermore, fibrotic Muller glia were over-represented in snRNA-seq samples, while reactive Muller glia were in scRNA-seq samples. Trajectory analyses were similar between the two methods, allowing for the combined analysis of the scRNA-seq and snRNA-seq datasets. ConclusionsThese findings highlight limitations of both scRNA-seq and snRNA-seq analysis and imply that use of both techniques can more accurately identify transcriptional networks critical for aberrant fibrogenesis in PVR.

neuroscience↗

bioRxiv2021

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

Gene regulatory networks (GRNs), consisting of transcription factors and their target cis- regulatory sequences, control neurogenesis and cell fate specification in the developing central nervous system, but their organization is poorly characterized. In this study, we performed integrated single-cell RNA- and scATAC-seq analysis in both mouse and human retina to profile dynamic changes in gene expression, chromatin accessibility and transcription factor footprinting during retinal neurogenesis. We identified multiple interconnected, evolutionarily-conserved GRNs consisting of cell type-specific transcription factors that both activate expression of genes within their own network and often inhibit expression of genes in other networks. These GRNs control state transitions within primary retinal progenitors that underlie temporal patterning, regulate the transition from primary to neurogenic progenitors, and drive specification of each major retinal cell type. We confirmed the prediction of this analysis that the NFI transcription factors Nfia, Nfib, and Nfix selectively activate expression of genes that promote late-stage temporal identity in primary retinal progenitors. We also used GRNs to identify additional transcription factors that promote (Insm1/2) and inhibit (Tbx3, Tcf7l1/2) rod photoreceptor specification in postnatal retina. This study provides an inventory of cis- and trans-acting factors that control retinal development, identifies transcription factors that control the temporal identity of retinal progenitors and cell fate specification, and will potentially guide cell-based therapies aimed at replacing retinal neurons lost due to disease.

neuroscience↗

bioRxiv2021

Transcriptomic profiling of human orbital fat and differentiating orbital fibroblasts

Structured AbstractO_ST_ABSPurposeC_ST_ABSOrbital fat hyperplasia has a central role in the manifestations of thyroid-associated orbitopathy (TAO). To better understand the pathways involved in adipogenesis in TAO, we have used transcriptomic methods to analyze gene expression in control and TAO patients, as well as in differentiating orbital fibroblasts (OFs). MethodsWe performed bulk RNA sequencing (RNA-Seq) on intraconal orbital fat to compare gene expression in control and TAO patients. We treated cultured OFs derived from TAO patients with media containing dexamethasone, insulin, rosiglitazone, and isobutylmethylxanthine (IBMX) to induce adipogenesis. We used single nuclear RNA-Seq (snRNA-Seq) profiling of treated OFs to compare gene expression over time in order to identify pathways that are involved in orbital adipogenesis in vitro and compared the dynamic patterns of gene expression identify differences in gene expression in control and TAO orbital fat. ResultsOrbital fat from TAO and control patients segregate with principal component analysis (PCA). Numerous signaling pathways are enriched in orbital fat isolated from TAO patients. SnRNA-Seq of orbital fibroblasts undergoing adipogenesis reveals differential expression of adipocyte-specific genes over the developmental time course. Furthermore, genes that are enriched in TAO orbital fat are also upregulated in orbital adipocytes that differentiate in vitro, while genes that are enriched in control orbital fat are enriched in orbital fibroblasts prior to differentiation. ConclusionsDifferentiating orbital fibroblasts serve as a model to study orbital fat hyperplasia seen in TAO. We demonstrate that the insulin-like growth factor-1 receptor (IGF-1R) and Wnt signaling pathways are differentially expressed early in orbital adipogenesis. PrecisTo understand the pathways involved in adipogenesis in TAO, we used transcriptomic methods to analyze gene expression in control and TAO patients, as well as in differentiating OFs. We demonstrate that the IGF-1R and Wnt signaling pathways are differentially expressed during orbital adipogenesis.

molecular biology↗

bioRxiv2021

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, 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.

developmental biology↗

bioRxiv2018

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 allows researchers to summarize, visualize, and query alternative splicing patterns in public RNA-Seq data. ASCOT enables rapid identification of splice-variants across tens of thousands of bulk and single-cell RNA-Seq datasets in human and mouse. To demonstrate the utility of ASCOT, we first focused on the nervous system and identified many alternative exons used only by a single neuronal subtype. We then leveraged datasets from the ENCODE and GTEx consortiums to study the unique splicing patterns of rod photoreceptors and found that PTBP1 knockdown combined with overexpression of MSI1 and PCBP2 activates rod-specific exons in HepG2 liver cancer cells. Furthermore, we observed that MSI1 targets intronic UAG motifs proximal to the 5 splice site and interacts synergistically with PTBP1 downregulation. Finally, we show that knockdown of MSI1 in the retina abolishes rod-specific splicing. This work demonstrates how large-scale analysis of public RNA-Seq datasets can yield key insights into cell type-specific control of RNA splicing and underscores the importance of considering both annotated and unannotated splicing events. ASCOT splicing and gene expression data tables, software, and interactive browser are available at http://ascot.cs.jhu.edu.

neuroscience↗

bioRxiv2018

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.

bioengineering↗