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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC Davis Electronic Theses and Dissertations (2021)

The retina is the light sensitive tissue at the back of the eye that processes the essential first steps of vision and contains several populations of neurons. Retinal Ganglion Cell (RGC) axons form the optic nerve, which is the only connection between the retina and the brain. Glaucoma is a leading cause of irreversible vision loss, affecting millions of people annually, and advanced stage glaucoma patients are blinded as a result of RGC degeneration. Since the human central nervous system possesses no functional regenerative capacity, there is ample interest in developing cell replacement therapies for optic neuropathies like glaucoma. In this dissertation, I describe work that I accomplished with my colleagues, both unpublished and published, that is relevant to human medicine and basic science alike. In my unpublished work, I generated a novel induced Pluripotent Stem Cell line that labels RGCs with a green fluorescent protein and attempted to engineer these cells with an inducible transgenic platform to expand the production of RGCs, but discovered potent transgenic silencing. My published work covers a range of topics; with colleagues, I developed a novel red fluorescent protein RGC reporter mouse that labels 70% of RGCs in the mouse retina from the earliest points of RGC development through adulthood. Additionally, this reporter can be visualized live and, since it uses a red fluorescent protein, can easily be combined with any green fluorescent protein reporter without crossing fluorescent signals. I have also shown that an endogenous glial cell of the retina, Müller glia, secrete factors that promote stem cell-derived RGC survival in vitro, which may help identify methods to promote transplanted cell survival in future therapies. I analyze the landscape of microRNA (miRNA) expression in the developing non-human primate fovea with the La Torre lab to study how the fovea, which is a region of the retina essential for acute human vision, develops. Finally, with my mentor Dr. Anna La Torre, I review the state of the RGC transplantation field to identify areas that require technical achievement to grow closer to establishing a reliable method of RGC transplantation in vivo.

    Cover page: MicroRNAs in Early Retinal Development and Their Application in Retinal Organoids to Treat Retinal Ganglion Cell Degenerative Conditions
    • Thesis
    • Peer Reviewed
    UC Davis Electronic Theses and Dissertations (2024)

    Sight relies on the integrity of the photoreceptor cells of the retina (rods and cones), and damage to these cells results in irreversible vision loss. Several degenerative diseases affect cones specifically, including age-related macular degeneration (AMD), one of the leading causes of blindness worldwide. A promising therapeutic strategy to restore vision in these patients is photoreceptor replacement, but our ability to produce cones in vitro is limited. Humans and other primates have a specialized region of the central retina called the fovea—the region affected by AMD—that contains the highest density of cones. This cone dominance is a sharp contrast to the majority of the retina, where rods outnumber cones 20:1. All the neurons of the retina (including rods and cones) are born from a single population of multipotent progenitors. However, the molecular mechanisms that dictate why the foveal progenitors differentiate into high yields of cones and not rods are unknown. A more complete understanding of these factors can be used to optimize existing stem cell protocols and increase cone production in vitro. In my collection of published work, I explored various molecular mechanisms that contribute to neural fate specification, including molecules of the retinoic acid pathway and microRNAs (miRNAs). My research focused primarily in the retina. I began by reviewing foveal development and curating the current knowledge of the steps towards its specification and maturation. The mechanisms driving foveal development are widely unknown, so I sought to study the fovea in the human and nonhuman primate. The La Torre lab is just three miles down the road from the California National Primate Research Center, priming us for access to nonhuman primate tissue across gestation. With these rhesus macaque tissues, I characterized retinal development and identified key stages of neurogenesis. I also showed that the expression patterns of molecules in and associated with the retinoic acid pathway, including CPY26A and FGF8, are not conserved between chicken and primate. Whereas in the chicken retina, these molecules contribute to the specification of the high acuity area, they are not fovea-specific in the primate retina. I continued to explore the role of miRNAs in fate specification through analyzing of the miRNA landscape across the developing nonhuman primate retina. I further validated those miRNAs found to be highly differentially expressed in the human retina. I reviewed oscillation patterns of miRNAs and the role in retinal development. Given that the cortex shares a similar developmental pattern with the retina, also having a progenitor population that all the neurons descend from in a stereotypic sequence, I explored how the Notch pathway and miRNAs influence fate specification in the mouse brain. Together, these studies identify a subset of the molecular mechanisms that drive neural differentiation and will inform future research on increasing the cone production in stem cell protocols with an ultimate goal of restoring vision in patients affected by optic neuropathies.

      Cover page: A multi-species investigation of neural fate specification
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2022)
      The dentate gyrus (DG) is an essential part of the hippocampal formation and participates in the majority of hippocampal functions. The DG is also one of the few structures in the mammalian central nervous system that produces adult-born neurons and, in humans, alterations in adult neurogenesis are associated with stress and depression. Given the importance of DG in hippocampal function, it is imperative to understand the molecular mechanisms driving DG development and homeostasis. The E3 ubiquitin ligase Cullin-5/RBX2 (CRL5) is a multiprotein complex involved in neuron migration and localization in the nervous system, but its role during development and in the adult DG remain elusive. Here, we show that CRL5 participates in mossy fiber pruning, DG layering, adult neurogenesis, and overall physical activity in mice. During DG development, RBX2 depletion causes an overextension of the DG mossy fiber infrapyramidal bundle (IPB). We further demonstrate that the increased activity in Reelin/DAB1 or ARF6 signaling, observed in RBX2 knockout mice, is not responsible for the lack of IPB pruning. Knocking out RBX2 also affects granule cell and neural progenitor localization and these defects were rescued by downregulating the Reelin/DAB1 signaling. Finally, we show that absence of RBX2 increases the number neural progenitors and adult neurogenesis. Importantly, RBX2 knockout mice exhibit higher levels of physical activity, uncovering a potential mechanism responsible for the increased adult neurogenesis in the RBX2 mutant DG. Overall, we present evidence of CRL5 regulating mossy fiber pruning and layering during development and opposing adult neurogenesis in the adult DG.
        Cover page: The E3 Ubiquitin Ligase CRL5 Regulates Dentate Gyrus Morphogenesis, Adult Neurogenesis, and Animal Behavior
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2024)
        The fovea centralis (fovea) is a specialized region of the primate retina that plays crucial roles in high-resolution visual acuity and color perception. The fovea is characterized by a high density of cone photoreceptors and no rods, and unique anatomical properties that contribute to its remarkable visual capabilities. Early histological analyses identified some of the key events that contribute to foveal development, but the mechanisms that direct the specification of this area are not understood. Recently, the expression of the retinoic acid-metabolizing enzyme CYP26A1 has become a hallmark of some of the retinal specializations found in vertebrates, including the primate fovea and the high-acuity area in avian species. In chickens, the retinoic acid pathway regulates the expression of FGF8 to then direct the development of a rod-free area. Similarly, high levels of CYP26A1, CDKN1A, and NPVF expression have been observed in the primate macula using transcriptomic approaches. However, which retinal cells express these genes and their expression dynamics in the developing primate eye remain unknown. Here, we systematically characterize the expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF during the development of the rhesus monkey retina, from early stages of development in the first trimester until the third trimester (near term). Our data suggest that some of the markers previously proposed to be fovea-specific are not enriched in the progenitors of the rhesus monkey fovea. In contrast, CYP26A1 is expressed at high levels in the progenitors of the fovea, while it localizes in a subpopulation of macular Müller glia cells later in development. Together these data provide invaluable insights into the expression dynamics of several molecules in the nonhuman primate retina and highlight the developmental advancement of the foveal region.
          Cover page: Expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF in the developing rhesus monkey retina
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2019)
          Brn3b (Pou4f2) is a class-4 POU domain transcription factor known to play central roles in the development of different neuronal populations of the Central Nervous System, including retinal ganglion cells (RGCs), the neurons that connect the retina with the visual centers of the brain. Here, we have used CRISPR-based genetic engineering to generate a Brn3b-mCherry reporter mouse without altering the endogenous expression of Brn3b. In our mouse line, mCherry faithfully recapitulates normal Brn3b expression in the retina, the optic tracts, the midbrain tectum, and the trigeminal ganglia. The high sensitivity of mCherry also revealed novel expression of Brn3b in the neuroectodermal cells of the optic stalk during early stages of eye development. Importantly, the fluorescent intensity of Brn3b-mCherry in our reporter mice allows for noninvasive live imaging of RGCs using Scanning Laser Ophthalmoscopy (SLO), providing a novel tool for longitudinal monitoring of RGCs.
            Cover page: A Novel Reporter Mouse Uncovers Endogenous Brn3b Expression
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2020)
            The small GTPase ARL4C participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The ARL4C signaling cascade starts by the recruitment of the ARF-GEF cytohesins to the plasma membrane, which, in turn, bind and activate the small GTPase ARF6. However, the role of ARL4C-cytohesin-ARF6 signaling during hippocampal development remains elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 (CRL5) controls the stability of ARL4C and its signaling effectors to regulate hippocampal morphogenesis. Both RBX2 knockout and Cullin 5 knockdown cause hippocampal pyramidal neuron mislocalization and development of multiple apical dendrites. We used quantitative mass spectrometry to show that ARL4C, Cytohesin-1/3, and ARF6 accumulate in the RBX2 mutant telencephalon. Furthermore, we show that depletion of ARL4C rescues the phenotypes caused by Cullin 5 knockdown, whereas depletion of CYTH1 or ARF6 exacerbates overmigration. Finally, we show that ARL4C, CYTH1, and ARF6 are necessary for the dendritic outgrowth of pyramidal neurons to the superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered ARL4C, CYTH1, and ARF6 as CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.
              Cover page: CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2022)

              Purpose

              To compare the timing and efficiency of the development of Macaca mulatta, a nonhuman primate (NHP), induced pluripotent stem cell (rhiPSC) derived retinal organoids to those derived from human embryonic stem cells (hESCs).

              Results

              Generation of retinal organoids was achieved from both human and several NHP pluripotent stem cell lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle-like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had laminated structure and were composed of mature retinal cell types including cone and rod photoreceptors. Single-cell RNA sequencing was conducted at two time points; this allowed identification of cell types and developmental trajectory characterization of the developing organoids. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While the culture of NHP-derived iPSCs is relatively difficult compared to that of human stem cells, the generation of retinal organoids from NHP iPSCs is feasible and may be less time-consuming due to an intrinsically faster timing of retinal differentiation.

              Conclusions

              Retinal organoids produced from rhesus monkey iPSCs using established protocols differentiate through the stages of organoid development faster than those derived from human stem cells. The production of NHP retinal organoids may be advantageous to reduce experimental time for basic biology studies in retinogenesis as well as for preclinical trials in NHPs studying retinal allograft transplantation.
                Cover page: Retinal organoids derived from rhesus macaque iPSCs undergo accelerated differentiation compared to human stem cells
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2021)
                Rod and cone photoreceptors differ in their shape, photopigment expression, synaptic connection patterns, light sensitivity, and distribution across the retina. Although rods greatly outnumber cones, human vision is mostly dependent on cone photoreceptors since cones are essential for our sharp visual acuity and color discrimination. In humans and other primates, the fovea centralis (fovea), a specialized region of the central retina, contains the highest density of cones. Despite the vast importance of the fovea for human vision, the molecular mechanisms guiding the development of this region are largely unknown. MicroRNAs (miRNAs) are small post-transcriptional regulators known to orchestrate developmental transitions and cell fate specification in the retina. Here, we have characterized the transcriptional landscape of the developing rhesus monkey retina. Our data indicates that non-human primate fovea development is significantly accelerated compared to the equivalent retinal region at the other side of the optic nerve head, as described previously. Notably, we also identify several miRNAs differentially expressed in the presumptive fovea, including miR-15b-5p, miR-342-5p, miR-30b-5p, miR-103-3p, miR-93-5p as well as the miRNA cluster miR-183/-96/-182. Interestingly, miR-342-5p is enriched in the nasal primate retina and in the peripheral developing mouse retina, while miR-15b is enriched in the temporal primate retina and increases over time in the mouse retina in a central-to-periphery gradient. Together our data constitutes the first characterization of the developing rhesus monkey retinal miRNome and provides novel datasets to attain a more comprehensive understanding of foveal development.
                  Cover page: MicroRNA Signatures of the Developing Primate Fovea
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2023)
                  Chronic neurodegeneration and acute injuries lead to neuron losses via diverse processes. We compared retinal ganglion cell (RGC) responses between chronic glaucomatous conditions and the acute injury model. Among major RGC subclasses, αRGCs and intrinsically photosensitive RGCs (ipRGCs) preferentially survive glaucomatous conditions, similar to findings in the retina subject to axotomy. Focusing on an αRGC intrinsic factor, Osteopontin (secreted phosphoprotein 1 [Spp1]), we found an ectopic neuronal expression of Osteopontin (Spp1) in other RGCs subject to glaucomatous conditions. This contrasted with the Spp1 downregulation subject to axotomy. αRGC-specific Spp1 elimination led to significant αRGC loss, diminishing their resiliency. Spp1 overexpression led to robust neuroprotection of susceptible RGC subclasses under glaucomatous conditions. In contrast, Spp1 overexpression did not significantly protect RGCs subject to axotomy. Additionally, SPP1 marked adult human RGC subsets with large somata and SPP1 expression in the aqueous humor correlated with glaucoma severity. Our study reveals Spp1's role in mediating neuronal resiliency in glaucoma.
                    Cover page: Osteopontin drives retinal ganglion cell resiliency in glaucomatous optic neuropathyCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2017)

                    Purpose

                    Previous studies have demonstrated the ability of retinal cells derived from human embryonic stem cells (hESCs) to survive, integrate into the host retina, and mediate light responses in murine mouse models. Our aim is to determine whether these cells can also survive and integrate into the retina of a nonhuman primate, Saimiri sciureus, following transplantation into the subretinal space.

                    Methods

                    hESCs were differentiated toward retinal neuronal fates using our previously published technique and cultured for 60 to 70 days. Differentiated cells were further treated with 20 μM N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) for a period of 5 days immediately prior to subretinal transplantation. Differentiated cells were labeled with a lentivirus expressing GFP. One million cells (10,000 cells/μL) were injected into the submacular space into a squirrel monkey eye, using an ab externo technique.

                    Results

                    RetCam imaging demonstrated the presence and survival of human donor cells 3 months after transplantation in the S. sciureus eye. Injected cells consolidated in the temporal macula. GFP+ axonal projections were observed to emanate from the central consolidation of cells at 1 month, with some projecting into the optic nerve by 3 months after transplantation.

                    Conclusions

                    Human ES cell-derived retinal neurons injected into the submacular space of a squirrel monkey survive at least 3 months postinjection without immunosuppression. Some donor cells appeared to integrate into the host inner retina, and numerous donor axonal projections were noted throughout, with some projecting into the optic nerve.

                    Translational relevance

                    These data illustrate the feasibility of hESC-derived retinal cell replacement in the nonhuman primate eye.
                      Cover page: Transplantation of Human Embryonic Stem Cell-Derived Retinal Cells into the Subretinal Space of a Non-Human PrimateChao et al.
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