Biology subjects

Cepko, C.

Publications and source records attributed to Cepko, C..

14 recordsLinked to original sources

bioRxiv2026

Gene expression in Anolis sagrei suggests conservation of patterning mechanisms drives formation of two high acuity areas

As humans we enjoy high acuity color vision. A specialized area in our retinas, the fovea, endows us with this ability. Some vertebrates, such as the Anolis sagrei (brown anole), have two such areas. Whether different species use conserved developmental mechanism(s), and whether the same mechanism is used in one organism to pattern both foveae, are herein addressed in the brown anole. We observed that the expression of canonical axis patterning genes is conserved in the anole retina. Retinoic acid and Fgf signaling pathways were found to define two discrete domains corresponding to emerging foveae. Comparative spatial analyses of early gene expression patterns among anole, chick, and human revealed conserved patterns in the anlage of the high acuity areas of each species. Variation in foveal number could arise through spatial redeployment of conserved patterning programs.

developmental biology↗

bioRxiv2026

Distinct neurogenic progenitor cell populations balance cell type production in the embryonic mouse retina

Mammalian vision depends on the reliable production of over 100 neural cell types from multipotent retinal progenitor cells. How so many cell types are generated in the correct proportions throughout the retina remains incompletely understood. Focusing on the early embryonic period in mice, we found that retinal neurogenesis is organized through discrete neurogenic progenitor cell (NPC) populations with distinct fate biases. NPCs expressing Galanin primarily produce retinal ganglion cells and amacrine cells, whereas Olig2-expressing NPCs produce cones and horizontal cells in the same temporal window. Clonal analysis revealed that these two fate-biased NPC populations arise predominantly from asymmetric, renewing progenitor cell divisions that produce one fate-biased NPC daughter cell. Differential Notch signaling regulates the production of these NPC populations, with Galanin+ and Olig2+ NPCs representing NotchHigh and NotchLow states, respectively. Manipulating Notch signaling was sufficient to toggle their production. The expression of Notch signaling components across NPCs is consistent with a lateral inhibition mechanism, in which fate-biased NPCs promote neighboring cells to adopt a complementary fate-biased NPC state. These findings suggest that the robust production of diverse retinal cell types is achieved through local feedback that balances discrete, fate-biased NPC populations.

developmental biology↗

bioRxiv2026

Integration of in situ hybridization and scRNA-seq data provides a 2D topographical map of the developing retina across species

Precise regional patterning is fundamental to tissue organization, yet the spatial logic that governs it remains poorly defined for many tissues. In the vertebrate retina, molecular domains along the dorsoventral and nasotemporal axes provide positional cues for regional specializations such as the high-acuity area (HAA). We combined multiplexed in situ hybridization data with single-cell transcriptomic data to create quantitative two-dimensional maps of developing retinal cells. In the developing chicken retina, this approach resolved sharp expression boundaries of genes involved in patterning, and revealed novel candidates enriched in the anlagen of the HAA. Comparative analysis of chicken, mouse, and human data demonstrated conserved axis-based programs, but distinct fine-scale organization consistent with presence/absence of an HAA. Here, we show that spatial reconstruction from scRNA-seq data, anchored by experimental benchmarks, enables comparative 2D topographic mapping of gene expression across species and provides a generalizable strategy to investigate the spatial logic of molecular organization in developing tissues. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/697548v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@e9b84aorg.highwire.dtl.DTLVardef@4207deorg.highwire.dtl.DTLVardef@112bc79org.highwire.dtl.DTLVardef@113e1d3_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIQuantification of multiplexed RNA-FISH signals generated reproducible spatial expression maps in the developing chicken retina. C_LIO_LIResolution of relative boundaries for early patterning genes revealed nested expression domains marking the boundaries of the developing HAA. C_LIO_LIRNA-FISH experimental data using genes with axis-specific expression allowed the generation of DV and NT scores which enabled the localization of each cell in a scRNA-seq dataset onto a 2D topographical map of the developing retina. C_LIO_LI2D topographic maps derived from scRNA-seq data revealed novel HAA-enriched genes in the developing chicken retina. C_LIO_LICross-species comparisons showed conserved DV/NT programs but divergent fine-scale organization in retinal development. C_LIO_LICyp26c1 expression pattern was validated in human retina correlating with the location of the future HAA. C_LI

developmental biology↗

bioRxiv2025

Tbx genes influence early gene expression and photoreceptor patterning in the chick retina

The retina, our sensory organ for vision, displays distinct photoreceptor distributions and can have a specialized region for high visual acuity. The establishment of these spatial patterns during development depends on regionally restricted transcription factors and signaling molecules. Members of the T-box family of transcription factors, Tbx2, Tbx3, and Tbx5, show such restricted patterns of expression, with a dorsal high to ventral low pattern in the early retina. Their potential regulatory interactions and roles in patterning have not been fully explored. Here, we investigated their regulatory interactions by overexpression (OE) and knockdown (KD) approaches. We found that Tbx2, Tbx3, and Tbx5, directly or indirectly regulate each other as well as other early, patterned genes, including Fgf8, Cyp26a1, Cyp26c1, Cyp1b1, Raldh1, Ventroptin, and Bmp2. KD of any one of these Tbx genes increased rod photoreceptor density in a specific region of the retina, whereas Tbx2 loss additionally reduced the number of UV cones throughout the retina. Notably, KD of Tbx2, Tbx3, or Tbx5 consistently resulted in a smaller rod-free zone (RFZ), a domain within the high acuity area. These findings demonstrate that T-box transcription factors form a coordinated regulatory network that governs regional gene expression and photoreceptor patterning. HighlightsO_LITbx2, Tbx3, and Tbx5 regulate each other during chick retinal development C_LIO_LITbx2, Tbx3, and Tbx5 regulate the expression of early patterned genes, including Fg8, Cyp26c1, Cyp26a1, Cyp1b1, and Bmp2 C_LIO_LIKnockdown of either Tbx2/3/5 increased rod numbers in the equatorial retina and reduced the size of the rod-free zone (RFZ) C_LIO_LITbx2 knockdown decreased UV cone numbers dorsally, ventrally and in the RFZ C_LIO_LITbx2, Tbx3, and Tbx5 had no impact on green or red cone abundance C_LI

developmental biology↗

bioRxiv2025

Programmable orthogonal and rapid DNA strand displacement for fluidic-exchange-free highly multiplexed fluorescent imaging

Multiplexed fluorescent imaging methods are essential for studying cellular function by visualization of biomolecules in cells and tissues with high-resolution spatial information, but most suffer from limitations such as low multiplexity because of spectral overlap between the used fluorophores. Fluidic exchange based sequential multiplexed imaging overcomes this limitation but requires time-consuming incubation and washing steps. We introduce a novel multiplexed fluorescent imaging method that uses rapid, orthogonal DNA strand displacement reactions to enable unlimited multiplexed fluorescent imaging without fluidic exchange. The signal switching between targets is achieved by strand displacement with added non-fluorescent DNA displacer strands, no fluidic washing step is required for sequential multiplexed imaging. We experimentally screened a set of rapid and orthogonal DNA displacement sequences for probe design and applied it for RNA imaging, which takes less than 30 seconds per round to complete in fixed cells. Because of the vast sequence design space of DNA probes, theoretically unlimited multiplexity can be achieved. Using 25 developed rapid orthogonal probes, we achieved 25-plex RNA imaging in a single fluorophore channel in fixed cells within 20 minutes. To further demonstrate robustness and practical usage of DIRSE-based imaging, we showed 24-plex RNA imaging with the method in retinal tissues and resolved different cell types. This DIRSE mechanism significantly simplifies the high-plex fluorescent imaging process with pre-programmed DNA probes and has broad biotechnical applications for future medicine and diagnostics.

bioengineering↗

bioRxiv2025

AAV NRF2 Gene Therapy Preserves Retinal Structure and Function in Rodent Models of Oxidative Damage

Dry age-related macular degeneration is the most frequent cause of visual impairment in individuals over age 50 in developed countries. It is characterized by subretinal deposits of oxidized proteins and lipids and results in progressive loss of high acuity vision. One major risk factor is smoking, which causes oxidative stress in many tissues, including the eye. We previously showed that an adeno-associated viral vector expressing human NRF2 (AAV8/Best1-Nrf2), a transcription factor that regulates responses to oxidative damage, slowed degeneration in mouse models of another blinding disorder, retinitis pigmentosa, which also includes oxidative stress. Here, our AAV8/Best1-Nrf2 vector was tested in a model of oxidative stress wherein sodium iodate was injected systemically, as this is often used to model dry age-related macular degeneration. Sodium iodate causes acute oxidative damage to supporting cells of the retina, the retinal pigment epithelial cells, and ultimately leads to photoreceptor death. Subretinal injection of AAV8/Best1-Nrf2 led to protection of the retinal pigment epithelium and photoreceptors, as well as preservation of visual function, in rat and mouse sodium iodate models. AAV8/Best1-Nrf2 may serve as an effective gene-agnostic therapy for diseases with oxidative stress, including dry age-related macular degeneration. ETOCOxidative stress occurs in diseases that lead to vision loss, including age-related macular degeneration (AMD). AMD is commonly modeled by injection of sodium iodate (S.I.), an oxidizing agent. AAV-mediated delivery of Nrf2, a transcription factor that regulates oxidative stress, protects the eye from S.I. damage in rat and mouse models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/663952v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@357e57org.highwire.dtl.DTLVardef@1f9986dorg.highwire.dtl.DTLVardef@186537corg.highwire.dtl.DTLVardef@1d31aad_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

High temporal frequency light response in mouse retina is mediated by ON and OFF bipolar cells and requires FAT3 signaling

Vision is initiated by the reception of light by photoreceptors and subsequent processing via downstream retinal neurons. Proper cellular organization depends on the multi-functional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here we investigated the retinal function of Fat3 mutant mice and found decreases in physiological and perceptual responses to high frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. The role of FAT3 in the response to high temporal frequency flashes depends upon its ability to transduce an intracellular signal. Mechanistically, FAT3 binds to the synaptic protein PTP{sigma}, intracellularly, and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. These findings expand the repertoire of FAT3s functions and reveal its importance in bipolar cells for high frequency light response.

neuroscience↗

bioRxiv2021

Retinoic acid signaling mediates peripheral cone photoreceptor survival in a mouse model of retina degeneration

Retinitis Pigmentosa (RP) is a wide array of progressive, debilitating visual disorders caused by mutations in a diverse set of genes. In both human patients and mouse models of RP, rod photoreceptor dysfunction leads to loss of night vision, and is followed by secondary cone photoreceptor dysfunction and degeneration, leading to loss of daylight color vision. A strategy to prevent secondary cone death could provide a generalized RP therapy to preserve daylight color vision regardless of the underlying mutation. In mouse models of RP, cones in the far peripheral retina survive long-term, despite complete rod loss. The mechanism for such peripheral cone survival had not been explored. Here, we found that active retinoic acid (RA) signaling in peripheral Muller glia is both sufficient and necessary for the extended cone survival. RA depletion by conditional knockout of RA synthesis enzymes, or overexpression of an RA degradation enzyme, abrogated peripheral cone survival. Conversely, constitutive activation of RA signaling in the central retina promoted long-term cone survival. These results indicate that RA signaling mediates the prolonged peripheral cone survival in the rd1 mouse model of retinal degeneration, and provide a basis for a generic strategy for cone survival in the many diseases that lead to loss of cone-mediated vision.

developmental biology↗

bioRxiv2021

Spatiotemporal patterns of neuronal subtype genesis suggest hierarchical development of retinal diversity

How do neuronal subtypes emerge during development? Recent molecular studies have expanded our knowledge of existing neuronal diversity. However, the genesis of neuronal subtypes remains elusive and previous studies have been limited by a lack of quantitative methods for simultaneous detection of subtype diversity in situ. The bipolar interneurons of the mammalian retina represent a diverse neuronal class, characterized by distinct functions, morphologies, and recently discovered transcriptional profiles. Here, we developed a comprehensive spatiotemporal map of bipolar subtype genesis in the retina. Combining multiplexed detection of 16 RNA markers with timed delivery of EdU and BrdU, we analyzed more than 30,000 single cells in full retinal sections to classify all bipolar subtypes and their birthdates. We found that bipolar subtype birthdates are ordered and follow a centrifugal developmental axis. Spatial analysis revealed a striking oscillatory wave pattern of bipolar subtype birthdates, and lineage analyses suggest clonal restriction on homotypic subtype production. These results inspired a hierarchical model of neuronal subtype genesis in the mammalian retina, with the wave pattern of subtype birthdates arising from early asymmetric cell divisions among founding retinal progenitor cells. Our results provide an outline of the developmental logic that generates diverse neuronal subtypes, and establishes a framework for studying subtype diversification.

developmental biology↗

bioRxiv2020

Otx2 and Oc1 directly regulate the transcriptional program of cone photoreceptor development

The vertebrate retina is a highly organized structure of approximately 110 cell types. Retinal progenitor cells (RPCs) produce these cell types in a temporal order that is highly conserved. While some RPCs produce many cell types, some terminally dividing RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). Here, we compared the transcriptomes and chromatin profiles of such a restricted cone/HC RPC with those of other RPCs. We identified many cis-regulatory modules (CRMs) active in cone/HC RPCs and developing cones. We then showed that Otx2 and Oc1 directly regulate the activity of multiple CRMs genome-wide, including near genes important for cone development, such as Rxrg and Neurod1. In addition, we found that Otx2 regulates itself. These results suggest that Otx2 and Oc1 have a broader role than previously appreciated, and deepen our understanding of retinal development, which may benefit therapies for retinal diseases.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

A Simple Enhancement for Gibson Isothermal Assembly

Gibson Isothermal Assembly has become a widespread cloning method, with a multitude of advantages over traditional cut-and-paste cloning. It allows for scarless assembly of multiple fragments simultaneously and has become widely used for molecular cloning. We have found that a simple change to the formulation of the reaction mix, the addition of a single-stranded DNA binding protein, can substantially improve both the accuracy and efficiency of assembly, especially as the number of fragments being assembled increases. In addition, when creating this Enhanced Gibson Isothermal Assembly reaction mix in-house with homemade DNA ligase, the cost of the reaction can be reduced to less than $10 per milliliter.

molecular biology↗

bioRxiv2020

A high-density narrow-field inhibitory retinal interneuron with direct coupling to Müller glia

Amacrine cells are interneurons comprising the most diverse cell type in the mammalian retina. They help encode visual features such as edges or directed motion by mediating excitatory and inhibitory interactions between input (i.e. bipolar) and output (i.e. ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, neurovascular control and metabolic regulation. Here, we report that a previously poorly characterized, but relatively abundant, inhibitory amacrine cell type in the mouse retina is coupled directly to Muller glia. Electron microscopic reconstructions of this amacrine type revealed extensive associations with Muller glia, whose processes often completely ensheathe the neurites of this amacrine cell type. Microinjections of small tracer molecules into the somas of these amacrine cells led to selective labelling of nearby Muller glia, leading us to suggest the name "Muller glia-coupled amacrine cell" or MAC. Our electrophysiological data also indicate that MACs release glycine at conventional chemical synapses with amacrine, bipolar and retinal ganglion cells (RGCs), and viral transsynaptic tracing showed connections to several known RGC types. Visually-evoked responses revealed a strong preference for light increments; these "ON" responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling to other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function. Significance StatementGap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system, and play a myriad of roles including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells are rare and poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Muller glia.

neuroscience↗

bioRxiv2019

Cell type- and stage-specific expression of Otx2 is coordinated by a cohort of transcription factors and multiple cis-regulatory modules in the retina

Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is an advantageous strategy. However, the mechanisms that control the activation of TFs in different cell types and at different stages of development remain unclear. The neural retina serves as a model of the development of a complex tissue. We used this system to analyze how expression of the homeobox TF, Orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during retinogenesis. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. These include mature bipolar interneurons, photoreceptors, and retinal progenitor/precursor cells. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages, as revealed by ATAC-seq and DNase-seq analyses. We conclude that retinal cells utilize a cohort of TFs with different expression patterns, and multiple CRMs with different chromatin configurations, to precisely regulate the expression of Otx2 in a cell type- and stage-specific manner in the retina.

developmental biology↗

bioRxiv2019

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.

neuroscience↗