High-efficiency base editing in the retina in primates and human tissues.

Stargardt disease is a currently untreatable, inherited neurodegenerative disease that leads to macular degeneration and blindness due to loss-of-function mutations in the ABCA4 gene. We have designed a dual adeno-associated viral vector encoding a split-intein adenine base editor to correct the most common mutation in ABCA4 (c.5882G>A, p.

A genome-wide scan of non-coding RNAs and enhancers for refractive error and myopia.

Refractive error (RE) and myopia are complex polygenic conditions with the majority of genome-wide associated genetic variants in non-exonic regions. Given this, and the onset during childhood, gene-regulation is expected to play an important role in its pathogenesis. This prompted us to explore beyond traditional gene finding approaches.

Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue.

Pyramidal neurons form active, transient, multilayered circuits perturbed by autism-associated mutations at the inception of neocortex.

Cortical circuits are composed predominantly of pyramidal-to-pyramidal neuron connections, yet their assembly during embryonic development is not well understood. We show that mouse embryonic Rbp4-Cre cortical neurons, transcriptomically closest to layer 5 pyramidal neurons, display two phases of circuit assembly in vivo. At E14.

Rare variant analyses across multiethnic cohorts identify novel genes for refractive error.

Refractive error, measured here as mean spherical equivalent (SER), is a complex eye condition caused by both genetic and environmental factors. Individuals with strong positive or negative values of SER require spectacles or other approaches for vision correction. Common genetic risk factors have been identified by genome-wide association studies (GWAS), but a great part of the refractive error heritability is still missing.

Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq.

We present FLASH-seq (FS), a full-length single-cell RNA sequencing (scRNA-seq) method with increased sensitivity and reduced hands-on time compared to Smart-seq3. The entire FS protocol can be performed in ~4.5 hours, is simple to automate and can be easily miniaturized to decrease resource consumption.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry.

Restoring light sensitivity using tunable near-infrared sensors.

Enabling near-infrared light sensitivity in a blind human retina may supplement or restore visual function in patients with regional retinal degeneration. We induced near-infrared light sensitivity using gold nanorods bound to temperature-sensitive engineered transient receptor potential (TRP) channels. We expressed mammalian or snake TRP channels in light-insensitive retinal cones in a mouse model of retinal degeneration.

Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans.

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies.

Whole-Brain Functional Ultrasound Imaging Reveals Brain Modules for Visuomotor Integration.

Large numbers of brain regions are active during behaviors. A high-resolution, brain-wide activity map could identify brain regions involved in specific behaviors. We have developed functional ultrasound imaging to record whole-brain activity in behaving mice at a resolution of ∼100 μm.

Virus stamping for targeted single-cell infection in vitro and in vivo.

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces.

Distinct subcomponents of mouse retinal ganglion cell receptive fields are differentially altered by light adaptation.

The remarkable dynamic range of vision is facilitated by adaptation of retinal sensitivity to ambient lighting conditions. An important mechanism of sensitivity adaptation is control of the spatial and temporal window over which light is integrated. The retina accomplishes this by switching between parallel synaptic pathways with differing kinetics and degrees of synaptic convergence.

The ON Crossover Circuitry Shapes Spatiotemporal Profile in the Center and Surround of Mouse OFF Retinal Ganglion Cells.

Retinal ganglion cells (RGCs) are often grouped based on their functional properties. Many of these functional properties, such as receptive field (RF) size, are driven by specific retinal circuits. In this report, we determined the role of the ON bipolar cell (BC) mediated crossover circuitry in shaping the center and surround of OFF RGCs.

Space-time codependence of retinal ganglion cells can be explained by novel and separable components of their receptive fields.

Reverse correlation methods such as spike-triggered averaging consistently identify the spatial center in the linear receptive fields (RFs) of retinal ganglion cells (GCs). However, the spatial antagonistic surround observed in classical experiments has proven more elusive. Tests for the antagonistic surround have heretofore relied on models that make questionable simplifying assumptions such as space-time separability and radial homogeneity/symmetry.

Effects of Chronic and Acute Intraocular Pressure Elevation on Scotopic and Photopic Contrast Sensitivity in Mice.

Purpose: To compare the impact of intraocular pressure (IOP) elevation on scotopic and photopic contrast sensitivity in mice.

Methods: We chronically elevated the IOP of wild-type mice via injection of polystyrene beads or acutely via injection of highly cohesive sodium hyaluronate. Some eyes with chronically elevated IOP were treated with either topical brimonidine tartrate 0.

Connexin 36 and rod bipolar cell independent rod pathways drive retinal ganglion cells and optokinetic reflexes.

Rod pathways are a parallel set of synaptic connections which enable night vision by relaying and processing rod photoreceptor light responses. We use dim light stimuli to isolate rod pathway contributions to downstream light responses then characterize these contributions in knockout mice lacking rod transducin-α (Trα), or certain pathway components associated with subsets of rod pathways. These comparisons reveal that rod pathway driven light sensitivity in retinal ganglion cells (RGCs) is entirely dependent on Trα, but partially independent of connexin 36 (Cx36) and rod bipolar cells.

The NH2-terminal and COOH-terminal fragments of dentin matrix protein 1 (DMP1) localize differently in the compartments of dentin and growth plate of bone.

Multiple studies have shown that dentin matrix protein 1 (DMP1) is essential for bone and dentin mineralization. After post-translational proteolytic cleavage, DMP1 exists within the extracellular matrix of bone and dentin as an NH2-terminal fragment, a COOH-terminal fragment, and the proteoglycan form of the NH2-terminal fragment (DMP1-PG). To begin to assess the biological function of each fragment, we evaluated the distribution of both fragments in the rat tooth and bone using antibodies specific to the NH2-terminal and COOH-terminal regions of DMP1 and confocal microscopy.

Proinflammatory cytokine expression in cyclooxygenase-2-deficient primary osteoblasts.

Many of the proinflammatory effects of interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha) are mediated through cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2). The purpose of this study was to examine the expression of IL-1beta and TNF-alpha in COX-2-deficient osteoblasts during inflammation. Primary osteoblasts prepared from wild-type (WT) and COX-2 knockout (K/O) mice were exposed to lipopolysaccharide (LPS) and endodontic obturation materials.

The role of cyclooxygenase-2 (COX-2) in inflammatory bone resorption.

Prostaglandin E2 (PGE(2)) is an important inflammatory mediator that plays an essential role in the development and progression of periradicular diseases. Cyclooxygenase-2 (COX-2) is the inducible enzyme responsible for increased PGE(2) levels during inflammation and other pathologic processes. The purpose of this study was to determine the role of COX-2-mediated PGE(2) synthesis in osteoclast formation in response to endodontic pathogens and materials.