Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics.

Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class.

Embryonic Origin of Postnatal Neural Stem Cells.

Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

A nanobody-based system using fluorescent proteins as scaffolds for cell-specific gene manipulation.

Fluorescent proteins are commonly used to label cells across organisms, but the unmodified forms cannot control biological activities. Using GFP-binding proteins derived from Camelid antibodies, we co-opted GFP as a scaffold for inducing formation of biologically active complexes, developing a library of hybrid transcription factors that control gene expression only in the presence of GFP or its derivatives. The modular design allows for variation in key properties such as DNA specificity, transcriptional potency, and drug dependency. Production of GFP controlled cell-specific gene expression and facilitated functional perturbations in the mouse retina and brain. Further, retrofitting existing transgenic GFP mouse and zebrafish lines for GFP-dependent transcription enabled applications such as optogenetic probing of neural circuits. This work establishes GFP as a multifunctional scaffold and opens the door to selective manipulation of diverse GFP-labeled cells across transgenic lines. This approach may also be extended to exploit other intracellular products as cell-specific scaffolds in multicellular organisms.

Thermal-plex: fluidic-free, rapid sequential multiplexed imaging with DNA-encoded thermal channels.

Multiplexed fluorescence imaging is typically limited to three- to five-plex on standard setups. Sequential imaging methods based on iterative labeling and imaging enable practical higher multiplexing, but generally require a complex fluidic setup with several rounds of slow buffer exchange (tens of minutes to an hour for each exchange step). We report the thermal-plex method, which removes complex and slow buffer exchange steps and provides fluidic-free, rapid sequential imaging. Thermal-plex uses simple DNA probes that are engineered to fluoresce sequentially when, and only when, activated with transient exposure to heating spikes at designated temperatures (thermal channels). Channel switching is fast (<30 s) and is achieved with a commercially available and affordable on-scope heating device. We demonstrate 15-plex RNA imaging (five thermal × three fluorescence channels) in fixed cells and retina tissues in less than 4 min, without using buffer exchange or fluidics. Thermal-plex introduces a new labeling method for efficient sequential multiplexed imaging.

Abortive and productive infection of CNS cell types following in vivo delivery of VSV.

Viral infection is frequently assayed by ongoing expression of viral genes. These assays fail to identify cells that have been exposed to the virus but limit or inhibit viral replication. To address this limitation, we used a dual-labeling vesicular stomatitis virus (DL-VSV), which has a deletion of the viral glycoprotein gene, to allow evaluation of primary infection outcomes. This virus encodes Cre, which can stably mark any cell with even a minimal level of viral gene expression. Additionally, the virus encodes GFP, which distinguishes cells with higher levels of viral gene expression, typically due to genome replication. Stereotactic injections of DL-VSV into the murine brain showed that different cell types had very different responses to the virus. Almost all neurons hosted high levels of viral gene expression, while glial cells varied in their responses. Astrocytes (Sox9+) were predominantly productively infected, while oligodendrocytes (Sox10+) were largely abortively infected. Microglial cells (Iba1+) were primarily uninfected. Furthermore, we monitored the early innate immune response to viral infection and identified unique patterns of interferon (IFN) induction. Shortly after infection, microglia were the main producers of IFNb, whereas later, oligodendrocytes were the main producers. IFNb+ cells were primarily abortively infected regardless of cell type. Last, we investigated whether IFN signaling had any impact on the outcome of primary infection and did not observe significant changes, suggesting that intrinsic factors are likely responsible for determining the outcome of primary infection.

Chromophore supply modulates cone function and survival in retinitis pigmentosa mouse models.

Retinitis pigmentosa (RP) is an ocular disease characterized by the loss of night vision, followed by the loss of daylight vision. Daylight vision is initiated in the retina by cone photoreceptors, which are gradually lost in RP, often as bystanders in a disease process that initiates in their neighboring rod photoreceptors. Using physiological assays, we investigated the timing of cone electroretinogram (ERG) decline in RP mouse models. A correlation between the time of loss of the cone ERG and the loss of rods was found. To investigate a potential role of the visual chromophore supply in this loss, mouse mutants with alterations in the regeneration of the retinal chromophore, 11-cis retinal, were examined. Reducing chromophore supply via mutations in Rlbp1 or Rpe65 resulted in greater cone function and survival in a RP mouse model. Conversely, overexpression of Rpe65 and Lrat, genes that can drive the regeneration of the chromophore, led to greater cone degeneration. These data suggest that abnormally high chromophore supply to cones upon the loss of rods is toxic to cones, and that a potential therapy in at least some forms of RP is to slow the turnover and/or reduce the level of visual chromophore in the retina.

Characterization of the development of the high-acuity area of the chick retina.

The fovea is a small region within the central retina that is responsible for our high acuity daylight vision. Chickens also have a high acuity area (HAA), and are one of the few species that enables studies of the mechanisms of HAA development, due to accessible embryonic tissue and methods to readily perturb gene expression. To enable such studies, we characterized the development of the chick HAA using single molecule fluorescent in situ hybridization (smFISH), along with more classical methods. We found that Fgf8 provides a molecular marker for the HAA throughout development and into adult stages, allowing studies of the cellular composition of this area over time. The radial dimension of the ganglion cell layer (GCL) was seen to be the greatest at the HAA throughout development, beginning during the period of neurogenesis, suggesting that genesis, rather than cell death, creates a higher level of retinal ganglion cells (RGCs) in this area. In contrast, the HAA acquired its characteristic high density of cone photoreceptors post-hatching, which is well after the period of neurogenesis. We also confirmed that rod photoreceptors are not present in the HAA. Analyses of cell death in the developing photoreceptor layer, where rods would reside, did not show apoptotic cells, suggesting that lack of genesis, rather than death, created the "rod-free zone" (RFZ). Quantification of each cone photoreceptor subtype showed an ordered mosaic of most cone subtypes. The changes in cellular densities and cell subtypes between the developing and mature HAA provide some answers to the overarching strategy used by the retina to create this area and provide a framework for future studies of the mechanisms underlying its formation.

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.

We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.

Development and diversification of bipolar interneurons in the mammalian retina.

The bipolar interneurons of the mammalian retina have evolved as a diverse set of cells with distinct subtype characteristics, which reflect specialized contributions to visual circuitry. Fifteen subtypes of bipolar interneurons have been identified in the mouse retina, each with characteristic gene expression, morphology, and light responses. This review provides an overview of the developmental events that underlie the generation of the diverse bipolar cell class, summarizing the current knowledge of genetic programs that establish and maintain bipolar subtype fates, as well as the events that shape the final distribution of bipolar subtypes. With much left to be discovered, bipolar interneurons present an ideal model system for studying the interplay between cell-autonomous and non-cell-autonomous mechanisms that influence neuronal subtype development within the central nervous system.

Enhancer transcription identifies cis-regulatory elements for photoreceptor cell types.

Identification of cell type-specific cis-regulatory elements (CREs) is crucial for understanding development and disease, although identification of functional regulatory elements remains challenging. We hypothesized that context-specific CREs could be identified by context-specific non-coding RNA (ncRNA) profiling, based on the observation that active CREs produce ncRNAs. We applied ncRNA profiling to identify rod and cone photoreceptor CREs from wild-type and mutant mouse retinas, defined by presence or absence, respectively, of the rod-specific transcription factor (TF) NrlNrl-dependent ncRNA expression strongly correlated with epigenetic profiles of rod and cone photoreceptors, identified thousands of candidate rod- and cone-specific CREs, and identified motifs for rod- and cone-specific TFs. Colocalization of NRL and the retinal TF CRX correlated with rod-specific ncRNA expression, whereas CRX alone favored cone-specific ncRNA expression, providing quantitative evidence that heterotypic TF interactions distinguish cell type-specific CRE activity. We validated the activity of novel Nrl-dependent ncRNA-defined CREs in developing cones. This work supports differential ncRNA profiling as a platform for the identification of cell type-specific CREs and the discovery of molecular mechanisms underlying TF-dependent CRE activity.

Cell type- and stage-specific expression of Otx2 is regulated by multiple transcription factors and 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 necessary. We analyzed how the expression of the homeobox TF, orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the mouse retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. 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. We conclude that retinal cells use a cohort of TFs with different expression patterns and multiple CRMs with different chromatin configurations to regulate the expression of Otx2 precisely.

SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification.

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.

SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues.

Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here we introduce signal amplification by exchange reaction (SABER), which endows oligonucleotide-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplified RNA and DNA FISH signals (5- to 450-fold) in fixed cells and tissues. We also applied 17 orthogonal amplifiers against chromosomal targets simultaneously and detected mRNAs with high efficiency. We then used 10-plex SABER-FISH to identify in vivo introduced enhancers with cell-type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.

FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system.

Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.

Soluble CX3CL1 gene therapy improves cone survival and function in mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a disease that initially presents as night blindness due to genetic deficits in the rod photoreceptors of the retina. Rods then die, causing dysfunction and death of cone photoreceptors, the cell type that mediates high acuity and color vision, ultimately leading to blindness. We investigated immune responses in mouse models of RP and found evidence of microglia activation throughout the period of cone degeneration. Using adeno-associated vectors (AAVs), delivery of genes encoding microglial regulatory signals led to the identification of AAV serotype 8 (AAV8) soluble CX3CL1 (sCX3CL1) as a promising therapy for degenerating cones. Subretinal injection of AAV8-sCX3CL1 significantly prolonged cone survival in three strains of RP mice. Rescue of cones was accompanied by improvements in visual function. AAV8-sCX3CL1 did not affect rod survival, microglia localization, or inflammatory cytokine levels in the retina. Furthermore, although RNA sequencing of microglia demonstrated marked transcriptional changes with AAV8-sCX3CL1, pharmacological depletion of up to ∼99% of microglia failed to abrogate the effect of AAV8-sCX3CL1 on cone survival. These findings indicate that AAV8-sCX3CL1 can rescue cones in multiple mouse models of RP via a pathway that does not require normal numbers of microglia. Gene therapy with sCX3CL1 is a promising mutation-independent approach to preserve vision in RP and potentially other forms of retinal degeneration.

AAV cis-regulatory sequences are correlated with ocular toxicity.

Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1ß were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.

The brain parenchyma has a type I interferon response that can limit virus spread.

The brain has a tightly regulated environment that protects neurons and limits inflammation, designated "immune privilege." However, there is not an absolute lack of an immune response. We tested the ability of the brain to initiate an innate immune response to a virus, which was directly injected into the brain parenchyma, and to determine whether this response could limit viral spread. We injected vesicular stomatitis virus (VSV), a transsynaptic tracer, or naturally occurring VSV-derived defective interfering particles (DIPs), into the caudate-putamen (CP) and scored for an innate immune response and inhibition of virus spread. We found that the brain parenchyma has a functional type I interferon (IFN) response that can limit VSV spread at both the inoculation site and among synaptically connected neurons. Furthermore, we characterized the response of microglia to VSV infection and found that infected microglia produced type I IFN and uninfected microglia induced an innate immune response following virus injection.

In Vivo Functional Imaging of Retinal Neurons Using Red and Green Fluorescent Calcium Indicators.

Adaptive optics retinal imaging of fluorescent calcium indicators is a minimally invasive method used to study retinal physiology over extended periods of time. It has potential for discovering novel retinal circuits, tracking retinal function in animal models of retinal disease, and assessing vision restoration therapy. We previously demonstrated functional adaptive optics imaging of retinal neurons in the living eye using green fluorescent calcium indicators; however, the use of green fluorescent indicators presents challenges that stem from the fact that they are excited by short-wavelength light. Using red fluorescent calcium indicators such as jRGECO1a, which is excited with longer-wavelength light (~560 nm), makes imaging approximately five times safer than using short-wavelength light (~500 nm) used to excite green fluorescent calcium indicators such as GCaMP6s. Red fluorescent indicators also provide alternative wavelength imaging regimes to overcome cross talk with the sensitivities of intrinsic photoreceptors and blue light-activated channelrhodopsins. Here we evaluate jRGECO1a for in vivo functional adaptive optics imaging of retinal neurons using single-photon excitation in mice. We find that jRGECO1a provides similar fidelity as the established green indicator GCaMP6s.

Alternative splicing produces high levels of noncoding isoforms of bHLH transcription factors during development.

During development, multiple cell types within a tissue often arise from a common pool of progenitor cells (PCs). PCs typically expand in number, while simultaneously producing post-mitotic cells (PMCs). This balance is partly regulated by transcription factors that are expressed within PCs, such as the basic helix-loop-helix (bHLH) gene mouse atonal homolog 7 (Math5), which is expressed in retinal PCs. Here we report that alternative splicing (AS) of Math5 serves as another layer of regulation of Math5 activity. Specifically, Math5, a single exon gene, is alternatively spliced such that the major isoform lacks the entire coding sequence. Similarly, neurogenin 3 (Ngn3), a Math5 paralog expressed in pancreatic PCs, is also alternatively spliced such that the major isoform fails to code for Ngn3 protein. The consequence of reducing the abundance of protein-coding isoforms is likely crucial, as we found that introduction of coding isoforms leads to a reduction in cycling PCs. Thus, AS can limit the number of PCs expressing key regulatory proteins that control PC expansion versus PMC production.

Notch1 is required in newly postmitotic cells to inhibit the rod photoreceptor fate.

Several models of cell fate determination can be invoked to explain how single retinal progenitor cells (RPCs) produce different cell types in a terminal division. To gain insight into this process, the effects of the removal of a cell fate regulator, Notch1, were studied in newly postmitotic cells using a conditional allele of Notch1 (N1-CKO) in mice. Almost all newly postmitotic N1-CKO cells became rod photoreceptors, whereas wild-type (WT) cells achieved a variety of fates. Single cell profiling of wild-type and N1-CKO retinal cells transitioning from progenitor to differentiated states revealed differential expression of inhibitor of DNA binding factors Id1 and Id3, as well as Notch-regulated ankyrin repeat protein (Nrarp). Misexpression of Id1 and Id3 was found to be sufficient to drive production of Müller glial cells and/or RPCs. Moreover, Id1 and Id3 were shown to partially rescue the production of bipolar and Müller glial cells in the absence of Notch1 in mitotic and newly postmitotic cells. Misexpression of Nrarp, a downstream target gene and inhibitor of the Notch signaling pathway, resulted in the overproduction of rod photoreceptors at the expense of Müller glial cells. These data demonstrate that cell fate decisions can be made in newly postmitotic retinal cells, and reveal some of the regulators downstream of Notch1 that influence the choice of rod and non-rod fates. Taken together, our results begin to address how different signals downstream from a common pathway lead to different fate outcomes.