A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain.

The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties1-3. Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions-in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.

Modification of Müller Glial Cell Fate and Proliferation with the Use of Small Molecules.

In recent years, reprogramming Müller glia by overexpressing Ascl1 and other transcription factors has shown promise for the regeneration of postmitotic retinal neurons, primarily bipolar cells, following injury. Müller glial proliferation and efficiency of neuronal differentiation can be modified by the use of small molecules in various systems. The molecules and pathways studied thus far share remarkable consistency with astrocytes. In this mini review, we provide an overview on the modulation of Müller glial proliferation and cell fate using small molecules in injury and reprogramming. We also compare these observations to what has been observed in astrocytes.

MicroRNAs miR-25, let-7 and miR-124 regulate the neurogenic potential of Müller glia in mice.

Müller glial cells (MG) generate retinal progenitor (RPC)-like cells after injury in non-mammalian species, although this does not occur in the mammalian retina. Studies have profiled gene expression in these cells to define genes that may be relevant to their differences in neurogenic potential. However, less is known about differences in micro-RNA (miRNA) expression. In this study, we compared miRNAs from RPCs and MG to identify miRNAs more highly expressed in RPCs, and others more highly expressed in MG. To determine whether these miRNAs are relevant to the difference in neurogenic potential between these two cell types, we tested them in dissociated cultures of MG using either mimics or antagomiRs to increase or reduce expression, respectively. Among the miRNAs tested, miR-25 and miR-124 overexpression, or let-7 antagonism, induced Ascl1 expression and conversion of ∼40% of mature MG into a neuronal/RPC phenotype. Our results suggest that the differences in miRNA expression between MG and RPCs contribute to their difference in neurogenic potential, and that manipulations in miRNAs provide a new tool with which to reprogram MG for retinal regeneration.

Müller Cell Biological Processes Associated with Leukemia Inhibitory Factor Expression.

Müller cells provide support to photoreceptors under many conditions of stress and degeneration. Leukemia inhibitory factor is known to be expressed in Müller cells, which is necessary to promote photoreceptor survival in stress. We hypothesize that Müller cells that express LIF are undergoing other biological processes or functions which may benefit photoreceptors in disease. In this study, we analyze an existing single Müller cell microarray dataset to determine which processes are upregulated in Müller cells that express LIF, by correlating LIF expression to the expression of other genes using a robust correlation method. Some enriched processes include divalent inorganic cation homeostasis, negative regulation of stem cell proliferation, and gamma-glutamyl transferase activity.

Co-Expression of Wild-Type and Mutant S163R C1QTNF5 in Retinal Pigment Epithelium.

The pathogenic mutation S163R in C1QTNF5 causes a disorder known as autosomal dominant late-onset retinal degeneration (L-ORD), characterized by the presence of thick extracellular sub-RPE deposits, similar histopathologically to those found in AMD patients. We have previously shown that the S163R C1QTNF5 mutant forms globular aggregates within the RPE in vivo following its AAV-mediated expression in the RPE and exhibits a reversely polarized distribution, being routed toward the basal rather than apical RPE. We show here that when both wild-type and mutant S163R C1QTNF5 are simultaneously delivered subretinally to mouse RPE cells, the mutant impairs the wild-type protein secretion from the RPE, and both proteins are dispersed toward the basal and lateral RPE membrane. This result has mechanistic and therapeutic implications for L-ORD disorder.

A multiplexed, single-cell sequencing screen identifies compounds that increase neurogenic reprogramming of murine Muller glia.

Retinal degeneration in mammals causes permanent loss of vision, due to an inability to regenerate naturally. Some non-mammalian vertebrates show robust regeneration, via Muller glia (MG). We have recently made significant progress in stimulating adult mouse MG to regenerate functional neurons by transgenic expression of the proneural transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited. With the goal of improving this in mammals, we designed a small molecule screen using sci-Plex, a method to multiplex up to thousands of single nucleus RNA-seq conditions into a single experiment. We used this technology to screen a library of 92 compounds, identified, and validated two that promote neurogenesis in vivo. Our results demonstrate that high-throughput single-cell molecular profiling can substantially improve the discovery process for molecules and pathways that can stimulate neural regeneration and further demonstrate the potential for this approach to restore vision in patients with retinal disease.

Evaluating Methods for the Prediction of Cell Type-Specific Enhancers in the Mammalian Cortex.

Identifying cell type-specific enhancers in the brain is critical to building genetic tools for investigating the mammalian brain. Computational methods for functional enhancer prediction have been proposed and validated in the fruit fly and not yet the mammalian brain. We organized the 'Brain Initiative Cell Census Network (BICCN) Challenge: Predicting Functional Cell Type-Specific Enhancers from Cross-Species Multi-Omics' to assess machine learning and feature-based methods designed to nominate enhancer DNA sequences to target cell types in the mouse cortex. Methods were evaluated based on in vivo validation data from hundreds of cortical cell type-specific enhancers that were previously packaged into individual AAV vectors and retro-orbitally injected into mice. We find that open chromatin was a key predictor of functional enhancers, and sequence models improved prediction of non-functional enhancers that can be deprioritized as opposed to pursued for in vivo testing. Sequence models also identified cell type-specific transcription factor codes that can guide designs of in silico enhancers. This community challenge establishes a benchmark for enhancer prioritization algorithms and reveals computational approaches and molecular information that are crucial for the identification of functional enhancers for mammalian cortical cell types. The results of this challenge bring us closer to understanding the complex gene regulatory landscape of the mammalian brain and help us design more efficient genetic tools and potential gene therapies for human neurological diseases.

A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We used transcriptomic and epigenomic cortical cell type taxonomies from mouse and human to define marker genes and putative enhancers and created a large toolkit of transgenic lines and enhancer AAVs for selective targeting of cortical cell populations. We report evaluation of fifteen new transgenic driver lines, two new reporter lines, and >800 different enhancer AAVs covering most subclasses of cortical cells. The tools reported here as well as the scaled process of tool creation and modification enable diverse experimental strategies towards understanding mammalian cortex and brain function.

Enhancer AAV toolbox for accessing and perturbing striatal cell types and circuits.

We present an enhancer AAV toolbox for accessing and perturbing striatal cell types and circuits. Best-in-class vectors were curated for accessing major striatal neuron populations including medium spiny neurons (MSNs), direct and indirect pathway MSNs, as well as Sst-Chodl, Pvalb-Pthlh, and cholinergic interneurons. Specificity was evaluated by multiple modes of molecular validation, three different routes of virus delivery, and with diverse transgene cargos. Importantly, we provide detailed information necessary to achieve reliable cell type specific labeling under different experimental contexts. We demonstrate direct pathway circuit-selective optogenetic perturbation of behavior and multiplex labeling of striatal interneuron types for targeted analysis of cellular features. Lastly, we show conserved in vivo activity for exemplary MSN enhancers in rat and macaque. This collection of striatal enhancer AAVs offers greater versatility compared to available transgenic lines and can readily be applied for cell type and circuit studies in diverse mammalian species beyond the mouse model.

Single-cell ATAC-seq of fetal human retina and stem-cell-derived retinal organoids shows changing chromatin landscapes during cell fate acquisition.

We previously used single-cell transcriptomic analysis to characterize human fetal retinal development and assessed the degree to which retinal organoids recapitulate normal development. We now extend the transcriptomic analyses to incorporate single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), a powerful method used to characterize potential gene regulatory networks through the changes in accessible chromatin that accompany cell-state changes. The combination of scATAC-seq and single-cell RNA sequencing (scRNA-seq) provides a view of developing human retina at an unprecedented resolution. We identify key transcription factors relevant to specific fates and the order of the transcription factor cascades that define each of the major retinal cell types. The changing chromatin landscape is largely recapitulated in retinal organoids; however, there are differences in Notch signaling and amacrine cell gene regulation. The datasets we generated constitute an excellent resource for the continued improvement of retinal organoid technology and have the potential to inform and accelerate regenerative medicine approaches to retinal diseases.

Reprogramming Müller glia to regenerate ganglion-like cells in adult mouse retina with developmental transcription factors.

Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in nonmammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. Here, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in Müller glia, including Ascl1, Pou4f2, Islet1, and Atoh1. Using immunohistochemistry, single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin sequencing, and electrophysiology, we find that retinal ganglion-like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental retinal ganglion cell TFs.

Efficient stimulation of retinal regeneration from Müller glia in adult mice using combinations of proneural bHLH transcription factors.

Regenerative neuroscience aims to stimulate endogenous repair in the nervous system to replace neurons lost from degenerative diseases. Recently, we reported that overexpressing the transcription factor Ascl1 in Müller glia (MG) is sufficient to stimulate MG to regenerate functional neurons in the adult mouse retina. However, this process is inefficient, and only a third of the Ascl1-expressing MG generate new neurons. Here, we test whether proneural transcription factors of the Atoh1/7 class can further promote the regenerative capacity of MG. We find that the combination of Ascl1:Atoh1 is remarkably efficient at stimulating neurogenesis, even in the absence of retinal injury. Using electrophysiology and single-cell RNA sequencing (scRNA-seq), we demonstrate that Ascl1:Atoh1 generates a diversity of retinal neuron types, with the majority expressing characteristics of retinal ganglion cells. Our results provide a proof of principle that combinations of developmental transcription factors can substantially improve glial reprogramming to neurons and expand the repertoire of regenerated cell fates.

Microglia Suppress Ascl1-Induced Retinal Regeneration in Mice.

The innate immune system plays key roles in tissue regeneration. For example, microglia promote neurogenesis in Müller glia in birds and fish after injury. Although mammalian retina does not normally regenerate, neurogenesis can be induced in mouse Müller glia by Ascl1, a proneural transcription factor. We show that in mice, microglia inhibit the Ascl1-mediated retinal regeneration, suggesting that the innate immune system limits the regenerative response to injury.

Developmental changes in the accessible chromatin, transcriptome and Ascl1-binding correlate with the loss in Müller Glial regenerative potential.

Diseases and damage to the retina lead to losses in retinal neurons and eventual visual impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have robust regeneration from Müller glia (MG). Recently, we have shown that driving expression of Ascl1 in adult mouse MG stimulates neural regeneration. The regeneration observed in the mouse is limited in the variety of neurons that can be derived from MG; Ascl1-expressing MG primarily generate bipolar cells. To better understand the limits of MG-based regeneration in mouse retinas, we used ATAC- and RNA-seq to compare newborn progenitors, immature MG (P8-P12), and mature MG. Our analysis demonstrated developmental differences in gene expression and accessible chromatin between progenitors and MG, primarily in neurogenic genes. Overexpression of Ascl1 is more effective in reprogramming immature MG, than mature MG, consistent with a more progenitor-like epigenetic landscape in the former. We also used ASCL1 ChIPseq to compare the differences in ASCL1 binding in progenitors and reprogrammed MG. We find that bipolar-specific accessible regions are more frequently linked to bHLH motifs and ASCL1 binding. Overall, our analysis indicates a loss of neurogenic gene expression and motif accessibility during glial maturation that may prevent efficient reprogramming.

STAT Signaling Modifies Ascl1 Chromatin Binding and Limits Neural Regeneration from Muller Glia in Adult Mouse Retina.

Müller glia (MG) serve as sources for retinal regeneration in non-mammalian vertebrates. We find that this process can be induced in mouse MG, after injury, by transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are generated only from a subset of MG. Identifying factors that limit Ascl1-mediated MG reprogramming could make this process more efficient. In this study, we test whether injury-induced STAT activation hampers the ability of Ascl1 to reprogram MG into retinal neurons. Single-cell RNA-seq shows that progenitor-like cells derived from Ascl1-expressing MG have a higher level of STAT signaling than do those cells that become neurons. Ascl1-ChIPseq and ATAC-seq show that STAT potentially directs Ascl1 to developmentally inappropriate targets. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of MG to generate neurons.

Damage-associated molecular pattern recognition is required for induction of retinal neuroprotective pathways in a sex-dependent manner.

Retinal degeneration is a common cause of irreversible blindness and is caused by the death of retinal light-sensitive neurons called photoreceptors. At the onset of degeneration, stressed photoreceptors cause retinal glial cells to secrete neuroprotective factors that slow the pace of degeneration. Leukemia inhibitory factor (LIF) is one such factor that is required for endogenous neuroprotection. Photoreceptors are known to release signals of cellular stress, called damage-associated molecular patterns (DAMPs) early in degeneration, and we hypothesized that receptors for DAMPs or pattern recognition receptors (PRRs) play a key role in the induction of LIF and neuroprotective stress responses in retinal glial cells. Toll-like receptor 2 (TLR2) is a well-established DAMP receptor. In our experiments, activation of TLR2 protected both male and female mice from light damage, while the loss of TLR2 in female mice did not impact photoreceptor survival. In contrast, induction of protective stress responses, microglial phenotype and photoreceptor survival were strongly impacted in male TLR2-/- mice. Lastly, using publicly available gene expression data, we show that TLR2 is expressed highly in resting microglia prior to injury, but is also induced in Müller cells in inherited retinal degeneration.