Molecular mechanisms controlling vertebrate retinal patterning, neurogenesis, and cell fate specification.

This review covers recent advances in understanding the molecular mechanisms controlling neurogenesis and specification of the developing retina, with a focus on insights obtained from comparative single cell multiomic analysis. We discuss recent advances in understanding the mechanisms by which extrinsic factors trigger transcriptional changes that spatially pattern the optic cup (OC) and control the initiation and progression of retinal neurogenesis. We also discuss progress in unraveling the core evolutionarily conserved gene regulatory networks (GRNs) that specify early- and late-state retinal progenitor cells (RPCs) and neurogenic progenitors and that control the final steps in determining cell identity. Finally, we discuss findings that provide insight into regulation of species-specific aspects of retinal patterning and neurogenesis, including consideration of key outstanding questions in the field.

Fatty acid-binding proteins and fatty acid synthase influence glial reactivity and promote the formation of Müller glia-derived progenitor cells in the chick retina.

A recent comparative transcriptomic study of Müller glia (MG) in vertebrate retinas revealed that fatty acid binding proteins (FABPs) are among the most highly expressed genes in chick ( Hoang et al., 2020). Here, we investigate how FABPs and fatty acid synthase (FASN) influence glial cells in the chick retina. During development, FABP7 is highly expressed by retinal progenitor cells and maturing MG, whereas FABP5 is upregulated in maturing MG. PMP2 (FABP8) is expressed by oligodendrocytes and FABP5 is expressed by non-astrocytic inner retinal glial cells, and both of these FABPs are upregulated by activated MG. In addition to suppressing the formation of Müller glia-derived progenitor cells (MGPCs), we find that FABP-inhibition suppresses the proliferation of microglia. FABP-inhibition induces distinct changes in single cell transcriptomic profiles, indicating transitions of MG from resting to reactive states and suppressed MGPC formation, with upregulation of gene modules for gliogenesis and decreases in neurogenesis. FASN-inhibition increases the proliferation of microglia and suppresses the formation of MGPCs. We conclude that fatty acid metabolism and cell signaling involving fatty acids are important in regulating the reactivity and dedifferentiation of MG, and the proliferation of microglia and MGPCs.

A diffusion MRI-based spatiotemporal continuum of the embryonic mouse brain for probing gene-neuroanatomy connections.

The embryonic mouse brain undergoes drastic changes in establishing basic anatomical compartments and laying out major axonal connections of the developing brain. Correlating anatomical changes with gene-expression patterns is an essential step toward understanding the mechanisms regulating brain development. Traditionally, this is done in a cross-sectional manner, but the dynamic nature of development calls for probing gene-neuroanatomy interactions in a combined spatiotemporal domain. Here, we present a four-dimensional (4D) spatiotemporal continuum of the embryonic mouse brain from E10.5 to E15.5 reconstructed from diffusion magnetic resonance microscopy (dMRM) data. This study achieved unprecedented high-definition dMRM at 30- to 35-µm isotropic resolution, and together with computational neuroanatomy techniques, we revealed both morphological and microscopic changes in the developing brain. We transformed selected gene-expression data to this continuum and correlated them with the dMRM-based neuroanatomical changes in embryonic brains. Within the continuum, we identified distinct developmental modes comprising regional clusters that shared developmental trajectories and similar gene-expression profiles. Our results demonstrate how this 4D continuum can be used to examine spatiotemporal gene-neuroanatomical interactions by connecting upstream genetic events with anatomical changes that emerge later in development. This approach would be useful for large-scale analysis of the cooperative roles of key genes in shaping the developing brain.

Profiling the human protein-DNA interactome reveals ERK2 as a transcriptional repressor of interferon signaling.

Protein-DNA interactions (PDIs) mediate a broad range of functions essential for cellular differentiation, function, and survival. However, it is still a daunting task to comprehensively identify and profile sequence-specific PDIs in complex genomes. Here, we have used a combined bioinformatics and protein microarray-based strategy to systematically characterize the human protein-DNA interactome. We identified 17,718 PDIs between 460 DNA motifs predicted to regulate transcription and 4,191 human proteins of various functional classes. Among them, we recovered many known PDIs for transcription factors (TFs). We identified a large number of unanticipated PDIs for known TFs, as well as for previously uncharacterized TFs. We also found that over three hundred unconventional DNA-binding proteins (uDBPs)--which include RNA-binding proteins, mitochondrial proteins, and protein kinases--showed sequence-specific PDIs. One such uDBP, ERK2, acts as a transcriptional repressor for interferon gamma-induced genes, suggesting important biological roles for such proteins.

Notch Inhibition Promotes Regeneration and Immunosuppression Supports Cone Survival in a Zebrafish Model of Inherited Retinal Dystrophy.

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as retinitis pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Müller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, in zebrafish models of retinal degeneration, photoreceptor death does not induce the MG to reprogram and regenerate lost cells. Here, we used male and female zebrafish cep290 mutants to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to prelesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.SIGNIFICANCE STATEMENT Inherited retinal degenerations (IRDs) are genetic diseases that lead to the progressive loss of photoreceptors and the permanent loss of vision. Zebrafish can regenerate photoreceptors after acute injury by reprogramming Müller glia (MG) into stem-like cells that produce retinal progenitors, but this regenerative process fails to occur in zebrafish models of IRDs. Here, we show that Notch pathway inhibition can promote photoreceptor regeneration in models of progressive degeneration and that immunosuppression can prevent photoreceptor loss. These results offer insight into the pathways that promote MG-dependent regeneration and the role of inflammation in photoreceptor degeneration.

NF-κB signaling regulates the formation of proliferating Müller glia-derived progenitor cells in the avian retina.

Retinal regeneration is robust in some cold-blooded vertebrates, but this process is ineffective in warm-blooded vertebrates. Understanding the mechanisms that suppress the reprogramming of Müller glia into neurogenic progenitors is key to harnessing the regenerative potential of the retina. Inflammation and reactive microglia are known to influence the formation of Müller glia-derived progenitor cells (MGPCs), but the mechanisms underlying this interaction are unknown. We used a chick in vivo model to investigate nuclear factor kappa B (NF-κB) signaling, a critical regulator of inflammation, during the reprogramming of Müller glia into proliferating progenitors. We find that components of the NF-κB pathway are dynamically regulated by Müller glia after neuronal damage or treatment with growth factors. Inhibition of NF-κB enhances, whereas activation suppresses, the formation of proliferating MGPCs. Following microglia ablation, the effects of NF-κB-agonists on MGPC-formation are reversed, suggesting that signals provided by reactive microglia influence how NF-κB impacts Müller glia reprogramming. We propose that NF-κB is an important signaling 'hub' that suppresses the reprogramming of Müller glia into proliferating MGPCs and this 'hub' coordinates signals provided by reactive microglia.

A single cell transcriptional atlas of early synovial joint development.

Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

LRLoop: a method to predict feedback loops in cell-cell communication.

MOTIVATION: Intercellular communication (i.e. cell-cell communication) plays an essential role in multicellular organisms coordinating various biological processes. Previous studies discovered that feedback loops between two cell types are a widespread and vital signaling motif regulating development, regeneration and cancer progression. While many computational methods have been developed to predict cell-cell communication based on gene expression datasets, these methods often predict one-directional ligand-receptor interactions from sender to receiver cells and are not suitable to identify feedback loops. RESULTS: Here, we describe ligand-receptor loop (LRLoop), a new method for analyzing cell-cell communication based on bi-directional ligand-receptor interactions, where two pairs of ligand-receptor interactions are identified that are responsive to each other and thereby form a closed feedback loop. We first assessed LRLoop using bulk datasets and found our method significantly reduces the false positive rate seen with existing methods. Furthermore, we developed a new strategy to assess the performance of these methods in single-cell datasets. We used the between-tissue interactions as an indicator of potential false-positive prediction and found that LRLoop produced a lower fraction of between-tissue interactions than traditional methods. Finally, we applied LRLoop to the single-cell datasets obtained from retinal development. We discovered many new bi-directional ligand-receptor interactions among individual cell types that potentially control proliferation, neurogenesis and/or cell fate specification. AVAILABILITY AND IMPLEMENTATION: An R package is available at https://github.com/Pinlyu3/LRLoop. The source code can be found at figshare (https://doi.org/10.6084/m9.figshare.20126138.v1). The datasets can be found at figshare (https://doi.org/10.6084/m9.figshare.20126021.v1). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep-wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep.

Corrigendum: Lhx6-positive GABA-releasing neurons of the zona incerta promote sleep.

This corrects the article DOI: 10.1038/nature23663.

NFkB-signaling promotes glial reactivity and suppresses Müller glia-mediated neuron regeneration in the mammalian retina.

Müller glia (MG) in mammalian retinas are incapable of regenerating neurons after damage, whereas the MG in lower vertebrates regenerate functional neurons. Identification of cell signaling pathways and gene regulatory networks that regulate MG-mediated regeneration is key to harnessing the regenerative potential of MG. Here, we study how NFkB-signaling influences glial responses to damage and reprogramming of MG into neurons in the rodent retina. We find activation of NFkB and dynamic expression of NFkB-associated genes in MG after damage, however damage-induced NFkB activation is inhibited by microglia ablation. Knockout of NFkB in MG suppressed the accumulation of immune cells after damage. Inhibition of NFkB following NMDA-damage significantly enhanced the reprogramming of Ascl1-overexpressing MG into neuron-like cells. scRNA-seq of retinal glia following inhibition of NFkB reveals coordination with signaling via TGFß2 and suppression of NFI and Id transcription factors. Inhibition of Smad3 signal transducer or Id transcription factors increased numbers of neuron-like cells produced by Ascl1-overexpressing MG. We conclude that NFkB is a key signaling hub that is activated in MG after damage, mediates the accumulation of immune cells, and suppresses the neurogenic potential of MG.

Epigenetic hallmarks of age-related macular degeneration are recapitulated in a photosensitive mouse model.

Age-related macular degeneration (AMD) is a chronic, multifactorial disorder and a leading cause of blindness in the elderly. Characterized by progressive photoreceptor degeneration in the central retina, disease progression involves epigenetic changes in chromatin accessibility resulting from environmental exposures and chronic stress. Here, we report that a photosensitive mouse model of acute stress-induced photoreceptor degeneration recapitulates the epigenetic hallmarks of human AMD. Global epigenomic profiling was accomplished by employing an Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq), which revealed an association between decreased chromatin accessibility and stress-induced photoreceptor cell death in our mouse model. The epigenomic changes induced by light damage include reduced euchromatin and increased heterochromatin abundance, resulting in transcriptional and translational dysregulation that ultimately drives photoreceptor apoptosis and an inflammatory reactive gliosis in the retina. Of particular interest, pharmacological inhibition of histone deacetylase 11 (HDAC11) and suppressor of variegation 3-9 homolog 2 (SUV39H2), key histone-modifying enzymes involved in promoting reduced chromatin accessibility, ameliorated light damage in our mouse model, supporting a causal link between decreased chromatin accessibility and photoreceptor degeneration, thereby elucidating a potential new therapeutic strategy to combat AMD.

Zeb2 regulates the balance between retinal interneurons and Müller glia by inhibition of BMP-Smad signaling.

The interplay between signaling molecules and transcription factors during retinal development is key to controlling the correct number of retinal cell types. Zeb2 (Sip1) is a zinc-finger multidomain transcription factor that plays multiple roles in central and peripheral nervous system development. Haploinsufficiency of ZEB2 causes Mowat-Wilson syndrome, a congenital disease characterized by intellectual disability, epilepsy and Hirschsprung disease. In the developing retina, Zeb2 is required for generation of horizontal cells and the correct number of interneurons; however, its potential function in controlling gliogenic versus neurogenic decisions remains unresolved. Here we present cellular and molecular evidence of the inhibition of Müller glia cell fate by Zeb2 in late stages of retinogenesis. Unbiased transcriptomic profiling of control and Zeb2-deficient early-postnatal retina revealed that Zeb2 functions in inhibiting Id1/2/4 and Hes1 gene expression. These neural progenitor factors normally inhibit neural differentiation and promote Müller glia cell fate. Chromatin immunoprecipitation (ChIP) supported direct regulation of Id1 by Zeb2 in the postnatal retina. Reporter assays and ChIP analyses in differentiating neural progenitors provided further evidence that Zeb2 inhibits Id1 through inhibition of Smad-mediated activation of Id1 transcription. Together, the results suggest that Zeb2 promotes the timely differentiation of retinal interneurons at least in part by repressing BMP-Smad/Notch target genes that inhibit neurogenesis. These findings show that Zeb2 integrates extrinsic cues to regulate the balance between neuronal and glial cell types in the developing murine retina.

Cannabinoid signaling promotes the de-differentiation and proliferation of Müller glia-derived progenitor cells.

Endocannabinoids (eCB) are lipid-based neurotransmitters that are known to influence synaptic function in the visual system. eCBs are also known to suppress neuroinflammation in different pathological states. However, nothing is known about the roles of the eCB system during the transition of Müller glia (MG) into proliferating progenitor-like cells in the retina. Accordingly, we used the chick and mouse model to characterize expression patterns of eCB-related genes and applied pharmacological agents to investigate how the eCB system impacts glial reactivity and the capacity of MG to become Müller glia-derived progenitor cells (MGPCs). We queried single cell RNA-seq libraries to identify eCB-related genes and identify cells with dynamic patterns of expression in damaged retinas. MG and inner retinal neurons expressed the eCB receptor CNR1, as well as enzymes involved in eCB metabolism. In the chick, intraocular injections of cannabinoids, 2-Arachidonoylglycerol (2-AG) and Anandamide (AEA), stimulated the formation of MGPCs. Cannabinoid Receptor 1 (CNR1)-agonists and Monoglyceride Lipase-inhibitor promoted the formation of MGPCs, whereas CNR1-antagonist and inhibitors of eCB synthesis suppressed this process. In damaged mouse retinas where MG activate NFkB-signaling, activation of CNR1 decreased and inhibition of CNR1 increased NFkB, whereas levels of neuronal cell death were unaffected. Surprisingly, retinal microglia were largely unaffected by increases or decreases in eCB-signaling in both chick and mouse retinas. We conclude that the eCB system in the retina influences the reactivity of MG and the formation of proliferating MGPCs, but does not influence the reactivity of immune cells in the retina.

Midkine is neuroprotective and influences glial reactivity and the formation of Müller glia-derived progenitor cells in chick and mouse retinas.

Recent studies suggest midkine (MDK) is involved in the development and regeneration of the zebrafish retina. We investigate the expression patterns of MDK and related factors, roles in neuronal survival, and influence upon the formation of Müller glia-derived progenitor cells (MGPCs) in chick and mouse model systems. By using single-cell RNA-sequencing, we find that MDK and pleiotrophin (PTN), a MDK-related cytokine, are upregulated by Müller glia (MG) during later stages of development in chick. While PTN is downregulated, MDK is dramatically upregulated in mature MG after retinal damage or FGF2 and insulin treatment. By comparison, MDK and PTN are downregulated by MG in damaged mouse retinas. In both chick and mouse retinas, exogenous MDK induces expression of cFos and pS6 in MG. In the chick, MDK significantly decreases numbers dying neurons, reactive microglia, and proliferating MGPCs, whereas PTN has no effect. Inhibition of MDK-signaling with Na3 VO4 blocks neuroprotective effects with an increase in the number of dying cells and negates the pro-proliferative effects on MGPCs in damaged retinas. Inhibitors of PP2A and Pak1, which are associated with MDK-signaling through integrin ß1, suppressed the formation of MGPCs in damaged chick retinas. In mice, MDK promotes a small but significant increase in proliferating MGPCs in damaged retinas and potently decreases the number of dying cells. We conclude that MDK expression is dynamically regulated in Müller glia during embryonic maturation, following retinal injury, and during reprogramming into MGPCs. MDK mediates glial activity, neuronal survival, and the re-programming of Müller glia into proliferating MGPCs.

Ldb1- and Rnf12-dependent regulation of Lhx2 controls the relative balance between neurogenesis and gliogenesis in the retina.

Precise control of the relative ratio of retinal neurons and glia generated during development is essential for visual function. We show that Lhx2, which encodes a LIM-homeodomain transcription factor essential for specification and differentiation of retinal Müller glia, also plays a crucial role in the development of retinal neurons. Overexpression of Lhx2 with its transcriptional co-activator Ldb1 triggers cell cycle exit and inhibits both Notch signaling and retinal gliogenesis. Lhx2/Ldb1 overexpression also induces the formation of wide-field amacrine cells (wfACs). In contrast, Rnf12, which encodes a negative regulator of LDB1, is necessary for the initiation of retinal gliogenesis. We also show that Lhx2-dependent neurogenesis and wfAC formation requires Ascl1 and Neurog2, and that Lhx2 is necessary for their expression, although overexpression of Lhx2/Ldb1 does not elevate expression of these proneural bHLH factors. Finally, we demonstrate that the relative level of the LHX2-LDB1 complex in the retina decreases in tandem with the onset of gliogenesis. These findings show that control of Lhx2 function by Ldb1 and Rnf12 underpins the coordinated differentiation of neurons and Müller glia in postnatal retina.

Clarin-1 expression in adult mouse and human retina highlights a role of Müller glia in Usher syndrome.

Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The selective estrogen receptor modulator raloxifene mitigates the effect of all-trans-retinal toxicity in photoreceptor degeneration.

The retinoid cycle is a metabolic process in the vertebrate retina that continuously regenerates 11-cis-retinal (11-cisRAL) from the all-trans-retinal (atRAL) isomer. atRAL accumulation can cause photoreceptor degeneration and irreversible visual dysfunction associated with incurable blinding retinal diseases, such as Stargardt disease, retinitis pigmentosa (RP), and atrophic age-related macular degeneration (AMD). The underlying cellular mechanisms leading to retinal degeneration remain uncertain, although previous studies have shown that atRAL promotes calcium influx associated with cell apoptosis. To identify compounds that mitigate the effects of atRAL toxicity, here we developed an unbiased and robust image-based assay that can detect changes in intracellular calcium levels in U2OS cells. Using our assay in a high-throughput screen of 2,400 compounds, we noted that selective estrogen receptor modulators (SERMs) potently stabilize intracellular calcium and thereby counteract atRAL-induced toxicity. In a light-induced retinal degeneration mouse model (Abca4-/-Rdh8-/-), raloxifene (a benzothiophene-type scaffold SERM) prevented the onset of photoreceptor apoptosis and thus protected the retina from degeneration. The minor structural differences between raloxifene and one of its derivatives (Y 134) had a major impact on calcium homeostasis after atRAL exposure in vitro, and we verified this differential impact in vivo In summary, the SERM raloxifene has structural and functional neuroprotective effects in the retina. We propose that the highly sensitive image-based assay developed here could be applied for the discovery of additional drug candidates preventing photoreceptor degeneration.