Stimulation of functional neuronal regeneration from Müller glia in adult mice.

Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage in zebrafish and is necessary for regeneration. Although Ascl1 is not expressed in mammalian MG after injury, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.

Conservation of trans-acting circuitry during mammalian regulatory evolution.

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.

A transient wave of BMP signaling in the retina is necessary for Müller glial differentiation.

The primary glial cells in the retina, the Müller glia, differentiate from retinal progenitors in the first postnatal week. CNTF/LIF/STAT3 signaling has been shown to promote their differentiation; however, another key glial differentiation signal, BMP, has not been examined during this period of Müller glial differentiation. In the course of our analysis of the BMP signaling pathway, we observed a transient wave of Smad1/5/8 signaling in the inner nuclear layer at the end of the first postnatal week, from postnatal day (P) 5 to P9, after the end of neurogenesis. To determine the function of this transient wave, we blocked BMP signaling during this period in vitro or in vivo, using either a BMP receptor antagonist or noggin (Nog). Either treatment leads to a reduction in expression of the Müller glia-specific genes Rlbp1 and Glul, and the failure of many of the Müller glia to repress the bipolar/photoreceptor gene Otx2. These changes in normal Müller glial differentiation result in permanent disruption of the retina, including defects in the outer limiting membrane, rosette formation and a reduction in functional acuity. Our results thus show that Müller glia require a transient BMP signal at the end of neurogenesis to fully repress the neural gene expression program and to promote glial gene expression.

Transgenic expression of the proneural transcription factor Ascl1 in Müller glia stimulates retinal regeneration in young mice.

Müller glial cells are the source of retinal regeneration in fish and birds; although this process is efficient in fish, it is less so in birds and very limited in mammals. It has been proposed that factors necessary for providing neurogenic competence to Müller glia in fish and birds after retinal injury are not expressed in mammals. One such factor, the proneural transcription factor Ascl1, is necessary for retinal regeneration in fish but is not expressed after retinal damage in mice. We previously reported that forced expression of Ascl1 in vitro reprograms Müller glia to a neurogenic state. We now test whether forced expression of Ascl1 in mouse Müller glia in vivo stimulates their capacity for retinal regeneration. We find that transgenic expression of Ascl1 in adult Müller glia in undamaged retina does not overtly affect their phenotype; however, when the retina is damaged, the Ascl1-expressing glia initiate a response that resembles the early stages of retinal regeneration in zebrafish. The reaction to injury is even more pronounced in Müller glia in young mice, where the Ascl1-expressing Müller glia give rise to amacrine and bipolar cells and photoreceptors. DNaseI-seq analysis of the retina and Müller glia shows progressive reduction in accessibility of progenitor gene cis-regulatory regions consistent with the reduction in their reprogramming. These results show that at least one of the differences between mammal and fish Müller glia that bears on their difference in regenerative potential is the proneural transcription factor Ascl1.

Ezh2 maintains retinal progenitor proliferation, transcriptional integrity, and the timing of late differentiation.

Epigenetic regulation, including histone modification, is a critical component of gene regulation, although precisely how this contributes to the development of complex tissues such as the neural retina is still being explored. We show that during retinal development in mouse, there are dynamic patterns of expression of the polycomb repressive complex 2 (PRC2) catalytic subunit EZH2 in retinal progenitors and some differentiated cells, as well as dynamic changes in the histone modification H3K27me3. Using conditional knockout of Ezh2 using either Pax6-αCre or Six3-Cre, we find selective reduction in postnatal retinal progenitor proliferation, disruption of retinal lamination, and enhanced differentiation of several late born cell types in the early postnatal retina, including photoreceptors and Müller glia, which are ultimately increased in number and become reactive. RNA-seq identifies many non-retinal genes upregulated with loss of Ezh2, including multiple Hox genes and the cell cycle regulator Cdkn2a, which are established targets of EZH2-mediated repression. ChIP analysis confirms loss of the H3K27me3 modification at these loci. Similar gene upregulation is observed in retinal explants treated with an EZH2 chemical inhibitor. There is considerable overlap with EZH2-regulated genes reported in non-neural tissues, suggesting that EZH2 can regulate similar genes in multiple lineages. Our findings reveal a conserved role for EZH2 in constraining the expression of potent developmental regulators to maintain lineage integrity and retinal progenitor proliferation, as well as regulating the timing of late differentiation.

ASCL1 reprograms mouse Muller glia into neurogenic retinal progenitors.

Non-mammalian vertebrates have a robust ability to regenerate injured retinal neurons from Müller glia (MG) that activate the gene encoding the proneural factor Achaete-scute homolog 1 (Ascl1; also known as Mash1 in mammals) and de-differentiate into progenitor cells. By contrast, mammalian MG have a limited regenerative response and fail to upregulate Ascl1 after injury. To test whether ASCL1 could restore neurogenic potential to mammalian MG, we overexpressed ASCL1 in dissociated mouse MG cultures and intact retinal explants. ASCL1-infected MG upregulated retinal progenitor-specific genes and downregulated glial genes. Furthermore, ASCL1 remodeled the chromatin at its targets from a repressive to an active configuration. MG-derived progenitors differentiated into cells that exhibited neuronal morphologies, expressed retinal subtype-specific neuronal markers and displayed neuron-like physiological responses. These results indicate that a single transcription factor, ASCL1, can induce a neurogenic state in mature MG.

P53 is required for the developmental restriction in Müller glial proliferation in mouse retina.

Müller glia are normally mitotically quiescent cells, but in certain pathological states they can re-enter the mitotic cell cycle. While several cell cycle regulators have been shown to be important in this process, a role for the tumor suppressor, p53, has not been demonstrated. Here, we investigated a role for p53 in limiting the ability of Müller glia to proliferate in the mature mouse retina. Our data demonstrate that Müller glia undergo a developmental restriction in their potential to proliferate. Retinal explants or dissociated cultures treated with EGF become mitotically quiescent by the end of the second postnatal week. In contrast, Müller glia from adult trp53-/+ or trp53-/- mice displayed a greater ability to proliferate in response to EGF stimulation in vitro. The enhanced proliferative ability of trp53 deficient mice correlates with a decreased expression of the mitotic inhibitor Cdkn1a/p21(cip) and an increase in c-myc, a transcription factor that promotes cell cycle progression. These data show that p53 plays an essential role in limiting the potential of Müller glia to re-enter the mitotic cycle as the retina matures during postnatal development.

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.

De novo design of protein logic gates.

The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, NOR, XNOR, and NOT gates built from de novo-designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the TIM3 gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.

Open chromatin dynamics in prosensory cells of the embryonic mouse cochlea.

Hearing loss is often due to the absence or the degeneration of hair cells in the cochlea. Understanding the mechanisms regulating the generation of hair cells may therefore lead to better treatments for hearing disorders. To elucidate the transcriptional control mechanisms specifying the progenitor cells (i.e. prosensory cells) that generate the hair cells and support cells critical for hearing function, we compared chromatin accessibility using ATAC-seq in sorted prosensory cells (Sox2-EGFP+) and surrounding cells (Sox2-EGFP-) from E12, E14.5 and E16 cochlear ducts. In Sox2-EGFP+, we find greater accessibility in and near genes restricted in expression to the prosensory region of the cochlear duct including Sox2, Isl1, Eya1 and Pou4f3. Furthermore, we find significant enrichment for the consensus binding sites of Sox2, Six1 and Gata3-transcription factors required for prosensory development-in the open chromatin regions. Over 2,200 regions displayed differential accessibility with developmental time in Sox2-EGFP+ cells, with most changes in the E12-14.5 window. Open chromatin regions detected in Sox2-EGFP+ cells map to over 48,000 orthologous regions in the human genome that include regions in genes linked to deafness. Our results reveal a dynamic landscape of open chromatin in prosensory cells with potential implications for cochlear development and disease.

Molecular Anatomy of the Developing Human Retina.

Clinical and genetic heterogeneity associated with retinal diseases makes stem-cell-based therapies an attractive strategy for personalized medicine. However, we have limited understanding of the timing of key events in the developing human retina, and in particular the factors critical for generating the unique architecture of the fovea and surrounding macula. Here we define three key epochs in the transcriptome dynamics of human retina from fetal day (D) 52 to 136. Coincident histological analyses confirmed the cellular basis of transcriptional changes and highlighted the dramatic acceleration of development in the fovea compared with peripheral retina. Human and mouse retinal transcriptomes show remarkable similarity in developmental stages, although morphogenesis was greatly expanded in humans. Integration of DNA accessibility data allowed us to reconstruct transcriptional networks controlling photoreceptor differentiation. Our studies provide insights into human retinal development and serve as a resource for molecular staging of human stem-cell-derived retinal organoids.

Retinal regeneration in birds and mice.

Retinal regeneration from resident Müller glia cells is robust in teleost fish, but is severely limited in birds and mammals. After neurotoxic injury, chick Müller glia can proliferate, and activate neurogenic genes, but they display limited capacity to differentiate into neurons. Developmental signaling molecules enhance this process. Regeneration of retinal neurons in rodents is even more limited. However, studies show evidence of proliferation and neurogenic gene expression after injury, with stronger effects in rats than mice, and differences between mouse strains. Mitogenic growth factors and Wnt signaling potentiate the proliferative response, while misexpression of the proneural transcription factor, Ascl1, reprograms to generate neurons from Müller glial in vitro, and stimulates neuronal regeneration in young mice, in vivo.

DNase I hypersensitivity analysis of the mouse brain and retina identifies region-specific regulatory elements.

BACKGROUND: The brain, spinal cord, and neural retina comprise the central nervous system (CNS) of vertebrates. Understanding the regulatory mechanisms that underlie the enormous cell-type diversity of the CNS is a significant challenge. Whole-genome mapping of DNase I-hypersensitive sites (DHSs) has been used to identify cis-regulatory elements in many tissues. We have applied this approach to the mouse CNS, including developing and mature neural retina, whole brain, and two well-characterized brain regions, the cerebellum and the cerebral cortex. RESULTS: For the various regions and developmental stages of the CNS that we analyzed, there were approximately the same number of DHSs; however, there were many DHSs unique to each CNS region and developmental stage. Many of the DHSs are likely to mark enhancers that are specific to the specific CNS region and developmental stage. We validated the DNase I mapping approach for identification of CNS enhancers using the existing VISTA Browser database and with in vivo and in vitro electroporation of the retina. Analysis of transcription factor consensus sites within the DHSs shows distinct region-specific profiles of transcriptional regulators particular to each region. Clustering developmentally dynamic DHSs in the retina revealed enrichment of developmental stage-specific transcriptional regulators. Additionally, we found reporter gene activity in the retina driven from several previously uncharacterized regulatory elements surrounding the neurodevelopmental gene Otx2. Identification of DHSs shared between mouse and human showed region-specific differences in the evolution of cis-regulatory elements. CONCLUSIONS: Overall, our results demonstrate the potential of genome-wide DNase I mapping to cis-regulatory questions regarding the regional diversity within the CNS. These data represent an extensive catalogue of potential cis-regulatory elements within the CNS that display region and temporal specificity, as well as a set of DHSs common to CNS tissues. Further examination of evolutionary conservation of DHSs between CNS regions and different species may reveal important cis-regulatory elements in the evolution of the mammalian CNS.

Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution.

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes.

Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla.

The thymic medulla is generally held to be a specialized environment for negative selection. However, many self-reactive thymocytes first encounter ubiquitous self-antigens in the cortex. Cortical epithelial cells are vital for positive selection, but whether such cells can also promote negative selection is controversial. We used the HY(cd4) model, where T cell receptor for antigen (TCR) expression is appropriately timed and a ubiquitous self-antigen drives clonal deletion in male mice. We demonstrated unambiguously that this deletion event occurs in the thymic cortex. However, the kinetics in vivo indicated that apoptosis was activated asynchronously relative to TCR activation. We found that radioresistant antigen-presenting cells and, specifically, cortical epithelial cells do not efficiently induce apoptosis, although they do cause TCR activation. Rather, thymocytes undergoing clonal deletion were preferentially associated with rare CD11c(+) cortical dendritic cells, and elimination of such cells impaired deletion.

Thymic emigration revisited.

Conventional alphabeta T cell precursors undergo positive selection in the thymic cortex. When this is successful, they migrate to the medulla and are exposed to tissue-specific antigens (TSA) for purposes of central tolerance, and they undergo maturation to become functionally responsive T cells. It is commonly understood that thymocytes spend up to 2 wk in the medulla undergoing these final maturation steps before emigrating to peripheral lymphoid tissues. In addition, emigration is thought to occur via a stochastic mechanism whereby some progenitors leave early and others leave late-a so-called "lucky dip" process. However, recent research has revealed that medullary thymocytes are a heterogeneous mix of naive alphabeta T cell precursors, memory T cells, natural killer T cells, and regulatory T cells. Given this, we revisited the question of how long it takes naive alphabeta T cell precursors to emigrate. We combined the following three approaches to study this question: BrdU labeling, intrathymic injection of a cellular tag, and RAG2p-GFP reporter mice. We established that, on average, naive alphabeta T cell precursors emigrate only 4-5 d after becoming single-positive (SP) thymocytes. Furthermore, emigration occurs via a strict "conveyor belt" mechanism, where the oldest thymocytes leave first.