Identification of cis-regulatory modules for adeno-associated virus-based cell-type-specific targeting in the retina and brain.

Adeno-associated viruses (AAVs) targeting specific cell types are powerful tools for studying distinct cell types in the central nervous system (CNS). Cis-regulatory modules (CRMs), e.g., enhancers, are highly cell-type-specific and can be integrated into AAVs to render cell type specificity. Chromatin accessibility has been commonly used to nominate CRMs, which have then been incorporated into AAVs and tested for cell type specificity in the CNS. However, chromatin accessibility data alone cannot accurately annotate active CRMs, as many chromatin-accessible CRMs are not active and fail to drive gene expression in vivo. Using available large-scale datasets on chromatin accessibility, such as those published by the ENCODE project, here we explored strategies to increase efficiency in identifying active CRMs for AAV-based cell-type-specific labeling and manipulation. We found that prescreening of chromatin-accessible putative CRMs based on the density of cell-type-specific transcription factor binding sites (TFBSs) can significantly increase efficiency in identifying active CRMs. In addition, generation of synthetic CRMs by stitching chromatin-accessible regions flanking cell-type-specific genes can render cell type specificity in many cases. Using these straightforward strategies, we generated AAVs that can target the extensively studied interneuron and glial cell types in the retina and brain. Both strategies utilize available genomic datasets and can be employed to generate AAVs targeting specific cell types in CNS without conducting comprehensive screening and sequencing experiments, making a step forward in cell-type-specific research.

Early cis-regulatory events in the formation of retinal horizontal cells.

During retinal development, multipotent and restricted progenitor cells generate all of the neuronal cells of the retina. Among these are horizontal cells, which are interneurons that modulate the light-induced signal from photoreceptors. This study utilizes the identification of novel cis-regulatory elements as a method to examine the gene regulatory networks that direct the development of horizontal cells. Here we describe a screen for cis-regulatory elements, or enhancers, for the horizontal cell-associated genes PTF1A, ONECUT1 (OC1), TFAP2A (AP2A), and LHX1. The OC1ECR22 and Tfap2aACR5 elements were shown to be potential enhancers for OC1 and TFAP2A, respectively, and to be specifically active in developing horizontal cells. The OC1ECR22 element is activated by PTF1A and RBPJ, which translates to regulation of OC1 expression and suggests that PTF1A is a direct activator of OC1 expression in developing horizontal cells. The region within the Tfap2aACR5 element that is responsible for its activation was determined to be a 100 bp sequence named Motif 4. Both OC1ECR22 and Tfap2aACR5 are negatively regulated by the nuclear receptors THRB and RXRG, as is the expression of OC1 and AP2A, suggesting that nuclear receptors may have a role in the negative regulation of horizontal cell development.

Fate-restricted retinal progenitor cells adopt a molecular profile and spatial position distinct from multipotent progenitor cells.

During development, multipotent retinal progenitor cells generate a large number of unique cell types. Recent evidence suggests that there are fate-restricted progenitor cell states in addition to multipotent ones. Here we report a transcriptomic analysis of fate- restricted progenitor cells biased to produce cone photoreceptors and horizontal cells, marked by the THRB cis-regulatory element ThrbCRM1. Comparison to a control population enriched in multipotent progenitor cells identified several genes considered to be pan-progenitor, such as VSX2, LHX2, and PAX6, as downregulated in these fate- restricted retinal progenitor cells. This differential regulation occurs in chick and in a different restricted progenitor population in mouse suggesting that this is a conserved feature of progenitor dynamics during retinal development. S-phase labeling also revealed that nuclear positions of restricted progenitor populations occupy distinct spatial niches within the developing chick retina. Using a conserved regulatory element proximal to the VSX2 gene, a potential negative feedback mechanism from specific transcription factors enriched in cone/horizontal cell progenitor cells was identified. This study identifies conserved molecular and cellular changes that occur during the generation of fate restricted retinal progenitor cells from multipotent retinal progenitor cells.

Drosophila semaphorin2b is required for the axon guidance of a subset of embryonic neurons.

BACKGROUND: The process of axon guidance is important in establishing functional neural circuits. The differential expression of cell-autonomous axon guidance factors is crucial for allowing axons of different neurons to take unique trajectories in response to spatially and temporally restricted cell non-autonomous axon guidance factors. A key motivation in the field is to provide adequate explanations for axon behavior with respect to the differential expression of these factors. RESULTS: We report the characterization of a predicted secreted semaphorin family member, semaphorin2b (Sema-2b) in Drosophila embryonic axon guidance. Misexpression of Sema-2b in neurons causes highly penetrant axon guidance phenotypes in specific longitudinal and motoneuron pathways; however, expression of Sema-2b in muscles traversed by these motoneurons has no effect on axon guidance. In Sema-2b loss-of-function embryos, specific motoneuron and interneuron axon pathways display guidance defects. Specific visualization of the neurons that normally express Sema-2b reveals that this neuronal cohort is strongly affected by Sema-2b loss-of-function alleles. CONCLUSIONS: While secreted semaphorins have been implicated as cell non-autonomous chemorepellants in a variety of contexts, here we report previously undescribed Sema-2b loss-of-function and misexpression phenotypes that are consistent with a cell-autonomous role for Sema-2b.

Enhanced Plasmid-Based Transcriptional Activation in Developing Mouse Photoreceptors.

Rod photoreceptor formation in the postnatal mouse is a widely used model system for studying mammalian photoreceptor development. This experimental paradigm provides opportunities for both gain and loss-of-function studies which can be accomplished through in vivo plasmid delivery and electroporation. However, the cis-regulatory elements used to implement this approach have not been fully evaluated or optimized for the unique transcriptional environment of photoreceptors. Here we report that the use of a photoreceptor cis-regulatory element from the Crx gene in combination with broadly active promoter elements can increase the targeting of developing rod photoreceptors in the mouse. This can lead to greater reporter expression, as well as enhanced misexpression and loss-of-function phenotypes in these cells. This study also highlights the importance of identifying and testing relevant cis-regulatory elements when planning cell subtype specific experiments. The use of the specific hybrid elements in this study will provide a more efficacious gene delivery system to study mammalian photoreceptor formation.

Identification of a retina-specific Otx2 enhancer element active in immature developing photoreceptors.

The homeodomain protein, Otx2, is a critical regulator of vertebrate photoreceptor genesis. However, the genetic elements that define the expression of Otx2 during photoreceptor development are unknown. Therefore, we sought to identify an Otx2 enhancer element that functions in photoreceptor development in order to better understand this specification event. Using the technique of electroporation, we tested a number of evolutionarily conserved elements (ECRs) for expression in the developing retina, and identified ECR2 as having robust activity in the retina. We have characterized this element using a number of assays, including Cre-fate mapping experiments. We found that ECR2 recapitulates expression/function of Otx2 primarily in newly postmitotic photoreceptor cells (PRs), as well as in a subset of retinal progenitor cells (RPCs). ECR2 was also found to be expressed in a subset of horizontal cells (HCs), in keeping with the role of Otx2 in HC development. Furthermore, we determined that the ECR2 element is not active in other Otx2-positive cells such as retinal bipolar cells (BPs), retinal pigmented epithelium (RPE), or the tectum, suggesting that the transcriptional networks controlling Otx2 expression in these cells are unique from those of developing PRs and HCs. These results reveal a distinct molecular state in dividing retinal cells and their newly postmitotic progeny, and provide genetic access to an early and critical transcriptional node involved in the genesis of vertebrate PRs.

Notch signaling represses cone photoreceptor formation through the regulation of retinal progenitor cell states.

Notch signaling is required to repress the formation of vertebrate cone photoreceptors and to maintain the proliferative potential of multipotent retinal progenitor cells. However, the mechanism by which Notch signaling controls these processes is unknown. Recently, restricted retinal progenitor cells with limited proliferation capacity and that preferentially generate cone photoreceptors have been identified. Thus, there are several potential steps during cone genesis that Notch signaling could act. Here we use cell type specific cis-regulatory elements to localize the primary role of Notch signaling in cone genesis to the formation of restricted retinal progenitor cells from multipotent retinal progenitor cells. Localized inhibition of Notch signaling in restricted progenitor cells does not alter the number of cones derived from these cells. Cell cycle promotion is not a primary effect of Notch signaling but an indirect effect on progenitor cell state transitions that leads to depletion of the multipotent progenitor cell population. Taken together, this suggests that the role of Notch signaling in cone photoreceptor formation and proliferation are both mediated by a localized function of Notch in multipotent retinal progenitor cells to repress the formation of restricted progenitor cells.

Robust marking of photoreceptor cells and pinealocytes with several reporters under control of the Crx gene.

Crx is a member of the Otx family of homeobox genes with expression restricted to vertebrate retinal photoreceptor and bipolar cells as well as the pinealocytes of the pineal organ. To facilitate the visualization of Crx-expressing cells, we generated transgenic mice expressing several reporters under the control of the Crx regulatory sequences present within a bacterial artificial chromosome (BAC). These mice expand the transgenic mouse collection, which uses photoreceptor regulatory elements for reporter gene expression by providing a broader repertoire of reporter genes. In addition, because Crx is expressed very soon after a cell fated to be a photoreceptor cell becomes postmitotic, they provide a means for early identification of immature photoreceptor cells.

OTX2 represses sister cell fate choices in the developing retina to promote photoreceptor specification.

During vertebrate retinal development, subsets of progenitor cells generate progeny in a non-stochastic manner, suggesting that these decisions are tightly regulated. However, the gene-regulatory network components that are functionally important in these progenitor cells are largely unknown. Here we identify a functional role for the OTX2 transcription factor in this process. CRISPR/Cas9 gene editing was used to produce somatic mutations of OTX2 in the chick retina and identified similar phenotypes to those observed in human patients. Single cell RNA sequencing was used to determine the functional consequences OTX2 gene editing on the population of cells derived from OTX2-expressing retinal progenitor cells. This confirmed that OTX2 is required for the generation of photoreceptors, but also for repression of specific retinal fates and alternative gene regulatory networks. These include specific subtypes of retinal ganglion and horizontal cells, suggesting that in this context, OTX2 functions to repress sister cell fate choices.

Cis-regulatory analysis of Onecut1 expression in fate-restricted retinal progenitor cells.

BACKGROUND: The vertebrate retina consists of six major classes of neuronal cells. During development, these cells are generated from a pool of multipotent retinal progenitor cells (RPCs) that express the gene Vsx2. Fate-restricted RPCs have recently been identified, with limited mitotic potential and cell fate possibilities compared to multipotent RPCs. One population of fate-restricted RPCs, marked by activity of the regulatory element ThrbCRM1, gives rise to both cone photoreceptors and horizontal cells. These cells do not express Vsx2, but co-express the transcription factors (TFs) Onecut1 and Otx2, which bind to ThrbCRM1. The components of the gene regulatory networks that control the transition from multipotent to fate-restricted gene expression are not known. This work aims to identify and evaluate cis-regulatory elements proximal to Onecut1 to identify the gene regulatory networks involved in RPC fate-restriction. METHOD: We identified regulatory elements through ATAC-seq and conservation, followed by reporter assays to screen for activity based on temporal and spatial criteria. The regulatory elements of interest were subject to deletion and mutation analysis to identify functional sequences and evaluated by quantitative flow cytometry assays. Finally, we combined the enhancer::reporter assays with candidate TF overexpression to evaluate the relationship between the TFs, the enhancers, and early vertebrate retinal development. Statistical tests included ANOVA, Kruskal-Wallis, or unpaired t-tests. RESULTS: Two regulatory elements, ECR9 and ECR65, were identified to be active in ThrbCRM1(+) restricted RPCs. Candidate bHLH binding sites were identified as critical sequences in both elements. Overexpression of candidate bHLH TFs revealed specific enhancer-bHLH interactions. Nhlh1 overexpression expanded ECR65 activity into the Vsx2(+) RPC population, and overexpression of NeuroD1/NeuroG2/NeuroD4 had a similar effect on ECR9. Furthermore, bHLHs that were able to activate ectopic ECR9 reporter were able to induce endogenous Otx2 expression. CONCLUSIONS: This work reports a large-scale screen to identify spatiotemporally specific regulatory elements near the Onecut1 locus. These elements were used to identify distinct populations in the developing retina. In addition, fate-restricted regulatory elements responded differentially to bHLH factors, and suggest a role for retinal bHLHs upstream of the Otx2 and Onecut1 genes during the formation of restricted RPCs from multipotent RPCs.

Quantitative analysis of the ThrbCRM1-centered gene regulatory network.

Enhancer activity is determined by both the activity and occupancy of transcription factors as well as the specific sequences they bind. Experimental investigation of this dynamic requires the ability to manipulate components of the system, ideally in as close to an in vivo context as possible. Here we use electroporation of plasmid reporters to define critical parameters of a specific cis-regulatory element, ThrbCRM1, during retinal development. ThrbCRM1 is associated with cone photoreceptor genesis and activated in a subset of developing retinal cells that co-express the Otx2 and Onecut1 (OC1) transcription factors. Variation of reporter plasmid concentration was used to generate dose response curves and revealed an effect of binding site availability on the number and strength of cells with reporter activity. Critical sequence elements of the ThrbCRM1 element were defined using both mutagenesis and misexpression of the Otx2 and OC1 transcription factors in the developing retina. Additionally, these experiments suggest that the ThrbCRM1 element is co-regulated by Otx2 and OC1 even under conditions of sub-optimal binding of OC1.

Identification of Genes With Enriched Expression in Early Developing Mouse Cone Photoreceptors.

Purpose: The early transcriptional events that occur in newly generated cone photoreceptors are not well described. Knowledge of these events is critical to provide benchmarks for in vitro-derived cone photoreceptors and to understand the process of cone and rod photoreceptor diversification. We sought to identify genes with differential gene expression in embryonic mouse cone photoreceptors. Methods: The specificity of expression of the LHX4 transcription factor in developing cone photoreceptors was examined using immunofluorescence visualization in both mouse and chicken retinas. A LHX4 transgenic reporter line with high specificity for developing mouse cone photoreceptors was identified and used to purify early-stage cone photoreceptors for profiling by single-cell RNA sequencing. Comparisons were made to previous datasets targeting photoreceptors. Results: The LHX4 transcription factor and a transgenic reporter were determined to be highly specific to early developing cone photoreceptors in the mouse. Single-cell transcriptional profiling identified new genes with enriched expression in cone photoreceptors relative to concurrent cell populations. Comparison to previous profiling datasets allowed for further characterization of these genes across developmental time, species, photoreceptor type, and gene regulatory network. Conclusions: The LHX4 gene is highly enriched in developing cone photoreceptors as are several new genes identified through transcriptional profiling, some of which are expressed in subclusters of cones. Many of these cone-enriched genes do not show obvious de-repression in profiling of retinas mutant for the rod-specific transcription factor NRL, highlighting differences between endogenous cones and those induced in NRL mutants.

Lineage tracing analysis of cone photoreceptor associated cis-regulatory elements in the developing chicken retina.

During vertebrate retinal development, transient populations of retinal progenitor cells with restricted cell fate choices are formed. One of these progenitor populations expresses the Thrb gene and can be identified by activity of the ThrbCRM1 cis-regulatory element. Short-term assays have concluded that these cells preferentially generate cone photoreceptors and horizontal cells, however developmental timing has precluded an extensive cell type characterization of their progeny. Here we describe the development and validation of a recombinase-based lineage tracing system for the chicken embryo to further characterize the lineage of these cells. The ThrbCRM1 element was found to preferentially form photoreceptors and horizontal cells, as well as a small number of retinal ganglion cells. The photoreceptor cell progeny are exclusively cone photoreceptors and not rod photoreceptors, confirming that ThrbCRM1 progenitor cells are restricted from the rod fate. In addition, specific subtypes of horizontal cells and retinal ganglion cells were overrepresented, suggesting that ThrbCRM1 progenitor cells are not only restricted for cell type, but for cell subtype as well.

Identification and characterization of early photoreceptor cis-regulatory elements and their relation to Onecut1.

BACKGROUND: Cone and rod photoreceptors are two of the primary cell types affected in human retinal disease. Potential strategies to combat these diseases are the use of gene therapy to rescue compromised photoreceptors or to generate new functional photoreceptors to replace those lost in the diseased retina. Cis-regulatory elements specific to cones, rods, or both types of photoreceptors are critical components of successful implementation of these two strategies. The purpose of this study was to identify and characterize the cell type specificity and activity of cis-regulatory elements active in developing photoreceptors. METHODS: Cis-regulatory elements were introduced into the developing chicken and mouse retina by electroporation. Characterization of reporter activity in relation with cell type markers was determined using confocal microscopy. In addition, two high-throughput flow cytometry assay were developed to assess whether these elements were downstream of Onecut1 in the photoreceptor specification network. RESULTS: The majority of cis-regulatory elements were active in both cone and rod photoreceptors and were largely uninfluenced by a Onecut1 dominant-negative construct. Elements associated with the Thrb, Nr2e3, and Rhodopsin genes showed highly enriched activity in cones or rods, and were affected by interference in Onecut1 signaling. Rhodopsin promoter activity was the most highly influenced by Onecut1 activity and its induction could be modulated by the Maf family transcription factor L-Maf. Nr2e3 elements were observed to have activity in cone photoreceptors and Nr2e3 protein was expressed in developing cone photoreceptors, suggesting a role for this predominant rod gene in cone photoreceptor development. CONCLUSIONS: The analysis presented here provides an experimental framework to determine the specificity and strength of photoreceptor elements within specific genetic networks during development. The Onecut1 transcription factor is one such factor that influences the gene regulatory networks specific to cones and rods, but not those that are common to both.

Novel insights into the regulation of the timeless protein.

In the Drosophila circadian clock, period (per) and its partner, timeless (tim), play a central role in the negative limb of an autoregulatory feedback loop. Unlike per, the dosage of which affects the frequency (tau) of the circadian cycle, we found that increasing copies of the tim gene has no effect on clock period length. The use of the tim promoter to express per results in a shortening of circadian period, also indicating that the regulation of tim is different from that of per. Drosophila TIM is similar to the mammalian circadian protein mPER2 in that it shuttles independently between the nucleus and cytoplasm both in vivo and in vitro. Contrary to the current model that PER and TIM heterodimerization is a prerequisite for their nuclear entry, PER is not required to transport TIM into nuclei, although it influences TIM localization and vice versa. Blocking nuclear export led to increased nuclear expression of TIM in S2 cells and in wild-type and per01 larvae, suggesting that PER may be required for nuclear retention of TIM. Unlike PER, nuclear TIM alone has no ability to repress transcription. We propose that TIM drives cycles of PER expression by regulating its stability, and in turn, PER retains TIM in the nucleus, either for the regulation of its own stability or for a novel nuclear role of TIM.

A gene regulatory network controls the binary fate decision of rod and bipolar cells in the vertebrate retina.

Gene regulatory networks (GRNs) regulate critical events during development. In complex tissues, such as the mammalian central nervous system (CNS), networks likely provide the complex regulatory interactions needed to direct the specification of the many CNS cell types. Here, we dissect a GRN that regulates a binary fate decision between two siblings in the murine retina, the rod photoreceptor and bipolar interneuron. The GRN centers on Blimp1, one of the transcription factors (TFs) that regulates the rod versus bipolar cell fate decision. We identified a cis-regulatory module (CRM), B108, that mimics Blimp1 expression. Deletion of genomic B108 by CRISPR/Cas9 in vivo using electroporation abolished the function of Blimp1. Otx2 and RORß were found to regulate Blimp1 expression via B108, and Blimp1 and Otx2 were shown to form a negative feedback loop that regulates the level of Otx2, which regulates the production of the correct ratio of rods and bipolar cells.

Otx2 and Onecut1 promote the fates of cone photoreceptors and horizontal cells and repress rod photoreceptors.

Cone photoreceptors carry out phototransduction in daylight conditions and provide the critical first step in color vision. Despite their importance, little is known about the developmental mechanisms involved in their generation, particularly how they are determined relative to rod photoreceptors, the cells that initiate vision in dim light. Here, we report the identification of a cis-regulatory module (CRM) for the thyroid hormone receptor beta (Thrb) gene, an early cone marker. We found that ThrbCRM1 is active in progenitor cells biased to the production of cones and an interneuronal cell type, the horizontal cell (HC). Molecular analysis of ThrbCRM1 revealed that it is combinatorially regulated by the Otx2 and Onecut1 transcription factors. Onecut1 is sufficient to induce cells with the earliest markers of cones and HCs. Conversely, interference with Onecut1 transcriptional activity leads to precocious rod development, suggesting that Onecut1 is critically important in defining cone versus rod fates.

Analysis of thyroid response element activity during retinal development.

Thyroid hormone (TH) signaling components are expressed during retinal development in dynamic spatial and temporal patterns. To probe the competence of retinal cells to mount a transcriptional response to TH, reporters that included thyroid response elements (TREs) were introduced into developing retinal tissue. The TREs were placed upstream of a minimal TATA-box and two reporter genes, green fluorescent protein (GFP) and human placental alkaline phosphatase (PLAP). Six of the seven tested TREs were first tested in vitro where they were shown to drive TH-dependent expression. However, when introduced into the developing retina, the TREs reported in different cell types in both a TH-dependent and TH-independent manner, as well as revealed specific spatial patterns in their expression. The role of the known thyroid receptors (TR), TRα and TRß, was probed using shRNAs, which were co-electroporated into the retina with the TREs. Some TREs were positively activated by TR+TH in the developing outer nuclear layer (ONL), where photoreceptors reside, as well as in the outer neuroblastic layer (ONBL) where cycling progenitor cells are located. Other TREs were actively repressed by TR+TH in cells of the ONBL. These data demonstrate that non-TRs can activate some TREs in a spatially regulated manner, whereas other TREs respond only to the known TRs, which also read out activity in a spatially regulated manner. The transcriptional response to even simple TREs provides a starting point for understanding the regulation of genes by TH, and highlights the complexity of transcriptional regulation within developing tissue.