Biology subjects

Reh, T. A.

Publications and source records attributed to Reh, T. A..

16 recordsLinked to original sources

bioRxiv2026

Long-read single cell transcriptomics uncovers isoform preferences in developing human retina

Retinal development has been extensively studied and key transcriptional regulators that drive fate decisions have been identified for major cell classes. These findings were confirmed and deepened in recent years with the advance of single cell RNA sequencing (Scrase). However, many processes that guide progenitor to postmitotic cell differentiation remain elusive, especially since some genes seem to yield different cell populations without apparent correlation with expression level or timing. Here, differential transcript isoform usage might play a role in diversifying the function of developmental genes. In short-read based scRNAseq, isoforms can only be identified if a read maps to a unique sequence or exon junction. However, due to the sparsity and very short reads, these events are extremely rare. We combined a commercial scRNAseq kit, that produces barcoded, full-length cDNA with Oxford Nanopore Technologies based long-read sequencing to generate the first single cell long-read sequencing dataset of fetal human retina. It can help elucidate the role of alternative splicing in retinal development and guide the design of transcript-specific gene therapies for retinal regeneration.

developmental biology↗

bioRxiv2026

Identification of transgene insertion sites via short- and long-read whole genome sequencing.

Transgenic mouse lines are essential to uncover organ or system level genotype-phenotype relationships. The generation of such lines via transgene addition may lead to the insertion into unknown genomic loci potentially leading not only to the disruption of native genes but also attenuation of transgene expression. Additionally, this often results in the inability to determine transgene zygosity which in turn complicates breeding and interpretation of experimental results. In this study we present two whole genome sequencing based pipelines that allow the identification and genotyping of even complex multi transgenic inserts. As they use widely available reagents and bioinformatic tools, they can easily be applied to develop genotyping strategies in potentially any species.

molecular biology↗

bioRxiv2025

CUT&TIME captures the history of open chromatin indeveloping neurons

Chromatin structure plays a central role in defining cell identity by regulating gene expression. During development, shifts in chromatin structure facilitate changes in gene expression needed to specify distinct cell types. To understand how changes in chromatin structure influence the developmental trajectory of neural progenitor cells, we developed CUT&TIME, a technique that uses a hyperactive 6-methyl adenosine (6mA) methyltransferase pulsed in living cells to map historical chromatin accessibility genome-wide in single cells. We show that CUT&TIME produces a record of the chromatin landscape during neurogenesis in the developing retina, specifically as neural progenitors produce the major projection neuron type, retinal ganglion cells (RGCs). We further show that this method is compatible with single cell profiling technologies, which allows us to visualize and capture the diversity of chromatin states that produce RGCs. Additionally, we identify changes in promoter accessibility associated with the transition from progenitor to RGC. Together, these data demonstrate that CUT&TIME captures a historical record of chromatin structure, which can be used to identify early changes in accessibility associated with cell-fate commitment.

developmental biology↗

bioRxiv2025

Dynamic expression of ASCL1 drives neurogenesis from infant and adult macaque Müller glia into immature retinal ganglion cells

Strategies to stimulate the regeneration of neurons in the adult central nervous system can offer universal solutions for neurodegenerative diseases. Taking lessons from naturally regenerating species, such as the zebrafish, we have previously shown that vector-mediated expression of proneural transcription factors can stimulate neurogenesis from the resident Muller glia (MG) population in the adult mouse retina, both in vitro and in vivo. To bring this closer to translation, we now show that vector-mediated expression of the proneural transcription factor ASCL1 can reprogram adult macaque MG into functional neurons. To this end, we established purified MG cultures and show they retain a mature transcriptomic profile that correlates with foveal and peripheral MG. Importantly, MG-derived neurons express retinal ganglion cell markers, can fire action potentials and have a transcriptome that overlaps with developing human and adult macaque retinal ganglion cells. To refine this approach for clinical application, we incorporated microRNA-124 target sites in the reprogramming cassette and show that this restricts expression to MG in mixed primary cultures and intact explant cultures of adult macaque retina. Regulating ASCL1 expression with microRNA-124 target sites maintained the reprogramming efficiency from adult MG cultures and improved the yield of RGC-like neurons from infant MG cultures. Most importantly, with this vector cassette we successfully reprogrammed macaque MG from both adult and infant retina into HuC/D+ neurons. Our findings demonstrate that ASCL1 can induce neurogenesis from macaque MG across ages and provide a targeted, effective strategy for potential clinical translation in retinal repair.

neuroscience↗

bioRxiv2025

DIO3 coordinates photoreceptor development timing and fate stability in human retinal organoids

The mechanisms governing the generation of neuronal subtypes at distinct times and proportions during human retinal development are poorly understood. While thyroid hormone (TH) signaling specifies cone photoreceptor subtypes, how this regulation changes over time remains unclear. To address this question, we studied the expression and function of type 3 iodothyronine deiodinase (DIO3), an enzyme that degrades TH, in human retinal organoids. We show that DIO3 is a master regulator of human photoreceptor developmental timing and cell fate stability. DIO3 is highly expressed in retinal progenitor cells (RPCs) and decreases as these cells asynchronously differentiate into neurons, progressively reducing TH degradation and increasing TH signaling. DIO3 mutant organoids display precocious development of S cones, L/M cones, and rods, increased photoreceptor (PR) density, and adoption of L/M cone fate characteristics by S cones and rods. Our multiomics and chimeric organoid experiments show that cell autonomous and non-autonomous mechanisms locally coordinate and maintain DIO3 expression and TH signaling levels among cells. Computational modeling reveals a mechanism that couples TH levels and fate specification, providing robustness to photoreceptor development as compared to a probabilistic, cell-intrinsic mechanism. Based on our findings, we propose an hourglass hypothesis, in which the proportion of progenitors to neurons decreases over time to relieve TH degradation, which triggers development of PR subtypes at specific times. Our study identifies how local regulation of thyroid hormone signaling influences neural cell fate specification, which may be a consideration for designing regenerative therapies.

developmental biology↗

bioRxiv2024

Stimulating the regenerative capacity of the human retina with proneural transcription factors in 3D cultures

Retinal diseases often lead to degeneration of specific retinal cell types with currently limited therapeutic options to replace the lost neurons. Previous studies have reported that overexpression of ASCL1 or combinations of proneural factors in Muller glia (MG) induces regeneration of functional neurons in the adult mouse retina. Recently, we applied the same strategy in dissociated cultures of fetal human MG and although we stimulated neurogenesis from MG, our effect in 2D cultures was modest and our analysis of newborn neurons was limited. In this study, we aimed to improve our MG reprogramming strategy in a more intact retinal environment. For this purpose, we used an in vitro culture system of human fetal retinal tissue and adult human postmortem retina. To stimulate reprogramming, we used lentiviral vectors to deliver constructs with a glial-specific promoter (HES1) driving ASCL1 alone or in combination with additional developmental transcription factors such as ATOH1 and NEUROD1. Combining IHC, scRNA-seq and electrophysiology, we show for the first time that human MG can generate new neurons even in adults. This work constitutes a key step towards a future clinical application of this regenerative medicine approach for retinal degenerative disorders.

developmental biology↗

bioRxiv2024

AAV-mediated expression of proneural factors stimulates neurogenesis from adult Müller glia in vivo.

The lack of regeneration in the human central nervous system (CNS) has major health implications. To address this, we previously used transgenic mouse models to show that neurogenesis can be stimulated in the adult mammalian retina by driving regeneration programs that other species activate following injury. Expression of specific proneural factors in adult Muller glia causes them to re-enter the cell cycle and give rise to new neurons following retinal injury. To bring this strategy closer to clinical application, we now show that neurogenesis can also be stimulated when delivering these transcription factors to Muller glia using adeno-associated viral (AAV) vectors. AAV-mediated neurogenesis phenocopies the neurogenesis we observed from transgenic animals, with different proneural factor combinations giving rise to distinct neuronal subtypes in vivo. Vector-borne neurons are morphologically, transcriptomically and physiologically similar to bipolar and amacrine/ganglion-like neurons. These results represent a key step forward in developing a cellular reprogramming approach to regenerative medicine in the CNS.

neuroscience↗

bioRxiv2024

Ciliary marginal zone of the developing human retina maintains retinal progenitor cells until late gestational stages

Non-mammalian vertebrates maintain a proliferative stem cell population at the far periphery of their retina called the ciliary marginal zone (CMZ), which gives rise to all retinal cell types and contributes to retinal regeneration upon injury. Humans do not maintain a proliferative CMZ into adulthood; however, it is not known how long in development this region continues to generate new neurons. Here, we identify a population of cells in the far peripheral retina of the fetal human that continues to proliferate long after the rest of the retina is quiescent. Single cell RNA-sequencing and EdU tracing at late time points in development reveal that this region has features of the non-mammalian CMZ, including the capacity to produce both early and late born cell types at late developmental stages, and a longer cell cycle than more centrally located retinal progenitor cells (RPCs). Moreover, while more central RPCs exit the cell cycle with the addition of a TGF{beta}-inhibitor, we show that early RPCs within the CMZ do not. These findings define the late stages of neurogenesis in human retinal development, and present a unique model system to study the fetal CMZ in humans.

developmental biology↗

bioRxiv2023

Synchronized long-read genome, methylome, epigenome, and transcriptome for resolving a Mendelian condition

Resolving the molecular basis of a Mendelian condition (MC) remains challenging owing to the diverse mechanisms by which genetic variants cause disease. To address this, we developed a synchronized long-read genome, methylome, epigenome, and transcriptome sequencing approach, which enables accurate single-nucleotide, insertion-deletion, and structural variant calling and diploid de novo genome assembly, and permits the simultaneous elucidation of haplotype-resolved CpG methylation, chromatin accessibility, and full-length transcript information in a single long-read sequencing run. Application of this approach to an Undiagnosed Diseases Network (UDN) participant with a chromosome X;13 balanced translocation of uncertain significance revealed that this translocation disrupted the functioning of four separate genes (NBEA, PDK3, MAB21L1, and RB1) previously associated with single-gene MCs. Notably, the function of each gene was disrupted via a distinct mechanism that required integration of the four omes to resolve. These included nonsense-mediated decay, fusion transcript formation, enhancer adoption, transcriptional readthrough silencing, and inappropriate X chromosome inactivation of autosomal genes. Overall, this highlights the utility of synchronized long-read multi-omic profiling for mechanistically resolving complex phenotypes.

genetics↗

bioRxiv2023

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.

neuroscience↗

bioRxiv2023

Single-cell sequencing of individual retinal organoids reveals determinants of cell fate heterogeneity

With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA-seq multiplexing method to retinal organoids. We demonstrate that sci-Plex and 10x methods produce highly concordant cell class compositions and then expand sci-Plex to analyze the cell class composition of 410 organoids upon modulation of critical developmental pathways. Leveraging individual organoid data, we develop a method to measure organoid heterogeneity, and we identify that activation of Wnt signaling early in retinal organoid cultures increases retinal cell classes up to six weeks later. Our data show sci-Plexs potential to dramatically scale-up the analysis of treatment conditions on relevant human models.

genomics↗

bioRxiv2023

Foveolar cone subtype patterning in human retinal organoids

The mechanisms that generate patterns of cell types unique to humans are poorly understood. In the central region of the human retina, the high-acuity foveola is notable, in part, for its dense packing of green (M) and red (L) cones and absence of blue (S) cones. To identify mechanisms that promote M/L and suppress S cone patterning in the foveola, we examined human fetal retinas and differentiated human retinal organoids. During development, sparse S-opsin-expressing cones are initially observed in the foveola. Later in fetal development, the foveola contains a mix of cones that either co-express S- and M/L-opsins or exclusively express M/L-opsin. In adults, only M/L cones are present. Two signaling pathway regulators are highly and continuously expressed in the central retina: Cytochrome P450 26 subfamily A member 1 (CYP26A1), which degrades retinoic acid (RA) and Deiodinase 2 (DIO2), which promotes thyroid hormone (TH) signaling. Both CYP26A1 null mutant organoids and high RA conditions increased the number of S cones and reduced the number of M/L cones in human retinal organoids. In contrast, sustained TH signaling promoted the generation of M/L-opsin-expressing cones and induced M/L-opsin expression in S-opsin-expressing cones, showing that cone fate is plastic. Our data suggest that CYP26A1 degrades RA to specify M/L cones and limit S cones and that continuous DIO2 expression sustains high levels of TH to convert S cones into M/L cones, resulting in the foveola containing only M/L cones. Since the foveola is highly susceptible to impairment in diseases such as macular degeneration, a leading cause of vision loss, our findings inform organoid design for potential therapeutic applications.

developmental biology↗

bioRxiv2022

Introduced chemokine gradients guide transplanted and regenerated retinal neurons toward their natural position in the retina

Ongoing cell replacement studies and clinical trials have demonstrated the need to control donor and newborn cell behavior within their target tissue. Here we present a methodology to guide stem cell-derived and endogenously regenerated neurons by engineering the microenvironment. Being an "approachable part of the brain," the eye provides a unique opportunity to study donor neuron fate, migration, and integration within the central nervous system. Glaucoma and other optic neuropathies lead to the permanent loss of retinal ganglion cells (RGCs) - the neurons in the retina that transfer all visual information from the eye to the brain. Cell transplantation and transdifferentiation strategies have been proposed to restore RGCs, and one of the significant barriers to successful RGC integration into the existing retinal circuitry is cell migration towards their natural position in the retina. Here we describe a framework for identifying, selecting, and applying chemokines to direct cell migration in vivo within the retina. We have performed an in silico analysis of the single-cell transcriptome of the developing human retina and identified six receptor-ligand candidates to guide stem cell-derived or newborn neurons. The lead candidates were then tested in functional in vitro assays for their ability to guide stem cell-derived RGCs. For the in vivo studies, donor and newborn neurons were differentiated in human and mouse retinal organoids or endogenously reprogrammed with proneuronal transcription factors, respectively. An exogenous stromal cell-derived factor-1 (SDF1) gradient led to a 2.7-fold increase in donor RGC migration into the ganglion cell layer and a 3.3-fold increase in the displacement of newborn RGCs out of the inner nuclear layer. Furthermore, by altering the migratory profile of donor RGCs toward multipolar migration, overall migration was improved in mature retinal tissues. Together, these results highlight the ability and importance of engineering the tissue microenvironment and the individual cells for research and clinical applications in gene and cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/510158v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@1856140org.highwire.dtl.DTLVardef@1b3ca26org.highwire.dtl.DTLVardef@9c9b34org.highwire.dtl.DTLVardef@19187c0_HPS_FORMAT_FIGEXP M_FIG C_FIG In brief, the "in silico - in vitro - in vivo" funnel holds significant potential for identifying targets to control cellular processes in research and clinical applications. In this report, Soucy et al. describes a framework for identifying, selecting, and applying chemokines to direct retinal ganglion cell migration in vivo within the adult mouse retina.

bioengineering↗

bioRxiv2021

NFkB-signaling suppresses Müller glia-mediated neuron regeneration in the mammalian retina

Muller glia (MG) in mammalian retinas are incapable of regenerating neurons after damage, whereas the MG in lower vertebrates regenerate functional neurons. Identification 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 NFkB activity 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 TGF{beta}2 and suppression of NFI and Id transcription factors. Inhibition of Smad3 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.

neuroscience↗

bioRxiv2019

Cis-Regulatory Accessibility Directs Muller Glial Development and Regenerative Capacity

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 Muller glia (MG). Recently, we have shown that driving expression of Ascl1 in adult mouse MG stimulates neurogenesis similar to fish 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 with MG. Our analysis demonstrated striking similarities between MG and progenitors, with losses in regulatory motifs for neurogenesis genes. Young MG were found to have intermediate expression profiles and accessible DNA, which is mirrored in the ability of Ascl1 to direct bipolar neurogenesis in young MG. When comparing what makes bipolar and photoreceptor cells distinct from glial cells, we find that bipolar-specific accessible regions are more frequently linked to bHLH motifs and Ascl1 binding, indicating that Ascl1 preferentially binds to bipolar regions. Overall, our analysis indicates a loss of neurogenic gene expression and motif accessibility during glial maturation that may prevent efficient reprogramming.

developmental biology↗

bioRxiv2019

STAT pathway activation limits the Ascl1-mediated chromatin remodeling required for neural regeneration from Muller glia in adult mouse retina.

Muller glia can serve as a source for retinal regeneration in some non-mammalian vertebrates. Recently we found that this process can be induced in mouse Muller glia after injury, by combining transgenic expression of the proneural transcription factor Ascl1 and the HDAC inhibitor TSA. However, new neurons are only generated from a subset of Muller glia in this model, and identifying factors that limit Ascl1-mediated MG reprogramming could potentially make this process more efficient, and potentially useful clinically. One factor that limits neurogenesis in some non-mammalian vertebrates is the STAT pathway activation that occurs in Muller glia in response to injury. In this report, we tested whether injury induced STAT activation hampers the ability of Ascl1 to reprogram Muller glia into retinal neurons. Using a STAT inhibitor, in combination with our previously described reprogramming paradigm, we found a large increase in the ability of Muller glia to generate neurons, similar to those we described previously. Single-cell RNA-seq showed that the progenitor-like cells derived from Ascl1-expressing Muller glia have a higher level of STAT signaling than those that become neurons. Using Ascl1 ChIP-seq and DNase-seq, we found that developmentally inappropriate Ascl1 binding sites (that were unique to the overexpression context) had enrichment for the STAT binding motif. This study provides evidence that STAT pathway activation reduces the efficiency of Ascl1-mediated reprogramming in Muller glia, potentially by directing Ascl1 to inappropriate targets.

developmental biology↗