Gene regulatory networks controlling temporal patterning, neurogenesis, and cell fate specification in the mammalian retina

Lyu, Pin; Hoang, Thanh; Santiago, Clayton; Thomas, Eric D.; Timms, Andrew E.; Appel, Haley; Gimmen, Megan Y.; Le, Nguyet; Jiang, Lizhi; Kim, D. W.; Chen, S.; Espinoza, David; Tegler, A.; Weir, Keiko; Clark, Brian S.; Cherry, Timothy; Qian, Jiang; Blackshaw, Seth

doi:10.1101/2021.07.31.454200

Cited by 14 publications

(22 citation statements)

References 110 publications

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“…Constructs tested included a GFAP-mCherry control and a range of different transcription factors that have been previously reported to reprogram Müller glia or were candidates for doing so based on their role in regulating neurogenesis and/or retinal neuron specification (Fig. 1A) (Mears et al, 2001; Sharma et al, 2019; Puls et al, 2020; Lyu et al, 2021; Todd et al, 2021; Xiao et al, 2021). To genetically label Müller glia prior to AAV infection, we conducted 3 consecutive intraperitoneal injections of 4-hydroxytamoxifen (4-OHT) from P21-P23 in GlastCreER;Sun1-GFP mice (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina

Appel

Pannullo

et al. 2022

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Direct reprogramming of retinal Muller glia is a promising avenue for replacing photoreceptors and retinal ganglion cells lost to retinal dystrophies. However, questions have recently been raised about the accuracy of studies claiming efficient glia-to-neuron reprogramming in retina that were conducted using GFAP mini promoter-driven adeno-associated virus (AAV) vectors. In this study, we have addressed these questions using GFAP mini promoter-driven AAV constructs to simultaneously overexpress the mCherry reporter and candidate transcription factors predicted to induce glia-to-neuron conversion, in combination with prospective genetic labeling of retinal Muller glia using inducible Cre-dependent GFP reporters. We find that, while control GFAP-mCherry constructs express faithfully in Muller glia, 5 out of 7 transcription factor overexpression constructs tested are predominantly expressed in amacrine and retinal ganglion cells. However, genetic cell lineage analysis shows no evidence for glia-to-neuron conversion. These findings demonstrate strong insert-dependent effects on AAV-based GFAP mini promoter specificity that preclude its use in inferring cell lineage relationships when studying glia-to-neuron conversion in retina.

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Section: Resultsmentioning

confidence: 99%

“…3), or the bipolar/photoreceptor cell-specific marker Otx2 (Fig. 4) (Lyu et al, 2021). All other constructs tested – including GFAP-Neurod1-mCherry, GFAP-Mybl1-mCherry, GFAP-Insm1-mCherry, GFAP-Atoh7-Ascl1-mCherry, and GFAP-Atoh7-Brn3b-mCherry – showed little mCherry expression in Sun1-GFP-positive Müller glia (Fig.…”

Section: Resultsmentioning

confidence: 99%

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina

Appel

Pannullo

et al. 2022

Preprint

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“…Meanwhile, formation of the superficial and deep, but not intermediate, plexus layers requires the activity of the hypoxic response pathway (via the von Hippel-Lindau factor (Vhl) found in amacrine and horizontal cells (Usui et al, 2015). In a recent study that used single cell PCR to generate a genetic regulatory network in the mouse retina, Tbx3 was found in amacrine and Müller glia (Lyu et al, 2021). Overexpression of TBX3 in retinal progenitors at P0 led to an increase of amacrine, retinal ganglion cells, Müller glia, and bipolar cells, but repressed formation of rod photoreceptors (Lyu et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

“…In a recent study that used single cell PCR to generate a genetic regulatory network in the mouse retina, Tbx3 was found in amacrine and Müller glia (Lyu et al, 2021). Overexpression of TBX3 in retinal progenitors at P0 led to an increase of amacrine, retinal ganglion cells, Müller glia, and bipolar cells, but repressed formation of rod photoreceptors (Lyu et al, 2021). Therefore, in future studies, we will determine the effect of Tbx3 loss on all types of retinal neurons.…”

Section: Discussionmentioning

confidence: 99%

Loss of Tbx3 in mouse eye causes retinal angiogenesis defects reminiscent of human disease

Derbyshire

Akula

Wong

et al. 2022

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Neural tissue formation requires coordinated, interwoven development of both neural and vascular cells. Spatially distinct regions of the central nervous system (CNS) express factors that are predicted to affect vasculature in one area but not another, yet an example of this idea has not been found. Due to its accessibility, the eye is an excellent tissue to study this question. The factor TBX3 is required for frog eye development, yet its role in mammalian eye formation was not known. To determine how Tbx3 loss-of-function affected eye formation, we crossed the validated Tbx3-floxed mice with the optic cup-Cre recombinase driver, BAC-Dkk3-CRE. We confirmed earlier expression studies of Tbx3 RNA and protein in the embryonic dorsal optic vesicle and, postnatally, in sporadic clusters of cells of the inner nuclear layer. Surprisingly, we also found TBX3 in astrocyte precursors at P0. With Tbx3 loss, blood vessels were malformed throughout the retina, but with more severe effects seen in the dorsal retina. Astrocyte precursors were reduced in number and failed to form a lattice at the dorsal edge. We next examined retinal ganglion cells, as they have been shown to play a critical role in retinal angiogenesis. We found that a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) were missing melanopsin in the dorsal half of the retina. In previous studies, loss of melanopsin has been linked to hyaloid artery persistence, which we also observed in the Tbx3 cKO retina. Together, these results show that TBX3 is required for retinal angiogenesis.Summary StatementTBX3 is a multifunctional protein important for frog eye development. This is the first study showing that TBX3 is critical for mammalian eye formation, controlling the function of key cells required for angiogenesis.

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“…In previous studies, we demonstrated that IReNA can be used to integrate scRNA-seq and ATAC-seq to reconstruct gene regulatory networks controlling retinal regeneration and retinal development [7,14]. Through network analysis using IReNA, we identified modular gene regulatory networks and key transcription factors regulating different cell states in retinal Müller glia in zebrafish and mice.…”

Section: Discussionmentioning

confidence: 99%

IReNA: integrated regulatory network analysis of single-cell transcriptomes and chromatin accessibility profiles

Jiang¹,

Blackshaw²,

Qian³

et al. 2021

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While single-cell RNA sequencing (scRNA-seq) is widely used to profile gene expression, few methods are available to infer gene regulatory networks using scRNA-seq data. Here, we developed and extended IReNA (Integrated Regulatory Network Analysis) to perform regulatory network analysis using scRNA-seq profiles. Four features are developed for IReNA. First, regulatory networks are divided into different modules which represent distinct biological functions. Second, transcription factors significantly regulating each gene module can be identified. Third, regulatory relationships among modules can be inferred. Fourth, IReNA can integrate ATAC-seq data into regulatory network analysis. If ATAC-seq data is available, both transcription factor footprints and binding motifs are used to refine regulatory relationships among co-expressed genes. Using public datasets, we showed that integrated network analysis of scRNA-seq data with ATAC-seq data identified a higher fraction of known regulators than scRNA-seq data alone. Moreover, IReNA provided a better performance of network analysis than currently available methods. Beyond the reconstruction of regulatory networks, IReNA can modularize regulatory networks, and identify key regulators and significant regulatory relationships for modules, facilitating the systems-level understanding of biological regulatory mechanisms. The R package IReNA is available at https://github.com/jiang-junyao/IReNA.Key pointsWe have developed IReNA, a new method for reconstructing regulatory networks using either scRNA-seq and ATAC-seq data or scRNA-seq data alone.IReNA can establish modular regulatory networks to identify key regulatory factors and statistically significant regulatory relationships among modules.Through analyzing three public scRNA-seq datasets, IReNA shows better performance on identifying known regulators than Rcistarget, the most widely used alternative method.

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Gene regulatory networks controlling temporal patterning, neurogenesis, and cell fate specification in the mammalian retina

Cited by 14 publications

References 110 publications

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina

Loss of Tbx3 in mouse eye causes retinal angiogenesis defects reminiscent of human disease

IReNA: integrated regulatory network analysis of single-cell transcriptomes and chromatin accessibility profiles

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