Regulation of retinal amacrine cell generation by miR-216b and Foxn3

The mammalian retina contains a complex mixture of different types of neurons. We find that microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b directed the formation of additional amacrine cells and reduced bipolar neurons in the developing retina. We identify the Foxn3 mRNA as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3, a transcription factor, in the postnatal developing retina by RNAi increased the formation of amacrine cells and reduced bipolar cell formation. Foxn3 disruption by CRISPR in embryonic retinal explants also increased amacrine cell formation, while Foxn3 overexpression inhibited amacrine cell formation prior to Ptf1a expression. Co-expression of Foxn3 partially reversed the effects of ectopic miR-216b on retinal cell formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates Foxn3 and other genes in amacrine cells.

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Regulation of retinal amacrine cell generation by miR-216b and Foxn3

10.1101/2020.10.28.358069 ◽

2020 ◽

Author(s):

Huanqing Zhang ◽

Pei Zhuang ◽

Ryan M. Welchko ◽

Manhong Dai ◽

Fan Meng ◽

...

Keyword(s):

Amacrine Cell ◽

Ectopic Expression ◽

Cell Formation ◽

Complex Mixture ◽

Amacrine Cells ◽

Retinal Cell ◽

Mouse Retina ◽

Mammalian Retina ◽

Different Types ◽

Retinal Explants

AbstractThe mammalian retina contains a complex mixture of different types of neurons. We find that the microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in the retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b, or the related miR-216a, can direct the formation of additional amacrine cells in the developing retina. In addition, we observe the loss of bipolar neurons in the retina after miR-216b expression. We identify the mRNA for the transcriptional regulator Foxn3 as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3 in the postnatal developing retina by RNAi also increases the formation of amacrine cells and reduces bipolar cell formation, while overexpression of Foxn3 inhibits amacrine cell formation prior to the expression of Ptf1a. Disruption of Foxn3 by CRISPR in embryonic retinal explants also reduces amacrine cell formation. Co-expression of Foxn3 can partially reverse the effects of ectopic miR-216b on retinal cell type formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates expression of Foxn3 and other genes in amacrine cells.

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Molecular identification of sixty-three amacrine cell types completes a mouse retinal cell atlas

10.1101/2020.03.10.985770 ◽

2020 ◽

Cited By ~ 2

Author(s):

Wenjun Yan ◽

Mallory A. Laboulaye ◽

Nicholas M. Tran ◽

Irene E. Whitney ◽

Inbal Benhar ◽

...

Keyword(s):

Transcription Factors ◽

Single Cell ◽

High Throughput ◽

Amacrine Cells ◽

Cell Types ◽

Retinal Cell ◽

Mouse Retina ◽

Single Cell Rna Sequencing ◽

Neuronal Types ◽

The Brain

ABSTRACTAmacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing (scRNA-seq) to profile >32,000 ACs from mouse retina, and applied computational methods to identify 63 AC types. We identified molecular markers for each type, and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types express markers for the canonical inhibitory neurotransmitters GABA or glycine, but several express neither or both. In addition, many express one or more neuropeptides, and two express glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological and morphological analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in mouse retina.SIGNIFICANCE STATEMENTThe mouse retina is a leading model for analyzing the development, structure, function and pathology of neural circuits. A complete molecular atlas of retinal cell types provides an important foundation for these studies. We used high-throughput single-cell RNA sequencing (scRNA-seq) to characterize the most heterogeneous class of retinal interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types identified previously as well as many novel types. We provide evidence for use of multiple neurotransmitters and neuropeptides and identify transcription factors expressed by groups of closely related types. Combining these results with those obtained previously, we proposed that the mouse retina contains 130 neuronal types, and is therefore comparable in complexity to other regions of the brain.

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The initiation of Otx2 expression in the developing mouse retina requires a unique enhancer and either Ascl1 or Neurog2 activity

Development ◽

10.1242/dev.199399 ◽

2021 ◽

Author(s):

Michael L. Kaufman ◽

Noah B. Goodson ◽

Ko Uoon Park ◽

Michael Schwanke ◽

Emma Office ◽

...

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Cell Formation ◽

Mouse Retina ◽

Bhlh Transcription Factor ◽

Enhancer Element ◽

Retinal Progenitor ◽

Helix Loop Helix ◽

Bhlh Factors ◽

Simultaneous Loss

During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation. How these retinal progenitor cells initially activate Otx2 expression is unclear. To address this, we investigated the cis-regulatory network that controls Otx2 expression. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. CRISPR/Cas9 mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor binding sites were required for its activity. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Our findings suggest that Ascl1 and Neurog2 act redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. We observed redundancy or compensation at both the transcriptional and enhancer utilization levels, suggesting that the mechanisms governing Otx2 regulation in the retina are flexible and robust.

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The LIM protein complex establishes a retinal circuitry of visual adaptation by regulating Pax6 α-enhancer activity

eLife ◽

10.7554/elife.21303 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 9

Author(s):

Yeha Kim ◽

Soyeon Lim ◽

Taejeong Ha ◽

You-Hyang Song ◽

Young-In Sohn ◽

...

Keyword(s):

Protein Complex ◽

Transforming Growth Factor ◽

Amacrine Cell ◽

Amacrine Cells ◽

Visual Adaptation ◽

Mouse Retina ◽

Visual Responses ◽

Enhancer Activity ◽

Lim Protein ◽

Retinal Circuitry

The visual responses of vertebrates are sensitive to the overall composition of retinal interneurons including amacrine cells, which tune the activity of the retinal circuitry. The expression of Paired-homeobox 6 (PAX6) is regulated by multiple cis-DNA elements including the intronic α-enhancer, which is active in GABAergic amacrine cell subsets. Here, we report that the transforming growth factor ß1-induced transcript 1 protein (Tgfb1i1) interacts with the LIM domain transcription factors Lhx3 and Isl1 to inhibit the α-enhancer in the post-natal mouse retina. Tgfb1i1-/- mice show elevated α-enhancer activity leading to overproduction of Pax6ΔPD isoform that supports the GABAergic amacrine cell fate maintenance. Consequently, the Tgfb1i1-/- mouse retinas show a sustained light response, which becomes more transient in mice with the auto-stimulation-defective Pax6ΔPBS/ΔPBS mutation. Together, we show the antagonistic regulation of the α-enhancer activity by Pax6 and the LIM protein complex is necessary for the establishment of an inner retinal circuitry, which controls visual adaptation.

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Ionotropic glutamate receptors of amacrine cells of the mouse retina

Visual Neuroscience ◽

10.1017/s0952523806231079 ◽

2006 ◽

Vol 23 (1) ◽

pp. 79-90 ◽

Cited By ~ 15

Author(s):

OLIVIA N. DUMITRESCU ◽

DARIO A. PROTTI ◽

SRIPARNA MAJUMDAR ◽

HANNS ULRICH ZEILHOFER ◽

HEINZ WÄSSLE

Keyword(s):

Nmda Receptors ◽

Glutamate Receptors ◽

Amacrine Cell ◽

Fluorescent Protein ◽

Lucifer Yellow ◽

Amacrine Cells ◽

Ionotropic Glutamate Receptors ◽

Mouse Retina ◽

Wide Field ◽

Transgenic Mouse Line

The mammalian retina contains approximately 30 different morphological types of amacrine cells, receiving glutamatergic input from bipolar cells. In this study, we combined electrophysiological and pharmacological techniques in order to study the glutamate receptors expressed by different types of amacrine cells. Whole-cell currents were recorded from amacrine cells in vertical slices of the mouse retina. During the recordings the cells were filled with Lucifer Yellow/Neurobiotin allowing classification as wide-field or narrow-field amacrine cells. Amacrine cell recordings were also carried out in a transgenic mouse line whose glycinergic amacrine cells express enhanced green fluorescent protein (EGFP). Agonist-induced currents were elicited by exogenous application of NMDA, AMPA, and kainate (KA) while holding cells at −75 mV. Using a variety of specific agonists and antagonists (NBQX, AP5, cyclothiazide, GYKI 52466, GYKI 53655, SYM 2081) responses mediated by AMPA, KA, and NMDA receptors could be dissected. All cells (n= 300) showed prominent responses to non-NMDA agonists. Some cells expressed AMPA receptors exclusively and some cells expressed KA receptors exclusively. In the majority of cells both receptor types could be identified. NMDA receptors were observed in about 75% of the wide-field amacrine cells and in less than half of the narrow-field amacrine cells. Our results confirm that different amacrine cell types express distinct sets of ionotropic glutamate receptors, which may be critical in conferring their unique temporal responses to this diverse neuronal class.

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Bistratified starburst amacrine cells in Sox2 conditional knockout mouse retina display ON and OFF responses

Journal of Neurophysiology ◽

10.1152/jn.00322.2018 ◽

2018 ◽

Vol 120 (4) ◽

pp. 2121-2129 ◽

Cited By ~ 1

Author(s):

Todd L. Stincic ◽

Patrick W. Keeley ◽

Benjamin E. Reese ◽

W. Rowland Taylor

Keyword(s):

Transcription Factor ◽

Plexiform Layer ◽

Amacrine Cells ◽

Bipolar Cells ◽

Conditional Knockout ◽

Physiological Data ◽

Mouse Retina ◽

Inner Plexiform Layer ◽

Environmental Signals ◽

Starburst Amacrine Cells

Cell-intrinsic factors, in conjunction with environmental signals, guide migration, differentiation, and connectivity during early development of neuronal circuits. Within the retina, inhibitory starburst amacrine cells (SBACs) comprise ON types with somas in the ganglion cell layer (GCL) and dendrites stratifying narrowly in the inner half of the inner plexiform layer (IPL) and OFF types with somas in the inner nuclear layer (INL) and dendrites stratifying narrowly in the outer half of the IPL. The transcription factor Sox2 is crucial to this subtype specification. Without Sox2, many ON-type SBACs destined for the GCL settle in the INL while many that reach the GCL develop bistratified dendritic arbors. This study asked whether ON-type SBACs in Sox2-conditional knockout retinas exhibit selective connectivity only with ON-type bipolar cells or their bistratified morphology allows them to connect to both ON and OFF bipolar cells. Physiological data demonstrate that these cells receive ON and OFF excitatory inputs, indicating that the ectopically stratified dendrites make functional synapses with bipolar cells. The excitatory inputs were smaller and more transient in Sox2-conditional knockout compared with wild type; however, inhibitory inputs appeared largely unchanged. Thus dendritic stratification, rather than cellular identification, may be the major factor that determines ON vs. OFF connectivity. NEW & NOTEWORTHY Conditional knockout of the transcription factor Sox2 during early embryogenesis converts a monostratifying starburst amacrine cell into a bistratifying starburst cell. Here we show that these bistratifying starburst amacrine cells form functional synaptic connections with both ON and OFF bipolar cells. This suggests that normal ON vs. OFF starburst connectivity may not require distinct molecular specification. Proximity alone may be sufficient to allow formation of functional synapses.

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Onecut Regulates Core Components of the Molecular Machinery for Neurotransmission in Photoreceptor Differentiation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.602450 ◽

2021 ◽

Vol 9 ◽

Author(s):

Quirino Attilio Vassalli ◽

Chiara Colantuono ◽

Valeria Nittoli ◽

Anna Ferraioli ◽

Giulia Fasano ◽

...

Keyword(s):

Transcription Factor ◽

Cell Formation ◽

Transcriptome Profiling ◽

Photoreceptor Cells ◽

Cell Types ◽

Progressive Increase ◽

Retinal Cell ◽

Genetic Mechanisms ◽

Core Components ◽

Multicellular Organisms

Photoreceptor cells (PRC) are neurons highly specialized for sensing light stimuli and have considerably diversified during evolution. The genetic mechanisms that underlie photoreceptor differentiation and accompanied the progressive increase in complexity and diversification of this sensory cell type are a matter of great interest in the field. A role of the homeodomain transcription factor Onecut (Oc) in photoreceptor cell formation is proposed throughout multicellular organisms. However, knowledge of the identity of the Oc downstream-acting factors that mediate specific tasks in the differentiation of the PRC remains limited. Here, we used transgenic perturbation of the Ciona robusta Oc protein to show its requirement for ciliary PRC differentiation. Then, transcriptome profiling between the trans-activation and trans-repression Oc phenotypes identified differentially expressed genes that are enriched in exocytosis, calcium homeostasis, and neurotransmission. Finally, comparison of RNA-Seq datasets in Ciona and mouse identifies a set of Oc downstream genes conserved between tunicates and vertebrates. The transcription factor Oc emerges as a key regulator of neurotransmission in retinal cell types.

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POU6F2 Positive Retinal Ganglion Cells a Novel Group of ON-OFF Directionally Selective Subtypes in the Mouse Retina

10.1101/2020.02.28.968503 ◽

2020 ◽

Author(s):

Ying Li ◽

Jiaxing Wang ◽

Rebecca King ◽

Eldon E. Geisert

Keyword(s):

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Yellow Fluorescent Protein ◽

Corneal Thickness ◽

Amacrine Cells ◽

Molecular Signature ◽

Retinal Cell ◽

Mouse Retina ◽

Inner Plexiform Layer ◽

Retinal Ganglion

AbstractPurposePreviously we identified POU6F2 as a genetic link between central corneal thickness (CCT) and risk of open-angle glaucoma. The present study is designed to characterize the POU6F2-positive retinal ganglion cells (RGCs).MethodsThe Thy1-YFP-H mouse was used to identify the structure of POU6F2-positive RGCs in the retina. In the retina of the Thy1-YFP-H mouse approximately 3% of the RGCs were labeled with yellow fluorescent protein. These retinas were stained for POU6F2 to identify the morphology of the POU6F2 subtypes in 3D reconstructions of the labeled RGCs. Multiple retinal cell markers were also co-stained with POU6F2 to characterize the molecular signature of the POU6F2-positive RGCs. DBA/2J glaucoma models were used to test the role of POU6F2 in injury.ResultsIn the retina POU6F2 labels 32.9% of the RGCs in the DBA/2J retina (16.1% heavily and 16.8% lightly labeled). In 3D constructions of Thy1-YFP-H positive RGCs, the heavily labeled POU6F2-positive cells had dendrites in the inner plexiform layer that were bistratified and appeared to be ON-OFF directionally selective cells. The lightly labeled POU6F2 RGCs displayed 3 different dendritic distributions, with dendrites in the ON sublaminae only, OFF sublaminae only, or bistratified. The POU6F2-positive cells partially co-stained with Cdh6. The POU6F2-positive cells do not co-stain with CART and SATB2 (markers for ON-OFF directionally selective RGC), SMI32 (a marker for alpha RGCs), or ChAT and GAD67(markers for amacrine cells). The POU6F2-positive cells were sensitive to injury. In DBA/2J glaucoma model, at 8 months of age there was a 22% loss of RGCs (labeled with RBPMS) while there was 73% loss of the heavily labeled POU6F2 RGCs.ConclusionsPOU6F2 is a marker for a novel group of RGC subtypes that are ON-OFF directionally selective RGCs that are sensitive to glaucomatous injury.

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The somal patterning of the AII amacrine cell mosaic in the mouse retina is indistinguishable from random simulations matched for density and constrained by soma size

Visual Neuroscience ◽

10.1017/s0952523817000347 ◽

2018 ◽

Vol 35 ◽

Cited By ~ 1

Author(s):

PATRICK W. KEELEY ◽

BENJAMIN E. REESE

Keyword(s):

Amacrine Cell ◽

Amacrine Cells ◽

Bipolar Cells ◽

Horizontal Cells ◽

Mouse Retina ◽

Retinal Neurons ◽

Soma Size ◽

Aii Amacrine Cells ◽

Aii Amacrine ◽

Cholinergic Amacrine Cells

AbstractThe orderly spacing of retinal neurons is commonly regarded as a characteristic feature of retinal nerve cell populations. Exemplars of this property include the horizontal cells and the cholinergic amacrine cells, where individual cells minimize the proximity to like-type neighbors, yielding regularity in the patterning of their somata. Recently, two types of retinal bipolar cells in the mouse retina were shown to exhibit an order in their somal patterning no different from density-matched simulations constrained by soma size but being otherwise randomly distributed. The present study has now extended this finding to a type of retinal amacrine cell, the AII amacrine cell. Voronoi domain analysis revealed the patterning in the population of AII amacrine somata to be no different from density-matched and soma-size-constrained random simulations, while analysis of the density recovery profile showed AII amacrine cells to exhibit a minimal intercellular spacing identical to that for those random simulations: AII amacrine somata were positioned side-by-side as often as chance would predict. Regularity indexes and packing factors (PF) were far lower than those achieved by either the horizontal cells or cholinergic amacrine cells, with PFs also being comparable to those derived from the constrained random simulations. These results extend recent findings that call into question the widespread assumption that all types of retinal neurons are assembled as regular somal arrays, and have implications for the way in which AII amacrine cells must distribute their processes to ensure a uniform coverage of the retinal surface.

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Transcription factor PRDM8 is required for rod bipolar and type 2 OFF-cone bipolar cell survival and amacrine subtype identity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1505870112 ◽

2015 ◽

Vol 112 (23) ◽

pp. E3010-E3019 ◽

Cited By ~ 16

Author(s):

Cynthia C. Jung ◽

Denize Atan ◽

David Ng ◽

Lynda Ploder ◽

Sarah E. Ross ◽

...

Keyword(s):

Transcription Factor ◽

Cell Survival ◽

Amacrine Cell ◽

Cell Function ◽

Ganglion Cells ◽

Mouse Retina ◽

Cell Specification ◽

Subtype Differentiation ◽

And Function

Retinal bipolar (BP) cells mediate the earliest steps in image processing in the visual system, but the genetic pathways that regulate their development and function are incompletely known. We identified PRDI-BF1 and RIZ homology domain containing 8 (PRDM8) as a highly conserved transcription factor that is abundantly expressed in mouse retina. During development and in maturity, PRDM8 is expressed strongly in BP cells and a fraction of amacrine and ganglion cells. To determine whether Prdm8 is essential to BP cell development or physiology, we targeted the gene in mice. Prdm8EGFP/EGFP mice showed nonprogressive b-wave deficits on electroretinograms, consistent with compromised BP cell function or circuitry resembling the incomplete form of human congenital stationary night blindness (CSNB). BP cell specification was normal in Prdm8EGFP/EGFP retina as determined by VSX2+ cell numbers and retinal morphology at postnatal day 6. BP subtype differentiation was impaired, however, as indicated by absent or diminished expression of BP subtype-specific markers, including the putative PRDM8 regulatory target PKCα (Prkca) and its protein. By adulthood, rod bipolar (RB) and type 2 OFF-cone bipolar (CB) cells were nearly absent from Prdm8-null mice. Although no change was detected in total amacrine cell (AC) numbers, increased PRKCA+ and cholinergic ACs and decreased GABAergic ACs were seen, suggesting an alteration in amacrine subtype identity. These findings establish that PRDM8 is required for RB and type 2 OFF-CB cell survival and amacrine subtype identity, and they present PRDM8 as a candidate gene for human CSNB.

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