Mouse Retinal Cell Atlas: Molecular Identification of Over Sixty Amacrine Cell Types
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Research Report: Regular Manuscript MOUSE RETINAL CELL ATLAS: MOLECULAR IDENTIFICATION OF OVER SIXTY AMACRINE CELL TYPES https://doi.org/10.1523/JNEUROSCI.0471-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0471-20.2020 Received: 27 February 2020 Revised: 7 May 2020 Accepted: 13 May 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 MOUSE RETINAL CELL ATLAS: MOLECULAR IDENTIFICATION OF OVER SIXTY 2 AMACRINE CELL TYPES 3 4 Wenjun Yan1*, Mallory A. Laboulaye1*, Nicholas M. Tran1*, Irene E. Whitney1, Inbal 5 Benhar2 and Joshua R. Sanes1^ 6 1. Center for Brain Science and Department of Molecular and Cellular Biology, Harvard 7 University, Cambridge, MA 02138 8 2. Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 9 02142 10 *equal contribution 11 ^author for correspondence. Email: [email protected] 12 13 Abbreviated title: Mouse retinal cell atlas 14 15 Length: 28 pages, 117 words in significance statement, 238 words in abstract, 649 16 words in introduction, 1463 words in discussion, 10 figures, 2 tables 17 18 Acknowledgments: We thank Drs. Thomas Bourgeron (Institut Pasteur), Isabelle Cloez- 19 Tayarani (Institut Pasteur), and James YH Li (University of Connecticut) for providing 20 some mouse tissue used in this study; and Drs. Julia Kaltschmidt (Stanford University), 21 Louis Reichardt (Simons Foundation), Chinfei Chen (Harvard Medical school), Lisa 22 Goodrich (Harvard Medical school) for providing mice. This work was supported by the 23 NIH (NS029169, MH105960, K99EY029360), and Human Frontiers Science Program). 24 25 Conflicts of interest: The authors declare no conflicts of interest 26 1 27 ABSTRACT 28 Amacrine cells (ACs) are a diverse class of interneurons that modulate input from 29 photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively 30 sensitive to particular visual features, which are then relayed to the brain. While many 31 AC types have been identified morphologically and physiologically, they have not been 32 comprehensively classified or molecularly characterized. We used high-throughput 33 single-cell RNA sequencing (scRNA-seq) to profile >32,000 ACs from mice of both 34 sexes and applied computational methods to identify 63 AC types. We identified 35 molecular markers for each type and used them to characterize the morphology of 36 multiple types. We show that they include nearly all previously known AC types as well 37 as many that had not been described. Consistent with previous studies, most of the AC 38 types expressed markers for the canonical inhibitory neurotransmitters GABA or 39 glycine, but several expressed neither or both. In addition, many expressed one or more 40 neuropeptides, and two express glutamatergic markers. We also explored 41 transcriptomic relationships among AC types and identified transcription factors 42 expressed by individual or multiple closely related types. Noteworthy among these were 43 Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, 44 respectively. Together, these results provide a foundation for developmental and 45 functional studies of ACs, as well as means for genetically accessing them. Along with 46 previous molecular, physiological and morphological analyses, they establish the 47 existence of at least 130 neuronal types and nearly 140 cell types in mouse retina. 48 49 SIGNIFICANCE STATEMENT 50 The mouse retina is a leading model for analyzing the development, structure, function 51 and pathology of neural circuits. A complete molecular atlas of retinal cell types 52 provides an important foundation for these studies. We used high-throughput single-cell 53 RNA sequencing (scRNA-seq) to characterize the most heterogeneous class of retinal 54 interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types 55 identified previously as well as many novel types. We provide evidence for use of 56 multiple neurotransmitters and neuropeptides and identify transcription factors 57 expressed by groups of closely related types. Combining these results with those 58 obtained previously, we proposed that the mouse retina contains ~130 neuronal types, 59 and is therefore comparable in complexity to other regions of the brain. 60 61 INTRODUCTION 62 63 The mouse retina is an experimentally tractable system for analyzing principles of 64 central nervous system circuit development, structure, and function (Sanes and 65 Masland, 2015; Seabrook et al., 2017). In addition, it is a prominent animal model for 66 assessing mechanisms underlying retinal diseases, the major cause of irreversible 67 blindness. An atlas of mouse retinal cell types would be a valuable resource for 2 68 pursuing such studies. High-throughput single-cell RNA sequencing (scRNA-seq) is a 69 promising method for achieving this goal: it enables comprehensive identification and 70 molecular characterization of the cell types that comprise complex tissues, as well as a 71 framework for incorporating structural and physiological data required for generating a 72 definitive atlas (Zeng and Sanes, 2017). Moreover, it provides molecular markers that 73 facilitate development of genetic strategies to access and manipulate specific cell types 74 within neural circuits. 75 76 In an initial study, we used scRNA-seq to profile ~45,000 cells from mouse retina, 77 recovering the 6 major classes of cells present in vertebrate retinas: photoreceptors 78 (PRs) that sense light; three classes of interneurons (horizontal cells, bipolar cells and 79 amacrine cells – HCs, BCs and ACs) that receive and process information from 80 photoreceptors; retinal ganglion cells (RGCs) that receive information from interneurons 81 and transmit it to central targets; and Müller glial cells (Macosko et al., 2015) (Figure 82 1A). This study was unable, however, to resolve all of the cell types into which the 83 classes are divided: only 33 neuronal groups were recovered, even though the number 84 of authentic types had been estimated to exceed 60 (Masland, 2012). The reason was 85 that ~80% of retinal cells are rod photoreceptors (Jeon et al., 1998), leaving the less 86 abundant but more heterogenous classes under sampled (BCs, ACs, and RGCs), 87 precluding the recovery of rare types or resolution of types with similar gene expression 88 profiles. Accordingly, we set out to enrich BCs, RGCs, and ACs so we could profile 89 them in sufficient numbers. For BCs and RGCs, we documented the existence of 15 90 and 46 types, respectively (Shekhar et al., 2016; Tran et al., 2019). These numbers 91 correspond well to those obtained from recent high-throughput physiological, 92 ultrastructural and molecular studies (Baden et al., 2016; Bae et al., 2018; Franke et al., 93 2017; Greene et al., 2016; Rheaume et al., 2018). 94 95 Here, we present an analysis of ACs. ACs receive synaptic input from BCs and other 96 ACs, and provide output to BCs, other ACs and RGCs. They modify the visual signals 97 that travel from photoreceptors to RGCs via BCs, thereby shaping the visual features to 98 which each RGC type responds. Several AC types have been shown to play specific 99 roles in retinal computation; for example, some render RGCs selectively responsive to 100 motion in particular directions, and others capable of distinguishing local from global 101 motion (Werblin., 2010; Vaney et al., 2012; Krishnaswamy et al., 2014; Lee et al., 2016; 102 Tian et al., 2016; Diamond, 2017; Wei, 2018). These diverse roles require multiple AC 103 types; indeed, they are generally thought to be the most heterogeneous retinal class 104 (MacNeil and Masland, 1998; Lin and Masland, 2006) (Figure 1B). 105 106 Our transcriptomic analysis revealed 63 AC types, enabling us to identify markers for all 107 and characterize morphology for many of them. Because ACs are known to display 108 remarkable heterogeneity in neurotransmitter phenotype, we systematically analyzed 109 expression of neurotransmitter biosynthetic enzymes and neuropeptide precursors, 110 providing evidence for the presence of at least 20 small molecule or peptide transmitters 111 in ACs, with potential use of multiple transmitters in the majority of them. We also 112 analyzed transcriptional relationships among types and identified transcription factors 3 113 expressed by closely related types. They include Meis2 and Tcf4, expressed by most 114 GABAergic and glycinergic types, respectively. 115 116 Combined with results from other classes, our inventory of ACs provides what we 117 believe to be a nearly complete mouse retinal cell atlas, comprising approximately 140 118 cell types. Thus, at least in this respect, the retina is about as complicated as any other 119 part of the brain. 120 121 MATERIALS AND METHODS 122 123 Animals 124 Animals were used in accordance with NIH guidelines and protocols approved by the 125 Institutional Animal Care and Use Committee (IACUC) at Harvard University. The 126 following knock-in and transgenic mouse lines were obtained from Jackson 127 Laboratories: Chx10-cre-GFP (Rowan and Cepko, 2004; Stock No:005105; Chx10 is 128 now named Vsx2), Slc17a7-IRES2-Cre (Harris et al., 2014 Stock No: 023527), Cck- 129 IRES-Cre (Taniguchi et. al, 2011; Stock No: 012706), Penk-IRES2-Cre (generated at 130 the Allen Institute; JAX Stock No: 025112), Sst-IRES-Cre (Stock No: 013044) and Thy1- 131 mitoCFP-P (Misgeld et al, 2007; Stock No: 007967). Contactin 5-lacZ and Contactin 6- 132 lacZ lines were from Sudo and colleagues (Li et al., 2003; Takeda et al., 2003) via Julia 133 Kaltschmidt and Thomas Bourgeron, respectively. NeuroD6-cre knock-in mice 134 (Goebbels et al., 2006) were obtained from K. Nave via L. Reichardt. The Gbx2- 135 CreERT2-IRES-GFP line (Chen et al., 2009) was a generous gift from James Y.