Research Article: New Research | Development Peripheral Nerve Single Cell Analysis Identifies Mesenchymal Ligands that Promote Axonal Growth https://doi.org/10.1523/ENEURO.0066-20.2020 Cite as: eNeuro 2020; 10.1523/ENEURO.0066-20.2020 Received: 24 February 2020 Revised: 20 April 2020 Accepted: 23 April 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.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 Toma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Peripheral Nerve Single Cell Analysis Identifies Mesenchymal Ligands that Promote 2 Axonal Growth 3 4 Jeremy S. Toma1, Konstantina Karamboulas1*, Matthew J. Carr 1,2*, Adelaida Kolaj1,3, Scott A. 5 Yuzwa1, Neemat Mahmud 1,3, Mekayla A. Storer1, David R. Kaplan1,2,4 and Freda D. Miller1-4 6 7 Program in Neurosciences and Mental Health1, Hospital for Sick Children, Toronto, Canada 8 M5G 1L7, Institute of Medical Sciences2, Departments of Physiology3 and Molecular Genetics4, 9 University of Toronto, Toronto, Canada M5G 1A8. 10 11 *These authors contributed equally. 12 13 Abbreviated Title: scRNA-seq identifies nerve ligands 14 Author Contributions: JST, DRK and FDM designed research; JST, MJC, AK, and NM 15 performed research; JST, KK, SAY, NM, MAS and FDM analyzed data; and JST, DRK and 16 FDM wrote the paper. 17 18 Correspondence should be addressed to: Freda D. Miller (Email: [email protected]) 19 20 Number of figures: 8 21 Number of tables: 10 22 Number of multimedia/extended data: 6 23 Number of words for abstract: 179 24 Number of words for significance statement: 116 25 Number of words for introduction: 648 26 Number of words for discussion: 1859 27 28 Acknowledgements: This work was funded by CIHR and the CFREF "Medicine by Design" to 29 FDM and DRK. JST and MAS were funded by CIHR fellowships, MJC by a CIHR studentship, 30 and SAY by a Lap-Chee Tsui Restracomp fellowship during the course of these studies. We 31 thank Dennis Aquino, Jon Krieger, Troy Ketela and Konstantin Feinberg for advice and 32 technical assistance. Current address for SAY: Department of Laboratory Medicine and 33 Pathobiology, University of Toronto, Toronto, Canada M5S 1A8. 34 35 Conflict of interest: The authors report no conflict of interest. 36 1 37 ABSTRACT 38 Peripheral nerves provide a supportive growth environment for developing and regenerating 39 axons and are essential for maintenance and repair of many non-neural tissues. This capacity has 40 largely been ascribed to paracrine factors secreted by nerve-resident Schwann cells. Here, we 41 used single cell transcriptional profiling to identify ligands made by different injured rodent 42 nerve cell types and have combined this with cell surface mass spectrometry to computationally 43 model potential paracrine interactions with peripheral neurons. These analyses show that 44 peripheral nerves make many ligands predicted to act on peripheral and central nervous system 45 neurons, including known and previously uncharacterized ligands. While Schwann cells are an 46 important ligand source within injured nerves, more than half of the predicted ligands are made 47 by nerve-resident mesenchymal cells, including the endoneurial cells most closely associated 48 with peripheral axons. At least three of these mesenchymal ligands, ANGPT1, CCL11 and 49 VEGFC, promote growth when locally applied on sympathetic axons. These data therefore 50 identify an unexpected paracrine role for nerve mesenchymal cells and suggest that multiple cell 51 types contribute to creating a highly pro-growth environment for peripheral axons. 52 53 2 54 SIGNIFICANCE STATEMENT 55 This work expands our understanding of the cellular sources of ligands in the injured peripheral 56 nerve that are potentially important for promoting axon growth. Here, we used single cell RNA 57 sequencing (scRNA-seq) to reveal that Schwann cells and, surprisingly, nerve mesenchymal 58 cells are primary sources of ligands in the injured nerve. We then combined injured nerve 59 scRNA-seq data with proteomic and transcriptomic data from sensory and sympathetic neurons 60 and used a systems-biology/modeling approach to predict novel mesenchymal cell-derived 61 factors that may promote peripheral axon growth. We tested some of these predictions and found 62 three factors, ANGPT1, CCL11 and VEGFC, that promoted outgrowth of cultured sympathetic 63 axons, supporting a potential role for mesenchymal-derived factors in axon growth. 64 3 65 66 INTRODUCTION 67 Following injury, mammalian peripheral neurons can regenerate and reinnervate their 68 target tissues. Their ability to do so is thought to be a consequence of a peripheral nerve 69 environment that is highly supportive of axonal growth. Support for this idea comes from classic 70 studies with central nervous system (CNS) neurons, which normally fail to regenerate following 71 brain or spinal cord injury but will regrow their axons when peripheral nerve segments are 72 transplanted into the damaged region (David and Aguayo, 1981; reviewed in Benowitz et al., 73 2017). Intriguingly, peripheral nerves are also important for maintenance, repair and 74 regeneration of the non-neural tissues that they innervate. For example, normal peripheral 75 innervation is essential for mammalian hair follicle and hematopoietic stem cells (Brownell et 76 al., 2011; Yamazaki et al., 2011), for cardiac and dermal repair (Mahmoud et al., 2015; Johnston 77 et al., 2013; 2016), and for amphibian limb (reviewed in Kumar and Brockes, 2012) and murine 78 digit tip regeneration (Johnston et al., 2016). 79 The supportive peripheral nerve environment has largely been ascribed to growth factors 80 made by nerve cells (reviewed in Terenghi, 1999; Fledrich et al., 2019). These nerve-derived 81 ligands have been particularly well-studied with regard to axonal development and regeneration 82 (Chen et al., 2007; Fledrich et al., 2019), although several studies have shown that they are also 83 important for limb and digit tip regeneration (Johnston et al., 2016; Kumar et al., 2007). These 84 growth factors are thought to be Schwann cell-derived since transplantation of Schwann cells 85 alone is enough to promote CNS axon regeneration (reviewed in Bunge, 2016) and murine digit 86 tip regeneration (Johnston et al., 2016). In addition to growth factors, the peripheral nerve 87 provides an extracellular matrix environment that is highly conducive to axonal growth, 88 particularly by contrast to the CNS, where known axon growth inhibitors prevail (Chen et al., 89 2007; Benowitz et al., 2017). This supportive substrate is also thought to derive in part from 90 Schwann cells, which generate a basal lamina and synthesize ECM proteins and cell adhesion 91 molecules (Gardiner, 2011; Muir, 2010). 92 These studies all indicate that Schwann cells play an important role in establishing a 93 nerve environment that is supportive of axonal growth. However, the nerve is a structurally- 94 complex tissue containing many different cell types, including vasculature-associated cells, 95 immune cells such as tissue-resident macrophages, and mesenchymal cells of both mesodermal 4 96 and neural crest origin. In this regard, one recent study identified four transcriptionally and 97 spatially-distinct populations of Pdgfra-positive mesenchymal cells within the injured peripheral 98 nerve, including endoneurial mesenchymal cells that are tightly-associated with Schwann cells 99 and axons (Carr et al., 2019). These nerve mesenchymal cells were shown to directly contribute 100 to the repair and regeneration of mesenchymal target tissues including the digit tip, bone and 101 dermis. Nerve mesenchymal cells have also been shown to play an essential role in forming 102 bridges over gaps in injured nerves (reviewed in Cattin and Lloyd, 2016). Together, these 103 findings raise the possibility that mesenchymal cells might also be important for axonal growth 104 in the peripheral nerve. 105 Here, we provide support for this concept, using an unbiased systems biology approach to 106 define the sciatic nerve ligand environment. We show, using single cell profiling, that under both 107 homeostatic and injury conditions, mesenchymal cells and Schwann cells are the predominant 108 sources of peripheral nerve ligands, including known and uncharacterized ligands, and that there 109 is induction of ligand expression in both these cell types following injury. Moreover, using mass 110 spectrometry, transcriptional profiling and computational modeling, we show that peripheral 111 neurons and CNS retinal ganglion neurons express receptors for many of these ligands. Finally, 112 we validate three of these ligands, ANGPT1, CCL11 and VEGFC, as being synthesized and 113 secreted by Pdgfra-positive nerve mesenchymal cells and show that they can promote growth 114 when applied to axons of peripheral sympathetic neurons. Thus, our data support a model where 115 nerve mesenchymal cells and Schwann cells collaborate to establish a generally supportive 116 growth environment in the peripheral nerve. 117 5 118 MATERIALS AND METHODS 119 120 Animals. All animal procedures were performed in accordance with Canadian Council on 121 Animal Care regulations as approved by the Hospital for Sick Children animal care committee. 122 Sprague-Dawley rats (purchased from Charles River, Canada) used in this study ranged from 123 E15 to young adult (6 weeks old) and CD1 mice (purchased from Charles River, Canada) ranged 124 in age from 8-12 weeks old. All rats and mice were healthy throughout the duration of the study 125 and had free access to chow and water in a 12 hour light-dark cycle room.