Midkine-A Is Required for Cell Cycle Progression of Müller Glia Glia

Research Articles: Development/Plasticity/Repair Midkine-a is required for cell cycle progression of Müller glia glia during neuronal regeneration in the vertebrate retina https://doi.org/10.1523/JNEUROSCI.1675-19.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1675-19.2019 Received: 15 July 2019 Revised: 27 November 2019 Accepted: 17 December 2019 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 © 2019 Nagashima and a 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. Title: Midkine-a is required for cell cycle progression of Müller glia glia during neuronal regeneration in the vertebrate retina Abbreviated title: Midkine-a regulates the cell cycle in Müller glia. Mikiko Nagashimaa, Travis S. D’Cruza, Antoinette E. Dankua, Doneen Hessea, Christopher Sifuentesb, Pamela A. Raymondb, Peter F. Hitchcocka* aDepartment of Ophthalmology and Visual Sciences, bDepartment of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48105, USA *Correspondence: [email protected] Number of pages: 42 Number of figures: 10 Number of tables: 1 Number of movies: 4 Number of extended data tables: 2 Number of words for Abstract: 211 words Number of words for Significance Statement: 113 Number of words for Introduction: 642 words Number of words for Discussion: 1442 words Conflict of interest: The authors declare no competing financial interests. Acknowledgements: This work was supported by grants from the National Institutes of Health (NEI) - R01EY07060 and P30EYO7003 (PFH) and an unrestricted grant from the Research to Prevent Blindness, New York. Fish lines and reagents provided by ZIRC were supported by NIH-NCRR Grant P40 RR01. The authors thank Dilip Pawar and Alex LeSage for zebrafish maintenance and technical assistance. 1 1 Abstract 2 In the retina of zebrafish, Müller glia have the ability to reprogram into stem cells capable of 3 regenerating all classes of retinal neurons and restoring visual function. Understanding the 4 cellular and molecular mechanisms controlling the stem cell properties of Müller glia in zebrafish 5 may provide cues to unlock the regenerative potential in the mammalian nervous system. 6 Midkine is a cytokine/ growth factor with multiple roles in neural development, tissue repair and 7 disease. In midkine-a loss-of-function mutants, of both sexes, Müller glia initiate the appropriate 8 reprogramming response to photoreceptor death by increasing expression of stem cell- 9 associated genes, and entering the G1 phase of the cell cycle. However, transition from G1 to S 10 phase is blocked in the absence of Midkine-a, resulting in significantly reduced proliferation and 11 selective failure to regenerate cone photoreceptors. Failing to progress through the cell cycle, 12 Müller glia undergo reactive gliosis, a pathological hallmark in the injured central nervous 13 system of mammals. Finally, we determined that the Midkine-a receptor, Anaplastic Lymphoma 14 Kinase, is upstream of the HLH regulatory protein, Id2a, and of the retinoblastoma gene, p130, 15 which regulates progression through the cell cycle. These results demonstrate that Midkine-a 16 functions as a core component of the mechanisms that regulate proliferation of stem cells in the 17 injured central nervous system. 2 18 Significance Statement 19 The death of retinal neurons and photoreceptors is a leading cause of vision loss. 20 Regenerating retinal neurons is a therapeutic goal. Zebrafish can regenerate retinal 21 neurons from intrinsic stem cells, Müller glia, and are a powerful model to understand 22 how stem cells might be used therapeutically. Midkine-a, an injury-induced growth 23 factor/cytokine that is expressed by Müller glia following neuronal death, is required for 24 Müller glia to progress through the cell cycle. The absence of Midkine-a suspends 25 proliferation and neuronal regeneration. With cell cycle progression stalled, Müller glia 26 undergo reactive gliosis, a pathological hallmark of the mammalian retina. This work 27 provides a unique insight into mechanisms that control the cell cycle during neuronal 28 regeneration. 3 29 Introduction 30 Cell division is an essential biological process during development, homeostasis and repair. In 31 the central nervous system of adult mammals, stem cells reside in specialized niches, and these 32 cells maintain the ability to divide and generate new neurons (Kriegstein and Alvarez-Buylla, 33 2009; Ming and Song, 2011). In the vertebrate retina, Müller glia harbor molecular features of 34 stem and progenitor cells (Dyer and Cepko, 2000). In mammals, Müller glia respond to injury by 35 partial dedifferentiation and entering the G1 phase of the cell cycle (Bringmann et al., 2006). 36 However, in general, this reprogramming does not lead to cell division, and structural 37 remodeling and the loss of retinal homeostasis are the typical sequalae (Bringmann et al., 2009; 38 Karl and Reh, 2010; Hamon et al., 2016). Importantly, in the limited instances where 39 regeneration does occur, new neurons functionally integrate into existing synaptic circuits 40 (Jorstad et al., 2017; Yao et al., 2018b), indicating that in the mammalian retina the limitations of 41 neuronal regeneration hinge on a more complete neurogenic response in Müller glia. 42 In zebrafish, Müller glia can adopt the features of stem cells (Karl and Reh, 2010; 43 Goldman, 2014; Gorsuch and Hyde, 2014; Lenkowski and Raymond, 2014; Hamon et al., 44 2016). In uninjured retinas, Müller glia reside in a quiescent state and function to maintain retinal 45 homeostasis. Neuronal death triggers Müller glia to reprogram into a stem cell-like state, enter 46 the cell cycle, and undergo a single asymmetric division to produce rapidly dividing, multipotent 47 retinal progenitors with the ability to regenerate retinal neurons (Nagashima et al., 2013; 48 Lenkowski and Raymond, 2014). Several signaling pathways have been identified that regulate 49 the initial response of Müller glia (Karl and Reh, 2010; Goldman, 2014; Gorsuch and Hyde, 50 2014; Lenkowski and Raymond, 2014; Hamon et al., 2016). Ascl1, Lin28, and Stat3 have been 51 identified as “core” transcriptional regulators that govern signaling cascades required for Müller 52 glia to divide (Fausett and Goldman, 2006; Ramachandran et al., 2010; Nelson et al., 2012). 53 Midkine is a growth factor/cytokine that has multiple roles in neural development, repair, 54 and disease (Sakamoto and Kadomatsu, 2012; Winkler and Yao, 2014; Sorrelle et al., 2017). In 4 55 malignant tumors, Midkine promotes proliferation and metastasis (Muramatsu, 2011), and is 56 also involved in CNS inflammation (Muramatsu, 2011; Weckbach et al., 2011; Herradon et al., 57 2019). The diverse functions of Midkine are transduced through receptors, which may function 58 individually or as members of a multi-protein complex (Muramatsu, 2011; Weckbach et al., 59 2011; Xu et al., 2014). During retinal development in zebrafish, midkine-a is expressed by 60 retinal progenitors and functions to govern elements of the cell cycle (Calinescu et al., 2009b; 61 Uribe and Gross, 2010; Luo et al., 2012). Post-mitotic neurons downregulate midkine-a. Retinal 62 injury rapidly induces midkine-a in Müller glia (Calinescu et al., 2009b; Gramage et al., 2014, 63 2015). Induction of midkine-a following injury has been reported for a variety of tissues with the 64 capacity to regenerate (Ochiai et al., 2004; Lien et al., 2006), suggesting that Midkine may 65 universally regulate aspects of tissue regeneration. The molecular mechanisms whereby 66 Midkine governs regeneration are not well understood. 67 Using a Midkine-a loss-of-function mutant, we demonstrate that following a retinal injury 68 Midkine-a is required for reprogrammed Müller glia to progress from G1 to S phases of the cell 69 cycle. Following photoreceptor death, Müller glia in Midkine-a mutants reprogram into a stem 70 cell state and enter G1 phase of the cell cycle. However, for the vast majority of Müller glia 71 subsequent entry into the S phase and mitotic division are blocked, resulting in failure to 72 regenerate cone photoreceptors. Further, Midkine-a is required for the upregulation of id2a, 73 which inhibits the retinoblastoma (Rb) family of cell cycle inhibitors. In addition, the G1-arrested 74 Müller glia undergo reactive gliotic remodeling, hallmarks of pathology in the mammalian retina. 75 Finally, we provide evidence that activation of the Midkine receptor, Anaplastic Lymphoma 76 Kinase (Alk), is required for proliferation in Müller glia. 77 Materials and Methods 78 Zebrafish 79 Fish were maintained at 28C on a 14/10 h light/ dark cycle with standard husbandry procedures. 80 Adult wild-type, AB-strain zebrafish (Danio rerio; ZIRC, University of Oregon, Eugene, OR) and 5 81 the transgenic reporter line, Tg(gfap:eGFP)mi2002 (Bernardos and Raymond, 2006) were of either 82 sex and used between 6 and12 months of age. All animal procedures were approved by the 83 Institutional Animal Care and Use Committee at the University of Michigan. 84 CRISPR-Cas9 mediated targeted mutation of midkine-a 85 Targeted mutations in the midkine-a locus were introduced using CRISPR-Cas9 (Hwang et al., 86 2013). Briefly, ZiFit software (zifit.partners.org) was used to identify guide RNA target sequence 87 for midkine-a. Oligos to the target sequencing (GGC AGC TGC GTG GCC AAT AAC GG) were 88 annealed and subcloned into the pT7 gRNA vector (Nasevicius and Ekker, 2000; Hwang et al., 89 2013; Jao et al., 2013). The subcloned vector was digested with BamHI and sgRNA was 90 transcribed with MEGAshortscript T7 kit (Thermo Fisher Scientific, Waltham, MA).

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