Research Articles: Development/Plasticity/Repair Rgma-induced Neo1 proteolysis promotes neural tube morphogenesis https://doi.org/10.1523/JNEUROSCI.3262-18.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.3262-18.2019 Received: 30 December 2018 Revised: 1 July 2019 Accepted: 31 July 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 the authors 1 Title: Rgma-induced Neo1 proteolysis promotes neural tube morphogenesis 2 3 Abbreviated Title: Neo1 signaling drives neural tube development 4 5 Author names and affiliation: Sharlene Brown1*, Pradeepa Jayachandran1*, Maraki 6 Negesse1, Valerie Olmo1, Eudorah Vital1 and Rachel Brewster1$ 7 1. Department of Biological Sciences, University of Maryland Baltimore County, 8 * These authors contributed equally to this work 9 $ Corresponding author: [email protected] 10 11 Number of pages: 46 12 Number of figures: 10 13 Multimedia: 2 14 Word Count: 15 Abstract: 250 - Significance statement 120 - Introduction: 632 - Discussion: 1,440 16 17 Acknowledgements 18 Research was supported by NIH/NIGMS grant # GM085290-02S1 to V. Olmo and # 19 GM085290 to R. Brewster; S. Brown was funded by a U.S. Department of Education 20 GAANN Fellowship, grant # P200A120017 and a Meyerhoff Graduate Fellowship 21 funded by NIH/NIGMS grant # GM055036; M. Negesse was funded by NIGMS/NIH T32 22 GM066706, NSF 1500511 and NIH/NIGMS GM055036; E. Vital was supported by a 23 grant to UMBC from the Howard Hughes Medical Institute through the Precollege and 1 24 Undergraduate Science Education Program, grant # 52008090. The Leica SP5 confocal 25 microscope was purchased with funds from the National Science Foundation, grant # 26 DBI-0722569. We thank the following people for their technical assistance: Robyn 27 Goodman for cell transplantation experiments; Neus Sanchez-Alberola, Julie Wolf, 28 Yiannis Balanos and Nilusha Jayasinghe for their contribution to CRISPR/Cas9 tools. 29 30 The authors declare no competing financial interests. 31 32 2 33 Abstract 34 Neuroepithelial cell (NEC) elongation is one of several key cell behaviors that mediate 35 the tissue-level morphogenetic movements that shape the neural tube (NT), the 36 precursor of the brain and spinal cord. However, the upstream signals that promote 37 NEC elongation have been difficult to tease apart from those regulating apico-basal 38 polarity and hinge point formation, due to their confounding interdependence. The 39 Repulsive Guidance Molecule a (Rgma)/ Neogenin 1 (Neo1) signaling pathway plays a 40 conserved role in NT formation (neurulation) and is reported to regulate both NEC 41 elongation and apico-basal polarity, through signal transduction events that have not 42 been identified. We examine here the role of Rgma/Neo1 signaling in zebrafish (sex 43 unknown), an organism that does not use hinge points to shape its hindbrain, thereby 44 enabling a direct assessment of the role of this pathway in NEC elongation. We confirm 45 that Rgma/Neo1 signaling is required for microtubule (MT)-mediated NEC elongation 46 and demonstrate via cell transplantation that Neo1 functions cell autonomously to 47 promote elongation. However, in contrast to previous findings, our data do not support a 48 role for this pathway in establishing apical junctional complexes. Lastly, we provide 49 evidence that Rgma promotes Neo1 glycosylation and intramembrane proteolysis, 50 resulting in the production of a transient, nuclear intracellular fragment (NeoICD). Partial 51 rescue of Neo1a and Rgma knockdown embryos by overexpressing neoICD suggests 52 that this proteolytic cleavage is essential for neurulation. Based on these observations, 53 we propose that Rgma-induced Neo1 proteolysis orchestrates NT morphogenesis by 54 promoting NEC elongation independently of the establishment of apical junctional 55 complexes. 3 56 57 Statement of Significance 58 The neural tube, the central nervous system precursor, is shaped during neurulation. 59 Neural tube defects (NTDs) occur frequently, yet underlying genetic risk factors are 60 poorly understood. Neuroepithelial cell (NEC) elongation is essential for proper 61 completion of neurulation. Thus, connecting NEC elongation with the molecular 62 pathways that control this process is expected to reveal novel NTD risk factors and 63 increase our understanding of NT development. Effectors of cell elongation include 64 microtubules and microtubule-associated proteins, however upstream regulators remain 65 controversial due to the confounding interdependence of cell elongation and 66 establishment of apico-basal polarity. Here, we reveal that Rgma-Neo1 signaling 67 controls NEC elongation independently of the establishment of apical junctional 68 complexes and identify Rgma-induced Neo1 proteolytic cleavage as a key upstream 69 signaling event. 70 71 4 72 Introduction 73 The central nervous system derives from the neural tube (NT) that is formed during 74 neurulation. Hallmarks of primary neurulation include the thickening of the neural plate 75 (NP), narrowing and lengthening of the NP, elevation of the lateral borders of the NP to 76 form neural folds, fusion of the neural folds at the dorsal midline and separation of the 77 neural folds from the overlying non-neural ectoderm (Colas and Schoenwolf, 2001). 78 Significant inroads have been made into understanding the cellular basis of these 79 tissue-level changes, however connecting specific cellular behaviors to the signaling 80 pathways that control them has proven more challenging. 81 The zebrafish is ideally suited to study the cellular basis of morphogenesis, 82 owing to the early accessibility and transparency of its embryo. Despite its 83 mesenchymal-like appearance, the zebrafish NP is shaped into a tube by organized 84 epithelial infolding, akin to primary neurulation (Papan and Campos-Ortega, 1994). The 85 transient structure formed by infolding, termed the neural keel, becomes a solid neural 86 rod by mid-somitogenesis and matures into a NT with a clearly defined midline and 87 lumen following cavitation (Papan and Campos-Ortega, 1994). 88 During neurulation cells undergo significant changes in shape, from cuboidal to 89 columnar, as they elongate along their apico-basal axis (Schroeder, 1970; Burnside, 90 1971; Karfunkel, 1974; Schoenwolf and Franks, 1984). Cell elongation is required for 91 thickening the NP of amniotes, resolving the bilayered NP of zebrafish and Xenopus 92 embryos into a monolayered neuroepithelium via radial intercalation (Hong and 93 Brewster, 2006; Kee et al., 2008) and elevating the neural folds, in absence of which the 94 NT fails to close (Karfunkel, 1971, 1972; Suzuki et al., 2012). Apical constriction, 5 95 another essential cell behavior, causes NECs to adopt a wedge shape (Suzuki et al., 96 2012), resulting in the formation of one median and two dorsolateral hinge points (Shum 97 and Copp, 1996), around which the NP of amniotes bends and folds. 98 The upstream signaling events that control NEC elongation are not well 99 understood. In a landmark study (Kee et al., 2008) Rgma and its receptor Neo1 were 100 identified as upstream regulators of NEC elongation and establishment of apico-basal 101 polarity. However, the interdependence of both of these processes in Xenopus (Suzuki 102 et al., 2012) prevented a direct assessment of the role of Rgma-Neo1 signaling in 103 controlling either process. 104 neo1 encodes a transmembrane receptor belonging to the immunoglobulin 105 superfamily. RGMs are a family of glycosylphosphatidylinositol (GPI)-anchored proteins 106 that function as membrane-bound, short-range guidance cues or as secreted, long- 107 range signals (Tassew et al., 2012). Rgma-Neo1 signaling plays a conserved role in 108 neurulation, as knockdown of the ligand or/and receptor prevents NT closure in mice 109 (Niederkofler et al., 2004) and Xenopus (Kee et al., 2008). The signaling events 110 activated downstream of Rgma-Neo1 interaction that promote cell elongation and NT 111 closure are currently unknown, however previous studies have identified several 112 putative signaling mechanisms. Both Rgma and Neo1 are known to modulate bone 113 morphogenetic protein signaling (Babitt et al., 2005; Zhou et al., 2010; Tian and Liu, 114 2013). RGMs can also directly bind to Neo1, triggering structural changes to the actin 115 cytoskeleton through the Rho family of small guanosine-5’-triphosphate-hydrolyzing 116 GTPases (Hata et al., 2006; Conrad et al., 2007). Furthermore, Neo1 is sequentially 117 cleaved by α- and γ-secretases in response to Rgma binding, leading to the release of a 6 118 Neo1 intracellular domain (NeoICD) that regulates transcription (Goldschneider et al., 119 2008; van Erp et al., 2015). Neo1 proteolytic cleavage has been implicated in axonal 120 pathfinding and neuronal migration (van Erp et al., 2015; Banerjee et al., 2016). 121 In zebrafish embryos, NEC elongation precedes the establishment of apical 122 junctional complexes (Hong and Brewster, 2006), making it an ideal model for teasing 123 apart the signals required for cell elongation specifically. This study aims to determine 124 the role of Rgma-Neo1 in NEC elongation and to identify the signal transduction events 125 triggered by ligand-receptor interaction that impinge on neurulation. 126 7 127 Materials and Methods 128 Husbandry, care, and use of zebrafish 129 Wild type (WT) zebrafish (Danio rerio) of the AB strain were reared and manipulated 130 using protocols approved by the Institutional Animal Care and Use Committee (IACUC) 131 at the University of Maryland Baltimore County. Fish were maintained in UV-irradiated, 132 filtered running water and exposed to 14:10 light:dark cycle. Male and female fish were 133 separated by a partition that was removed after first light to initiate spawning of fry 134 (embryos). Embryos were collected and staged according to previously described 135 methods (Kimmel et al., 1995).