The Notch Ligand Jagged1 Is Required for the Formation

Research Articles: Development/Plasticity/Repair The Notch Ligand Jagged1 is Required for the Formation, Maintenance, and Survival of Hensen’s Cells in the Mouse Cochlea https://doi.org/10.1523/JNEUROSCI.1192-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1192-20.2020 Received: 14 May 2020 Revised: 16 October 2020 Accepted: 23 October 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 Title: The Notch Ligand Jagged1 is Required for the Formation, Maintenance, and 2 Survival of Hensen’s Cells in the Mouse Cochlea. 3 4 Abbreviated Title: Jagged1 is required for Hensen’s cell development 5 Elena Chrysostomou1, *, Luyi Zhou3, *, Yuanzhao L. Darcy3, Kaley A. Graves3, Angelika 6 Doetzlhofer1, 2 $, and Brandon C. Cox3,4, $ 7 8 1. The Solomon H. Snyder Department of Neuroscience and 2.Center for Hearing and 9 Balance, Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins 10 University School of Medicine, Baltimore, Maryland, 21205 11 3. Departments of Pharmacology and 4. Otolaryngology, Southern Illinois University 12 School of Medicine, Springfield, Illinois, 62702 13 * Denotes co-first authors, $ denotes co-corresponding authors 14 15 Corresponding authors email addresses: 16 Angelika Doetzlhofer: [email protected] 17 Brandon C. Cox: [email protected] 18 19 Number of pages: 56 20 Number of Figures: 7 21 Number of Tables: 1 22 Number of words in Abstract: 203 23 Number of words in Introduction: 644 1 24 Number of words in Discussion: 1,615 25 Extended data (tables): 4 26 27 CONFLICT OF INTEREST STATEMENT 28 Brandon C. Cox, PhD is a consultant for Turner Scientific, LLC, and Otonomy, Inc. 29 Other authors do not have anything to declare. 30 31 ACKNOWLEDGEMENTS 32 We thank Dr. William Richardson at University College London for providing the Fgfr3- 33 iCreERT2 mouse line, and Dr. Julian Lewis at Cancer Research UK London Research 34 Institute for providing one of the Jag1fx/fx mouse lines. This work was supported by 35 grants from NIDCD [R01DC011571 (AD) and R01DC014441 (BC)] and the Office of the 36 Assistant Secretary of Defense for Health Affairs [W81XWH-15-1-0475 (BC)]. The 37 Southern Illinois University School of Medicine Research Imaging Facility is supported 38 by a grant from the Office of Naval Research (N00014-15-1-2866). 39 40 41 42 2 43 ABSTRACT 44 During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important 45 role in the specification of the prosensory region, which gives rise to sound-sensing hair 46 cells and neighboring supporting cells (SCs). While JAG1’s expression is maintained in 47 SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we 48 demonstrate that JAG1 is essential for the formation and maintenance of Hensen’s 49 cells, a highly specialized SC-subtype located at the edge of the auditory epithelium. 50 Using Sox2CreER/+::Jag1loxP/loxP mice of both genders, we show that Jag1 deletion at the 51 onset of differentiation, at embryonic day 14.5, disrupted Hensen’s cell formation. 52 Similar loss of Hensen’s cells was observed when Jag1 was deleted after Hensen’s cell 53 formation at postnatal day (P) 0/P1 and fate-mapping analysis revealed that in the 54 absence of Jag1, some Hensen’s cells die, but others convert into neighboring Claudius 55 cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial 56 function and protein synthesis were downregulated in the sensory epithelium of P0 57 cochlea lacking Jag1. Finally, using Fgfr3-iCreERT2::Jag1loxP/loxP mice to delete Jag1 at 58 P0, we observed a similar loss of Hensen’s cells and found that adult Jag1 mutant mice 59 have hearing deficits at the low frequency range. 60 61 SIGNIFICANCE STATEMENT 62 Hensen’s cells play an essential role in the development and homeostasis of the 63 cochlea. Defects in the biophysical or functional properties of Hensen’s cells have been 64 linked to auditory dysfunction and hearing loss. Despite their importance, surprisingly 65 little is known about the molecular mechanisms that guide their development. 3 66 Morphological and fate-mapping analyses in our study revealed that in the absence of 67 the Notch ligand JAGGED1, Hensen’s cells died or converted into Claudius cells, which 68 are specialized epithelial-like cells outside the sensory epithelium. Confirming a link 69 between JAGGED1 and cell survival, transcriptional profiling showed that JAGGED1 70 maintains genes critical for mitochondrial function and tissue homeostasis. Finally, 71 auditory phenotyping revealed that JAGGED1’s function in supporting cells is necessary 72 for low frequency hearing. 73 74 75 INTRODUCTION 76 Canonical Notch signaling, which plays essential roles in the development of 77 sensory structures in the vertebrate inner ear is initiated by the binding of a DSL 78 (Delta/Serrate/LAG-2)-type membrane-bound ligand to the transmembrane Notch 79 receptor located on neighboring cells. Ligand binding leads to the release of the 80 intercellular domain of the Notch receptor, which as part of the RBPJ-Mastermind 81 transcription complex, activates transcription of downstream target genes. Mammals 82 have four Notch receptors (Notch1-4) and five DSL-type ligands [Delta-like (Dll) 1, 3, 83 and 4 and Jagged (Jag) 1 and 2] (Kopan and Ilagan, 2009). 84 In the inner ear, canonical Notch signaling is best known for its roles in lateral 85 inhibition and lateral induction (Eddison et al., 2000; Daudet and Zak, 2020). Notch- 86 mediated lateral induction, a positive feedback mechanism in which the signaling and 87 signal-receiving cells acquire the same cell fate, first defines and maintains the 88 prosensory domains that give rise to specialized sensory epithelia composed of hair 89 cells (HCs) and surrounding supporting cells (SCs) (Daudet and Lewis, 2005; Hartman 4 90 et al., 2010; Pan et al., 2010; Neves et al., 2011). The Notch ligand JAGGED1 (JAG1) 91 and the downstream Notch effector RBPJ are essential for the early role of Notch 92 signaling in prosensory development. Early loss of JAG1 or RBPJ abolishes, or greatly 93 reduces, the pool of inner ear prosensory progenitors (Brooker et al., 2006; Kiernan et 94 al., 2006; Basch et al., 2011; Yamamoto et al., 2011; Petrovic et al., 2014). 95 As prosensory progenitor cells differentiate into HCs and SCs during late 96 embryogenesis, Notch signaling plays a key role in limiting the number of HCs that form 97 in a process termed lateral inhibition. Activated by ATOH1, a transcriptional activator 98 and master regulator of HC formation (Bermingham et al., 1999; Chen et al., 2002; 99 Woods et al., 2004), nascent HCs express the Notch ligands Dll1, Dll3, and Jag2 (Adam 100 et al., 1998; Lanford et al., 1999; Morrison et al., 1999; Hartman et al., 2007). 101 DLL1/JAG2 mediated activation of Notch1 receptor signaling in adjacent prosensory 102 progenitor cells limits these cells to a SC fate (Lanford et al., 1999; Kiernan et al., 2005; 103 Brooker et al., 2006). 104 Important effectors of this Notch-mediated HC-repressive function are members 105 of the HES/HEY family. HES/HEY proteins are transcriptional repressors, which 106 antagonize HC formation by repressing Atoh1 expression and ATOH1 activity (Zheng 107 and Gao, 2000; Zine et al., 2001; Li et al., 2008; Doetzlhofer et al., 2009; Tateya et al., 108 2011). 109 We recently uncovered that canonical Notch signaling, in addition to its role in 110 HC fate repression, is required for the differentiation and survival of cochlear SCs 111 (Campbell et al., 2016). However, the Notch ligand(s) and receptor(s) involved in this 112 process are unknown. A potential candidate for this function is the Notch ligand JAG1. 5 113 JAG1, which initially is expressed by cochlear and vestibular prosensory progenitors, 114 continues to be highly expressed in SCs (Morrison et al., 1999). While other Notch 115 ligands are down-regulated during the first postnatal week, as cochlear HC and SC 116 mature, SC-specific JAG1 expression continues throughout adulthood where its function 117 is unknown (Murata et al., 2006; Hartman et al., 2007; Oesterle et al., 2008; Maass et 118 al., 2015). 119 In the present study, we investigated the role of JAG1 in differentiating and 120 maturing SCs. Our analysis revealed that deletion of Jag1 at the onset of cochlear 121 differentiation alters the patterning and cellular morphology of SCs and leads to a down- 122 regulation of genes involved in mitochondrial function and protein synthesis within the 123 sensory epithelium. Furthermore, using morphological analyses and fate-mapping, we 124 show that Jag1 deletion prior to and after cochlear differentiation resulted in loss of a 125 specialized SC subtype called Hensen’s cells. Functional analysis of adult Jag1 mutant 126 animals after Jag1 deletion at the perinatal stage revealed mild hearing deficits at low 127 frequencies. Together, our results suggest that Jag1-mediated Notch signaling in 128 cochlear SCs is critical for the formation, maintenance, and survival of Hensen’s cells. 129 130 MATERIALS AND METHODS 131 Animals 132 Sox2CreERT2/+ mice (Arnold et al., 2011) (stock #17593; RRID:IMSR_JAX:017593) and 133 Rosa26loxP-stop-loxP-tdTomato (Rosa26tdTomato) mice (Madisen et al., 2010) (stock #7914; 134 RRID:IMSR_JAX:007914) were purchased from The Jackson Laboratory (Bar Harbor, 135 ME). Fgfr3-iCreERT2 mice (Rivers et al., 2008; Young et al., 2010) were provided by Dr.

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