Brain injury activates complex inflammatory signals in dying neurons, surviving neurons, and glia. Here, we establish that inflammation regulates the regeneration of photoreceptors in the zebrafish retina and determine the cellular expression and function of the inflammatory protease, matrix metalloproteinase 9 (Mmp-9), during this regenerative neurogenesis. Following photoreceptor ablation, anti-inflammatory treatment suppresses the number of injury-induced progenitors and regenerated photoreceptors. Upon photoreceptor injury, mmp-9 is induced in Müller glia and Müller glia-derived photoreceptor progenitors. Deleting mmp-9 results in over production of injury-induced progenitors and regenerated photoreceptors, but over time the absence of Mmp-9 compromises the survival of the regenerated cones. At all time-points studied, the levels of tnf-α are significantly elevated in mutant retinas.Anti-inflammatory treatment in mutants rescues the defects in cone survival. These data provide a link between injury-induced inflammation in the vertebrate CNS, Mmp-9 function during neuronal regeneration and the requirement of Mmp-9 for the survival of regenerated cones.

51Brain injury activates complex inflammatory signals in dying neurons, surviving neurons, and 52 glia. Here, we establish that inflammation regulates the regeneration of photoreceptors in the 53 zebrafish retina and determine the cellular expression and function of the inflammatory 54 protease, matrix metalloproteinase 9 (Mmp-9), during this regenerative neurogenesis. Animals 55 of either sex were used in this study. Following photoreceptor ablation, anti-inflammatory 56 treatment suppresses the number of injury-induced progenitors and regenerated 57 photoreceptors. Upon photoreceptor injury, mmp-9 is induced in Müller glia, the intrinsic retinal 58 stem cell, and Müller glia-derived photoreceptor progenitors. Deleting mmp-9 results in over 59 production of injury-induced progenitors and regenerated photoreceptors, but over time the 60 absence of Mmp-9 compromises the maturation and survival of the regenerated cones. Anti-61 inflammatory treatment in mutants rescues the defects in cone maturation and survival. These 62 data provide a link between injury-induced inflammation in the vertebrate CNS, Mmp-9 function 63 during photoreceptor regeneration and the requirement of Mmp-9 for the survival of regenerated 64 cones. 65 66 67 68 69 70 71 72 73 74 75 3 Significance Statement 76 77 The innate immune system is activated by neuronal death, and recent studies demonstrate that 78 in zebrafish neuroinflammation is required for neuronal regeneration. The roles of inflammatory 79 cytokines are being investigated, however, the function of the inflammatory protease, matrix 80 metalloprotease Mmp-9, in neuronal regeneration is unknown. We show herein that in adult 81 zebrafish retinal inflammation governs the proliferative phase of the stem cell-based 82 regeneration of rod and cone photoreceptors and determine the specific roles for Mmp-9 in 83 photoreceptor regeneration. This study provides the first mechanistic insights into the potential 84 role of Mmp-9 in retinal regeneration and serves to link neuroinflammation, stem cell-based 85 regeneration of photoreceptors and human photoreceptor disease. 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 2013; Zhao et al., 2014). Following a photolytic lesion, dying photoreceptors secrete Tnf-a, to 129 which Müller glia respond by partial dedifferentiation, synthesis of Tnf-a and entry into the cell 130 cycle (Nelson et al., 2013). Mechanical lesions induce the expression of leptin and Il-6 family 131 cytokines in Müller glia and Müller glia-derived progenitors and is required for injury-induced 132 proliferation (Zhao et al., 2014).133 Matrix metalloproteinase 9 (Mmp-9) is a secreted protease that plays a prominent role in 134 tissue development and homeostasis by acting on extracellular molecules, including adhesion 135

Persistent rod genesis in the retinas of teleost fish was first described over 2 decades ago, but little is known regarding the underlying genetic and molecular mechanisms that govern this phenomenon. Because of its function in the developing mammalian retina and persistently mitotic adult tissues, we sought to characterize the cellular expression of the basic helix-loop-helix (bHLH) transcription factor neuroD in the persistently neurogenic retina of adult teleosts. We show here that, in the adult retina of the goldfish, neuroD is expressed by putative amacrine cells, nascent cones, and the mitotically active cells of the rod lineage. neuroD is the first gene shown to be expressed by rod precursors, the immediate antecedents of rod photoreceptors. In contrast to the vertebrate classes described previously, neuroD is not expressed in multipotent progenitors in the teleost retina. Combining neuroD in situ hybridizations with cell-cycle-specific markers suggests that, in rod precursors, neuroD expression is cell cycle specific.