The TGFβ/Notch axis facilitates Müller cell-to-epithelial transition to ultimately form a chronic glial scar

Abstract

Background Contrasting with zebrafish, retinal regeneration from Müller cells (MCs) is largely limited in mammals. There, MCs undergo reactive gliosis that consist of a hypertrophic response and ultimately results in vision loss. Transforming growth factor β (TGFβ) is essential for wound healing, including both scar formation and regeneration. However, targeting TGFβ may affect other physiological mechanisms, owing its pleiotropic nature. The regulation of various cellular activities by TGFβ relies on its interaction with other pathways including Notch. Here, we explore the interplay of TGFβ with Notch and how this regulates MC response to injury in zebrafish and mice. Furthermore, we aimed to characterize potential similarities between murine and human MCs during chronic reactive gliosis. Methods Focal damage to photoreceptors was induced with a 532 nm diode laser in TgBAC (gfap:gfap-GFP) zebrafish (ZF) and B6-Tg (Rlbp1-GFP) mice. Transcriptomics, immunofluorescence, and flow cytometry were employed for a comparative analysis of MC response to laser-induced injury between ZF and mouse. The laser-induced injury was paired with pharmacological treatments to inhibit either Notch (DAPT) or TGFβ (Pirfenidone) or TGFβ/Notch interplay (SIS3). To determine if the murine laser-induced injury model translates to the human system, we compared the related MC response to human donors with early retinal degeneration. Results Investigations into injury-induced changes in murine MCs revealed TGFβ/Notch interplay during reactive gliosis. We found that TGFβ1/2 and Notch1/2 interact via Smad3 to reprogram murine MCs towards an epithelial lineage and ultimately to form a glial scar. Similar to what we observed in mice, we confirmed the epithelial phenotype of human Müller cells during gliotic response. Conclusion This study indicates a pivotal role for TGFβ/Notch interplay in tuning MC stemness during injury response and provides novel insights into the remodeling mechanism during retinal degenerative diseases.

Heterogeneity in quiescent Müller glia in the uninjured zebrafish retina drive differential responses following photoreceptor ablation

“…However, Tgfβ signaling can have differing effects on Müller glia depending on the timing of expression; Tgfβ signaling is necessary for the expression of pro-regenerative genes immediately after injury, yet inhibition of Tgfβ during the regenerative phase leads to increased proliferation (Lenkowski et al, 2013;Tappeiner et al, 2016;Sharma et al, 2020). Interestingly, the activin ligand-encoding gene inhibin subunit beta (INHB) is associated with quiescent states of chick and zebrafish Müller glia (Hoang et al, 2020), but an activated or gliotic state in mammalian Müller glia (Hoang et al, 2020;Conedera et al, 2021). One method of regulating this pathway is through endocytosis of transmembrane receptors through invaginated plasma membrane pits called caveolae, with associated proteins including cav2 and cav1, which were unique to this cluster (Anderson et al, 1992;Di Guglielmo et al, 2003;Hartung et al, 2006).…”

“…In zebrafish, upregulation of cxcl14 has been observed in reactive oligodendrocyte progenitor cells following spinal cord injury (Tsata et al, 2020). The expression of immune-associated markers by Müller glia was intriguing as the presence of immune cells can influence Müller glia proliferation (Bradley, 2008;Nelson et al, 2013;Conner et al, 2014;Fischer et al, 2014;Gallina et al, 2014;White et al, 2017;Conedera et al, 2019;Mitchell et al, 2019;Todd 10.3389/fnmol.2023.1087136 Frontiers in Molecular Neuroscience 18 frontiersin.org Leach et al, 2021). While we also observed a recruitment of L-plastin labeled leukocytes to the photoreceptor layer following ablation, in the absence of Müller glia, this migration to the photoreceptor layer remained unchanged (data not shown).…”

“…Despite mammalian Müller glia showing evidence of cell cycle entry in response to retinal injury (Dyer and Cepko, 2000;Yoshida et al, 2004;Kase et al, 2006;Conedera et al, 2021), these cells do not replace lost neurons and instead form scarring at the site of injury, termed reactive gliosis (Wilhelmsson et al, 2004;Bringmann et al, 2006). Across vertebrates, the gene expression profiles for Müller glia are remarkably conserved (Bringmann et al, 2009a;Thomas et al, 2016), with neuroprotective (Harada et al, 2002;Hauck et al, 2006;Bringmann and Wiedemann, 2012;Furuya et al, 2012;Reichenbach and Bringmann, 2013) and homeostatic roles (Newman et al, 1984;Yamada et al, 1999;Bringmann et al, 2009b;MacDonald et al, 2015) conserved in vertebrate species.…”

Clcf1/Crlf1a-mediated signaling is neuroprotective and required for Müller glia proliferation in the light-damaged zebrafish retina

“…It has previously been shown within the mouse retina that the neuroprotective effects of CNTF require GP130 within Müller glia (Rhee et al, 2013), therefore changes induced in Müller glia in response to CNTFR ligands may provide the observed neuroprotective effects, rather than direct CNTFRmediated signalling within damaged cell types. CNTF has also previously been shown to interact with microglia (Kahn et al, 1995;Baek et al, 2018), which are known to be required during retinal regeneration (Conedera et al, 2019;Mitchell et al, 2019). Therefore, the potential interaction of Clcf1 and Crlf1a with microglia within the damaged retina may be one way in which these two cell types interact.…”

Successful regeneration of the adult zebrafish retina is dependent on inflammatory signaling

“…Likewise, the inflammation-associated factors Interleukin-11 and TGF-ß stimulate Müller glia cell cycle re-entry and NPC generation (Lenkowski et al, 2013;Zhao et al, 2014). Furthermore, microglia -the CNS tissue resident macrophages -support this initial regenerative response by secreting proinflammatory factors (Kizil et al, 2015;Conedera et al, 2019;Iribarne & Hyde, 2022;Zhang et al, 2020). However, to date the molecular pathways involved in damage recognition, stem cell proliferation, neuronal precursor cell amplification and differentiation during retinal regeneration are poorly understood; in particular, the role of inflammation is unclear (Lahne et al, 2020;Lenkowski & Raymond, 2014;Mitchell et al, 2018Mitchell et al, , 2019Iribarne & Hyde, 2022).…”