HB-EGF is Necessary and Sufficient for Müller Glia Dedifferentiation and Retina Regeneration

“N IH -PA Author M anuscript N IH -PA Author M anuscript N IH -PA Author M anuscript EGFR inhibitor PD153035 (Fry et al., 1994; Fry et al., 1997), the MAPK (ERK1 and 2) inhibitors PD98059 (Dudley et al., 1995) or SL327 (Atkins et al., 1998) and DMSO in separate experiments.”

“It is first synthesized as a transmembrane protein (pro-HB-EGF) and can be cleaved by metalloproteinases (MMPs) within a juxtamembrane domain on the cell surface to release soluble HB-EGF (Higashiyama et al., 1991; Higashiyama et al., 1992; Higashiyama and Nanba, 2005).”

“N IH -PA Author M anuscript N IH -PA Author M anuscript N IH -PA Author M anuscript EGFR inhibitor PD153035 (Fry et al., 1994; Fry et al., 1997), the MAPK (ERK1 and 2) inhibitors PD98059 (Dudley et al., 1995) or SL327 (Atkins et al., 1998) and DMSO in separate experiments.”

“N IH -PA Author M anuscript N IH -PA Author M anuscript N IH -PA Author M anuscript EGFR inhibitor PD153035 (Fry et al., 1994; Fry et al., 1997), the MAPK (ERK1 and 2) inhibitors PD98059 (Dudley et al., 1995) or SL327 (Atkins et al., 1998) and DMSO in separate experiments.”

“HB-EGF is a member of a family of ligands that activate the EGFR and include EGF, TGFα, β-cellulin, epiregulin and amphiregulin (Harris et al., 2003).”

“In general, MG of the mammalian retina exhibit a reactive gliotic response to injury that often causes more harm than good (Bringmann et al., 2009).”

“However, similar to the injured zebrafish retina, Notch signaling inhibits neural stem cell proliferation in the zebrafish and mouse brain (Chapouton et al., 2010; Imayoshi and Kageyama, 2011; Imayoshi et al., 2010).”

“…on the dedifferentiation of Müller glia (MG), a radial glia-like cell that normally does not generate neurons during development, but can regenerate neurons after retinal injury (Bernardos et al., 2007; Fausett and Goldman, 2006; Fimbel et al., 2007; Ramachandran, 2010a; Thummel et al., 2008).”

“…in the injured retina To investigate if Notch signaling is necessary for injury-dependent MG dedifferentiation and proliferation we took advantage of the γ-secretase inhibitor DAPT, which prevents Notch cleavage and subsequent signaling by its intracellular domain, NICD (Geling et al., 2002).”

“Pharmacological destabilization of β-catenin with pyrvinium (Thorne et al., 2010), dramatically reduced the number of HB-EGF induced BrdU+ progenitors (Figure 7C and 7D), and suppressed HB-EGF-dependent induction of regeneration-associated genes (Figure 7E).”

“Although MG populate the mammalian retina and share many characteristics with those found in zebrafish, they only exhibit a limited regenerative potential under special conditions (Karl et al., 2008; Ooto et al., 2004; Osakada et al., 2007; Takeda et al., 2008; Wan et al., 2008).”

“Human recombinant HB-EGF is encoded by amino acids 1–148 of pro-HB-EGF which is a membrane spanning protein that undergoes ectodomain shedding, releasing a soluble extracellular portion that contains an EGF-like domain that binds the EGFR (Schneider and Wolf, 2009).”

“The antibodies used in this study were diluted in PBS plus 0.1% Triton X-100 as previously described (Fausett and Goldman, 2006; Ramachandran et Dev Cell.”

“MAPK signaling was previously reported to be required for proliferation of MG-derived progenitors in the postnatal chick retina (Fischer et al., 2009a; Fischer et al., 2009b) and likely mediates the effects of EGF on MG proliferation in the injured mouse retina (Karl et al., 2008).”

“It is first synthesized as a transmembrane protein (pro-HB-EGF) and can be cleaved by metalloproteinases (MMPs) within a juxtamembrane domain on the cell surface to release soluble HB-EGF (Higashiyama et al., 1991; Higashiyama et al., 1992; Higashiyama and Nanba, 2005).”

“Although MG populate the mammalian retina and share many characteristics with those found in zebrafish, they only exhibit a limited regenerative potential under special conditions (Karl et al., 2008; Ooto et al., 2004; Osakada et al., 2007; Takeda et al., 2008; Wan et al., 2008).”

“…on the dedifferentiation of Müller glia (MG), a radial glia-like cell that normally does not generate neurons during development, but can regenerate neurons after retinal injury (Bernardos et al., 2007; Fausett and Goldman, 2006; Fimbel et al., 2007; Ramachandran, 2010a; Thummel et al., 2008).”

“…to those reported in the postnatal chick retina and adult mouse retina where Notch signaling stimulated proliferation of MG-derived progenitors (Das et al., 2006; Del Debbio et al., 2010; Ghai et al., 2010; Hayes et al., 2007) and, at least in the postnatal chick retina, inhibited progenitor…”

“…the idea that HB-EGF mediates the effects of retinal injury on the Wnt signaling pathway, we found that HB-EGF stimulated βcatenin nuclear accumulation in MG derived progenitors (Figure 7A) and gfp mRNA expression in MG of the Wnt signaling reporter fish TOP:dGFP (Figure 7B) (Dorsky et al., 2002).”

“DAPT was used at a final concentration of 40 μM (Chapouton et al., 2010).”

“Indeed, EGF stimulation of the injured mouse retina did not induce Ascl1 gene expression (Karl et al., 2008), which is an essential regeneration-associated gene in zebrafish (Fausett et al., 2008; Ramachandran et al., 2010a).”

“…of Notch inhibition and activation reported in the developing mouse retina where Notch activity stimulates retinal stem cells to precociously differentiate into MG and Notch inhibition led to an enhancement of photoreceptor production (Jadhav et al., 2006; Scheer et al., 2001; Yaron et al., 2006).”

“N IH -PA Author M anuscript N IH -PA Author M anuscript N IH -PA Author M anuscript include FGF2 and insulin that in combination stimulate MG proliferation in the postnatal chick retina (Fischer et al., 2002) and CNTF that stimulates MG proliferation in zebrafish (Kassen et al., 2009).”

“HB-EGF also activated the Wnt/β-catenin signaling cascade which we recently showed was necessary for MG proliferation (Ramachandran et al., 2011).”

“Although a number of genes have been identified that are necessary for MG dedifferentiation (Fausett and Goldman, 2006; Fausett et al., 2008; Kassen et al., 2007; Qin et al., 2009; Ramachandran et al., 2010a; Raymond et al., 2006; Thummel et al., 2010), mechanisms underlying their activation have remained elusive.”

“…of Notch inhibition and activation reported in the developing mouse retina where Notch activity stimulates retinal stem cells to precociously differentiate into MG and Notch inhibition led to an enhancement of photoreceptor production (Jadhav et al., 2006; Scheer et al., 2001; Yaron et al., 2006).”

“…on the dedifferentiation of Müller glia (MG), a radial glia-like cell that normally does not generate neurons during development, but can regenerate neurons after retinal injury (Bernardos et al., 2007; Fausett and Goldman, 2006; Fimbel et al., 2007; Ramachandran, 2010a; Thummel et al., 2008).”

“It is first synthesized as a transmembrane protein (pro-HB-EGF) and can be cleaved by metalloproteinases (MMPs) within a juxtamembrane domain on the cell surface to release soluble HB-EGF (Higashiyama et al., 1991; Higashiyama et al., 1992; Higashiyama and Nanba, 2005).”

“Although a number of genes have been identified that are necessary for MG dedifferentiation (Fausett and Goldman, 2006; Fausett et al., 2008; Kassen et al., 2007; Qin et al., 2009; Ramachandran et al., 2010a; Raymond et al., 2006; Thummel et al., 2010), mechanisms underlying their activation have remained elusive.”

“Although a number of genes have been identified that are necessary for MG dedifferentiation (Fausett and Goldman, 2006; Fausett et al., 2008; Kassen et al., 2007; Qin et al., 2009; Ramachandran et al., 2010a; Raymond et al., 2006; Thummel et al., 2010), mechanisms underlying their activation have remained elusive.”

“, 2007) and, at least in the postnatal chick retina, inhibited progenitor differentiation (Hayes et al., 2007).”

“GM6001 was previously shown to inhibit HB-EGF ectodomain shedding and signaling (Stoll et al., 2010; Xu et al., 2004).”

“Although MG populate the mammalian retina and share many characteristics with those found in zebrafish, they only exhibit a limited regenerative potential under special conditions (Karl et al., 2008; Ooto et al., 2004; Osakada et al., 2007; Takeda et al., 2008; Wan et al., 2008).”

“Although MG populate the mammalian retina and share many characteristics with those found in zebrafish, they only exhibit a limited regenerative potential under special conditions (Karl et al., 2008; Ooto et al., 2004; Osakada et al., 2007; Takeda et al., 2008; Wan et al., 2008).”

“This differential response in uninjured and injured mouse retina may be due to injury dependent activation of the EGFR (Close et al., 2006).”

“In addition, MG participate in phagocytosis of dying retinal photoreceptors (Bailey et al., 2010).”

“N IH -PA Author M anuscript N IH -PA Author M anuscript N IH -PA Author M anuscript include FGF2 and insulin that in combination stimulate MG proliferation in the postnatal chick retina (Fischer et al., 2002) and CNTF that stimulates MG proliferation in zebrafish (Kassen et al., 2009).”

“The observation that Notch signaling inhibits MG dedifferentiation and proliferation in the injured zebrafish retina was surprising in light of reports indicating Notch signaling stimulated proliferation of MG-derived progenitors in birds and mammals (Del Debbio et al., 2010; Ghai et al., 2010; Hayes et al., 2007).”

“MAPK signaling was previously reported to be required for proliferation of MG-derived progenitors in the postnatal chick retina (Fischer et al., 2009a; Fischer et al., 2009b) and likely mediates the effects of EGF on MG proliferation in the injured mouse retina (Karl et al., 2008).”

“MAPK signaling was previously reported to be required for proliferation of MG-derived progenitors in the postnatal chick retina (Fischer et al., 2009a; Fischer et al., 2009b) and likely mediates the effects of EGF on MG proliferation in the injured mouse retina (Karl et al., 2008).”

“[PubMed: 16949068] Del Debbio CB, Balasubramanian S, Parameswaran S, Chaudhuri A, Qiu F, Ahmad I. Notch and Wnt signaling mediated rod photoreceptor regeneration by Muller cells in adult mammalian retina.”

“However, similar to the injured zebrafish retina, Notch signaling inhibits neural stem cell proliferation in the zebrafish and mouse brain (Chapouton et al., 2010; Imayoshi and Kageyama, 2011; Imayoshi et al., 2010).”

“…of genes have been identified that are necessary for MG dedifferentiation (Fausett and Goldman, 2006; Fausett et al., 2008; Kassen et al., 2007; Qin et al., 2009; Ramachandran et al., 2010a; Raymond et al., 2006; Thummel et al., 2010), mechanisms underlying their activation have remained elusive.”

“Ascl1 impacts Notch signaling during retina development by regulating the expression of genes encoding Notch ligands (Nelson et al., 2009; Nelson and Reh, 2008).”

“1:ERT2CreERT2;β-actin2:loxPmCherry-loxP-gfp double transgenic fish (Boniface et al., 2009) to show that this induction was a result of increased her4 promoter activity in proliferating MG-derived progenitors (Figure S4D).”

“In situ hybridization was performed as described previously (Barthel and Raymond, 2000).”

“We tested recombinant soluble human HB-EGF because of its commercial availability and its ability to activate the zebrafish EGFR (Goishi et al., 2003).”

“N IH -PA Author M anuscript N IH -PA Author M anuscript N IH -PA Author M anuscript EGFR inhibitor PD153035 (Fry et al., 1994; Fry et al., 1997), the MAPK (ERK1 and 2) inhibitors PD98059 (Dudley et al., 1995) or SL327 (Atkins et al., 1998) and DMSO in separate experiments.”

“GM6001 was previously shown to inhibit HB-EGF ectodomain shedding and signaling (Stoll et al., 2010; Xu et al., 2004).”

“The HB-EGF/EGFR/MAPK/Ascl1a signaling cascade regulates injury-dependent induction of Notch signaling components Ascl1 impacts Notch signaling during retina development by regulating the expression of genes encoding Notch ligands (Nelson et al., 2009; Nelson and Reh, 2008).”

“Notch signaling components are also induced in the injured adult zebrafish retina (Raymond et al., 2006; Yurco and Cameron, 2007).”

“Other candidate molecules that appear to stimulate Müller glia proliferation in the zebrafish retina include Heparin-binding-Epidermal Growth Factor (HB-EGF) [113], Insulinoma associated 1a (Insm1a) [104], ADP [114], and free radicals [98], suggesting a possibly complicated process for full activation of regeneration.”

“Many of the initial steps in the response to injury that lead to regeneration in fish are also conserved in the mammalian retina (7).”

“Although inhibition of Notch signaling enhances Müller glia proliferation in the injured retina (Figure 2) [20 ,24 ], a number of studies have also indicated an increased expression of Notch signaling components in injury-responsive Müller glia [20 ,26,27].”

“ascl1a is upregulated in proliferating MG within six hours of injury, and Ascl1a knockdown blocks MG proliferation and de-differentiation into progenitors (Fausett et al., 2008; Ramachandran et al., 2010; Ramachandran et al., 2012; Wan et al., 2012).”

“Heparin-binding epidermal-like growth factor (HB-EGF) is necessary and sufficient to induce Mu¨ller glia proliferation (Wan et al., 2012).”

“…from multiple divisions of injury-induced Müller glia, and the key to successful retina regeneration was thought to rely on Müller glial dedifferentiation into a cycling population of progenitor cells (Karl and Reh, 2010; Ramachandran et al., 2010b; Ramachandran et al., 2011; Wan et al., 2012).”

“…Ascl1a‡‡, Tgif1§§, Six3b§§, Hb-egf¶ Tgf⧧, miR203***, Notch¶, let-7*** Newt midbrain Shh‡‡‡ Dopamine§§§ **Reimer et al., 2013; ‡Dias et al., 2012; §Nelson et al., 2013; ¶Wan et al., 2012; **Thummel et al., 2008; ‡‡Ramachandran et al., 2010a, 2011; §§Lenkowski et al., 2013; ***Rajaram et al.,…”

“As ERK1/2 are activated in Müller glia following injury, extracellular signals might be efficiently relayed via these kinases into a response that renders the epigenomic landscape active, thereby allowing for gene transcription (Wan et al. 2012).”

“through a feedback loop that includes Ascl1a (Wan et al., 2012),”

“We also observed radial clusters of EdU+ cells that looked similar to clonally expanding MG-derived progenitor cells in the zebrafish retina (Figure 3K, boxed region) (Powell et al., 2016; Raymond et al., 2006; Wan et al., 2012).”

“New rod photoreceptors are generated from resident proliferative cells in the inner nuclear layer of the central retina (Johns and Fernald, 1981; Johns, 1982; Julian et al., 1998; Otteson et al., 2001; Otteson and Hitchcock, 2003).”

“In the injured retina, egfr is expressed by differentiated Müller glia and dedifferentiated Müller glia/NPCs (Wan et al., 2012).”

“…regeneration, including pcna, pax6a, pax6b, lin28a, let-7 miRNA, mdka, mdkb, hspd1, mps1, apobec2a, apobec2b, hb-egf, and ascl1a (Thummel et al., 2008, 2010; Fausett et al., 2008; Calinescu et al., 2009; Qin et al., 2009; Ramachandran et al., 2010, 2011; Powell et al., 2012; Wan et al., 2012).”

“), Notch signalling in the zebrafish acts to suppress the formation of Müller glia-derived progenitors in the uninjured retina, whereas inhibition of this pathway leads to the expansion of the region of proliferating Müller glia cells (Wan et al., 2012).”

“and differentiate into the neuronal types that were lost (Vihtelic and Hyde, 2000; Fimbel et al., 2007; Yurco and Cameron, 2007; Ramachandran et al., 2010, 2011; Nelson et al., 2012; Wan et al., 2012).”

“It is worth noting that in the regenerating fish retina, Notch-signaling inhibits the expression of ascl1a and inhibits the proliferation of MGPCs (Wan et al., 2012), contrary to the roles of Notch during neural development and in the MGPCs in birds and mammals (reviewed by Gallina et al.”

“Radial glia throughout the zebrafish CNS persist as neural progenitors (Kroehne et al., 2011; Wan et al., 2012).”

“We maintained fish under standard conditions (Westerfield, 2000).”

“…signaling cascades and gene expression programs underlying MG reprogramming and retina regeneration have been identified (Ramachandran et al., 2010b, 2011, 2012; Thummel et al., 2010; Nelson et al., 2012, 2013; Conner et al., 2014; Rajaram et al., 2014; Wan et al., 2012, 2014; Zhao et al., 2014).”

“…of mature cell types without using a specific precursor population (Geurtzen et al., 2014; Jopling et al., 2010; Knopf et al., 2011; Poss et al., 2002; Ramachandran et al., 2010; Saera-Vila et al., 2015; Saera-Vila et al., 2016; Sousa et al., 2011; Stewart et al., 2012; Wan et al., 2012).”

“In addition, HB-EGF partially exerts its mitogenic effects by acting upstream of the Wnt-pathway (Wan et al., 2012).”

“We used a Tg(ascl1a:GFP) zebrafish line (Wan et al., 2012) to examine the response of ascl1a-expressing cells to telencephalic injury.”

“In addition, the subsequent EGFR activation in zebrafish also requires MAPK signaling (Wan et al., 2012).”

“Similarly, inhibition of FGF receptors or MAPK signaling suppresses the proliferation of D E V E LO P M E N T MGPCs in damaged retinas (Fischer et al., 2009b;Wan et al., 2014), whereas inhibition of MAPK does not suppress the activation of mTor (current study).”

“A number of specific factors have been identified as key regulators of this regenerative response to damage (Qin et al., 2009; Ramachandran et al., 2010; Bailey et al., 2011; Thummel et al., 2011; Ramachandran et al., 2011a,b; Ramachandran et al., 2012; Wan et al., 2012; Lenkowski et al., 2013; Nagashima et al., 2013;…”

“2F)—many of which have experimentally confirmed roles in zebrafish retinal regeneration.(14,23,24) We next performed gene ontology (GO) and pathway-level analyses with KEGG and Reactome tools as described in more detail in the Supplementary Methods.”

“In the regenerating adult retina, however, inactivation of Notch signaling is required to recruit Müller glia into the cell cycle (Wan et al., 2012; Conner et al., 2014).”

“This was supported by the finding that PAX6 gene expression is down-regulated by EGF to allow for proper corneal epithelial proliferation, while the overexpression of PAX6 in these cells attenuated EGF-induced proliferation [177].”

“Similarly, repression of Notch signaling with the gamma-secretase inhibitor DAPT expands the pool of progenitor cells following retinal poke injury (Wan et al., 2012), although it is not sufficient to stimulate proliferation in the absence of damage (Wan & Goldman, 2017).”

“HB-EGF is known to activate cell-signaling in Müller glia through the MAPK-pathway (Wan et al. 2012).”

“The maintenance of Notch signaling in MG of the adult zebrafish retina contributes to MG quiescence and Notch suppression is required for MG proliferation (Conner et al., 2014; Elsaeidi et al., 2018; Taylor et al., 2015; Wan and Goldman, 2017; Wan et al., 2012).”

“This differs from observations after physical damage to all retinal layers where, 4 days after retinal injury, notch1a was expressed in all BrdU-positive cells while delta genes were ‘‘preferentially expressed’’ in apposing BrdU-negative cells.37 The disparity between our study and that of Wan et al.37 may be due to t…”

“Knockdown of HB-EGF and ascl1a reduced the total amount of pax6b mRNA in the retina (Ramachandran et al., 2011; Wan et al., 2012) and stabilization of β-catenin upregulated pax6b levels (Ramachandran et al.”

“…of the Notch1a ICD with the Tg(hsp70l:gal4) ; Tg(UAS:myc-notch1a-intra) transgenic line reversed the effect of γ-secretase inhibition, indicating that induced and increased Müller glia proliferation was regulated by the inhibition of Notch signaling (Wan et al., 2012; Conner et al., 2014).”

“These include growth factors (Gramage et al., 2015; Russell, 2003; Wan et al., 2012; Zhao et al., 2014), cytokines (Wan et al., 2014; Zhao et al., 2014), gene transcription factors (Nelson et al., 2012; Ramachandran et al., 2010a, 2012b; Thummel et al., 2010; Wan et al., 2014), epigenome modifiers…”

“For example, EGF, HB-EGF, FGF2, and IGF1 signal through the MAPK pathway to stimulate M€uller glia to de-differentiate, proliferate and become MGPCs in fish (Wan et al., 2012, 2014), birds (Fischer et al., 2009a,b), and mammals (Karl et al., 2008).”

“In zebra-fish retinas, HB-EGF induces de-differentiation of M€uller glia into multipotent progenitors (Wan et al. 2012).”

“these two macroglia both express EGFR and respond to EGFR ligands [83,86,87], they may undergo similar molecular changes after injury (Figure 2).”

“The ascl1a:GFP–luciferase construct was described previously (Ramachandran et al, 2010a; Wan et al, 2012).”

“Recently, additional studies have demonstrated stemness increased in different microenvironments, and transcription factors such as Sox-2, Oct-4, Klf4 and Nanog were reprogrammed into both normal tissues including human dermal fibroblasts [42] and Müller glia cells [43], and tumors including lung cancer [44], colon ca…”

“Zebrafish retina regeneration is possible through the orchestration of various growth factors (Russell, 2003; Wan et al., 2012; Zhao et al., 2014b; Gramage et al., 2015), cytokines (Wan et al.”

“In contrast to zebrafish, in which HB-EGF induces proliferation of Müller glia in the undamaged retina [10], Müller glia did not proliferate when a Wnt protein, an EGF protein, or a GSK3 inhibitor was added to the undamaged rodent retina [3] (Osakada, unpublished data).”

“This is of interest because Wnt signaling was previously shown to play an important role in M€ uller glia dedifferentiation and proliferation in the regenerating zebrafish retina (Ramachandran et al., 2011; Wan et al., 2012).”

“…production of progenitor cells that are capable of restoring all lost cell types (Bernardos et al., 2007; Fausett and Goldman, 2006; Nagashima et al., 2013; Rajaram et al., 2014a, 2014b; Ramachandran et al., 2012; Thummel et al., 2008; Vihtelic and Hyde, 2000; Wan et al., 2012; Zhao et al., 2014).”

“glia reprogramming whereas PDGF is involved in the development of proliferative vitreoretinopathy (Andrews et al., 1999; Wan et al., 2012).”

“After hair cell damage in chick or axolotl lateral line neuromasts,macrophages are recruited to the lesion site in vitro and in vivo (Bhave et al. 1998,Warchol 1997).”

“These data add Shh to the growing list of cell signaling pathways that regulate Müller glial dedifferentiation and proliferation (Lenkowski et al., 2013; Nelson et al., 2013; Ramachandran et al., 2011; Thummel et al., 2010; Wan et al., 2012).”

“Following a penetrating injury, combinatorial treatments of insulin combined with either heparin binding EGFlike growth factor (HB-EGF; an EGF-related but more potent mitogen [129]) or FGF2 increase proliferation of Müller glia in the zebrafish retina, an effect that is reduced by inhibition of MAPK or JAK/STAT [74, 1…”

“…that were genetically manipulated to be oversensitive to Wnt signaling by knocking-out inhibitor genes, or used acute injury models (laser light, oxidative stress), or a non-cell typespecific Wnt activator (Zhou et al., 2010; Wan et al., 2012; Liu et al., 2013; Sanges et al., Neuropharmacology.”

“It has been suggested that cytokines, including tumor necrosis factor-α (TNF-α) and growth factors, such as heparinbinding epidermal growth factor-like growth factor a (Hbefga), are secreted in response to RGC injury and are necessary in the microenvironment for MGC reprogramming [7, 8].”

“Pan-MMP inhibitor GM6001 reduced proliferation, which was overcome by the administration of epidermal growth factor (EGF), suggesting that the EGF/EGFR pathway was dependent of MMP activity for reprogramming (Wan et al., 2012).”