Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina

Joshua Kramer; Joana Neves; Mia Koniikusic; Heinrich Jasper; Deepak A Lamba

doi:10.1371/journal.pone.0258872

. 2021 Oct 26;16(10):e0258872. doi: 10.1371/journal.pone.0258872

Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina

Joshua Kramer ^1,², Joana Neves ^2,³, Mia Koniikusic ², Heinrich Jasper ^2,^4,^*, Deepak A Lamba ^1,^2,^*

Editor: Tudor C Badea⁵

PMCID: PMC8547621 PMID: 34699550

Abstract

Retinal homeostasis relies on intricate coordination of cell death and survival in response to stress and damage. Signaling mechanisms that coordinate this process in the adult retina remain poorly understood. Here we identify Decapentaplegic (Dpp) signaling in Drosophila and its mammalian homologue Transforming Growth Factor-beta (TGFβ) superfamily, that includes TGFβ and Bone Morphogenetic Protein (BMP) signaling arms, as central mediators of retinal neuronal death and tissue survival following acute damage. Using a Drosophila model for UV-induced retinal damage, we show that Dpp released from immune cells promotes tissue loss after UV-induced retinal damage. Interestingly, we find a dynamic response of retinal cells to this signal: in an early phase, Dpp-mediated stimulation of Saxophone/Smox signaling promotes apoptosis, while at a later stage, stimulation of the Thickveins/Mad axis promotes tissue repair and survival. This dual role is conserved in the mammalian retina through the TGFβ/BMP signaling, as supplementation of BMP4 or inhibition of TGFβ using small molecules promotes retinal cell survival, while inhibition of BMP negatively affects cell survival after light-induced photoreceptor damage and NMDA induced inner retinal neuronal damage. Our data identify key evolutionarily conserved mechanisms by which retinal homeostasis is maintained.

Introduction

A functional and resilient visual system, durable to potential insults, is crucial for rapid interpretation of an animal’s surroundings. In both mammals and insects, the visual system contains a specific organization of structures; a lens to focus incoming light, a fluid filled vitreous, photoreceptors to sense and transmit light sensing signals to the optic lobes, pigmented cells to prevent diffraction, and immune cells adjacent to the tissue to mediate the damage response [1]. Photoreceptors that make up these systems become postmitotic in early maturation (24 hours after puparium formation (APF) in Drosophila [2]; the first postnatal week in mice [3,4], and no stem cell pool exists to replace them. As such, progressive loss of these cells due to injury can significantly impact the organ’s function. When the eye suffers damage, retinal tissue must strike a balance between repair, survival, and apoptosis. This homeostatic response is mediated in part by immune cell-derived cytokines and growth factors, which influence the balance between apoptosis and survival of damaged cells [5–7]. The exact interplay of these factors in the retinal damage response remains to be fully understood.

Drosophila is an excellent model for studying the interaction between immune cells and photoreceptors during retinal tissue repair. 90% of Drosophila hemocytes are macrophages (plasmatocytes), existing in clusters around every major organ including the eyecups [8]. Genetic or UV-C induced damage directed to the post-mitotic Drosophila pupal retina can induce reproducible, quantifiable damage that persists to adulthood and is sensitive to genetic perturbations, allowing dissection of pathways that mediate photoreceptor apoptosis and control survival [2,7,9]. Persistent DNA damage induced by UV has been shown to promote initiator caspase activity in photoreceptors and thus apoptosis [10–17].

After UV damage, hemocytes are recruited to the retina, where they are activated by the Pdgf1 orthologue Pvf1 in response to activation of the Dpp signal transducer Schnurri (Shn) in damaged retinal cells [9,18]. Pvf1, in turn, induces the neurotrophic factor Mesencephalic Astrocyte Derived Neurotrophic Factor (MANF), in hemocytes, regulating their transition to an anti-inflammatory, pro-repair phenotype [7] and this response is conserved in mice [7]. However, the precise role of Dpp/Shn signaling in coordinating the tissue repair response remains unclear.

Canonically, Dpp signals through two downstream type 1 receptors, Thickveins (Tkv) and Saxophone (Sax), which both form heterotetrameric complexes with the type II receptor Punt [19]. The downstream target of Tkv is Mad, while Sax phosphorylates Smox, resulting in its nuclear translocation [20]. A coordination of Tkv and Sax signaling has recently been described in the tissue damage response of the fly intestinal epithelium, where Sax/Smox signaling mediates activation of intestinal stem cell (ISC) proliferation in response to enteropathogen infection, while Tkv/Mad signaling promotes the return to quiescence of these cells [21]. This response is coordinated by the control of Tkv protein turnover in ISCs. Tkv degradation is reduced during ISC activation, allowing for Tkv protein accumulation and replacement of Sax in Tkv/Punt complexes [22].

In mammals, Dpp homologues include various members of the bone morphogenic protein (BMP) and transforming growth factor beta (TGFβ) family. Their downstream targets include Smad 1/5/9 (activated by BMP) and Smad 2/3 (activated by TGFβ). BMP 2/4 have been shown to act in an anti-inflammatory manner in multiple systems, biasing macrophages to their anti-inflammatory M2 subtype [23–26]. These proteins also stimulate Müller glia proliferation, promote survival of retinal ganglion cells after damage, and decrease microglial activation in vivo [27–30]. However, the timing and mechanism(s) by which Dpp/BMP signaling modulates repair in the retina is not fully understood. It is also unclear how these two homologous pathways interact in this tissue.

In this report, we characterize the role of Dpp/BMP/TGFβ signaling in the retinal damage response of Drosophila and mice. We find a conserved role for early Sax/Smox signaling and Smad 2/3 in promoting photoreceptor apoptosis after light damage, and a later role for Tkv/Mad and Smad 1/5/9 signaling in promoting survival. We further provide evidence for a role of immune cells as sources for the ligands of these pathways after injury. Our findings provide critical new insight into mechanisms that maintain retinal homeostasis after injury.

Results

Hemocyte derived Dpp controls the damaged retinal tissue response in Drosophila

Our previous findings had identified Shn as a critical mediator of the retinal damage response in flies (9), yet the role of Dpp signaling in that response remained unclear. To start assessing a possible role for Dpp signaling components in retinal apoptosis, we performed genetic interaction experiments using a previously characterized fly line overexpressing a constitutively active form of the Jun N-terminal Kinase Kinase (JNKK) Hemipterous (Hep) under the control of the photoreceptor and cone cell driver Sep-Gal4 [9,31–34]. In this line (Sep-Hep^ACT), Hep^ACT expression is initiated during development in postmitotic photoreceptor and cone cells in the third instar larval eye disc, inducing photoreceptor apoptosis through activation of the pro-apoptotic gene hid [32,35].

When Sep-Hep^ACT was crossed to flies expressing Sax-RNAi or Smox-RNAi under the control of UAS, a significant increase in surviving photoreceptors compared to control flies (crossed to UAS::mCherry-RNAi) was observed (Fig 1A and 1A’). Overexpression of the negative feedback inhibitor of Dpp signaling, Daughters against Dpp (Dad; [36,37]), also promoted survival when compared to control (Figs 1A’ and S1F). Conversely, we found that knocking down Tkv and Mad resulted in a significant decrease in surviving photoreceptors (Fig 1A and 1A’).

Fig 1 — (1A-A’) Representative images and quantitation of ratio of surviving photoreceptors to total in w;Sep::Gal4,UAS::HepACT flies crossed with RNAi lines of each genotype. Representative images and quantification of adult GMR::Gal4; /UAS::RNAi progeny following knockdown of is compared through ratio of UV-exposed to unexposed eye (1B-B’). Representative images and quantitation of the pMad (in maturing GMR::Gal4;Dad-nGFP/UAS::RNAi) and nuclear Smox (in GMR::Gal4;Smox::FLAG::GFP/UAS::RNAi) (1D-D’) expression in Elav+ (1C-C’) progeny eye with and without irradiation at 27, 30, 36 and 48hours post UV. Representative images and quantitation of total DCP1+ Elav+ cells (1E-E’) or DadnGFP+/Elav+ cells (1 E”-F’”) in GMR::Gal4;Dad-nGFP/UAS::mCherry^RNAi progeny with and without irradiation at various time points post exposure. Representative images and quantitation of total DCP1+ Elav+ cells (1FF’) or DadnGFP+Elav+ cells (1 F”-F’”) in maturing GMR::Gal4;Dad-nGFP/ Hml::Dpp^RNAi progeny compared to controls. Scale Bar: 20 μm. Experiments were conducted with n = 2 replicates and n = 3–9 eyes/timepoint. Error bars indicate s.e.m. P-values from Student’s t-test. *p<0.05, **p<0.01, **p<0.001.

To assess whether this antagonistic effect of Sax/Smox and Tkv/Mad signaling are also observed during UV-induced cell death, we used an assay in which the headcase of pupae is removed at 24 hours after puparium formation, and animals are irradiated on one side with 17.5 microjoules of UV-C radiation [2,32]. In this model, hemocytes are attracted to the retina after UV-C induced damage and are critical to limit excessive cell death. When animals emerge after development, the extent of apoptosis can be measured by quantifying the size of the irradiated eye in relation to the non-irradiated control eye. We used the glass multimer reporter driver (GMR-Gal4; [38]) to express RNAi targeting Dpp pathway components in all postmitotic cells of the retina, and found that Sax or Smox knockdown inhibits UV-induced tissue loss, while Tkv or Mad knockdown promote it (Fig 1B and 1B’). A similar protective effect was observed when we knocked down Dpp in hemocytes used a hemocyte-specific driver (hemolectin::Gal4; [39]) (Fig 1B) in support of previous studies from our lab and others demonstrating that hemocytes secrete Dpp [21,40–44]. These data further support a model in which Sax/Smox signaling promotes apoptosis and Tkv/Mad signaling inhibit apoptosis in the retina.

To directly investigate the effects of Dpp signaling in photoreceptors, we irradiated Drosophila pupa, waited an additional 24 hours as previously described [2,9], and analyzed retinae during the post maturation stage (27, 30, 36 and 48 hours post UV exposure; equivalent to 51, 54, 60 and 72 hours post puparium formation). We used expression phospho-Mad (pMad) to assess activation of the Tkv/Mad pathway in Elav+ photoreceptors [45] during each post maturation timepoint, under both non UV control (S1A and S1A’ Fig) and UV condition (Fig 1C). After UV treatment, pMad activity in Elav+ cells peaked at 36 hours (Fig 1C’). We further assessed Smox nuclear localization using a Smox::GFP knock-in line in which GFP-tagged Smox is expressed from the endogenous Smox locus [46] in non UV control condition (S1B and S1B’ Fig). Nuclear localization of Smox represents activation of the Sax/Smox response [21]. Compared to pMad, Smox undergoes nuclear translocation at the earliest time point we assessed, 27 hours post UV exposure (Fig 1D and 1D’). To assess whether the kinetics of Sox/Smox and Tkv/Mad activation correlates with the induction of apoptosis, we stained for cleaved Drosophila caspase-1 (DCP1), a short-prodomain caspase and crucial driver of cell death [47]. When compared to control animals, DCP1 activity peaks at 36 hours post UV exposure (Fig 1E and 1E’). Expression of Dad as reported by the DadnGFP reporter [48,49] also peaked in Elav+ cells at 36 hours following irradiation (Fig 1E” and 1E”‘).

These kinetics led us to hypothesize that Mad activation induced Dad transcription inhibits Sax/Smox signaling post-UV thereby limiting apoptosis. To test this, we examined the effects of Dpp, Tkv/Mad and Sax/Smox perturbations on UV-induced apoptosis. Knocking down Dpp in immune cells significantly reduced apoptosis (as determined by anti-DCP1 staining) when compared to controls (Fig 1F and 1F’), while also resulting in a significant increase in Dad activity at 48hours post UV (Fig 1F” and 1F”‘).

Knockdown of Dpp in immune cells under non UV state did not significantly differ from control level of apoptosis (S1C and S1C’ Fig). Knockdown of Sax or Smox, on the other hand, resulted in a significant decrease in apoptosis at the 30 and 36 hour time points (Fig 2A, 2A’, 2B and 2B’) compared to control, associated with a significant increase in Dad-nGFP expression (Fig 2A”–2B”)(S3A, S3A’–S3B, S3B’ Fig). Knocking down Tkv or Mad, in turn, significantly increased the number of DCP1+ cells at every time point (Fig 2C, 2C’–2D, 2D’), and decreased Dad-nGFP reporter activity (Figs 2C”–2D”; S3C, S3C’–S3D, S3D’). These observations were recapitulated when Tkv/Mad and Sax/Smox perturbations were targeted directly to photoreceptors and cone cells by Sep::Gal4 rather than to the whole retina (Figs 2E, 2E’–2F, 2F’ and S4). DCP1+ cell count under control Sep::Gal4 had similar results to GMR::Gal4 under UV and non UV states (S3D, S3D’ and S3D” Fig).

Fig 2 — Representative images and quantitation of total DCP1+ Elav+ cells (2-A’; 2B-B’) or DadnGFP+ Elav+ cells (2A”-S2A”‘; 2B”-S2B”‘) in maturing GMR::Gal4;Dad-nGFP/ Sax-RNAi and Smox-RNAi progeny compared to controls. Representative images and quantitation of total DCP1+ Elav+ cells (2C-C’; 2D-D’) or DadnGFP+ Elav+ cells (2C”-S2C”‘; 2D”-S2D”‘) in maturing GMR::Gal4;Dad-nGFP/ Tkv-RNAi and Mad-RNAi progeny compared to controls. Representative images and quantitation of total DCP1+ Elav+ cells (2-E-E’; 2F-F’) in maturing Sep::Gal4;/ Smox-RNAi and Mad-RNAi progeny compared to controls. Scale Bar: 20 μm. Experiments were conducted with n = 2 replicates and n = 3–5 eyes/timepoint. Error bars indicate s.e.m. P-values from Student’s t-test. *p<0.05, **p<0.01, **p<0.001.

Our data suggest a bimodal response of photoreceptors to Dpp after UV irradiation that is controlled by Sax and Tkv signaling. Additionally, suppression of Dad expression by Smox and subsequent activation of Dad expression by Mad contribute to the regulation of photoreceptor cell death and survival.

BMP and TGFβ signaling are activated in a time dependent manner following retinal stress and damage

As the Drosophila visual system has many similarities with the mammalian retina [50], we next asked whether the bimodal response of the two arms of the Dpp pathway to damage in the Drosophila eye is conserved in mammals. We specifically focused on the TGFβ superfamily, which has a close homology to the Drosophila Dpp canonical pathway and has shown to play a role in the immune response in multiple systems [51,52]. Two important members of the family, BMP and TGFβ, share close homology with Dpp [52,53]. Following binding-induced TGFβ type 1 receptor phosphorylation, they trigger phosphorylation and nuclear translocation of Smads (Smad 1/5/9 for BMP pathway or Smad 2/3 for TGFβs), and transcriptional activation of Smad target genes. Based on our studies in Drosophila, we hypothesized that the TGF∝/Smad2/3 arm of the pathway is responsible for the inflammatory or apoptotic response, and the BMP/Smad1/5/9 arm for the anti-inflammatory pro-repair response.

To test this hypothesis, and to elucidate the dynamics of TGFβ superfamily activation, we used light-induced photoreceptor stress and damage models previously used by various labs including ours [7,54,55]. Briefly, C57BL/6 mice were exposed to high-intensity (10,000 lux) light for 1.5 hours to induce retinal stress without overt retinal apoptosis. We then analyzed the phosphorylation of the mammalian homologues to Mad and Smox, Smad1/5/9 and Smad2/3 [56] comparing non-light exposed conditions to control (S5A Fig), 0, 6, 12, 24 and 36 hours post exposure. Upon exposing 3.5 month old C57BL/6 mice to light, we observed elevated levels of phosphorylated Smad 1/5/9 (BMP response) in the inner nuclear layer (INL) of the retina, starting at 6 hours post-light exposure, and peaking at 12 hours post exposure (Fig 3A and 3A’). Phosphorylation of Smad 2/3 (TGFβ activation) peaked earlier, at 6 hours post exposure (Fig 3B and 3B’).

Fig 3 — Representative retinal images and quantitation of control dark adapted 3.5 m/o C57BL/6 mice, or 0, 6, 12, and 24 hours post exposure to 10,000 lux for 1.5 hours stained for pSmad 1/5/9 (in green, 3A-A’) and pSmad 2/3 (in green,3B-B’). Similar analysis of pSmad 2/3 in light-damaged BALB/c mice retinal sections (3-C). Representative retinal images and quantitation in 3.5 m/o CD11b::DTR mice treated with either diphtheria toxin or PBS control and exposed to 10,000 Lux. Sections were stained pSmad 1/5/9 (green) (3D-D’). Arrows (red) highlight pSmad expression in the inner retinal in all images. Scale Bar: 30 μm. Experiments were conducted with n = 2 replicates and n = 3–5 mice/condition. Error bars indicate s.e.m. Pvalues from Student’s t-test. *p<0.05, **p<0.01, **p<0.001. PE = post-exposure. DAPI (blue) marks nuclei in all panels.

Next, we asked if the response differs in BALB/c mice, which are more sensitive to light damage and exhibits widespread apoptosis in the retina following light exposure [54,57]. BALB/c mice were exposed to 5,000 lux light for 1 hour to induce apoptosis as previously shown [7]. TUNEL assay was performed on retina 24 hours post exposure to confirm significant apoptosis (S5B Fig). Interestingly, even prior to light exposure, BALB/c mice exhibit persistent TGF-β activity (pSmad 2/3) in the inner nuclear layer, suggesting substantial retinal stress under normal light conditions (Fig 3C). Immediately after light exposure (0 hours), this activity increases transiently, but by 6 hours, the pSmad2/3 signal returns to pre-light exposure levels and is absent once apoptosis sets in at 24 hours (Fig 3C’). As opposed to C57BL/6 mice, BALB/c mice exhibited no phosphoSmad1/5/9 (BMP) activity in the inner retina either under control conditions or after light exposure at any of the timepoints assessed in our study (S5D Fig). These studies suggest that TGFβ signaling may be involved in the early response which is typically of inflammatory nature, while BMPs may play a role in a later response which tends to be protective and anti-apoptotic. The absence of BMP/SMAD1/5/9 response in BALB/c mice was further investigated below to test a cause or effect conundrum.

We and others have previously shown that the retinal damage response relies on factors secreted by activated immune cells [58–61]. We next tested whether the light stress-induced BMP response is impacted when immune cells are absent. We used CD11b::DTR mice wherein myeloid cells can be inducibly ablated using intraperitoneally injected diphtheria toxin (DT) [62,63]. We have previously shown that light stress results in photoreceptor apoptosis in CD11b::DTR mice despite them being on C57BL/6 background [7]. DT induced immune cell loss in CD11b::DTR mice also resulted in loss of pSmad 1/5/9 in the inner retina under apoptosis inducing light-damage condition (Fig 3D and 3D’ in comparison to PBS injected light damaged control retinas where pSmad 1/5/9 activation was seen in CRALBP+ Müller glial cells in (S5E Fig). These studies suggest that immune cells participate in the damage-induced BMP response in mammalian retinas.

Modulation of the BMP signaling pathway protects against damage induced retinal apoptosis

We hypothesized that BMP mediates an anti-inflammatory protective retinal damage response and sought to test this hypothesis by asking whether modulation of BMP signaling would alter retinal repair response. We used the NMDA excitotoxic damage model previously used by multiple labs to cause inner retinal damage [64–67] in addition to the light damage model. The excitotoxin NMDA damages the ganglion and amacrine cells in the retina and has been used to model retinal damage due to glaucoma, retinal ischemia and diabetic retinopathy [68–71]. To directly monitor BMP activity, we used BRE-eGFP transgenic mice. These mice report the transcriptional response of BMP-Smad activation through the BMP response element (BRE) [72]. In these mice, GFP expression was observed in CRALBP+ Müller glia following NMDA exposure (Fig 4A).

Fig 4 — 3.5 m/o BRE-eGFP reporter mice intravitreally injected with 25mM NMDA were treated with either; PBS control, mBMP4, Dorsomorphin (BMP Inhibitor), or SB431542 (TGFβ Inhibitor). At 48 hours, BRE-GFP (green) colocalization assessed and quantified with Muller glial marker, CRALBP (white) and plotted following various treatments in comparison to control NMDA (4A-A’). TUNEL assay (red) was performed under above conditions, with total TUNEL+ cells plotted in comparison to control (4B-B’). Effects of various treatments tested in light damage conditions following 20,000 Lux exposure for 1.5 hours with retina collected 48 hours post exposure. TUNEL assay was then performed and quantified in comparison to control (4C-C’). Retinal tissue analysis for immune markers IBA1 (white) and CD68 (green), with ratio of ramified morphology immune cells over total compared between conditions (4D-D’). Total number of activated CD68+ immune cells was also compared between treatment conditions (4D-D”). Scale Bar: 20 μm (4-A). 30 μm (4B-D). Experiments were conducted with n = 2 replicates and n = 3–5 mice/condition. Error bars indicate s.e.m.; P-values from Student’s t-test. *p<0.05, **p<0.01, **p<0.001. DAPI (blue) marks nuclei in all panels.

To modulate the BMP signaling pathway, mice were injected intravitreally with either recombinant BMP4 or the small molecule BMP inhibitor Dorsomorphin [28,73]. Upon comparing various treatments, we confirmed that GFP activity increases following BMP4 treatment and reduces with Dorsomorphin (Fig 4A and 4A’). TUNEL staining at 48-hours post-NMDA injection revealed that inhibition of BMP signaling increased apoptosis, while supplementation of BMP4 inhibited NMDA induced apoptosis (Fig 4B and 4B’). To assess whether this was a conserved retinal damage response or specific to NMDA damage, we assessed pathway modulation under light-induced photoreceptor damage conditions (20,000 Lux for 2 hours [74]). TUNEL staining at 48 hours post exposure revealed that inhibition of BMP signaling increases apoptosis and supplementation of recombinant BMP4 reduces it (4C,C’), similar to our observations in the NMDA damage model.

Inhibition of TGFβ signaling pathway protects against damage induced retinal apoptosis

Since our data suggested that TGFβ may be involved in the earlier inflammatory response to damage, we tested if inhibiting the pathway using the small molecule TGFβ inhibitor SB431542 [75] would be protective. We carried similar NMDA and light-damage experiments as above and co-treated the mice with 25 μM of SB431542 intravitreally. In both damage models, we observed that inhibition of the TGFβ pathway reduced retinal apoptosis compared to control mice (Fig 4B and 4C). Interestingly, we also observed a small reduction in BRE-GFP activity following TGFβ inhibition (Fig 4A) suggesting cross-regulation between the pathways.

Microglia activation can be modulated by manipulating BMP/ TGFβ pathways

Lastly, we asked whether microglia activation can be biased by modulation of TGFβ and BMP pathways. Recently, morphological changes in microglia have been shown to reflect activation [76]. A change from a ramified non-activated morphology to an amoeboid shape signifies activation, and the ratio of the two morphologies can be used as a measure of relative activation. Retinal tissue was stained for the pan-microglia marker IBA1 and the inflammatory activation marker CD68. We carried out fractional analysis comparing the ratio of ramified over total (ramified and amoeboid microglia) between conditions (Fig 4D) to determine the fraction of nonactivated microglia. Under PBS injected control conditions, all microglia have ramified morphology (S5C Fig) and NMDA damage leads to over 60% of microglia changing to amoeboid morphology (Fig 4D’). We found inhibition of BMP (Dorsomorphin treatment) led to further reduction in the ramified fraction (less than 20%), indicating increased activation, whereas supplementation of BMP4 or inhibition of TGFβ led to increases in ramified microglia, indicating reduced activation (Fig 4D’). Furthermore, the morphological changes in microglia closely correlated with CD68 expression (Fig 4D”).

Discussion

Our studies identify the morphogen Decapentaplegic (Dpp) and its mammalian homologues BMP/TGFβ as important regulators of retinal tissue survival post injury (Fig 5). Our findings suggest that in the fly, Dpp is secreted from hemocytes that previous studies have shown to be drawn to the injury site [9]. Hemocytes have also been shown to secrete Dpp in the fly intestine and embryo after injury [21,41]. We find that the response to these hemocyte derived ligands in the retina is dynamic: in Elav+ cells, Smox nuclear translocation is detected first after damage, while Mad phosphorylation occurs later and correlates with Dad::GFP expression, consistent with Mad-mediated induction of Dad, as described previously in literature [36,53,77]. Mad phosphorylation also correlates with peak levels of apoptosis, which are detected at 36 hours post UV, indicating that Mad activity is associated with the apoptotic state. Since loss of hemocyte Dpp, or of retinal Sox and Sax all result in reduced tissue loss, while loss of Tkv and Mad increased tissue loss, this suggests that hemocyte-derived Dpp induces retinal apoptosis by activating Sax/Smox signaling, while the later engagement of Tkv/Mad signaling is required to downregulate the apoptotic response. The selective engagement of Sax/Smox and Tkv/Mad signaling at different timepoints in the injury response is reminiscent of a similar dynamic observed in intestinal stem cells after bacterial infection [21,22].

Fig 5 — Based on our data, under immune cell secreted Dpp/TGFβ/BMP ligand activation, Sax/pSmox ~ TGFβR2/pSmad2/3 activate first driving retinal apoptosis, followed by Tkv/pMad ~ BMPR2/pSmad 1/5/9 promoting increased retinal survival post-injury.

As Dad is an inhibitory Smad, we hypothesize that Dad induction by Mad is required as a negative feedback signal to repress Smox signaling (S1-E). Dpp has been similarly shown to drive a proapoptotic response under damage in other tissues such as ovarian somatic cells, leg disk and retinal glia [78–80]. In ovarian cells, overexpression of Dpp was shown to drive activation of the pro-apoptotic genes reaper (rpr) and head involution defective (hid) [78]. Additionally, pSmox has been shown to induce cell and stage specific apoptosis of larval neurons in other Drosophila models [81]. Interestingly, knocking down Sax and Smox in the eye increased Dad-nGFP expression, while, as expected, knocking down Tkv and Mad significantly reduced Dad-nGFP activity. Sax/Smox signaling may thus partially repress Tkv/Mad signaling in the early phase of the injury response.

We also identified dynamic changes in the phosphorylation of the downstream effectors of the mammalian homologues to Dpp, BMP and TGFβ, in our mouse retinal stress and injury models. Under conditions of light-induced retinal stress based on our prior studies [7], we observed an initial TGFβ activation represented by early peak of pSmad2/3 followed by a peak in BMP (pSmad1/5/9) activity 6 hours later. Intriguingly, we observed an early Smox followed by a delayed Mad phosphorylation in UV-damaged flies as well, suggestive of conserved response in the arms of the pathway between the two species. These findings provide further support to homology between pSmad2/3—Smox, and Smad1/5/9—Mad, as described in other injury models in Drosophila as well as other species such as Haemonchus contortus [82–84]. We additionally observed that in our light damaged BALB/c mouse model which bears a RPE65 SNP associated with increased susceptibility to light stress [54,57], pSmad 2/3 activity exists under control conditions suggesting this arm of pathway is driven by retinal stress. Interestingly, there was no detection of Smad1/5/9 activity at any timepoint following light exposure. These results suggest that the BMP arm is protective and lack of activity of this arm of the pathway is associated with increased apoptosis.

Our data also demonstrate that knockdown of CD11b-positive immune cells in the retina significantly reduce the pSmad 1/5/9 activity in Müller glia during the light induced damage response (S5-F). This suggests that immune cells play a role in activation of the mammalian homologue of Tkv/Mad pathway, BMP. This could be by either directly secreting BMPs [85] or through intermediates by promoting BMP induced Müller glial activation [86].

Supplementation of the BMP4 and inhibition of TGFβ through the ALK inhibitor SB431542, reduce apoptosis after light and NMDA induced injury. Conversely, suppression of BMP activity through the inhibitor Dorsomorphin increases both apoptosis and total CD68+ cells, a known marker of immune cell mediated inflammation after injury [87]. Previous work has found similar findings in damaged retinal ganglion cells [27,28]. Additionally, NMDA damaged Müller glia from zebrafish, rodent and chick Müller glia derived progenitor cells (MGPCs), showing pSmad 1/5/9 promotes regeneration and pSmad 2/3 inhibits it [88–90]. Thus, our data further supports the tissue conserved response with an opportunity to modulate it to promote repair in clinical settings.

The opposing effects of apoptotic driving TGFβ/Smad2/3 and apoptosis inhibiting BMP/Smad1/5/9 on retinal injury are also correlated with different downstream target genes. BMP2/4 activity is closely associated with anti-apoptotic genes id1/2/3, sox9, ihx2, and wnt 10a/11/14/7b [91]. TGFβ is associated with TGFβ-inducible early response gene 1 (tieg1), Bcl-2interacting mediator of cell death (bim), death-associated protein kinase (DAP-kinase), TGFβ induced gene human clone 3 (bigh3) and repression of id2 [92–94]. Additionally, it been shown that TGFβ driven activation in injury can also drive delayed secondary necrosis in tissue, driving the cellular membrane permeable to macromolecules, inducing delayed inflammation and leading to the activation of cleaved caspase-1 (Mitchell et al., 2013) [95,96]. However, not all previous literature on TGFβ defines it as pro-apoptotic within the retina. Recent work ablating TGFβ signaling in retinal microglia using tamoxifen induced Tgfbr2^flox/flox mice lead to increased retinal degeneration and Muller cell gliosis [97]. Also, AAV8-TGFβ1 was recently shown to promote cone survival in rd1 mice [98]. This protective response required Tgfbr1 and Tgfbr2 activity or microglia which express them. This suggests that while TGFβ activity downstream of Tgfbr2 on microglia drives secretion of pro-survival effectors, other cell types including Muller glia may have different independent roles especially in acute damage.

These results highlight the critical role of Dpp/BMP/TGFβ in regulating retinal tissue repair. Future studies targeting downstream aspects of the mammalian pathway may have therapeutic implications to delay or repair the onset of inflammation and allay retinal damage.

Materials and methods

Drosophila stocks and culture

Fly stocks were raised on standard cornmeal- and molasses-based food. All experiments were performed at 25°C. Both sexes gave the same results in all experiments, unless otherwise described. Lines used were w;Sep::Gal4,UAS::HepACT, GMR::Gal4, GMR::Gal4;Dad-nGFP, Sep::Gal4,CD8-GFP, and GMR::Gal4;Smox::FLAG::GFP. All lines were crossed with UAS::Dad, UAS::Dad^RNAi, UAS::Sax^RNAi, UAS::Smox^RNAi, UAS::Tkv^RNAi, UAS::Mad^RNAi, UAS::Hml::Dpp^RNAi, or UAS::mCherry^RNAi, UAS-GFP and W₁₁₁₈ as controls.

Mice

All mice used in the described studies were housed and bred at the Association for Assessment and Accreditation of Laboratory Animal Care International accredited vivarium of the Buck Institute for Research on Aging, in a specific-pathogen-free facility, or in the UCSF Laboratory Animal Resource Center, in individually ventilated cages on a standard 12:12 light cycle. Anesthetic state was induced in mice by 1.5% isoflurane for the duration of injection experiments. In preparation for tissue collection, mice were placed in a new bedding lined cage and euthanized by displacement of air with 100% carbon dioxide for 5 minutes, and concluded with cervical dislocation to induce rapid loss of consciousness and death with a minimum of pain, distress or discomfort. All procedures were approved by the Buck Institute Institutional Animal Care and Use Committee or UCSF IACUC Animal Care Committee.

Intraocular injections in mice

For intravitreal injection, recombinant proteins or compounds in 1-μl volume were injected into the right eye using a graduated pulled glass pipette and a wire plunger (Wiretrol II, 5-0000-2005, Drummond Scientific Company) or directly by using a Hamilton 10uL pipette following isoflurane anesthesia.

Light and NMDA damage in mice

C57BL/6, BALB/c, or BRE-eGFP 4x backcrossed with C57BL/6 background were dark adapted for 18 hours, then intravitreally injected of control PBS or 25 mM NMDA, and recombinant protein of either; mBMP4, Dorsomorphin (BMP Inhibitor), or SB431542 (TGFβ Inhibitor). Animals were then exposed to either 5000–20,000 lux for 1–1.5 hours, or 25 mM NMDA. In case of light damage, mice were allowed to recover from anesthesia, returned to their cages, and housed in darkness until analysis. Retinal tissue was collected under control state, 0, 6, 12, 24 or 48 hours post exposure.

UV damage in Drosophila pupae retina and larvae

Pupae retinas were exposed to 17.5 microJoules of UV-C radiation 24 hours post puparium formation and either collected 5 days post adulthood or 24, 27, 30, 36, and 48 hours post exposure.

Histological analysis, imaging, and quantification methods

Retinal sections and macrophages were analyzed by immunohistochemistry (IHC) and other histological methods and imaged using a LSM 700 confocal laser-scanning microscope, images were processed sequentially on separate channels. All images were used for quantification purposes and processed with adobe photoshop software, ImageJ, Imaris 9.5.1, ZEN 3.2 and LAS X software. Eyes were fixed with 4% formaldehyde, PBS washed and placed in progressively increasing concentrations of PBS/Sucrose solution (5, 10, 15, 20%), mounted in O.C.T.(TissueTek) compound in -80 degrees C overnight, sectioned into 10 micron tissue on slide, prepared as (Lamba et al., 2010) and analyzed with the following antibodies: Elav-9F8A9 (1:200, rat, DSHB), pMad-EP823Y (1:300, rabbit, abcam), DCP1-Asp216 (1:100, rabbit, Cell Signaling), pSmad 1/5/9 (1:300, rabbit, Cell Signaling), pSmad 2/3 (1:300, rabbit, Cell Signaling), GFP (1:1000, rabbit, GeneTex), CRALBP (1:200, mouse, Santa Cruz), TUNEL, IBA1 (1:200, rabbit, Abcam), and CD68 (1:100, rat, BioLegend).

Statistical analysis

All counts are presented as average and standard error of mean (SEM). Statistical analysis was carried out using Microsoft Excel or GraphPad Prism 8.0.1, and Student’s t test or two-way analysis of variance (ANOVA) was used to determine statistical significance, assuming normal distribution and equal variance.

Supporting information

S1 Fig

Representative images and quantitation of control pMad+ Elav+ cells under all timepoints and non-UV WT genotype is compared (S1-A). Control nSmox+ Elav+ cells under all timepoints and non-UV WT genotype is compared (S1-B). GMR::Gal4;Hml::DppRNAi progeny total DCP1+ Elav+ cells is compared to control no UV animals (S1-C). Sep::Gal4;mCherry-RNAi control progeny is compared vs no UV and with GMR::Gal4 control progeny (S1-D). GMR::Gal4;UAS::Dad-RNAi is compared with control UV progeny (S1-E). Sep::Gal4;UAS-HepACT; UAS-GFP representative control image (S1-F). Scale Bar: 20 μm. Error bars indicate s.e.m.; P-values from Student’s t-test. *p<0.05, **p<0.01, **p<0.001.

(TIF)

Click here for additional data file.^{(3MB, tif)}

S2 Fig. Quantitation of Individual Drosophila eyes.

Representative images and quantitation of all experiments were collected below. Control UV WT progeny pMad+ Elav+ cells compared with non UV progeny (S2-A). UV exposed control nSmox+ Elav+ cells is compared with non UV progeny (S2-B). Total WT DCP1+ Elav+ cells quantitation compared with no UV (S2-C). Total Dad-nGFP+ Elav+ cells compared between UV and no UV WT progeny (S2-C’). Individual quantitation of total GMR::G4;Hml::Dpp^RNAi DCP1+ Elav+ cells and Dad-nGFP+ Elav+ cells compared with WT UV (S2-D). Error bars indicate s.e.m.; P-values from Student’s t-test. *p<0.05, **p<0.01, **p<0.001.

(TIF)

Click here for additional data file.^{(654.6KB, tif)}

S3 Fig. Quantitation of Individual Drosophila eyes continued.

Representative images and quantitation of all experiments were collected below. UV exposed GMR::G4;SaxRNAi individual progeny DCP1+ Elav+ cells and Dad-nGFP+ Elav+ cells are compared to UV WT (S3-A). UV exposed GMR::G4;SmoxRNAi individual progeny DCP1+ Elav+ cells and Dad-nGFP+ Elav+ cells are compared to UV WT (S3-B). UV exposed GMR::G4;TkvRNAi individual progeny DCP1+ Elav+ and Dad-nGFP+ Elav+ cells are compared to UV WT (S3-C). UV exposed GMR::G4;MadRNAi individual progeny DCP1+ Elav+ and Dad-nGFP+ Elav+ cells are compared to UV WT (S3-D). Scale Bar: 20 μm. Error bars indicate s.e.m.; P-values from Student’s t-test. *p<0.05, **p<0.01, **p<0.001.

(TIF)

Click here for additional data file.^{(3.6MB, tif)}

S4 Fig. UV exposed Sep::G4;Smox and MadRNAi individual progeny DCP1+ Elav+ cells to UV Sep WT.

Error bars indicate s.e.m.; P-values from Student’s t-test. *p<0.05, **p<0.01, **p<0.001.

(TIF)

Click here for additional data file.^{(814.3KB, tif)}

S5 Fig

pSmad 1/5/9 (BMP) and pSmad 2/3 (TGFB) control images of C57 animals (S5-A). TUNEL stain in red of BALB/c animals post light exposure, with DAPI in blue (S5-B). PBS WT Control unexposed retina with IBA1 in white and CD68 in green (S5-C). pSmad 1/5/9 representative images of BALB/c animals post light exposure at 0, 24 and 36 hours (S5-E). Representative images of DT injected CD11b::DTR mice with pSmad 1/5/9 (BMP) in red, and CRALBP in white (S5-E). Scale Bar: 30 μm.

(TIF)

Click here for additional data file.^{(5.6MB, tif)}

Acknowledgments

The authors thank members of the Lamba and Jasper labs for advice and technical assistance. We would like to thank the UCSF Vision Core for their support.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The research presented here is supported by NIH grants (R01 EY025779 and EY032197 to DL; AG057353 and EY018177 to HJ; P30 Vision Core grant to UCSF Dept of Ophthalmology), and the Research to Prevent Blindness (unrestricted grant to UCSF Dept of Ophthalmology). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dr. Jasper’s affiliation with Genetech, Inc provided support in the form of salaries for author HJ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

References

1.Katz B, Minke B. Drosophila photoreceptors and signaling mechanisms. Front Cell Neurosci. 2009. Jun 11;3:2. doi: 10.3389/neuro.03.002.2009 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Jassim OW, Fink JL, Cagan RL. Dmp53 protects the Drosophila retina during a developmentally regulated DNA damage response. EMBO J. 2003. Oct 15;22(20):5622–5632. doi: 10.1093/emboj/cdg543 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Young RW. Cell proliferation during postnatal development of the retina in the mouse. Brain Res. 1985. Aug;353(2):229–239. doi: 10.1016/0165-3806(85)90211-1 [DOI] [PubMed] [Google Scholar]
4.Close JL, Gumuscu B, Reh TA. Retinal neurons regulate proliferation of postnatal progenitors and Müller glia in the rat retina via TGF beta signaling. Development. 2005. Jul;132(13):3015–3026. doi: 10.1242/dev.01882 [DOI] [PubMed] [Google Scholar]
5.Agaisse H, Petersen UM, Boutros M, Mathey-Prevot B, Perrimon N. Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. Dev Cell. 2003. Sep;5(3):441–450. doi: 10.1016/s1534-5807(03)00244-2 [DOI] [PubMed] [Google Scholar]
6.Vlisidou I, Wood W. Drosophila blood cells and their role in immune responses. FEBS J. 2015. Apr;282(8):1368–1382. doi: 10.1111/febs.13235 [DOI] [PubMed] [Google Scholar]
7.Neves J, Zhu J, Sousa-Victor P, Konjikusic M, Riley R, Chew S, et al. Immune modulation by MANF promotes tissue repair and regenerative success in the retina. Science. 2016. Jul 1;353(6294):aaf3646. doi: 10.1126/science.aaf3646 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Gold KS, Brückner K. Macrophages and cellular immunity in Drosophila melanogaster. Semin Immunol. 2015;27(6):357–368. doi: 10.1016/j.smim.2016.03.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Kelsey EM, Luo X, Brückner K, Jasper H. Schnurri regulates hemocyte function to promote tissue recovery after DNA damage. J Cell Sci. 2012. Mar 15;125(Pt 6):1393–1400. doi: 10.1242/jcs.095323 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Brodsky MH, Nordstrom W, Tsang G, Kwan E, Rubin GM, Abrams JM. Drosophila p53 binds a damage response element at the reaper locus. Cell. 2000. Mar 31;101(1):103–113. doi: 10.1016/S0092-8674(00)80627-3 [DOI] [PubMed] [Google Scholar]
11.Jin S, Martinek S, Joo WS, Wortman JR, Mirkovic N, Sali A, et al. Identification and characterization of a p53 homologue in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2000;97(13):7301–7306. doi: 10.1073/pnas.97.13.7301 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Ollmann M, Young LM, Di Como CJ, Karim F, Belvin M, Robertson S, et al. Drosophila p53 is a structural and functional homolog of the tumor suppressor p53. Cell. 2000. Mar 31;101(1):91–101. doi: 10.1016/S0092-8674(00)80626-1 [DOI] [PubMed] [Google Scholar]
13.Sogame N, Kim M, Abrams JM. Drosophila p53 preserves genomic stability by regulating cell death. Proc Natl Acad Sci U S A. 2003;100(8):4696–4701. doi: 10.1073/pnas.0736384100 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Zhou L, Steller H. Distinct pathways mediate UV-induced apoptosis in Drosophila embryos. Dev Cell. 2003. Apr;4(4):599–605. doi: 10.1016/s1534-5807(03)00085-6 [DOI] [PubMed] [Google Scholar]
15.Hay BA, Guo M. Caspase-dependent cell death in Drosophila. Annu Rev Cell Dev Biol. 2006;22:623–650. doi: 10.1146/annurev.cellbio.21.012804.093845 [DOI] [PubMed] [Google Scholar]
16.Steller H. Regulation of apoptosis in Drosophila. Cell Death Differ. 2008. Jul;15(7):1132–1138. doi: 10.1038/cdd.2008.50 [DOI] [PubMed] [Google Scholar]
17.Denton D, Aung-Htut MT, Kumar S. Developmentally programmed cell death in Drosophila. Biochim Biophys Acta. 2013. Dec;1833(12):3499–3506. doi: 10.1016/j.bbamcr.2013.06.014 [DOI] [PubMed] [Google Scholar]
18.Yao L-C, Blitz IL, Peiffer DA, Phin S, Wang Y, Ogata S, et al. Schnurri transcription factors from Drosophila and vertebrates can mediate Bmp signaling through a phylogenetically conserved mechanism. Development. 2006. Oct;133(20):4025–4034. doi: 10.1242/dev.02561 [DOI] [PubMed] [Google Scholar]
19.Neal SJ, Dolezal D, Jusić N, Pignoni F. Drosophila ML-DmD17-c3 cells respond robustly to Dpp and exhibit complex transcriptional feedback on BMP signaling components. BMC Dev Biol. 2019. Jan 22;19(1):1. doi: 10.1186/s12861-019-0181-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Li C-Y, Guo Z, Wang Z. TGFbeta receptor saxophone non-autonomously regulates germline proliferation in a Smox/dSmad2-dependent manner in Drosophila testis. Dev Biol. 2007. Sep 1;309(1):70–77. doi: 10.1016/j.ydbio.2007.06.019 [DOI] [PubMed] [Google Scholar]
21.Ayyaz A, Li H, Jasper H. Haemocytes control stem cell activity in the Drosophila intestine. Nat Cell Biol. 2015. Jun;17(6):736–748. doi: 10.1038/ncb3174 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Tracy Cai X, Li H, Safyan A, Gawlik J, Pyrowolakis G, Jasper H. AWD regulates timed activation of BMP signaling in intestinal stem cells to maintain tissue homeostasis. Nat Commun. 2019. Jul 5;10(1):2988. doi: 10.1038/s41467-019-10926-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Talati M, West J, Zaynagetdinov R, Hong CC, Han W, Blackwell T, et al. BMP pathway regulation of and by macrophages. PLoS One. 2014. Apr 8;9(4):e94119. doi: 10.1371/journal.pone.0094119 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Dzik JM. Evolutionary roots of arginase expression and regulation. Front Immunol. 2014. Nov 7;5:544. doi: 10.3389/fimmu.2014.00544 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Ji T, Takabayashi H, Mao MY, Todisco A. 199 Regulation of BMP-4 Expression by Inflammatory Cytokines. Gastroenterology. 2014. May;146(5):S–52. [Google Scholar]
26.Shinohara M, Mao M, Keeley TM, El-Zaatari M, Lee H-J, Eaton KA, et al. Bone morphogenetic protein signaling regulates gastric epithelial cell development and proliferation in mice. Gastroenterology. 2010. Dec;139(6):2050–2060.e2. doi: 10.1053/j.gastro.2010.08.052 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Ueki Y, Reh TA. EGF stimulates Müller glial proliferation via a BMP-dependent mechanism. Glia. 2013. May 1;61(5):778–789. doi: 10.1002/glia.22472 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Ueki Y, Reh TA. Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo. PLoS One. 2012. Jun 6;7(6):e38690. doi: 10.1371/journal.pone.0038690 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Thompson A, Berry M, Logan A, Ahmed Z. Activation of the bmp4/smad1 pathway promotes retinal ganglion cell survival and axon regeneration. Invest Ophthalmol Vis Sci. 2019. Apr 1;60(5):1748–1759. doi: 10.1167/iovs.18-26449 [DOI] [PubMed] [Google Scholar]
30.Mitchell K, Shah JP, Tsytsikova LV, Campbell AM, Affram K, Symes AJ. LPS antagonism of TGF-β signaling results in prolonged survival and activation of rat primary microglia. J Neurochem. 2014. Apr;129(1):155–168. doi: 10.1111/jnc.12612 [DOI] [PubMed] [Google Scholar]
31.Jasper H, Benes V, Atzberger A, Sauer S, Ansorge W, Bohmann D. A genomic switch at the transition from cell proliferation to terminal differentiation in the Drosophila eye. Dev Cell. 2002. Oct;3(4):511–521. doi: 10.1016/s1534-5807(02)00297-6 [DOI] [PubMed] [Google Scholar]
32.Luo X, Puig O, Hyun J, Bohmann D, Jasper H. Foxo and Fos regulate the decision between cell death and survival in response to UV irradiation. EMBO J. 2007. Jan 24;26(2):380–390. doi: 10.1038/sj.emboj.7601484 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Therrien M, Wong AM, Kwan E, Rubin GM. Functional analysis of CNK in RAS signaling. Proc Natl Acad Sci USA. 1999. Nov 9;96(23):13259–13263. doi: 10.1073/pnas.96.23.13259 [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Kanda H, Miura M. Regulatory roles of JNK in programmed cell death. J Biochem. 2004. Jul;136(1):1–6. doi: 10.1093/jb/mvh098 [DOI] [PubMed] [Google Scholar]
35.Nielsen MD, Luo X, Biteau B, Syverson K, Jasper H. 14-3-3 Epsilon antagonizes FoxO to control growth, apoptosis and longevity in Drosophila. Aging Cell. 2008. Oct;7(5):688–699. doi: 10.1111/j.1474-9726.2008.00420.x [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Tsuneizumi K, Nakayama T, Kamoshida Y, Kornberg TB, Christian JL, Tabata T. Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature. 1997. Oct 9;389(6651):627–631. doi: 10.1038/39362 [DOI] [PubMed] [Google Scholar]
37.Kamiya Y, Miyazono K, Miyazawa K. Specificity of the inhibitory effects of Dad on TGFbeta family type I receptors, Thickveins, Saxophone, and Baboon in Drosophila. FEBS Lett. 2008. Jul 23;582(17):2496–2500. doi: 10.1016/j.febslet.2008.05.052 [DOI] [PubMed] [Google Scholar]
38.Moses K, Rubin GM. Glass encodes a site-specific DNA-binding protein that is regulated in response to positional signals in the developing Drosophila eye. Genes Dev. 1991. Apr;5(4):583–593. doi: 10.1101/gad.5.4.583 [DOI] [PubMed] [Google Scholar]
39.Goto A, Kadowaki T, Kitagawa Y. Drosophila hemolectin gene is expressed in embryonic and larval hemocytes and its knock down causes bleeding defects. Dev Biol. 2003. Dec;264(2):582–591. doi: 10.1016/j.ydbio.2003.06.001 [DOI] [PubMed] [Google Scholar]
40.Dey NS, Ramesh P, Chugh M, Mandal S, Mandal L. Correction: Dpp dependent Hematopoietic stem cells give rise to Hh dependent blood progenitors in larval lymph gland of Drosophila. Elife. 2019. Sep 11;8. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Eleftherianos I, Castillo JC, Patrnogic J. TGF-β signaling regulates resistance to parasitic nematode infection in Drosophila melanogaster. Immunobiology. 2016. Jul 25;221(12):1362–1368. doi: 10.1016/j.imbio.2016.07.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Chakrabarti S, Dudzic JP, Li X, Collas EJ, Boquete J-P, Lemaitre B. Remote control of intestinal stem cell activity by haemocytes in drosophila. PLoS Genet. 2016. May 27;12(5):e1006089. doi: 10.1371/journal.pgen.1006089 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Tian A, Wang B, Jiang J. Injury-stimulated and self-restrained BMP signaling dynamically regulates stem cell pool size during Drosophila midgut regeneration. Proc Natl Acad Sci USA. 2017. Mar 28;114(13):E2699–E2708. doi: 10.1073/pnas.1617790114 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Gervais L, Bardin AJ. Tissue homeostasis and aging: new insight from the fly intestine. Curr Opin Cell Biol. 2017. Jul 16;48:97–105. doi: 10.1016/j.ceb.2017.06.005 [DOI] [PubMed] [Google Scholar]
45.Suh GSB, Poeck B, Chouard T, Oron E, Segal D, Chamovitz DA, et al. Drosophila JAB1/CSN5 acts in photoreceptor cells to induce glial cells. Neuron. 2002. Jan 3;33(1):35–46. doi: 10.1016/s0896-6273(01)00576-1 [DOI] [PubMed] [Google Scholar]
46.Spokony Rebecca F, White K. Spokony insertions. FlyBase ID: FBrf0221557. 2013.
47.Xu D, Wang Y, Willecke R, Chen Z, Ding T, Bergmann A. The effector caspases drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila. Cell Death Differ. 2006. Oct;13(10):1697–1706. doi: 10.1038/sj.cdd.4401920 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Ninov N, Menezes-Cabral S, Prat-Rojo C, Manjón C, Weiss A, Pyrowolakis G, et al. Dpp signaling directs cell motility and invasiveness during epithelial morphogenesis. Curr Biol. 2010. Mar 23;20(6):513–520. doi: 10.1016/j.cub.2010.01.063 [DOI] [PubMed] [Google Scholar]
49.Malzer E, Dominicus CS, Chambers JE, Dickens JA, Mookerjee S, Marciniak SJ. The integrated stress response regulates BMP signalling through effects on translation. BMC Biol. 2018. Apr 3;16(1):34. doi: 10.1186/s12915-018-0503-x [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Sanes JR, Zipursky SL. Design principles of insect and vertebrate visual systems. Neuron. 2010. Apr 15;66(1):15–36. doi: 10.1016/j.neuron.2010.01.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Sanjabi S, Oh SA, Li MO. Regulation of the Immune Response by TGF-β: From Conception to Autoimmunity and Infection. Cold Spring Harb Perspect Biol. 2017. Jun 1;9(6). doi: 10.1101/cshperspect.a022236 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Upadhyay A, Moss-Taylor L, Kim M-J, Ghosh AC, O’Connor MB. TGF-β Family Signaling in Drosophila. Cold Spring Harb Perspect Biol. 2017. Sep 1;9(9). [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Hamaratoglu F, Affolter M, Pyrowolakis G. Dpp/BMP signaling in flies: from molecules to biology. Semin Cell Dev Biol. 2014. Aug;32:128–136. doi: 10.1016/j.semcdb.2014.04.036 [DOI] [PubMed] [Google Scholar]
54.Grimm C, Remé CE. Light damage as a model of retinal degeneration. Methods Mol Biol. 2013;935:87–97. doi: 10.1007/978-1-62703-080-9_6 [DOI] [PubMed] [Google Scholar]
55.White DA, Fritz JJ, Hauswirth WW, Kaushal S, Lewin AS. Increased sensitivity to light induced damage in a mouse model of autosomal dominant retinal disease. Invest Ophthalmol Vis Sci. 2007. May;48(5):1942–1951. doi: 10.1167/iovs.06-1131 [DOI] [PubMed] [Google Scholar]
56.Kashima R, Hata A. The role of TGF-β superfamily signaling in neurological disorders. Acta Biochim Biophys Sin (Shanghai). 2018. Jan 1;50(1):106–120. doi: 10.1093/abbs/gmx124 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Wenzel A, Grimm C, Samardzija M, Remé CE. The genetic modifier Rpe65Leu(450): effect on light damage susceptibility in c-Fos-deficient mice. Invest Ophthalmol Vis Sci. 2003. Jun;44(6):2798–2802. doi: 10.1167/iovs.02-1134 [DOI] [PubMed] [Google Scholar]
58.Minhas G, Sharma J, Khan N. Cellular Stress Response and Immune Signaling in Retinal Ischemia-Reperfusion Injury. Front Immunol. 2016. Oct 24;7:444. doi: 10.3389/fimmu.2016.00444 [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Okunuki Y, Mukai R, Pearsall EA, Klokman G, Husain D, Park D-H, et al. Microglia inhibit photoreceptor cell death and regulate immune cell infiltration in response to retinal detachment. Proc Natl Acad Sci USA. 2018. Jul 3;115(27):E6264–E6273. doi: 10.1073/pnas.1719601115 [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Okunuki Y, Mukai R, Nakao T, Tabor SJ, Butovsky O, Dana R, et al. Retinal microglia initiate neuroinflammation in ocular autoimmunity. Proc Natl Acad Sci USA. 2019. May 14;116(20):9989–9998. doi: 10.1073/pnas.1820387116 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Akhtar-Schäfer I, Wang L, Krohne TU, Xu H, Langmann T. Modulation of three key innate immune pathways for the most common retinal degenerative diseases. EMBO Mol Med. 2018;10(10). doi: 10.15252/emmm.201708450 [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Frieler RA, Mortensen RM. Immune cell and other noncardiomyocyte regulation of cardiac hypertrophy and remodeling. Circulation. 2015. Mar 17;131(11):1019–1030. doi: 10.1161/CIRCULATIONAHA.114.008788 [DOI] [PMC free article] [PubMed] [Google Scholar]
63.McPherson SW, Heuss ND, Lehmann U, Roehrich H, Abedin M, Gregerson DS. The retinal environment induces microglia-like properties in recruited myeloid cells. J Neuroinflammation. 2019. Jul 20;16(1):151. doi: 10.1186/s12974-019-1546-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Karl MO, Hayes S, Nelson BR, Tan K, Buckingham B, Reh TA. Stimulation of neural regeneration in the mouse retina. Proc Natl Acad Sci USA. 2008. Dec 9;105(49):19508–19513. doi: 10.1073/pnas.0807453105 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Lamba D, Karl M, Reh T. Neural regeneration and cell replacement: a view from the eye. Cell Stem Cell. 2008. Jun 5;2(6):538–549. doi: 10.1016/j.stem.2008.05.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Kuehn S, Rodust C, Stute G, Grotegut P, Meißner W, Reinehr S, et al. Concentration Dependent Inner Retina Layer Damage and Optic Nerve Degeneration in a NMDA Model. J Mol Neurosci. 2017. Dec;63(3–4):283–299. doi: 10.1007/s12031-017-0978-x [DOI] [PubMed] [Google Scholar]
67.Lambuk L, Iezhitsa I, Agarwal R, Bakar NS, Agarwal P, Ismail NM. Antiapoptotic effect of taurine against NMDA-induced retinal excitotoxicity in rats. Neurotoxicology. 2019;70:62–71. doi: 10.1016/j.neuro.2018.10.009 [DOI] [PubMed] [Google Scholar]
68.Niwa M, Aoki H, Hirata A, Tomita H, Green PG, Hara A. Retinal Cell Degeneration in Animal Models. Int J Mol Sci. 2015;17(1). [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Maekawa S, Sato K, Fujita K, Daigaku R, Tawarayama H, Murayama N, et al. The neuroprotective effect of hesperidin in NMDA-induced retinal injury acts by suppressing oxidative stress and excessive calpain activation. Sci Rep. 2017. Jul 31;7(1):6885. doi: 10.1038/s41598-017-06969-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
70.Ranaei Pirmardan E, Soheili Z-S, Samiei S, Ahmadieh H, Mowla SJ, Naseri M, et al. In vivo evaluation of PAX6 overexpression and NMDA cytotoxicity to stimulate proliferation in the mouse retina. Sci Rep. 2018. Dec 7;8(1):17700. doi: 10.1038/s41598-018-35884-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
71.Christensen I, Lu B, Yang N, Huang K, Wang P, Tian N. The Susceptibility of Retinal Ganglion Cells to Glutamatergic Excitotoxicity Is Type-Specific. Front Neurosci. 2019. Mar 15;13:219. doi: 10.3389/fnins.2019.00219 [DOI] [PMC free article] [PubMed] [Google Scholar]
72.Javier AL, Doan LT, Luong M, Reyes de Mochel NS, Sun A, Monuki ES, et al. Bmp indicator mice reveal dynamic regulation of transcriptional response. PLoS One. 2012. Sep 11;7(9):e42566. doi: 10.1371/journal.pone.0042566 [DOI] [PMC free article] [PubMed] [Google Scholar]
73.Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat Chem Biol. 2008. Jan;4(1):33–41. doi: 10.1038/nchembio.2007.54 [DOI] [PMC free article] [PubMed] [Google Scholar]
74.Zhang J, Kiser PD, Badiee M, Palczewska G, Dong Z, Golczak M, et al. Molecular pharmacodynamics of emixustat in protection against retinal degeneration. J Clin Invest. 2015. Jul 1;125(7):2781–2794. doi: 10.1172/JCI80950 [DOI] [PMC free article] [PubMed] [Google Scholar]
75.Tappeiner C, Maurer E, Sallin P, Bise T, Enzmann V, Tschopp M. Inhibition of the tgfβ pathway enhances retinal regeneration in adult zebrafish. PLoS One. 2016. Nov 23;11(11):e0167073. doi: 10.1371/journal.pone.0167073 [DOI] [PMC free article] [PubMed] [Google Scholar]
76.Karperien A, Ahammer H, Jelinek HF. Quantitating the subtleties of microglial morphology with fractal analysis. Front Cell Neurosci. 2013. Jan 30;7:3. doi: 10.3389/fncel.2013.00003 [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Inoue H, Imamura T, Ishidou Y, Takase M, Udagawa Y, Oka Y, et al. Interplay of signal mediators of decapentaplegic (Dpp): molecular characterization of mothers against dpp, Medea, and daughters against dpp. Mol Biol Cell. 1998. Aug;9(8):2145–2156. doi: 10.1091/mbc.9.8.2145 [DOI] [PMC free article] [PubMed] [Google Scholar]
78.Kang I, Choi Y, Jung S, Lim JY, Lee D, Gupta S, et al. Identification of target genes regulated by the Drosophila histone methyltransferase Eggless reveals a role of Decapentaplegic in apoptotic signaling. Sci Rep. 2018. May 8;8(1):7123. doi: 10.1038/s41598-018-25483-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
79.Manjón C, Sánchez-Herrero E, Suzanne M. Sharp boundaries of Dpp signalling trigger local cell death required for Drosophila leg morphogenesis. Nat Cell Biol. 2007. Jan;9(1):57–63. doi: 10.1038/ncb1518 [DOI] [PubMed] [Google Scholar]
80.Velarde SB, Quevedo A, Estella C, Baonza A. Dpp and Hedgehog promote the Glial response to neuronal damage in the developing Drosophila Visual system. BioRxiv. 2020. Feb 25; [DOI] [PMC free article] [PubMed] [Google Scholar]
81.Wang Z, Lee G, Vuong R, Park JH. Two-factor specification of apoptosis: TGF-β signaling acts cooperatively with ecdysone signaling to induce cell- and stage-specific apoptosis of larval neurons during metamorphosis in Drosophila melanogaster. Apoptosis. 2019;24(1112):972–989. doi: 10.1007/s10495-019-01574-4 [DOI] [PubMed] [Google Scholar]
82.Goldstein JA, Kelly SM, LoPresti PP, Heydemann A, Earley JU, Ferguson EL, et al. SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy. Hum Mol Genet. 2011. Mar 1;20(5):894–904. doi: 10.1093/hmg/ddq528 [DOI] [PMC free article] [PubMed] [Google Scholar]
83.Goldstein JA, Bogdanovich S, Beiriger A, Wren LM, Rossi AE, Gao QQ, et al. Excess SMAD signaling contributes to heart and muscle dysfunction in muscular dystrophy. Hum Mol Genet. 2014. Dec 20;23(25):6722–6731. doi: 10.1093/hmg/ddu390 [DOI] [PMC free article] [PubMed] [Google Scholar]
84.Li F-F, Gasser RB, Liu F, Shan J-N, Di W-D, He L, et al. Identification and characterization of an R-Smad homologue (Hco-DAF-8) from Haemonchus contortus. Parasit Vectors. 2020. Apr 3;13(1):164. doi: 10.1186/s13071-020-04034-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
85.Yousef H, Morgenthaler A, Schlesinger C, Bugaj L, Conboy IM, Schaffer DV. AgeAssociated Increase in BMP Signaling Inhibits Hippocampal Neurogenesis. Stem Cells. 2015. May;33(5):1577–1588. doi: 10.1002/stem.1943 [DOI] [PMC free article] [PubMed] [Google Scholar]
86.Dharmarajan S, Fisk DL, Sorenson CM, Sheibani N, Belecky-Adams TL. Microglia activation is essential for BMP7-mediated retinal reactive gliosis. J Neuroinflammation. 2017. Apr 5;14(1):76. doi: 10.1186/s12974-017-0855-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
87.Chistiakov DA, Killingsworth MC, Myasoedova VA, Orekhov AN, Bobryshev YV. CD68/macrosialin: not just a histochemical marker. Lab Invest. 2017;97(1):4–13. doi: 10.1038/labinvest.2016.116 [DOI] [PubMed] [Google Scholar]
88.Saika S. TGFbeta pathobiology in the eye. Lab Invest. 2006. Feb;86(2):106–115. doi: 10.1038/labinvest.3700375 [DOI] [PubMed] [Google Scholar]
89.Lenkowski JR, Qin Z, Sifuentes CJ, Thummel R, Soto CM, Moens CB, et al. Retinal regeneration in adult zebrafish requires regulation of TGFβ signaling. Glia. 2013. Oct;61(10):1687–1697. doi: 10.1002/glia.22549 [DOI] [PMC free article] [PubMed] [Google Scholar]
90.Todd L, Palazzo I, Squires N, Mendonca N, Fischer AJ. BMP- and TGFβ-signaling regulate the formation of Müller glia-derived progenitor cells in the avian retina. Glia. 2017. Jul 13;65(10):1640–1655. doi: 10.1002/glia.23185 [DOI] [PMC free article] [PubMed] [Google Scholar]
91.Genander M, Cook PJ, Ramsköld D, Keyes BE, Mertz AF, Sandberg R, et al. BMP signaling and its pSMAD1/5 target genes differentially regulate hair follicle stem cell lineages. Cell Stem Cell. 2014. Nov 6;15(5):619–633. doi: 10.1016/j.stem.2014.09.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
92.Bender H, Wang Z, Schuster N, Krieglstein K. TIEG1 facilitates transforming growth factor-beta-mediated apoptosis in the oligodendroglial cell line OLI-neu. J Neurosci Res. 2004. Feb 1;75(3):344–352. doi: 10.1002/jnr.10856 [DOI] [PubMed] [Google Scholar]
93.Betts-Obregon BS, Mondragon AA, Mendiola AS, LeBaron RG, Asmis R, Zou T, et al. TGFβ induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy. Eye (Lond). 2016. Dec;30(12):1639–1647. doi: 10.1038/eye.2016.179 [DOI] [PMC free article] [PubMed] [Google Scholar]
94.Jang C-W, Chen C-H, Chen C-C, Chen J, Su Y-H, Chen R-H. TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nat Cell Biol. 2002. Jan;4(1):51–58. doi: 10.1038/ncb731 [DOI] [PubMed] [Google Scholar]
95.Rock KL, Kono H. The inflammatory response to cell death. Annu Rev Pathol. 2008;3:99–126. doi: 10.1146/annurev.pathmechdis.3.121806.151456 [DOI] [PMC free article] [PubMed] [Google Scholar]
96.Appelbaum T, Santana E, Aguirre GD. Strong upregulation of inflammatory genes accompanies photoreceptor demise in canine models of retinal degeneration. PLoS One. 2017. May 9;12(5):e0177224. doi: 10.1371/journal.pone.0177224 [DOI] [PMC free article] [PubMed] [Google Scholar]
97.Ma W, Silverman SM, Zhao L, Villasmil R, Campos MM, Amaral J, et al. Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization. Elife. 2019. Jan 22;8. doi: 10.7554/eLife.42049 [DOI] [PMC free article] [PubMed] [Google Scholar]
98.Wang SK, Xue Y, Cepko CL. Microglia modulation by TGF-β1 protects cones in mouse models of retinal degeneration. J Clin Invest. 2020. Aug 3;130(8):4360–4369. doi: 10.1172/JCI136160 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0258872.r001

Decision Letter 0

Tudor C Badea

15 Jul 2021

PONE-D-21-17742

Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina

PLOS ONE

Dear Dr. Lamba,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Most reviewer comments refer to data clarity or interpretation. PLOSOne publishes clear-cut data (including well described experiments and appropriate controls), without emphasizing impact. Please address the concerns of reviewers, by reporting the missing information, adjusting your claims/statements to be in sync with the data, and/or rephrasing appropriately. In particular, please comment on the choice of controls in response to reviewer 1.

==============================

Please submit your revised manuscript by Aug 29 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tudor C Badea, M.D., M.A., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

3. In your Methods section, please provide additional information on the animal research and ensure you have included details on : (a) methods of sacrifice (b) methods of anesthesia and/or analgesia, and (c) efforts to alleviate suffering

4. Thank you for stating the following in the Financial Disclosure section:

"The research presented here is supported by NIH grants (R01 EY025779 and

EY032197 to DL; AG057353 and EY018177 to HJ; P30 Vision Core grant to UCSF

Dept of Ophthalmology), and the Research to Prevent Blindness (unrestricted grant to

UCSF Dept of Ophthalmology). The funders had no role in study design, data

collection and analysis, decision to publish, or preparation of the manuscript."

We note that one or more of the authors are employed by a commercial company: "Genentech, Inc."

a) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b) Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

5. Please include a caption for figure 5.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is paper “Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina” is a nice work by Kramer and colleagues. I have a few concerns to be addressed before it is final for publication.

Major comments

1. Autors say “Tkv turnover is reduced during ISC activation, allowing for Tkv protein accumulation and replacement of Sax in Tkv/Punt complexes(22).”

Reduced Tkv turnover would prevent its accumulation.

2. Autors say “Overexpression of the negative feedback inhibitor of Dpp signaling, Daughters against Dpp (Dad; (36,37)), also….”

Similar to knockdown experiment, ideal control for overexpression(UAS-GFP/mcherry) should also have been used.

3. Autors say “These kinetics are consistent with the hypothesis that Mad activation induces Dad transcription to inhibit Sax/Smox signaling in the eye, thus limiting apoptosis.”

Not necessarily implied by the result. In Figure D’ and E’’’, nsmox level continues to increase, even when dad levels decrease(from 27h to 30h)

4. In Figure 2, all the labels specify usage of WT, whereas it was mentioned earlier that control for knockdown experiment had UAS- mCherry RNAi. Labels have to be changed accordingly.

5. Autors say “These studies suggest that TGFβ signaling may be involved in the early inflammatory response, while BMPs may play a role in a later protective anti-apoptotic pro-repair response which BALB/c mice lack.”

Here, mere absence of BMP in Balb/c doesn’t imply its anti apoptotic/pro-repair pathway. It needn’t be the cause, it could be an effect.

Minor comments

1. There are typo errors in the manuscript, like in TGFβ, the symbol is replaced by a square at some places. May be a computer error too.

2. Some of the data need further clarification, like in the statement ‘These studies suggest that TGFβ signaling may be involved in the early inflammatory response, while BMPs may play a role in a later protective anti-apoptotic pro-repair response which BALB/c mice lack.’, the results are based on smad2/3 or smad1/5/9 nuclear localization only. If they have to prove the real role of these pathways in the stated contexts then showing with pharmacological inhibitors would be a better choice.

3. Image quality need to be improved, everything including text is so blurred that I cannot conclude anything after looking at them.

4. Protective effect of TGFβ inhibition on apoptosis is shown by sb431542 treatment, it would be better to show its flip side by overexpression of TGFβ signaling as shown in the BMP pathway.

Reviewer #2: In this manuscript the authors elegantly show using a Drosophila model for UV-induced retinal damage, that Dpp released from immune cells promotes tissue loss after UV-induced retinal damage. Interestingly they found that, Dpp-mediated stimulation of Saxophone / Smox signaling promotes apoptosis, while at a later stage, stimulation of the Thickveins / Mad axis promotes tissue repair and survival.

This seems to be a key evolutionary conserved mechanisms by which retinal homeostasis is maintained.

Critique

1. Figure 5 does not have a Figure legend.

2. Is unclear from the Figure Legends how many times the experiments were repeated and if they were run in duplicates. This should be introduced in the Figure Legend.

3. In Figure 1 A+ and B+ will benefit from larger font. This is valid for Figure 2.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Oct 26;16(10):e0258872. doi: 10.1371/journal.pone.0258872.r002

Author response to Decision Letter 0

17 Sep 2021

Dear Editors,

We thank the reviewers for their comments on the manuscript and have edited the manuscript to address their concerns.

Editorial Comments:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

>We have checked and ensured the submission meets the naming and style guidelines.

2. Please review your reference list to ensure that it is complete and correct.

>One paper was revised and that reference is used instead of the original. Otherwise all references are acceptable under PLOS ONE guidelines.

>We have updated the Methods to include the requested details on animal handling and euthanasia.

4. Question regarding Financial Disclosure and Competing Interests for Dr. Jasper w.r.t. Genentech affiliation.

>Updated funding and competing interest statements are below.

Funding Statement: The research presented here is supported by NIH grants (R01 EY025779 and EY032197 to DL; AG057353 and EY018177 to HJ; P30 Vision Core grant to UCSF Dept of Ophthalmology), and the Research to Prevent Blindness (unrestricted grant to UCSF Dept of Ophthalmology). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dr. Jasper’s affiliation with Genetech, Inc provided support in the form of salaries for author HJ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

Competing Interests Statement: I have read the journal's policy and the following authors of this manuscript have the following competing interests: Heinrich Jasper is an employee of Genentech Inc. This commercial affiliation does not alter author’s adherence to all PLOS ONE policies on sharing data and materials.

5. Please include a caption for figure 5.

>We apologize for the oversight and have now included a legend for the figure.

Review Comments to the Author

Major comments

1. Autors say “Tkv turnover is reduced during ISC activation, allowing for Tkv protein accumulation and replacement of Sax in Tkv/Punt complexes(22).” Reduced Tkv turnover would prevent its accumulation.

>We apologize for any confusion this statement caused. Our previous study cited here (#22, Cai et al 2019) showed that after ISC activation, Tkv degradation is reduced, resulting in accumulation of Tkv protein. We used Tkv turnover as synonymous with degradation, but understand that this can be misinterpreted. We now use the term ‘degradation’ instead.

2. Autors say “Overexpression of the negative feedback inhibitor of Dpp signaling, Daughters against Dpp (Dad; (36,37)), also….” Similar to knockdown experiment, ideal control for overexpression(UAS-GFP/mcherry) should also have been used.

>As requested, we have carried out that study and included the UAS-GFP control experimental data in Figure 1A, with representative image in S1F. We do not observe any significant differences between the UAS-GFP and UAS::mCherry-RNAi lines.

3. Autors say “These kinetics are consistent with the hypothesis that Mad activation induces Dad transcription to inhibit Sax/Smox signaling in the eye, thus limiting apoptosis.”

Not necessarily implied by the result. In Figure D’ and E’’’, nsmox level continues to increase, even when dad levels decrease(from 27h to 30h)

>We believe that the kinetics of Dad induction and Smox nuclear translocation are linked, but the readouts are not entirely comparable. With other words, the induction of Dad was measured using a GFP reporter line, which is likely to lag true Dad protein production (GFP fluorescence requires about 2 hours to be visible). We measure Smox nuclear translocation in real time, however. We therefore interpret the results as showing that while Dad is being induced between 27 and 30 (in the UV treated samples), nuclear Smox levels are decreasing. In the untreated samples, the relationship between Dad and nuclear smox is less clear, but the expression of Dad is also much lower than in the UV-exposed samples.

To clarify our thinking, we have now edited the sentence to the following: “These kinetics led us to hypothesize that Mad activation induces Dad transcription, resulting in inhibition of Smox nuclear translocation after UV exposure, limiting apoptosis.”

4. In Figure 2, all the labels specify usage of WT, whereas it was mentioned earlier that control for knockdown experiment had UAS- mCherry RNAi. Labels have to be changed accordingly.

>We have changed labels to illustrate that UAS-mCherry RNAi was used for control.

Here, mere absence of BMP in Balb/c doesn’t imply its anti apoptotic/pro-repair pathway. It needn’t be the cause, it could be an effect.

> The reviewer here raises a good point. We have now edited the statement to the following ”These studies suggest that TGFβ signaling may be involved in the early response which is typically of inflammatory nature, while BMPs may play a role in a later response which tends to be protective and anti-apoptotic. The absence of BMP/SMAD1/5/9 response in BALB/c mice was further investigated below to test a cause or effect conundrum.”

Additionally, experiments further in the manuscript answer this question using recombinant protein and pharmacological inhibitor injections in mice.

Minor comments

1. There are typo errors in the manuscript, like in TGFβ, the symbol is replaced by a square at some places. May be a computer error too.

>We did not find β symbol to square errors in our uploaded version of the manuscript and was likely an issue with PDF conversion. All miscellaneous typos were corrected.

> Since Smad 2/3 and Smad 1/5/9 are canonical downstream effectors for TGF and BMP signaling respectively, it is reasonable to hypothesize their role at early and late state of tissue response based on their activation and nuclear localization. To further clarify this, we have edited the statement to the following ”These studies suggest that TGFβ signaling may be involved in the early response which is typically of inflammatory nature, while BMPs may play a role in a later response which tends to be protective and anti-apoptotic. The absence of BMP/SMAD1/5/9 response in BALB/c mice was further investigated below to test a cause or effect conundrum.”

The pharmacological manipulations are described further in the manuscript.

3. Image quality need to be improved, everything including text is so blurred that I cannot conclude anything after looking at them.

> All uploaded images passed check. The Image quality in the preview PDF is poor quality. However, full figure images on the links in the PDF are of high resolution.

4. Protective effect of TGFβ inhibition on apoptosis is shown by sb431542 treatment, it would be better to show its flip side by overexpression of TGFβ signaling as shown in the BMP pathway.

> That is an excellent suggestion and obvious next step. However, due to strong effects of TGFβ on vasculature, those studies require careful consideration and will be carried out in future studies.

This seems to be a key evolutionary conserved mechanisms by which retinal homeostasis is maintained.

Critique

1. Figure 5 does not have a Figure legend.

>We apologize for the oversight and have now included a legend for the figure.

2. Is unclear from the Figure Legends how many times the experiments were repeated and if they were run in duplicates. This should be introduced in the Figure Legend.

> The requested details have now been included.

3. In Figure 1 A+ and B+ will benefit from larger font. This is valid for Figure 2.

>We have increased the font size as requested.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(21.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0258872.r003

Decision Letter 1

Tudor C Badea

7 Oct 2021

Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina

PONE-D-21-17742R1

Dear Dr. Lamba,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Tudor C Badea, M.D., M.A., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0258872.r004

Acceptance letter

Tudor C Badea

18 Oct 2021

PONE-D-21-17742R1

Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina

Dear Dr. Lamba:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tudor C Badea

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

(TIF)

Click here for additional data file.^{(3MB, tif)}

S2 Fig. Quantitation of Individual Drosophila eyes.

(TIF)

Click here for additional data file.^{(654.6KB, tif)}

S3 Fig. Quantitation of Individual Drosophila eyes continued.

(TIF)

Click here for additional data file.^{(3.6MB, tif)}

S4 Fig. UV exposed Sep::G4;Smox and MadRNAi individual progeny DCP1+ Elav+ cells to UV Sep WT.

Error bars indicate s.e.m.; P-values from Student’s t-test. *p<0.05, **p<0.01, **p<0.001.

(TIF)

Click here for additional data file.^{(814.3KB, tif)}

S5 Fig

(TIF)

Click here for additional data file.^{(5.6MB, tif)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(21.8KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0258872.ref001] 1.Katz B, Minke B. Drosophila photoreceptors and signaling mechanisms. Front Cell Neurosci. 2009. Jun 11;3:2. doi: 10.3389/neuro.03.002.2009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref002] 2.Jassim OW, Fink JL, Cagan RL. Dmp53 protects the Drosophila retina during a developmentally regulated DNA damage response. EMBO J. 2003. Oct 15;22(20):5622–5632. doi: 10.1093/emboj/cdg543 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref003] 3.Young RW. Cell proliferation during postnatal development of the retina in the mouse. Brain Res. 1985. Aug;353(2):229–239. doi: 10.1016/0165-3806(85)90211-1 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref004] 4.Close JL, Gumuscu B, Reh TA. Retinal neurons regulate proliferation of postnatal progenitors and Müller glia in the rat retina via TGF beta signaling. Development. 2005. Jul;132(13):3015–3026. doi: 10.1242/dev.01882 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref005] 5.Agaisse H, Petersen UM, Boutros M, Mathey-Prevot B, Perrimon N. Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. Dev Cell. 2003. Sep;5(3):441–450. doi: 10.1016/s1534-5807(03)00244-2 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref006] 6.Vlisidou I, Wood W. Drosophila blood cells and their role in immune responses. FEBS J. 2015. Apr;282(8):1368–1382. doi: 10.1111/febs.13235 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref007] 7.Neves J, Zhu J, Sousa-Victor P, Konjikusic M, Riley R, Chew S, et al. Immune modulation by MANF promotes tissue repair and regenerative success in the retina. Science. 2016. Jul 1;353(6294):aaf3646. doi: 10.1126/science.aaf3646 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref008] 8.Gold KS, Brückner K. Macrophages and cellular immunity in Drosophila melanogaster. Semin Immunol. 2015;27(6):357–368. doi: 10.1016/j.smim.2016.03.010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref009] 9.Kelsey EM, Luo X, Brückner K, Jasper H. Schnurri regulates hemocyte function to promote tissue recovery after DNA damage. J Cell Sci. 2012. Mar 15;125(Pt 6):1393–1400. doi: 10.1242/jcs.095323 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref010] 10.Brodsky MH, Nordstrom W, Tsang G, Kwan E, Rubin GM, Abrams JM. Drosophila p53 binds a damage response element at the reaper locus. Cell. 2000. Mar 31;101(1):103–113. doi: 10.1016/S0092-8674(00)80627-3 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref011] 11.Jin S, Martinek S, Joo WS, Wortman JR, Mirkovic N, Sali A, et al. Identification and characterization of a p53 homologue in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2000;97(13):7301–7306. doi: 10.1073/pnas.97.13.7301 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref012] 12.Ollmann M, Young LM, Di Como CJ, Karim F, Belvin M, Robertson S, et al. Drosophila p53 is a structural and functional homolog of the tumor suppressor p53. Cell. 2000. Mar 31;101(1):91–101. doi: 10.1016/S0092-8674(00)80626-1 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref013] 13.Sogame N, Kim M, Abrams JM. Drosophila p53 preserves genomic stability by regulating cell death. Proc Natl Acad Sci U S A. 2003;100(8):4696–4701. doi: 10.1073/pnas.0736384100 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref014] 14.Zhou L, Steller H. Distinct pathways mediate UV-induced apoptosis in Drosophila embryos. Dev Cell. 2003. Apr;4(4):599–605. doi: 10.1016/s1534-5807(03)00085-6 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref015] 15.Hay BA, Guo M. Caspase-dependent cell death in Drosophila. Annu Rev Cell Dev Biol. 2006;22:623–650. doi: 10.1146/annurev.cellbio.21.012804.093845 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref016] 16.Steller H. Regulation of apoptosis in Drosophila. Cell Death Differ. 2008. Jul;15(7):1132–1138. doi: 10.1038/cdd.2008.50 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref017] 17.Denton D, Aung-Htut MT, Kumar S. Developmentally programmed cell death in Drosophila. Biochim Biophys Acta. 2013. Dec;1833(12):3499–3506. doi: 10.1016/j.bbamcr.2013.06.014 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref018] 18.Yao L-C, Blitz IL, Peiffer DA, Phin S, Wang Y, Ogata S, et al. Schnurri transcription factors from Drosophila and vertebrates can mediate Bmp signaling through a phylogenetically conserved mechanism. Development. 2006. Oct;133(20):4025–4034. doi: 10.1242/dev.02561 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref019] 19.Neal SJ, Dolezal D, Jusić N, Pignoni F. Drosophila ML-DmD17-c3 cells respond robustly to Dpp and exhibit complex transcriptional feedback on BMP signaling components. BMC Dev Biol. 2019. Jan 22;19(1):1. doi: 10.1186/s12861-019-0181-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref020] 20.Li C-Y, Guo Z, Wang Z. TGFbeta receptor saxophone non-autonomously regulates germline proliferation in a Smox/dSmad2-dependent manner in Drosophila testis. Dev Biol. 2007. Sep 1;309(1):70–77. doi: 10.1016/j.ydbio.2007.06.019 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref021] 21.Ayyaz A, Li H, Jasper H. Haemocytes control stem cell activity in the Drosophila intestine. Nat Cell Biol. 2015. Jun;17(6):736–748. doi: 10.1038/ncb3174 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref022] 22.Tracy Cai X, Li H, Safyan A, Gawlik J, Pyrowolakis G, Jasper H. AWD regulates timed activation of BMP signaling in intestinal stem cells to maintain tissue homeostasis. Nat Commun. 2019. Jul 5;10(1):2988. doi: 10.1038/s41467-019-10926-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref023] 23.Talati M, West J, Zaynagetdinov R, Hong CC, Han W, Blackwell T, et al. BMP pathway regulation of and by macrophages. PLoS One. 2014. Apr 8;9(4):e94119. doi: 10.1371/journal.pone.0094119 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref024] 24.Dzik JM. Evolutionary roots of arginase expression and regulation. Front Immunol. 2014. Nov 7;5:544. doi: 10.3389/fimmu.2014.00544 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref025] 25.Ji T, Takabayashi H, Mao MY, Todisco A. 199 Regulation of BMP-4 Expression by Inflammatory Cytokines. Gastroenterology. 2014. May;146(5):S–52. [Google Scholar]

[pone.0258872.ref026] 26.Shinohara M, Mao M, Keeley TM, El-Zaatari M, Lee H-J, Eaton KA, et al. Bone morphogenetic protein signaling regulates gastric epithelial cell development and proliferation in mice. Gastroenterology. 2010. Dec;139(6):2050–2060.e2. doi: 10.1053/j.gastro.2010.08.052 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref027] 27.Ueki Y, Reh TA. EGF stimulates Müller glial proliferation via a BMP-dependent mechanism. Glia. 2013. May 1;61(5):778–789. doi: 10.1002/glia.22472 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref028] 28.Ueki Y, Reh TA. Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo. PLoS One. 2012. Jun 6;7(6):e38690. doi: 10.1371/journal.pone.0038690 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref029] 29.Thompson A, Berry M, Logan A, Ahmed Z. Activation of the bmp4/smad1 pathway promotes retinal ganglion cell survival and axon regeneration. Invest Ophthalmol Vis Sci. 2019. Apr 1;60(5):1748–1759. doi: 10.1167/iovs.18-26449 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref030] 30.Mitchell K, Shah JP, Tsytsikova LV, Campbell AM, Affram K, Symes AJ. LPS antagonism of TGF-β signaling results in prolonged survival and activation of rat primary microglia. J Neurochem. 2014. Apr;129(1):155–168. doi: 10.1111/jnc.12612 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref031] 31.Jasper H, Benes V, Atzberger A, Sauer S, Ansorge W, Bohmann D. A genomic switch at the transition from cell proliferation to terminal differentiation in the Drosophila eye. Dev Cell. 2002. Oct;3(4):511–521. doi: 10.1016/s1534-5807(02)00297-6 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref032] 32.Luo X, Puig O, Hyun J, Bohmann D, Jasper H. Foxo and Fos regulate the decision between cell death and survival in response to UV irradiation. EMBO J. 2007. Jan 24;26(2):380–390. doi: 10.1038/sj.emboj.7601484 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref033] 33.Therrien M, Wong AM, Kwan E, Rubin GM. Functional analysis of CNK in RAS signaling. Proc Natl Acad Sci USA. 1999. Nov 9;96(23):13259–13263. doi: 10.1073/pnas.96.23.13259 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref034] 34.Kanda H, Miura M. Regulatory roles of JNK in programmed cell death. J Biochem. 2004. Jul;136(1):1–6. doi: 10.1093/jb/mvh098 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref035] 35.Nielsen MD, Luo X, Biteau B, Syverson K, Jasper H. 14-3-3 Epsilon antagonizes FoxO to control growth, apoptosis and longevity in Drosophila. Aging Cell. 2008. Oct;7(5):688–699. doi: 10.1111/j.1474-9726.2008.00420.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref036] 36.Tsuneizumi K, Nakayama T, Kamoshida Y, Kornberg TB, Christian JL, Tabata T. Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature. 1997. Oct 9;389(6651):627–631. doi: 10.1038/39362 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref037] 37.Kamiya Y, Miyazono K, Miyazawa K. Specificity of the inhibitory effects of Dad on TGFbeta family type I receptors, Thickveins, Saxophone, and Baboon in Drosophila. FEBS Lett. 2008. Jul 23;582(17):2496–2500. doi: 10.1016/j.febslet.2008.05.052 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref038] 38.Moses K, Rubin GM. Glass encodes a site-specific DNA-binding protein that is regulated in response to positional signals in the developing Drosophila eye. Genes Dev. 1991. Apr;5(4):583–593. doi: 10.1101/gad.5.4.583 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref039] 39.Goto A, Kadowaki T, Kitagawa Y. Drosophila hemolectin gene is expressed in embryonic and larval hemocytes and its knock down causes bleeding defects. Dev Biol. 2003. Dec;264(2):582–591. doi: 10.1016/j.ydbio.2003.06.001 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref040] 40.Dey NS, Ramesh P, Chugh M, Mandal S, Mandal L. Correction: Dpp dependent Hematopoietic stem cells give rise to Hh dependent blood progenitors in larval lymph gland of Drosophila. Elife. 2019. Sep 11;8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref041] 41.Eleftherianos I, Castillo JC, Patrnogic J. TGF-β signaling regulates resistance to parasitic nematode infection in Drosophila melanogaster. Immunobiology. 2016. Jul 25;221(12):1362–1368. doi: 10.1016/j.imbio.2016.07.011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref042] 42.Chakrabarti S, Dudzic JP, Li X, Collas EJ, Boquete J-P, Lemaitre B. Remote control of intestinal stem cell activity by haemocytes in drosophila. PLoS Genet. 2016. May 27;12(5):e1006089. doi: 10.1371/journal.pgen.1006089 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref043] 43.Tian A, Wang B, Jiang J. Injury-stimulated and self-restrained BMP signaling dynamically regulates stem cell pool size during Drosophila midgut regeneration. Proc Natl Acad Sci USA. 2017. Mar 28;114(13):E2699–E2708. doi: 10.1073/pnas.1617790114 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref044] 44.Gervais L, Bardin AJ. Tissue homeostasis and aging: new insight from the fly intestine. Curr Opin Cell Biol. 2017. Jul 16;48:97–105. doi: 10.1016/j.ceb.2017.06.005 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref045] 45.Suh GSB, Poeck B, Chouard T, Oron E, Segal D, Chamovitz DA, et al. Drosophila JAB1/CSN5 acts in photoreceptor cells to induce glial cells. Neuron. 2002. Jan 3;33(1):35–46. doi: 10.1016/s0896-6273(01)00576-1 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref046] 46.Spokony Rebecca F, White K. Spokony insertions. FlyBase ID: FBrf0221557. 2013.

[pone.0258872.ref047] 47.Xu D, Wang Y, Willecke R, Chen Z, Ding T, Bergmann A. The effector caspases drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila. Cell Death Differ. 2006. Oct;13(10):1697–1706. doi: 10.1038/sj.cdd.4401920 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref048] 48.Ninov N, Menezes-Cabral S, Prat-Rojo C, Manjón C, Weiss A, Pyrowolakis G, et al. Dpp signaling directs cell motility and invasiveness during epithelial morphogenesis. Curr Biol. 2010. Mar 23;20(6):513–520. doi: 10.1016/j.cub.2010.01.063 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref049] 49.Malzer E, Dominicus CS, Chambers JE, Dickens JA, Mookerjee S, Marciniak SJ. The integrated stress response regulates BMP signalling through effects on translation. BMC Biol. 2018. Apr 3;16(1):34. doi: 10.1186/s12915-018-0503-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref050] 50.Sanes JR, Zipursky SL. Design principles of insect and vertebrate visual systems. Neuron. 2010. Apr 15;66(1):15–36. doi: 10.1016/j.neuron.2010.01.018 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref051] 51.Sanjabi S, Oh SA, Li MO. Regulation of the Immune Response by TGF-β: From Conception to Autoimmunity and Infection. Cold Spring Harb Perspect Biol. 2017. Jun 1;9(6). doi: 10.1101/cshperspect.a022236 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref052] 52.Upadhyay A, Moss-Taylor L, Kim M-J, Ghosh AC, O’Connor MB. TGF-β Family Signaling in Drosophila. Cold Spring Harb Perspect Biol. 2017. Sep 1;9(9). [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref053] 53.Hamaratoglu F, Affolter M, Pyrowolakis G. Dpp/BMP signaling in flies: from molecules to biology. Semin Cell Dev Biol. 2014. Aug;32:128–136. doi: 10.1016/j.semcdb.2014.04.036 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref054] 54.Grimm C, Remé CE. Light damage as a model of retinal degeneration. Methods Mol Biol. 2013;935:87–97. doi: 10.1007/978-1-62703-080-9_6 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref055] 55.White DA, Fritz JJ, Hauswirth WW, Kaushal S, Lewin AS. Increased sensitivity to light induced damage in a mouse model of autosomal dominant retinal disease. Invest Ophthalmol Vis Sci. 2007. May;48(5):1942–1951. doi: 10.1167/iovs.06-1131 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref056] 56.Kashima R, Hata A. The role of TGF-β superfamily signaling in neurological disorders. Acta Biochim Biophys Sin (Shanghai). 2018. Jan 1;50(1):106–120. doi: 10.1093/abbs/gmx124 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref057] 57.Wenzel A, Grimm C, Samardzija M, Remé CE. The genetic modifier Rpe65Leu(450): effect on light damage susceptibility in c-Fos-deficient mice. Invest Ophthalmol Vis Sci. 2003. Jun;44(6):2798–2802. doi: 10.1167/iovs.02-1134 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref058] 58.Minhas G, Sharma J, Khan N. Cellular Stress Response and Immune Signaling in Retinal Ischemia-Reperfusion Injury. Front Immunol. 2016. Oct 24;7:444. doi: 10.3389/fimmu.2016.00444 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref059] 59.Okunuki Y, Mukai R, Pearsall EA, Klokman G, Husain D, Park D-H, et al. Microglia inhibit photoreceptor cell death and regulate immune cell infiltration in response to retinal detachment. Proc Natl Acad Sci USA. 2018. Jul 3;115(27):E6264–E6273. doi: 10.1073/pnas.1719601115 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref060] 60.Okunuki Y, Mukai R, Nakao T, Tabor SJ, Butovsky O, Dana R, et al. Retinal microglia initiate neuroinflammation in ocular autoimmunity. Proc Natl Acad Sci USA. 2019. May 14;116(20):9989–9998. doi: 10.1073/pnas.1820387116 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref061] 61.Akhtar-Schäfer I, Wang L, Krohne TU, Xu H, Langmann T. Modulation of three key innate immune pathways for the most common retinal degenerative diseases. EMBO Mol Med. 2018;10(10). doi: 10.15252/emmm.201708450 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref062] 62.Frieler RA, Mortensen RM. Immune cell and other noncardiomyocyte regulation of cardiac hypertrophy and remodeling. Circulation. 2015. Mar 17;131(11):1019–1030. doi: 10.1161/CIRCULATIONAHA.114.008788 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref063] 63.McPherson SW, Heuss ND, Lehmann U, Roehrich H, Abedin M, Gregerson DS. The retinal environment induces microglia-like properties in recruited myeloid cells. J Neuroinflammation. 2019. Jul 20;16(1):151. doi: 10.1186/s12974-019-1546-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref064] 64.Karl MO, Hayes S, Nelson BR, Tan K, Buckingham B, Reh TA. Stimulation of neural regeneration in the mouse retina. Proc Natl Acad Sci USA. 2008. Dec 9;105(49):19508–19513. doi: 10.1073/pnas.0807453105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref065] 65.Lamba D, Karl M, Reh T. Neural regeneration and cell replacement: a view from the eye. Cell Stem Cell. 2008. Jun 5;2(6):538–549. doi: 10.1016/j.stem.2008.05.002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref066] 66.Kuehn S, Rodust C, Stute G, Grotegut P, Meißner W, Reinehr S, et al. Concentration Dependent Inner Retina Layer Damage and Optic Nerve Degeneration in a NMDA Model. J Mol Neurosci. 2017. Dec;63(3–4):283–299. doi: 10.1007/s12031-017-0978-x [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref067] 67.Lambuk L, Iezhitsa I, Agarwal R, Bakar NS, Agarwal P, Ismail NM. Antiapoptotic effect of taurine against NMDA-induced retinal excitotoxicity in rats. Neurotoxicology. 2019;70:62–71. doi: 10.1016/j.neuro.2018.10.009 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref068] 68.Niwa M, Aoki H, Hirata A, Tomita H, Green PG, Hara A. Retinal Cell Degeneration in Animal Models. Int J Mol Sci. 2015;17(1). [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref069] 69.Maekawa S, Sato K, Fujita K, Daigaku R, Tawarayama H, Murayama N, et al. The neuroprotective effect of hesperidin in NMDA-induced retinal injury acts by suppressing oxidative stress and excessive calpain activation. Sci Rep. 2017. Jul 31;7(1):6885. doi: 10.1038/s41598-017-06969-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref070] 70.Ranaei Pirmardan E, Soheili Z-S, Samiei S, Ahmadieh H, Mowla SJ, Naseri M, et al. In vivo evaluation of PAX6 overexpression and NMDA cytotoxicity to stimulate proliferation in the mouse retina. Sci Rep. 2018. Dec 7;8(1):17700. doi: 10.1038/s41598-018-35884-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref071] 71.Christensen I, Lu B, Yang N, Huang K, Wang P, Tian N. The Susceptibility of Retinal Ganglion Cells to Glutamatergic Excitotoxicity Is Type-Specific. Front Neurosci. 2019. Mar 15;13:219. doi: 10.3389/fnins.2019.00219 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref072] 72.Javier AL, Doan LT, Luong M, Reyes de Mochel NS, Sun A, Monuki ES, et al. Bmp indicator mice reveal dynamic regulation of transcriptional response. PLoS One. 2012. Sep 11;7(9):e42566. doi: 10.1371/journal.pone.0042566 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref073] 73.Yu PB, Hong CC, Sachidanandan C, Babitt JL, Deng DY, Hoyng SA, et al. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat Chem Biol. 2008. Jan;4(1):33–41. doi: 10.1038/nchembio.2007.54 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref074] 74.Zhang J, Kiser PD, Badiee M, Palczewska G, Dong Z, Golczak M, et al. Molecular pharmacodynamics of emixustat in protection against retinal degeneration. J Clin Invest. 2015. Jul 1;125(7):2781–2794. doi: 10.1172/JCI80950 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref075] 75.Tappeiner C, Maurer E, Sallin P, Bise T, Enzmann V, Tschopp M. Inhibition of the tgfβ pathway enhances retinal regeneration in adult zebrafish. PLoS One. 2016. Nov 23;11(11):e0167073. doi: 10.1371/journal.pone.0167073 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref076] 76.Karperien A, Ahammer H, Jelinek HF. Quantitating the subtleties of microglial morphology with fractal analysis. Front Cell Neurosci. 2013. Jan 30;7:3. doi: 10.3389/fncel.2013.00003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref077] 77.Inoue H, Imamura T, Ishidou Y, Takase M, Udagawa Y, Oka Y, et al. Interplay of signal mediators of decapentaplegic (Dpp): molecular characterization of mothers against dpp, Medea, and daughters against dpp. Mol Biol Cell. 1998. Aug;9(8):2145–2156. doi: 10.1091/mbc.9.8.2145 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref078] 78.Kang I, Choi Y, Jung S, Lim JY, Lee D, Gupta S, et al. Identification of target genes regulated by the Drosophila histone methyltransferase Eggless reveals a role of Decapentaplegic in apoptotic signaling. Sci Rep. 2018. May 8;8(1):7123. doi: 10.1038/s41598-018-25483-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref079] 79.Manjón C, Sánchez-Herrero E, Suzanne M. Sharp boundaries of Dpp signalling trigger local cell death required for Drosophila leg morphogenesis. Nat Cell Biol. 2007. Jan;9(1):57–63. doi: 10.1038/ncb1518 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref080] 80.Velarde SB, Quevedo A, Estella C, Baonza A. Dpp and Hedgehog promote the Glial response to neuronal damage in the developing Drosophila Visual system. BioRxiv. 2020. Feb 25; [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref081] 81.Wang Z, Lee G, Vuong R, Park JH. Two-factor specification of apoptosis: TGF-β signaling acts cooperatively with ecdysone signaling to induce cell- and stage-specific apoptosis of larval neurons during metamorphosis in Drosophila melanogaster. Apoptosis. 2019;24(1112):972–989. doi: 10.1007/s10495-019-01574-4 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref082] 82.Goldstein JA, Kelly SM, LoPresti PP, Heydemann A, Earley JU, Ferguson EL, et al. SMAD signaling drives heart and muscle dysfunction in a Drosophila model of muscular dystrophy. Hum Mol Genet. 2011. Mar 1;20(5):894–904. doi: 10.1093/hmg/ddq528 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref083] 83.Goldstein JA, Bogdanovich S, Beiriger A, Wren LM, Rossi AE, Gao QQ, et al. Excess SMAD signaling contributes to heart and muscle dysfunction in muscular dystrophy. Hum Mol Genet. 2014. Dec 20;23(25):6722–6731. doi: 10.1093/hmg/ddu390 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref084] 84.Li F-F, Gasser RB, Liu F, Shan J-N, Di W-D, He L, et al. Identification and characterization of an R-Smad homologue (Hco-DAF-8) from Haemonchus contortus. Parasit Vectors. 2020. Apr 3;13(1):164. doi: 10.1186/s13071-020-04034-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref085] 85.Yousef H, Morgenthaler A, Schlesinger C, Bugaj L, Conboy IM, Schaffer DV. AgeAssociated Increase in BMP Signaling Inhibits Hippocampal Neurogenesis. Stem Cells. 2015. May;33(5):1577–1588. doi: 10.1002/stem.1943 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref086] 86.Dharmarajan S, Fisk DL, Sorenson CM, Sheibani N, Belecky-Adams TL. Microglia activation is essential for BMP7-mediated retinal reactive gliosis. J Neuroinflammation. 2017. Apr 5;14(1):76. doi: 10.1186/s12974-017-0855-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref087] 87.Chistiakov DA, Killingsworth MC, Myasoedova VA, Orekhov AN, Bobryshev YV. CD68/macrosialin: not just a histochemical marker. Lab Invest. 2017;97(1):4–13. doi: 10.1038/labinvest.2016.116 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref088] 88.Saika S. TGFbeta pathobiology in the eye. Lab Invest. 2006. Feb;86(2):106–115. doi: 10.1038/labinvest.3700375 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref089] 89.Lenkowski JR, Qin Z, Sifuentes CJ, Thummel R, Soto CM, Moens CB, et al. Retinal regeneration in adult zebrafish requires regulation of TGFβ signaling. Glia. 2013. Oct;61(10):1687–1697. doi: 10.1002/glia.22549 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref090] 90.Todd L, Palazzo I, Squires N, Mendonca N, Fischer AJ. BMP- and TGFβ-signaling regulate the formation of Müller glia-derived progenitor cells in the avian retina. Glia. 2017. Jul 13;65(10):1640–1655. doi: 10.1002/glia.23185 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref091] 91.Genander M, Cook PJ, Ramsköld D, Keyes BE, Mertz AF, Sandberg R, et al. BMP signaling and its pSMAD1/5 target genes differentially regulate hair follicle stem cell lineages. Cell Stem Cell. 2014. Nov 6;15(5):619–633. doi: 10.1016/j.stem.2014.09.009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref092] 92.Bender H, Wang Z, Schuster N, Krieglstein K. TIEG1 facilitates transforming growth factor-beta-mediated apoptosis in the oligodendroglial cell line OLI-neu. J Neurosci Res. 2004. Feb 1;75(3):344–352. doi: 10.1002/jnr.10856 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref093] 93.Betts-Obregon BS, Mondragon AA, Mendiola AS, LeBaron RG, Asmis R, Zou T, et al. TGFβ induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy. Eye (Lond). 2016. Dec;30(12):1639–1647. doi: 10.1038/eye.2016.179 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref094] 94.Jang C-W, Chen C-H, Chen C-C, Chen J, Su Y-H, Chen R-H. TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nat Cell Biol. 2002. Jan;4(1):51–58. doi: 10.1038/ncb731 [DOI] [PubMed] [Google Scholar]

[pone.0258872.ref095] 95.Rock KL, Kono H. The inflammatory response to cell death. Annu Rev Pathol. 2008;3:99–126. doi: 10.1146/annurev.pathmechdis.3.121806.151456 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref096] 96.Appelbaum T, Santana E, Aguirre GD. Strong upregulation of inflammatory genes accompanies photoreceptor demise in canine models of retinal degeneration. PLoS One. 2017. May 9;12(5):e0177224. doi: 10.1371/journal.pone.0177224 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref097] 97.Ma W, Silverman SM, Zhao L, Villasmil R, Campos MM, Amaral J, et al. Absence of TGFβ signaling in retinal microglia induces retinal degeneration and exacerbates choroidal neovascularization. Elife. 2019. Jan 22;8. doi: 10.7554/eLife.42049 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0258872.ref098] 98.Wang SK, Xue Y, Cepko CL. Microglia modulation by TGF-β1 protects cones in mouse models of retinal degeneration. J Clin Invest. 2020. Aug 3;130(8):4360–4369. doi: 10.1172/JCI136160 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Dpp/TGFβ-superfamily play a dual conserved role in mediating the damage response in the retina

Joshua Kramer

Joana Neves

Mia Koniikusic

Heinrich Jasper

Deepak A Lamba

Roles

Abstract

Introduction

Results

Hemocyte derived Dpp controls the damaged retinal tissue response in Drosophila

Fig 1. Dpp inhibition controls JNK and UV driven apoptosis in Drosophila.

Fig 2. Following UV driven whole eye radiation damage, Sax and Smox RNAi inhibit apoptosis, while Mad and Tkv RNAi promote it.

BMP and TGFβ signaling are activated in a time dependent manner following retinal stress and damage

Fig 3. BMP and TGFβ activation occurs in a time dependent manner following light stress and damage.

Modulation of the BMP signaling pathway protects against damage induced retinal apoptosis

Fig 4. Modulation of the BMP/TGFβ signaling pathway affects tissue damage induced retinal apoptosis and immune activation.

Inhibition of TGFβ signaling pathway protects against damage induced retinal apoptosis

Microglia activation can be modulated by manipulating BMP/ TGFβ pathways

Discussion

Fig 5. Cartoon depicting the role of Dpp and TGFβ/BMP signaling as central controllers of photoreceptor death and survival after acute damage within the retina.

Materials and methods

Drosophila stocks and culture

Mice

Intraocular injections in mice

Light and NMDA damage in mice

UV damage in Drosophila pupae retina and larvae

Histological analysis, imaging, and quantification methods

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Tudor C Badea

Roles

Author response to Decision Letter 0

Decision Letter 1

Tudor C Badea

Roles

Acceptance letter

Tudor C Badea

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases