N6-methyladenosine (m6A) reader YTHDF2 is highly expressed in the mouse RGCs. Conditional knockout (cKO) of Ythdf2 in the retina leads to increased RGC dendrite branching, resulting in more synapses in the inner plexiform layer. Interestingly, the Ythdf2 cKO mice show improved visual acuity compared with control mice. We further demonstrate that Ythdf2 cKO in the retina protects RGCs from dendrite degeneration caused by the experimental acute glaucoma model. We identify the m6A-modified YTHDF2 target transcripts which mediate these effects. This study reveals mechanisms by which YTHDF2 restricts RGC dendrite development and maintenance. YTHDF2 and its target mRNAs might be valuable in developing new treatment approaches for glaucomatous eyes.The precise control of growth and maintenance of the retinal ganglion cell (RGC) dendrite arborization is critical for normal visual functions in mammals. However, the underlying mechanisms remain elusive. Here, we find that the  The mammalian retina is an ideal model system to study neuronal development and neural circuit formation. The retinal ganglion cells (RGCs) are the final and only output neurons in the vertebrate retina and their dendrites collect the electrical information concerning the visual signal from all other cells preceding them. One of the major focuses of research in the retina is to understand how RGC dendrite arborization arises during development . ExistinGlaucoma is one of the leading causes for blindness. The major risk factors for glaucoma include increased intraocular tension. Studies have shown that glaucoma causes pathological changes in RGC dendrites before axon degeneration and soma loss were detected in different model animals . Thus, eN6-methyladenosine (m6A) is the most widely distributed and extensively studied internal modification in mRNA (6A modification has been shown to regulate brain development and functions in the nervous system (6A erasers’) and methyltransferases (‘m6A writers’). Since the fate of m6A-modified transcripts is decoded by the m6A-binding proteins (‘m6A readers’), how the readers mediate these functions and what are their neural target mRNAs remain to be elucidated. In addition, more precisely controlled spatial-temporal ablation of the m6A readers instead of null knockout is required to elucidate their functions and mechanisms in nervous system. in mRNA . m6A mods system . By effe6A-dependent intrinsic limiting mechanism for RGC dendrite arborization and maintenance. Conditional knockout (cKO) of the m6A reader YTHDF2 in the developing mouse retina increases RGC dendrite branching and improves visual acuity. YTHDF2 also mediates acute ocular hypertension (AOH)-induced RGC degeneration, the experiment model for glaucoma, and Ythdf2 cKO in the retina alleviates AOH-induced RGC dendrite shrinking and neuronal loss. The regulation of RGC dendrite development and maintenance by YTHDF2 is mediated by two distinct groups of m6A-modified target mRNAs which encode proteins that promote dendrite arborization during development and maintain dendrite tree during injury, respectively. Therefore, our study reveals mechanisms by which YTHDF2 restricts RGC dendrite development and maintenance, which sheds light on developing new treatment approaches for glaucomatous eyes.In this study, we identified an m6A modification and its reader proteins play a role in the dendrite development, we utilized the retina as the model system. We first checked their expression patterns in the developing mouse retina. Immunostaining with a widely used m6A antibody demonstrated that RGCs had high m6A modification levels  of YTHDFs in RGC cultures in vitro  decrease of RGC dendrite branching compared with control shRNA  which labels a few RGCs . Since the IPL sublaminar targeting of RGC dendrites is critical for normal visual functions, we wondered whether the increased dendrite branching caused by few RGCs . As show control . Sublami control . These d control .Ythdf2 cKO changes IPL development. Immunostaining of P6 retina vertical sections using a MAP2 antibody demonstrated that IPL thickness significantly increased in Ythdf2 cKO retina (Ythdf2 cKO and control mice (-D). Quantification of MAP2 IF intensity in IPL suggested that the IPL of Ythdf2 cKO retina became denser with dendrites  show no difference between Ythdf2 cKO and control retinas  and 0.43 ± 0.0085 c/deg, respectively  in the retina followed by RNA sequencing of the elute (RIP-Seq). Two biological replicates of anti-YTHDF2 RIP-Seq identified 1638 transcripts  identified 114 proteins which were upregulated by YTHDF2 KD  gene generates three alternative splicing isoforms Kalrn7, Kalrn9, and Kalrn12 encoding guanine-nucleotide exchange factors for Rho GTPases, which have been shown to regulate hippocampal and cortical dendritic branching , Nanos (Nos), Glorund (Glo), and Smaug (Smg) regulate morphogenesis and branching of specific classes of dendritic arborization neurons through controlling translation of their target mRNAs including nanos mRNA itself , and the upregulated genes are of importance for discovering new treatment approaches . One of fter ONC . Here, wfl/flYthdf2 mice were reported previously , cpt-cAMP , forskolin , and insulin . EGF , BDNF , NT-3 , and FGF-basic  were freshly added before using.Retinal neurons were dissociated from E14.5 to 15.5 mouse embryos by papain in DPBS  following the previously described methods , and neuGCATCAAGGTGAACTTCAAGA-3’; shYthdf2: 5’-GGACGTTCCCAATAGCCAACT-3’; shYthdf1: 5’- GGACATTGGTACTTGGGATAA-3’; shYthdf3: 5’- GGATTTGGCAATGATACTTTG-3’; shMettl14#6: 5’-GCTGGACCTGGGATGATATTA-3’; shMettl14#7: 5’-CCCAGCTTGTACTTTGCTTTA-3’) were generated from pLKO.1-TRC and lentivirus preparation process was described previously  were generated using pHBAAV-U6-MCS-CMV-EGFP and packaged in serotype-9 by Hanbio (1.5 × 1012 genomic copies per ml). AAV overexpression plasmids of Hspa12a  and Islr2  were generated from pHBAAV-CMV-MCS-3flag-T2A-ZsGreen and packaged serotype-9 by Hanbio (1.2 × 1012 genomic copies per ml).Lentiviral KD plasmids encoding shRNA  was used in siRNA transfection following the manufacturer’s protocols. Culture medium was changed after 1 day of lentiviral shRNA infection or siRNA transfection. For lentiviral shRNA assay, puromycin  was added after 2 days of infection. Immunofluorescence, RNA, or protein preparation was performed after shRNA or siRNA worked for 3 days. For AAV intravitreal injection, P0-P1 mouse pups were anesthetized in ice and then eyes were pierced at the edge of corneal by 30G × 1/2 needle  under stereomicroscope. Then 1 μl AAV was intravitreally injected with 10 µl Syringe  following the pinhole. P15 or adult mice were anesthetized with 2.5% Avertin and then eyes were pierced at the side of corneal and the outer segment of sclera by 30G × 1/2 needle successively. Two μl AAV was intravitreally injected with 10 µl Syringe following the pinhole on the sclera. All subsequent experiments such as AOH operation and immunostaining were carried out after at least 3 weeks  and then used for reverse transcription by PrimeScript RT Master Mix (TaKaRa). Synthesized cDNA was used for qPCR by 2× ChamQ Universal SYBR qPCR Master Mix (Vazyme) on StepOnePlus Real-Time PCR System (ABI) or BioRad CFX96 Touch Real-Time PCR system. Primers used for qPCR are as following: mouse TGGAC-3’ ; mouse KGTAAG-3’ ; mouse KTGGGC-3’ ; mouse KCGCAC-3’ ; mouse SCTGTC-3’ ; mouse H6A , rabbit anti-melanopsin , rabbit anti-PKCα , rabbit anti-PSD95 , rabbit anti-Recoverin , rabbit anti-YTHDF2 , rabbit anti-YTHDF1 , rabbit anti-YTHDF3 , rabbit anti-Ubr4 , sheep anti-Chx10 , chicken anti-GFAP , goat anti-Lhx2 , rabbit anti-Lhx2 . After three times of PBS washing, sections were incubated in PBST for 1 hr at RT with secondary antibodies: Alexa 488 donkey anti-chicken , Alexa 488 donkey anti-guinea pig , Alexa 488 donkey anti-mouse , Alexa 488 donkey anti-rabbit , Alexa 555 donkey anti-goat , Alexa 555 donkey anti-mouse , Alexa 555 donkey anti-rabbit , Alexa 555 donkey anti-sheep , Alexa 555 goat anti-chicken , or Alexa 647 donkey anti-mouse  and then mounted with the VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratory).For tissue sections, mouse embryonic eyes were fixed with 4% PFA (Sigma) in 0.1 M phosphate buffer (PB) for 30–45 min at room temperature (RT); eyes of mouse pups (<P10) were pre-fixed briefly and then eyecups were dissected and fixed for 45 min-1 hr at RT; for P20-30 or adult mice, eyecups were dissected after myocardial perfusion with 0.9% NaCl, followed by fixation for 1 hr. After PBS (3 × 5 min) washing, tissues were dehydrated with 30% sucrose in 0.1 M PB overnight at 4°C, then embedded with OCT (SAKURA) and cryosectioned at 12 μm  with Leica CM1950 Cryostat. Tissue sections were permeabilized and blocked with 10% ChemiBLOCKER (Millipore) and 0.5% Triton X-100 (Sigma) in PBS (PBST) for 1 hr at RT and incubated in PBST overnight at 4°C with following primary antibodies: chicken anti-GFP , chicken anti-MAP2 , goat anti-VAChT , guinea pig anti-RBPMS , mouse anti-AP2α , mouse anti-Bassoon , mouse anti-Brn3a , mouse anti-Calbindin-D-28K , mouse anti-PKCα , rabbit anti-Strn (Striatin) , rabbit anti-CART , rabbit anti-mFor cultured neurons, after twice of PBS washing, cells were fixed for 15 min with 4% PFA in 0.1 M PB at RT, then washed with PBS three times and blocked in PBST for 20 min at RT. Antibody incubation conditions are the same as tissue sections.For wholemount immunostaining of retina, eyes were dissected after myocardial perfusion with 0.9% NaCl. Then retinas were separated from sclera and fixed with 4% PFA in 0.1 M PB for 1 hr at RT. Then retinas were blocked with 5% normal goat serum (Novus), 0.4% Triton X-100 in PBS overnight at 4°C. Primary antibodies such as chicken anti-GFP , mouse anti-Brn3a , mouse anti-SMI-32 , or rabbit anti-Melanopsin , rabbit anti-CART  were diluted in 5% normal goat serum, 0.4% Triton X-100 in PBS and incubated overnight at 4°C. Then retinas were incubated with Alexa 488 donkey anti-chicken , Alexa 488 donkey anti-mouse , Alexa 555 donkey anti-mouse  and Alexa 555 donkey anti-rabbit  secondary antibodies in 5% normal goat serum (Novus), 0.4% Triton X-100 in PBS and finally mounted with the VECTASHIELD Antifade Mounting Medium with DAPI.+/PSD-95+ excitatory synapses in IPL, the colocalization puncta was measured by ImageJ/Analyze/Puncta Analyzer as described previously  which was a widely used method in neurobiology to quantify the complexity of dendritic arbors using ImageJ . Retina Ythdf2 cKO and control mice aged about 6 weeks were dark-adapted overnight before experiment and used in the OMR assay following the previously reported protocols  through intravitreal injection 48 hr before sacrifice. After PFA perfusion, the brains were fixed with 4% PFA in 0.1 M PB overnight, dehydrated with 15% sucrose and 30% sucrose in 0.1 M PB overnight at 4°C sequentially, embedded with OCT for coronal section, and cryosectioned at 12 μm with Leica CM1950 Cryostat. After PBS washing, the sections were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratory). The images were captured on Tissue Genostics with identical settings for each group in the same experiment with the TissueFAXS 7.0 software.7 retinal neurons were subjected to each 100 μl lysis buffer. The amount of YTHDF2 antibody  and control IgG used for immunoprecipitation is 5 μg, respectively. RIP experimental steps, RNA sample preparation and sequencing, and sequence data analysis followed the procedures reported previously  following the manual with minor modifications. Briefly, 1 × 10eviously .Ythdf2 or shCtrl. Sample collection and lysis, protein and peptide preparation were performed following procedures reported previously  following the manual. m6A antibody  and corresponding control IgG were used in this experiment. The RNA samples pulled down from the experiment were used for RT-qPCR.Total retinal RNA was extracted from P0 WT mouse pups. Immunoprecipitation of mHspa12a and Islr2 3 days after AOH, dendritic complexity and RGC number 3–7 days after AOH.Mice were anesthetized with 5% chloral hydrate in normal saline (10 μl/g) based on body weight and the Compound Tropicamide Eye Drops were used to scatter pupil. The anterior chamber was penetrated using the 32G × 1/2’’ needles (TSK) and filled with the BBS Sterile Irrigating Solution (Alcon) which was hung at a high position to provide proper pressure. Intraocular pressure was measured with the Tonolab tonometer (icare) for every 10 min and maintained at 85–90 mmHg for 1 hr. Levofloxacin hydrochloride was used after the operation and mice were revived in a 37°C environment. Retinas were analyzed for gene expression of YTHDF2 1 day after AOH, gene expression of t test was performed on the basis of experimental design. The settings for all box and whisker plots are: 25th-75th percentiles (boxes), minimum and maximum (whiskers), and medians . A p value less than 0.05 was considered as statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.All experiments were conducted at a minimum of three independent biological replicates  or three mice/pups for each genotype/condition in the lab. Data are mean ± SEM. Statistical analysis was preformed using GraphPad Prism 7.0. When comparing the means of two groups, an unpaired or paired  6A reader YTHDF2 in regulating the dendritic arbor size of retinal ganglion cells (RGCs). You show that retina-specific loss of Ythdf2 leads to the expansion of RGC dendritic arbors in the horizontal plane and a widening of the inner plexiform layer (IPL), with Ythdf2 conditional knockouts showing a modest increase in visual acuity in an optomotor assay. You point to a number of factors downstream of YTHDF2 not previously known to be involved in retinal dendritic development, and propose a role for YTHDF2 in a glaucoma model, in which loss of YTHDF2 is shown to prevent RGC loss. This study presents a careful phenotypic analysis of manipulation of YTHDF2 and provides a foundation for studies on how YTHDF2-mediated mechanisms are integrated into programs of dendritic development and RGC survival.In this study, you propose a role for the m In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "The m6A reader YTHDF2 is a negative regulator for dendrite development and maintenance of retinal ganglion cells" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife for revision . The concerns include:The reviewers have provided thorough assessment of your study proposing a role for the m6A reader YTHDF2 in regulating retinal ganglion cell (RGCs) dendritic arbor size, and maintenance of RGCs, with implications for retinal IPL morphology and for glaucoma. Unfortunately, as you can read in the reviews, the reviewers had a number of concerns that would each require additional work in a longer time frame required by – Better phenotyping of the Ythdf2 cKO mouse model, including cross sections of retina in addition to horizontal views to more precisely measure layer dimensions, synapse number and dendritic extension; whether cells other than RGCs are affected; co-localization of YTHDF2 and RGC markers. Moreover, the connection between the dendrite development phenotype and the neuroprotective role of YTHDF2 is not clear from your analyses.– in vivo evidence for the putative targets of YTHDF2, rather than simply in vitro, a better understanding of how the products produced by these mRNAs – kalirin, Strn and Ubr4 – contribute to the increase in dendrite branching in the Ythdf2 knockout; the mechanism of YTHDF2 upregulation induced by AOH and loss of Brn3a+ RGCs thereof.– The use of the optomotor response is not an apt measure function, especially in the absence of connecting the response to the classes of direction-selective RGCs which one would expect might interface with OMR responses.– Ythdf2 effects in the acute ocular hypertension (AOH) model: Is this related to the downstream mRNAs, or some other more general effect of loss of this m6 reader? And how is this related to RGC survival at a mechanistic level (and thus the relation to glaucoma)?We hope that you can use the reviewer's comments to amend your manuscript for submission elsewhere.Reviewer #1:The authors report a functional-relevant role of YTHDF2 in mouse retina ganglion cells (RGCs) to negatively regulate dendrite development and maintenance of RGCs. Mechanistically, Kalrn, Strn, and Ubr4 mRNAs are selectively recognized and decay through the YTHDF2-mediated pathway, the subsequent downregulation of proteins encoded by those mRNAs negatively regulate dendrite development and maintenance of RGCs.This work highlights regulatory roles of N6-methyladenosine (m6A) and YTHDF2 protein in retina and suggested YTHDF2 as one possible target in AOH-induced RGC loss. A few concerns will need to be addressed.Concerns:1. The authors showed YTHDF2 mRNA is upregulated by AOH and argued a loss of YTHDF2 in retina results in less severe AOH-induced loss of Brn3a+ RGCs. The mechanism of YTHDF2 upregulation induced by AOH and loss of Brn3a+ RGCs mediated by YTHDF2 upregulation are unclear. The first question might be harder to address but the second one is within the scope of this work.2. In figure 7B, YTHDF2 expression in Brn3a+ RGCs was quantified. The figure showed that Brn3a was not detected in some cells. In figure 7C, mRNA level was quantified with an extraction method that did not separate Brn3a+ and Brn3a- cells.3. The authors showed AOH-induced loss of Brn3a+ RGCs in mouse models. Supportive evidence at the phenotype level such as less severe loss of visual acuity in YTHDF2 cKO mice are required.4. The authors showed knockdown of Kalrn, Strn, and Ubr4 are sufficient to impair dendrite growth. Rescue experiments will further back up their claim.5. The authors suggested that m6A-YTHDF2 axis regulates the stability of Kalrn, Strn, and Ubr4 mRNAs. The authors will need to show these RNAs contain m6A by m6A-IP-qPCR. The effect on Kalrn, Strn, and Ubr4 mRNAs upon m6A writer complex knockdown can also be shown.Reviewer #2:This manuscript investigates the role of the m6A reader YTHDF2 during retinal ganglion cell (RGC) dendrite development and branching. The data presented in this study shows that deletion of YTHDF2 in the retina led to increased RGC dendrite branching and improved visual acuity. Based on high throughput analysis, YTHDF2 is suggested to regulate dendrite branching by reducing the stability of kalirin, Strn and Ubr4 mRNAs. Lastly, loss of YTHDF2 is shown to prevent RGC loss in a glaucoma model. The experimental designs were presented clearly, and the results were interpreted with the appropriate statistical tests. However, the phenotype of the Ythdf2 cKO mouse model is not well characterized and there lacks in vivo evidence for the putative targets of YTHDF2. It is also difficult to explain the connection, if any, between the dendritic role of YTHDF2 and its neuroprotective function.1. The authors used RGC cultures to show that knockdown/knockout of YTHDF2 resulted in increased the dendrite branching based on the number of interactions in Sholl analysis. However, to determine the full extent of dendritic defect, it is important to perform detailed analysis of the dendrite structure, including the total dendritic length, number of total segments, maximum branch length, branch order and number of branches.2. It is suggested in the manuscript that loss of YTHDF2 in the retina only affects dendrite development of RGCs, but not other cell types. This is based on the simple counting of the number of RPCs at E15 and bipolar and amacrine cells at P15, glossing over the vast complexity of the visual circuitry in the retina. At the very least, the authors should confirm that differentiation of all major retinal neurons and proper formation of the ribbon synapses were unaffected in the cKO mouse model of YTHDF2, which would other confound the interpretation of the RGC and visual acuity phenotype.3. The in vivo characterisation of RGC dendrite morphology should be more thorough as previously mentioned, including the arbor shape, size and tiling pattern. The image quality of Melanopsin and SMI-32 staining is low. There are too many cells labelled, making it difficult to examine individual cells/dendrites. Higher resolution images from more sparsely labelled retina would be needed.4. The authors provide evidence of increased synaptic density in the IPL using PSD-95 staining in the cKO mice compared to the control litter mates. It is important to investigate if there are any changes to the synaptic density in the OPL. In addition, it is also important to study the number of successful synapse formation by co-staining the tissues with presynaptic markers such as bassoon with PSD-95 and count for co-localisation.5. The improved visual acuity in Six3Cre-mediated Ythdf2 cKO is solely attributed to the RGC dendrite defects. However, Six3Cre is widely expressed in the ventral telencephalon and ventral anterior hypothalamus, including the SCN and SPZ (PMID: 24767996). It is necessary to examine the retinocollicular projections of the RGC axons.6. The Optomotor response (OMR)-based assay is not the most appropriate test to test the response of ganglion cells since the improved OMR could be attributed to other cell types as previously mentioned. A more targeted approach such as pattern ERG could provide more evidence to their claims.7. The quality of the RIP-seq analysis is not clear. Is there an isotype antibody control? Are there biological replicates? What is the statistical significance of the top hits? There are similar concerns regarding the MS analysis and whether there is significant overlap between the target lists generated from RIP-Seq and MS studies.8. The authors provide evidence in the RGC culture that stabilisation of kalirin, Strn and Ubr4 mRNAs could contribute to the increase in dendrite branching in Ythdf2 knockout. The regulation of these genes by Ythdf2 need to be validated in Ythdf2 cKO retina at the level of mRNA by qRT-PCR and at the level of protein by western blot. Functionally, the authors need to investigate if knockdown or knockout of these genes in retina indeed affect dendrite branching or even rescue Ythdf2 cKO phenotype.9. Finally, there is not clear connection between the dendrite development phenotype and the neuroprotective role of YTHDF2. The authors need to clarify the rationale behind these experiments.Reviewer #3:In this study, the authors propose a role for the m6A reader YTHDF2 in regulating the dendritic arbor size of retinal ganglion cells (RGCs), and they extrapolate this to address issues in glaucoma and overall retina IPL morphology. They find that retina-specific loss of Ythdf2 leads to the expansion of RGC dendritic arbors in the horizontal plane and also a widening of the inner plexiform layer (IPL), with Ythdf2 cKOs showing a modest increase in visual acuity in an optomotor assay. They identify a number of factors downstream of YTHDF2 not previously known to be involved in retinal dendritic development, and they propose a role for Ythdf2 in a glaucoma model, providing a foundation for future studies into their functions in these contexts.eLife, and it is suited for publication in a more specialized journal following attention to many of the points raised below.Although these data are sufficient to establish a role for Ythdf2 in RGC dendritic development and other more general aspects of retina morphology, many of the authors' specific claims are weakened by issues with the experiments, their analyses, and by weak effect sizes. Even if the points raised below were addressed, it remains unclear from this study how Ythdf2-mediated mechanisms are integrated into programs of dendritic development and RGC survival, diminishing the impact of this work. This study would be strengthened by, for example, identifying a developmental program that modifies the target mRNAs found in this study to promote recognition by YTHDF2, and also by providing better understanding of how the products produced by these mRNAs actually regulate dendrite morphology and synapse formation. Overall, while there are a number of promising findings in this study, the work required to address weaknesses falls well outside of the limits usually recommended by 1. The cell-type specific tracing in Figure 3 is difficult to assess due to the extremely high background in the melanopsin images and unclear signal in the SMI-32 images. This analysis would be improved by sparse viral labeling, with a fluorophore provided by the virus used for tracing and the melanopsin & SMI-32 antibodies used solely for establishing RGC subtype identity. Further, the authors emphasize that the defects they observe here are in the plane of retinal ganglion cell (RGC) dendritic arbors, but this is an over-interpretation of these data. One wonders why the authors did not examine in cross section the lamination within the IPL of the melanopsin-positive ipRGCs, since this would provide, at least for this RGC subclass, a direct assessment of this issue. Finally, the Thy-1-O line GFP distribution is quite general, and it really is not the correct line to use if one wants to investigate defects in lamination.2. The authors repeatedly describe increased dendritic and synaptic density within the IPL in Ythdf2 cKOs, yet they only present absolute signal intensities to support these claims; without normalizing signal intensity to IPL width, it is impossible to determine whether changes in intensity are indeed due to increased density or from the IPL becoming enlarged with a similar density of dendrites and synapses. Further, they claim in the text that VGlut2 puncta numbers are changed in these mutants, however the analysis presented is only for general signal intensity. If claims are to be made regarding changes in synaptic specializations, higher resolution assessments of individual puncta using pre- and postsynaptic markers is required.3. While Ythdf2 cKOs do show a statistically significant improvement in visual acuity , the effect size is small enough to call into question how meaningful the improvement is. Further, what is the actual relationship between this modest improvement in optomotor response (OMR) and the morphological phenotypes observed here, in particular since they are not connected here to the classes of direction-selective RGCs one would expect might affect OMR responses.4. Much clearer co-localization between YTHDF2 and RGC markers is needed, both in vivo and in vitro, to clearly demonstrate association with RGCs and not displaced amacrine cells or other retinal cell types.5. The several mRNAs and their produces shown to be under YTHDF2 control do appear to play some role in regulating general RGC dendritic morphology, though of course from these data we do not know whether or not there is any RGC-subtype specificity. However, as presented, we really do not learn much at all about their mechanisms of action. At the very least, do they act in the same, or different, pathways….i.e. have any attempts been made to knock down more than one at a time and ask about independent pathway affiliations?6. It is not clear how to interpret the Ythdf2 effects in the acute ocular hypertension (AOH) model. Is this related to the downstream mRNAs, or some other more general effect of loss of this m6 reader? And how is this related to RGC survival at a mechanistic level?6A reader YTHDF2 is a negative regulator for dendrite development and maintenance of retinal ganglion cells" for further consideration by eLife. Your revised article has been evaluated by Catherine Dulac (Senior Editor) and Carol Mason as Reviewing Editor.Thank you for resubmitting your work entitled "The mThe manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below and in the appended reviews:Reviewer 2 (previous Reviewer 1) was satisfied with the revisions of this resubmission as was Reviewer 3 (previous Reviewer (3) although the latter reviewer adds that "…we still are far from a mechanistic understanding of how these gene products regulate RGC dendritic arborization, and whether or not there is any specificity of these effects with regard to RGC subtypes."Reviewer 1 (previous Reviewer 2) continues to have concerns: He summarized in the Public Review (this will not be made public as it is a resubmission) that "the phenotype of the Ythdf2 cKO mouse model is not well characterized and there lacks in vivo evidence for the putative targets of YTHDF2".Two items he previously requested were not addressed: (1) to perform either qPCR or western blots of Ythdf2 cKO retina to confirm the regulation of Kalirin, Strn and Ubr4 by Ythdf2, and (2) to show that overexpression of Hspa12a and Islr2 alleviates loss of RGCs in the AOH model (See point #8 of previous Reviewer 2’s review). We ask that you address these two items that were raised in the first review, to the best of your ability, given time constraints. ).Current Reviewer 1 has also raised additional points not in the first review, that could be answered mostly textually:1. Your stated exclusivity of YTHDF2 expression in RGCs, given that there is apparent staining in all cell layers in Suppl Figure 1B. He cited very "old"scRNAseq data indicating that YTHDF2 is found in all retinal cell types.2. To verify that YTHDF2 is neuronal: determining YTHDF2 expression in Muller glia and astrocytes.3. Addressing the utility of the Six3-Cre, by determining whether there is mosaic expression of Cre in the peripheral retina.Reviewer #1:The experimental designs were presented clearly and the results were interpreted with the appropriate statistical tests. However, the phenotype of the Ythdf2 cKO mouse model is not well characterized and there lacks in vivo evidence for the putative targets of YTHDF2. It is also difficult to explain the connection, if any, between the dendritic role of YTHDF2 and its neuroprotective function. Most importantly, whether Ythdf2 acts cell autonomously in the RGCs remains to be resolved.1. The premise of this study is that Ythdf2 is cell autonomously required in RGCs for dendritic development. In the rebuttal letter, the authors even went as far as claiming that "all RGCs express YTHDF2 and all YTHDF2-expressing cells in retina are RGCs". However, this statement is belied by the strong YTHDF2 staining outside the RGC layers in Figure 1—figure supplement 1B. The YTHDF2 expression in non-RGC cells can also be seen in Figure 1A, although for unknown reason the staining intensity was weaker. In fact, numerous RNAseq studies have now thoroughly catalogued gene expression patterns in the retina throughout development and YTHDF2 expression can be found among all retinal cell types even from the earliest scRNAseq data . The ubiquitous expression of YTHDF2 in the retina poses serious concerns regarding the usefulness of Six3-Cre mediated Ythdf2 cKO model and the validity of presumed Ythdf2 downstream targets in RGCs.2. The revised manuscript presents data to show that retinal progenitors, amacrine cells, bipolar cells, photoreceptors, or horizontal cells were not affected in Ythdf2 cKO retina. It is also important for the authors to examine Müller glia and astrocyte, especially the latter which is known to have a strong influence on RGC activity and health.3. The potential targets of YTHDF2 were mostly characterized in the RGC culture, which is not a good model for studying dendritic development because RGCs will evitable die in vitro. As previously requested by this reviewer, the authors need to perform either qPCR or western blos of Ythdf2 cKO retina to confirm the regulation of Kalirin, Strn and Ubr4 by Ythdf2. They also need to show whether siRNA knockdown or CRISPR knockout of these genes rescue the Ythdf2 cKO phenotype in vivo.4. The authors propose that the better maintenance of RGC dendrite arborization in Ythdf2 cKO retina make it resistant to RGC death in AOH model and this is mediated by Hspa12a and Islr2 genes. To support this claim, the authors need to show that overexpression of Hspa12a and Islr2 alleviates loss of RGCs in the AOH model.Reviewer #2:The authors have responded to my comments and suggestions with several new experiments and some additional explanations. Though the effects of YTHDF2 described here are pleiotropic across RGC cell types, these defects in dendritic arborization are better described in this revised manuscript. In addition, further attention to the mRNAs regulated by YTHDF2 is given, and though we still are far from a mechanistic understanding of how these gene products regulate RGC dendritic arborization, and whether or not there is any specificity of these effects with regard to RGC subtypes, I find this revision to be a substantial improvement over the initial submission.Reviewer #3:The authors have addressed my comments. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:The authors report a functional-relevant role of YTHDF2 in mouse retina ganglion cells (RGCs) to negatively regulate dendrite development and maintenance of RGCs. Mechanistically, Kalrn, Strn, and Ubr4 mRNAs are selectively recognized and decay through the YTHDF2-mediated pathway, the subsequent downregulation of proteins encoded by those mRNAs negatively regulate dendrite development and maintenance of RGCs.This work highlights regulatory roles of N6-methyladenosine (m6A) and YTHDF2 protein in retina and suggested YTHDF2 as one possible target in AOH-induced RGC loss. A few concerns will need to be addressed.Concerns:1. The authors showed YTHDF2 mRNA is upregulated by AOH and argued a loss of YTHDF2 in retina results in less severe AOH-induced loss of Brn3a+ RGCs. The mechanism of YTHDF2 upregulation induced by AOH and loss of Brn3a+ RGCs mediated by YTHDF2 upregulation are unclear. The first question might be harder to address but the second one is within the scope of this work.We agree with the reviewer that the mechanism of YTHDF2 upregulation induced by AOH is beyond the scope of this work, which may require further investigation as a future direction.+ RGCs mediated by YTHDF2 upregulation in AOH-treated retina, we tried to answer this question by expanding the scope of our study in the revised manuscript. We wanted to know whether YTHDF2 target mRNAs mediate these effects in the AOH models. AOH has been shown to cause pathological changes in RGC dendrites before axon degeneration or soma loss is detected in different model animals , suggesting that RGC soma loss might be following dendrite degeneration. So we tested whether YTHDF2 upregulation mediates RGC dendrite degeneration in AOH-treated retina. We found that two target mRNAs Hspa12a and Islr2 show upregulation in the adult Ythdf2 cKO retina compared with control . We further found that Hspa12a and Islr2 are downregulated in the retina after AOH operation , which is likely caused by upregulation of YTHDF2 in the AOH-treated retina . We therefore hypothesized that AOH upregulates YTHDF2 which in turn downregulates its targets Hspa12a and Islr2, thus causing RGC dendrite degeneration and soma loss.As for the mechanism of loss of Brn3aHspa12a and Islr2 might protect RGC dendrite from AOH triggered degeneration. We thus generated AAV harboring overexpression constructs of Hspa12a and Islr2 which were intravitreally injected to wild type retinas. After the AOH induction, the retinas overexpressing Hspa12a and Islr2 maintain significantly more complex RGC dendrite arbor compared with control AAV .If this is the case, overexpression of Hspa12a and Islr2, thus causing RGC dendrite degeneration and eventually resulting in soma lossThese data support a model that AOH upregulates YTHDF2 which in turn downregulates its targets 2. In figure 7B, YTHDF2 expression in Brn3a+ RGCs was quantified. The figure showed that Brn3a was not detected in some cells. In figure 7C, mRNA level was quantified with an extraction method that did not separate Brn3a+ and Brn3a- cells.Figure 1A, all RGCS marked by the pan-RGC marker RBPMS express YTHDF2 while all YTHDF2-expressing cells are RBPMS+ RGCs. These data suggest that all RGCs express YTHDF2 and all YTHDF2-expressing cells in retina are RGCs.We thank the reviewer for pointing out this. Brn3a is a major RGC marker but does not stain all RGCs. We also used the pan-RGC marker RBPMS. As shown ion 3. The authors showed AOH-induced loss of Brn3a+ RGCs in mouse models. Supportive evidence at the phenotype level such as less severe loss of visual acuity in YTHDF2 cKO mice are required.Ythdf2 cKO retina with AOH operation maintains significantly higher dendrite complexity compared with the glaucomatous eyes of fl/flYthdf2 control mice . In addition, the reduction of RGC number in the Ythdf2 cKO retina with AOH operation is less than control retina with AOH operation . These results support that Ythdf2 cKO protects retina from RGC dendrite degeneration and soma loss caused by AOH.We thank the reviewer for the suggestion. We have tried to do the OMR assay using the AOH model mice. Unfortunately, the AOH model mice are not eligible for the OMR assay to test their visual acuity. However, we did find that the 4. The authors showed knockdown of Kalrn, Strn, and Ubr4 are sufficient to impair dendrite growth. Rescue experiments will further back up their claim.Figure 2E-H and Figure 3, cKO of Ythdf2 led to increased dendrite branching of RGCs both in vitro and in vivo. Transfection of siRNAs against these target mRNAs rescued dendrite branching increases in cultured Ythdf2 cKO RGCs . We continued to generate and performed intravitreal injection of AAV viral shKalrn12 and shUbr4, which significantly rescued dendrite branching increases of CART+ ooDSGCs and SMI-32+ αRGCs in Ythdf2 cKO retina in vivo . These data suggest that these target mRNAs mediate YTHDF2-controlled RGC dendrite branching.We thank the reviewer for the suggestion. We have now further performed rescue experiments to examine whether these target mRNAs mediate YTHDF2-regulated RGC dendrite branching. As shown in 5. The authors suggested that m6A-YTHDF2 axis regulates the stability of Kalrn, Strn, and Ubr4 mRNAs. The authors will need to show these RNAs contain m6A by m6A-IP-qPCR. The effect on Kalrn, Strn, and Ubr4 mRNAs upon m6A writer complex knockdown can also be shown.6A modification of these mRNAs by m6A-IP-qPCR . We also examined whether m6A modification regulates the expression levels of these target mRNAs. As shown in Figure 7—figure supplement 1D, the mRNA levels of Kalrn7, Kalrn9, Kalrn12, Strn and Ubr4 were dramatically increased after KD of METTL14, supporting that the stability of these target mRNAs is controlled in an m6A-dependent manner.We thank the reviewer for the suggestion. We now verified the mReviewer #2:This manuscript investigates the role of the m6A reader YTHDF2 during retinal ganglion cell (RGC) dendrite development and branching. The data presented in this study shows that deletion of YTHDF2 in the retina led to increased RGC dendrite branching and improved visual acuity. Based on high throughput analysis, YTHDF2 is suggested to regulate dendrite branching by reducing the stability of kalirin, Strn and Ubr4 mRNAs. Lastly, loss of YTHDF2 is shown to prevent RGC loss in a glaucoma model. The experimental designs were presented clearly, and the results were interpreted with the appropriate statistical tests. However, the phenotype of the Ythdf2 cKO mouse model is not well characterized and there lacks in vivo evidence for the putative targets of YTHDF2. It is also difficult to explain the connection, if any, between the dendritic role of YTHDF2 and its neuroprotective function.1. The authors used RGC cultures to show that knockdown/knockout of YTHDF2 resulted in increased the dendrite branching based on the number of interactions in Sholl analysis. However, to determine the full extent of dendritic defect, it is important to perform detailed analysis of the dendrite structure, including the total dendritic length, number of total segments, maximum branch length, branch order and number of branches.Figure 1—figure supplement 1G,H, the total dendrite length showed significant increase after YTHDF2 while the length of maximum branch was not changed.We thank the reviewer for the suggestions. We have now performed more detailed analysis of the dendrite structure of cultured RGCs after KD of YTHDF2. As shown in Ythdf2 cKO retina in vivo. As shown in Figure 3—figure supplement 1A-E, the total dendrite length, number of branches, number of total segments, and number of segments in each branch order of melanopsin+ RGCs in the Ythdf2 cKO retina showed significant increases compared with control retina, while the length of maximum branch was not changed.We further focused on the physiological dendrite structures by checking the RGC dendrites in the 2. It is suggested in the manuscript that loss of YTHDF2 in the retina only affects dendrite development of RGCs, but not other cell types. This is based on the simple counting of the number of RPCs at E15 and bipolar and amacrine cells at P15, glossing over the vast complexity of the visual circuitry in the retina. At the very least, the authors should confirm that differentiation of all major retinal neurons and proper formation of the ribbon synapses were unaffected in the cKO mouse model of YTHDF2, which would other confound the interpretation of the RGC and visual acuity phenotype.Ythdf2 cKO retina , suggesting that YTHDF2 is not involved in the generation or development of these cells. This is in line with the low YTHDF2 expression in these cells. The RGC number or density was not affected in the Ythdf2 cKO retina , indicating that Ythdf2 cKO does not disturb RGC neurogenesis. These data suggest that differentiation of all major retinal neurons was unaffected in the Ythdf2 cKO mouse model.Retinal progenitors, amacrine cells, bipolar cells, photoreceptors, or horizontal cells were not affected in +/PSD-95+ in OPL show no difference between Ythdf2 cKO and control retinas .In addition, the numbers of the excitatory ribbon synapses marked by the colocalization of Bassoon3. The in vivo characterisation of RGC dendrite morphology should be more thorough as previously mentioned, including the arbor shape, size and tiling pattern. The image quality of Melanopsin and SMI-32 staining is low. There are too many cells labelled, making it difficult to examine individual cells/dendrites. Higher resolution images from more sparsely labelled retina would be needed.Figure 3C,E). As mentioned earlier, we performed more detailed analysis of the RGC dendrite structures in vivo. As shown in Figure 3—figure supplement 1A-E, the total dendrite length, number of branches, number of total segments, and number of segments in each branch order of melanopsin+ RGCs in the Ythdf2 cKO retina showed significant increases compared with control retina, while the length of maximum branch was not changed.We thank the reviewer for the suggestion. We now have repeated and chosen better representative images for melanopsin and SMI-32 staining of RGC dendrites in vivo in the revised manuscript . As a control, the numbers of the excitatory ribbon synapses marked by the colocalization of Bassoon+/PSD-95+ in OPL show no difference between Ythdf2 cKO and control retinas .We thank the reviewer for the suggestion. We now used co-staining of the presynaptic marker Bassoon and the postsynaptic marker PSD-95 to count the colocalization puncta of Bassoon5. The improved visual acuity in Six3Cre-mediated Ythdf2 cKO is solely attributed to the RGC dendrite defects. However, Six3Cre is widely expressed in the ventral telencephalon and ventral anterior hypothalamus, including the SCN and SPZ (PMID: 24767996). It is necessary to examine the retinocollicular projections of the RGC axons.Ythdf2 cKO and control mice , suggesting the guidance and central targeting of RGC axons are not affected in the Ythdf2 cKO.We thank the reviewer for the suggestion. We checked the targeting of optic nerves to the brain by anterograde labeling with cholera toxin subunit B (CTB) and found no difference of retinogeniculate or retinocollicular projections between 6. The Optomotor response (OMR)-based assay is not the most appropriate test to test the response of ganglion cells since the improved OMR could be attributed to other cell types as previously mentioned. A more targeted approach such as pattern ERG could provide more evidence to their claims.Ythdf2 cKO mediated by Six3-cre:We tried to find a way to do pattern ERG, but unfortunately we do not have this and could not find reachable resources to do this, either. However, based on the analysis we have done so far, we believe that the improved OMR is most likely attributed to the increased RGC dendrite branching and thicker and denser IPL with more synapses because all other parts and processes of retina are not affected except RGC dendrite in the Ythdf2 cKO in retina .(1) As mentioned earlier, the neurogenesis of all major neuron types in retina is not affected by Ythdf2 cKO retina were validated both in vitro and in vivo .(2) The increases of RGC dendrite branching in the Ythdf2 cKO retina . As a control, the thicknesses of other retinal layers  showed no difference between the Ythdf2 cKO and control mice .(3)IPL thickness significantly increased in the +/PSD-95+ excitatory synapses in IPL of Ythdf2 cKO retina are significantly larger than that of control retina . As a control, the numbers of the excitatory ribbon synapses marked by the colocalization of Bassoon+/PSD-95+ in OPL show no difference between Ythdf2 cKO and control retinas .The numbers of Bassoon7. The quality of the RIP-seq analysis is not clear. Is there an isotype antibody control? Are there biological replicates? What is the statistical significance of the top hits? There are similar concerns regarding the MS analysis and whether there is significant overlap between the target lists generated from RIP-Seq and MS studies.Supplementary file 1) and YTHDF2 KD/MS_upregulation (Supplementary file 2), we identified a group of potential YTHDF2 target mRNAs in RGCs (Supplementary file 3).We are sorry that the related information was not clear or missing. For the RIP experiment: Yes, we used a control IgG and performed two biological replicates. To determine which gene is enriched, we computed the FPKM from RIP elute to input and any fold change greater than 2  was considered enriched. For MS analysis, we performed three biological replicates, and proteins with fold changes greater than 1.3 and p values less than 0.05 were considered to be regulated by YTHDF2 KD with statistical significance. By overlapping the two gene lists screened from anti-YTHDF2 RIP-Seq . For validation, we performed IF using antibodies against Strn and Ubr4  and detected specific signals in the IPL which were increased in Ythdf2 cKO retina compared with control retina , suggesting that Strn and Ubr4 are upregulated in Ythdf2 cKO retina at the level of protein.MS analysis after YTHDF2 KD has shown that the protein levels of these target mRNAs were upregulated . We then checked the effects on RGC dendrite branching after KD of these target mRNAs by siRNAs in cultured RGCs. As shown in Figure 7B, KD of Kalrn7, Kalrn9, Kalrn12, Strn or Ubr4 led to significant decreases of RGC dendrite branching. We further examined whether these target mRNAs mediate YTHDF2regulated RGC dendrite branching. As shown in Figure 2E-H, and Figure 3, cKO of Ythdf2 led to increased dendrite branching of RGCs both in vitro and in vivo. Transfection of siRNAs against these target mRNAs rescued dendrite branching increases in cultured Ythdf2 cKO RGCs . We continued to perform intravitreal injection of AAV viral shKalrn12 and shUbr4, which significantly rescued dendrite branching increases of CART+ ooDSGCs and SMI-32+ αRGCs in Ythdf2 cKO retina in vivo . These data support that knockdown of these genes indeed reduces the RGC dendrite branching, and can rescue the Ythdf2 cKO phenotype both in vitro and in vivo.We continued to explore the functions of these YTHDF2 target mRNAs in RGC dendrite development. We first generated siRNAs against these transcripts .The pathological changes in RGC dendrites precede axon degeneration or soma loss in the glaucomatous eyes of different model animals . Our findings that Hspa12a and Islr2 show upregulation in the adult Ythdf2 cKO retina compared with control . We further found that Hspa12a and Islr2 are downregulated in the retina after AOH operation , which is likely caused by upregulation of YTHDF2 in the AOH-treated retina . We therefore hypothesized that AOH upregulates YTHDF2 which in turn downregulates its targets Hspa12a and Islr2, thus causing RGC dendrite degeneration and soma loss. If this is the case, overexpression of Hspa12a and Islr2 might protect RGC dendrite from AOH-triggered degeneration. We thus generated AAV harboring overexpression constructs of Hspa12a and Islr2 which were intravitreally injected to wild type retinas. After the AOH induction, the retinas overexpressing Hspa12a and Islr2 maintain significantly more complex RGC dendrite arbor compared with control AAV . These data support a model that AOH upregulates YTHDF2 which in turn downregulates its targets Hspa12a and Islr2, thus causing RGC dendrite degeneration and eventually resulting in soma loss.We found that two target mRNAs Kalrn7, Kalrn9, Kalrn12, Strn and Ubr4 mediate YTHDF2 functions to regulate RGC dendrite development. In adult mice, another set of target mRNAs Hspa12a and Islr2 mediate YTHDF2 function to regulate RGC dendrite maintenance. We now added more discussion and explanation on the connection of the two phases of YTHDF2 function in the revised manuscript.The mechanistic link between the dendrite development phenotype and the neuroprotective role of YTHDF2 is that YTHDF2 has two phases of function to control RGC dendrite development first and then maintenance through regulating two sets of target mRNAs. In early postnatal stages, the target mRNAs Reviewer #3:In this study, the authors propose a role for the m6A reader YTHDF2 in regulating the dendritic arbor size of retinal ganglion cells (RGCs), and they extrapolate this to address issues in glaucoma and overall retina IPL morphology. They find that retina-specific loss of Ythdf2 leads to the expansion of RGC dendritic arbors in the horizontal plane and also a widening of the inner plexiform layer (IPL), with Ythdf2 cKOs showing a modest increase in visual acuity in an optomotor assay. They identify a number of factors downstream of YTHDF2 not previously known to be involved in retinal dendritic development, and they propose a role for Ythdf2 in a glaucoma model, providing a foundation for future studies into their functions in these contexts.Although these data are sufficient to establish a role for Ythdf2 in RGC dendritic development and other more general aspects of retina morphology, many of the authors' specific claims are weakened by issues with the experiments, their analyses, and by weak effect sizes. Even if the points raised below were addressed, it remains unclear from this study how Ythdf2-mediated mechanisms are integrated into programs of dendritic development and RGC survival, diminishing the impact of this work. This study would be strengthened by, for example, identifying a developmental program that modifies the target mRNAs found in this study to promote recognition by YTHDF2, and also by providing better understanding of how the products produced by these mRNAs actually regulate dendrite morphology and synapse formation. Overall, while there are a number of promising findings in this study, the work required to address weaknesses falls well outside of the limits usually recommended by eLife, and it is suited for publication in a more specialized journal following attention to many of the points raised below.1. The cell-type specific tracing in Figure 3 is difficult to assess due to the extremely high background in the melanopsin images and unclear signal in the SMI-32 images. This analysis would be improved by sparse viral labeling, with a fluorophore provided by the virus used for tracing and the melanopsin & SMI-32 antibodies used solely for establishing RGC subtype identity. Further, the authors emphasize that the defects they observe here are in the plane of retinal ganglion cell (RGC) dendritic arbors, but this is an over-interpretation of these data. One wonders why the authors did not examine in cross section the lamination within the IPL of the melanopsin-positive ipRGCs, since this would provide, at least for this RGC subclass, a direct assessment of this issue. Finally, the Thy-1-O line GFP distribution is quite general, and it really is not the correct line to use if one wants to investigate defects in lamination.We thank the reviewer for pointing out this. To answer this question, we now have expanded the scope of our study in the revised manuscript.Ythdf2 cKO in retina promotes RGC dendrite branching during development inspired us to wonder whether YTHDF2 also regulates RGC dendrite maintenance in the acute glaucoma model caused by acute ocular hypertension (AOH).The pathological changes in RGC dendrites precede axon degeneration or soma loss in the glaucomatous eyes of different model animals . Our findings that Hspa12a and Islr2 show upregulation in the adult Ythdf2 cKO retina compared with control . We further found that Hspa12a and Islr2 are downregulated in the retina after AOH operation , which is likely caused by upregulation of YTHDF2 in the AOH-treated retina . We therefore hypothesized that AOH upregulates YTHDF2 which in turn downregulates its targets Hspa12a and Islr2, thus causing RGC dendrite degeneration and soma loss. If this is the case, overexpression of Hspa12a and Islr2 might protect RGC dendrite from AOH-triggered degeneration. We thus generated AAV harboring overexpression constructs of Hspa12a and Islr2 which were intravitreally injected to wild type retinas. After the AOH induction, the retinas overexpressing Hspa12a and Islr2 maintain significantly more complex RGC dendrite arbor compared with control AAV . These data support a model that AOH upregulates YTHDF2 which in turn downregulates its targets Hspa12a and Islr2, thus causing RGC dendrite degeneration and eventually resulting in soma loss.We found that two target mRNAs Kalrn7, Kalrn9, Kalrn12, Strn and Ubr4 mediate YTHDF2 functions to regulate RGC dendrite development. In adult mice, another set of target mRNAs Hspa12a and Islr2 mediate YTHDF2 function to regulate RGC dendrite maintenance. We now added more discussion and explanation on the connection of the two phases of YTHDF2 function in the revised manuscript.The mechanistic link between the dendrite development phenotype and the neuroprotective role of YTHDF2 is that YTHDF2 has two phases of function to control RGC dendrite development first and then maintenance through regulating two sets of target mRNAs. In early postnatal stages, the target mRNAs 2. The authors repeatedly describe increased dendritic and synaptic density within the IPL in Ythdf2 cKOs, yet they only present absolute signal intensities to support these claims; without normalizing signal intensity to IPL width, it is impossible to determine whether changes in intensity are indeed due to increased density or from the IPL becoming enlarged with a similar density of dendrites and synapses. Further, they claim in the text that VGlut2 puncta numbers are changed in these mutants, however the analysis presented is only for general signal intensity. If claims are to be made regarding changes in synaptic specializations, higher resolution assessments of individual puncta using pre- and postsynaptic markers is required.Ythdf2 cKO was not clear. The MAP2 IF intensity shown in Figure 4C was already normalized to the IPL area. Considering the increased IPL thickness, the absolute signal intensities (MAP2 IF intensity per area × IPL area) would be even larger in the Ythdf2 cKO compared with the control. We have now made this (MAP2 IF intensity per area in IPL) clearer in the revised manuscript.We are sorry that the information about increased dendritic and synaptic density within the IPL in +/PSD-95+. We found that the numbers of Bassoon+/PSD-95+ excitatory synapses in IPL of Ythdf2 cKO retina are significantly larger than that of control retina .We thank the reviewer for the suggestion on quantifying synaptic puncta. We now used co-staining of the presynaptic marker Bassoon and the postsynaptic marker PSD-95 to count the colocalization puncta of Bassoon3. While Ythdf2 cKOs do show a statistically significant improvement in visual acuity , the effect size is small enough to call into question how meaningful the improvement is. Further, what is the actual relationship between this modest improvement in optomotor response (OMR) and the morphological phenotypes observed here, in particular since they are not connected here to the classes of direction-selective RGCs one would expect might affect OMR responses.Ythdf2 cKO and control is small yet significant in both male and female mice in our study, and this difference is comparable with other studies in the field . Considering the fact that young wild-type (control) mice already have good visual acuity, this 0.02 cycle/degree increase is quite significant.The difference of 0.02 cycle/degree between young Ythdf2 cKO retinal cultures increased compared with control in vitro . Intravitreal injection of an AAV reporter expressing ZsGreen visualized the dendrite morphology of ooDSGCs marked by CART immunostaining in vivo . We found that ooDSGCs showed dramatically increased dendrite branching in Ythdf2 cKO retina compared with control retina . Thus, Ythdf2 cKO increased the dendrite branching of ooDSGCs both in vitro and in vivo, along with other data in this study supporting the improved optomotor response in the Ythdf2 cKO mice.We thank the reviewer for suggesting checking direction-selective RGCs to enhance the relationship between the improvement in optomotor response (OMR) and the morphological phenotypes. The ON-OFF directionally selective RGCs (ooDSGCs) respond preferentially to movement in particular directions. Expression of CART (cocaine- and amphetamine-regulated transcript), a neuropeptide, distinguishes ooDSGCs from other RGCs . We now found that the dendrite branching of ooDSGCs marked by CART/Brn3a co-staining in 4. Much clearer co-localization between YTHDF2 and RGC markers is needed, both in vivo and in vitro, to clearly demonstrate association with RGCs and not displaced amacrine cells or other retinal cell types.Figure 1A, all RGCS marked by the pan-RGC marker RBPMS express high YTHDF2 while all YTHDF2-expressing cells are RBPMS+ RGCs. These data demonstrate the co-localization between YTHDF2 and the pan-RGC marker.We now used the pan-RGC marker RBPMS. As shown ion 5. The several mRNAs and their produces shown to be under YTHDF2 control do appear to play some role in regulating general RGC dendritic morphology, though of course from these data we do not know whether or not there is any RGC-subtype specificity. However, as presented, we really do not learn much at all about their mechanisms of action. At the very least, do they act in the same, or different, pathways….i.e. have any attempts been made to knock down more than one at a time and ask about independent pathway affiliations?Figure 7—figure supplement 1F, the siCocktail further significantly reduced the RGC dendrite branching compared with each individual siRNA. These results suggest that these targets may work in different pathways to regulate the RGC dendrite morphology. We agree that the exploration of their working mechanisms is an important future direction.We thank the reviewer for the suggestion. We now have prepared a cocktail of siRNAs against these target mRNAs and performed the KD assay to check the effect on RGC dendrite morphology. As shown in 6. It is not clear how to interpret the Ythdf2 effects in the acute ocular hypertension (AOH) model. Is this related to the downstream mRNAs, or some other more general effect of loss of this m6 reader? And how is this related to RGC survival at a mechanistic level?Hspa12a and Islr2 show upregulation in the adult Ythdf2 cKO retina compared with control . We further found that Hspa12a and Islr2 are downregulated in the wildtype retina after AOH operation , which is likely caused by upregulation of YTHDF2 in the AOH-treated retina . We therefore hypothesized that AOH upregulates YTHDF2 which in turn downregulates its targets Hspa12a and Islr2, thus causing RGC dendrite degeneration. If this is the case, overexpression of Hspa12a and Islr2 might protect RGC dendrite from AOH-triggered degeneration. We thus generated AAV harboring overexpression constructs of Hspa12a and Islr2 which were intravitreally injected to wild type retinas. After the AOH induction, the retinas overexpressing Hspa12a and Islr2 maintain significantly more complex RGC dendrite arbor compared with control AAV .We are sorry that this was not clear in the original manuscript. To answer this question, we now have expanded the scope of our study in the revised manuscript. We found that two YTHDF2 target mRNAs Hspa12a and Islr2, thus causing RGC dendrite degeneration and eventually resulting in soma loss.AOH has been shown to cause pathological changes in RGC dendrites before axon degeneration or soma loss is detected in different model animals , suggesting that RGC soma loss might be following dendrite degeneration. Thus, these findings support a model that AOH upregulates YTHDF2 which in turn downregulates its targets SummaryAs a summary, we have now substantially expanded and improved our study to address all the concerns raised by the reviewers and summarized by the editor:Ythdf2 cKO mouse model by more precisely measuring the layer dimensions , the synapse number , the dendritic extension ; checking cells other than RGCs in retina ; confirming colocalization of YTHDF2 and RGC markers . We also explained the connection between the dendrite development phenotype and the neuroprotective role of YTHDF2 (please see our response to Reviewer #2’s major comment 9 for details).1. We have now done a better phenotyping of the Kalirin, Strn and Ubr4 indeed contribute to the increase in dendrite branching in the Ythdf2 knockout. We generated and performed intravitreal injection of AAV viral shKalrn12 and shUbr4, which significantly rescued dendrite branching increases of CART+ ooDSGCs and SMI-32+ αRGCs in Ythdf2 cKO retina in vivo . These data suggest that these target mRNAs mediate YTHDF2-controlled RGC dendrite branching. As for the mechanism of loss of Brn3a+ RGCs mediated by YTHDF2 upregulation in AOHtreated retina, please see our response to Reviewer #1’s comment 1 for details.2. We have now provided in vivo evidence to show that the YTHDF2 target mRNAs 3. We have now included the analysis of the direction-selective RGCs to enhance the relationship between the improvement in optomotor response (OMR) and the morphological phenotypes (please see our response to Reviewer #3’s comment 3 for details).Hspa12a and Islr2 (please see our response to Reviewer #3’s comment 6 for details).4. By expanding the scope of our study, we have now more clearly interpreted the YTHDF2 effects in the acute ocular hypertension (AOH) model, which are mediated by the two YTHDF2 target mRNAs Hspa12a and Islr2, thus causing RGC dendrite degeneration and eventually resulting in soma loss.5. We used a model to explain the relationship among glaucoma, YTHDF2, and RGC survival: AOH upregulates YTHDF2 which in turn downregulates its targets We think that the revised manuscript is substantially improved with these revisions and new data.We would like to thank the reviewers for their comments and suggestions.ReferencesDietrich, M., Hecker, C., Hilla, A., Cruz-Herranz, A., Hartung, H.P., Fischer, D., Green, A., and Albrecht, P. (2019). Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats. 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Invest Ophthalmol Vis Sci 39, 2304-2320.[Editors’ note: what follows is the authors’ response to the second round of review.]The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below and in the appended reviews:Reviewer 2 (previous Reviewer 1) was satisfied with the revisions of this resubmission as was Reviewer 3  continues to have concerns: He summarized in the Public Review (this will not be made public as it is a resubmission) that "the phenotype of the Ythdf2 cKO mouse model is not well characterized and there lacks in vivo evidence for the putative targets of YTHDF2".We thank the reviewer for pointing out these important future directions. We have now added related discussions in the revised manuscript.Two items he previously requested were not addressed:(1) To perform either qPCR or western blots of Ythdf2 cKO retina to confirm the regulation of Kalirin, Strn and Ubr4 by Ythdf2.Kalirin, Strn, Ubr4 using the Ythdf2 cKO and control retina, confirming that Kalirin, Strn, and Ubr4 mRNA levels were upregulated in the Ythdf2 cKO retina compared with control .We have now carried out qPCR for 2) To show that overexpression of Hspa12a and Islr2 alleviates loss of RGCs in the AOH model (See point #8 of previous Reviewer 2’s review). We ask that you address these two items that were raised in the first review, to the best of your ability, given time constraints. .Figure 8E,F). We now have repeated this experiment to further check loss of RGCs. As shown in the revised Figure 8G, retinas overexpressing Hspa12a and Islr2 maintain significantly more Brn3a+ RGCs than control AAV after the AOH induction, suggesting that overexpression of Hspa12a and Islr2 alleviates loss of RGCs in the AOH model.In the original manuscript, we have shown that the retinas overexpressing Hspa12a and Islr2 maintain significantly more complex RGC dendrite arbor compared with control AAV after the AOH induction . Conversely, the expression of YTHDF2 in other layers of the retina is much lower . Though the expression of YTHDF2 could be detected by scRNAseq in other retinal cell types in a previous report as the reviewer mentioned, their YTHDF2 expression levels are much lower than RGCs. More importantly, consistent with the minimal or no expression of YTHDF2 in other retinal layers/cells, all other retinal layers/cells except IPL are not affected in the Ythdf2 cKO retina .YTHDF2 is highly expressed in RGCs .GFAP is reliable marker for astrocytes in the mammalian retina . As shown in 3. Addressing the utility of the Six3-Cre, by determining whether there is mosaic expression of Cre in the peripheral retina.Figure 5—figure supplement 1A. The fact that the layer of Brn3a+ RGCs become thinner and thinner toward the edge of peripheral retina  makes the trace residual expression of YTHDF2 in the peripheral retina of Ythdf2 cKO mice negligible .As reported previously, there is minimal mosaic absence of expression of Six3-Cre in the peripheral retina as shown in gligible . Indeed,Reviewer #1:The experimental designs were presented clearly and the results were interpreted with the appropriate statistical tests. However, the phenotype of the Ythdf2 cKO mouse model is not well characterized and there lacks in vivo evidence for the putative targets of YTHDF2. It is also difficult to explain the connection, if any, between the dendritic role of YTHDF2 and its neuroprotective function. Most importantly, whether Ythdf2 acts cell autonomously in the RGCs remains to be resolved.1. The premise of this study is that Ythdf2 is cell autonomously required in RGCs for dendritic development. In the rebuttal letter, the authors even went as far as claiming that "all RGCs express YTHDF2 and all YTHDF2-expressing cells in retina are RGCs". However, this statement is belied by the strong YTHDF2 staining outside the RGC layers in Figure 1—figure supplement 1B. The YTHDF2 expression in non-RGC cells can also be seen in Figure 1A, although for unknown reason the staining intensity was weaker. In fact, numerous RNAseq studies have now thoroughly catalogued gene expression patterns in the retina throughout development and YTHDF2 expression can be found among all retinal cell types even from the earliest scRNAseq data . The ubiquitous expression of YTHDF2 in the retina poses serious concerns regarding the usefulness of Six3-Cre mediated Ythdf2 cKO model and the validity of presumed Ythdf2 downstream targets in RGCs.This has been addressed in the response to reviewer 1’s point 1 summarized by the editor.2. The revised manuscript presents data to show that retinal progenitors, amacrine cells, bipolar cells, photoreceptors, or horizontal cells were not affected in Ythdf2 cKO retina. It is also important for the authors to examine Müller glia and astrocyte, especially the latter which is known to have a strong influence on RGC activity and health.+ Müller glia and GFAP+ astrocytes in P20 Ythdf2 cKO retina. Neither of these two cell populations was affected in the Ythdf2 cKO retina , which is not surprising since these cells don’t express YTHDF2 We now have checked Lhx23. The potential targets of YTHDF2 were mostly characterized in the RGC culture, which is not a good model for studying dendritic development because RGCs will evitable die in vitro. As previously requested by this reviewer, the authors need to perform either qPCR or western blos of Ythdf2 cKO retina to confirm the regulation of Kalirin, Strn and Ubr4 by Ythdf2. They also need to show whether siRNA knockdown or CRISPR knockout of these genes rescue the Ythdf2 cKO phenotype in vivo.Figure 7D).This has been addressed in the response to the editor’s emphasized item (1). For the rescue experiments in vivo, these have been done .Reviewer #2:The authors have responded to my comments and suggestions with several new experiments and some additional explanations. Though the effects of YTHDF2 described here are pleiotropic across RGC cell types, these defects in dendritic arborization are better described in this revised manuscript. In addition, further attention to the mRNAs regulated by YTHDF2 is given, and though we still are far from a mechanistic understanding of how these gene products regulate RGC dendritic arborization, and whether or not there is any specificity of these effects with regard to RGC subtypes, I find this revision to be a substantial improvement over the initial submission.We thank the reviewer for pointing out these important future directions. We have now added related discussions in the revised manuscript.References36, 2391-2405. 10.1523/jneurosci.3145-15.2016.de Melo, J., Zibetti, C., Clark, B.S., Hwang, W., Miranda-Angulo, A.L., Qian, J., and Blackshaw, S. (2016). Lhx2 Is an Essential Factor for Retinal Gliogenesis and Notch Signaling. J Neurosci 25, 11595-11604. 10.1523/jneurosci.2837-05.2005.Quina, L.A., Pak, W., Lanier, J., Banwait, P., Gratwick, K., Liu, Y., Velasquez, T., O'Leary, D.D., Goulding, M., and Turner, E.E. (2005). Brn3a-expressing retinal ganglion cells project specifically to thalamocortical and collicular visual pathways. J Neurosci 51, 1-40. 10.1016/j.preteyeres.2015.06.003.Vecino, E., Rodriguez, F.D., Ruzafa, N., Pereiro, X., and Sharma, S.C. (2016). Glia-neuron interactions in the mammalian retina. Prog Retin Eye Res


Conclusion: NAFLD and MAFLD were significantly associated with a higher risk of colorectal adenomas, especially in females. NAFLD and MAFLD with advanced fibrosis were associated with an increased risk of colorectal adenoma. Colonoscopic examinations may be emphasized for patients with NAFLD/MAFLD, for women, or patients with the presence of hepatic fibrosis.Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease associated with various metabolic disorders. Metabolic dysfunction-associated fatty liver disease (MAFLD) emphasizes metabolic dysfunction in NAFLD. Although the relationship between NAFLD and colorectal adenomas has been suggested, the effect of MAFLD on colorectal adenoma has yet to be investigated. In this study, we examined the relationship between NAFLD/MAFLD and colorectal adenoma in comparison with other metabolic factors. Methods: Examinees who underwent colonoscopy and abdominal ultrasonography on the same day from January 2012 to December 2012 were included. NAFLD was diagnosed according to the findings of ultrasonography. The Fibrosis-4 (FIB-4) index was used as a surrogate marker for advanced hepatic fibrosis. A logistic regression model was used to analyze the risk of NAFLD/MAFLD for colorectal adenoma. Results: The prevalence of NAFLD and MAFLD was 37.5% and 32.8%, respectively. In the multivariate analysis, male sex, older age, diabetes, and smoking increased the risk of colorectal adenoma. NAFLD and MAFLD were the most important risk factors for colorectal adenoma only in females . NAFLD and MAFLD with an advanced fibrosis index were significantly associated with an increased risk of colorectal adenoma. (NAFLD: OR 1.38, 95% CI, 1.04–1.83,  Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide, with an increasing prevalence of up to 20–30% . AlthougCRC is the third most commonly diagnosed cancer and the fourth most common cause of cancer-related deaths worldwide . ColorecThus, we aimed to investigate the relationship of colorectal adenoma and both NAFLD and MAFLD in asymptomatic individuals undergoing screening colonoscopy. We also evaluated whether the severity of NAFLD/MAFLD was associated with the risk of these neoplasms.We used a previously conducted retrospective cohort . BrieflyFor NAFLD analysis, subjects who displayed any potential cause of chronic liver disease were excluded; 149 were positive for the hepatitis B virus, 29 were positive for the hepatitis C virus, and 219 had significant alcohol intake  [The study protocol was approved by the Institutional Review Board of Seoul National University Hospital (IRB No. H-2106-014-1223) and conformed to the ethical guidelines of the World Medical Association Declaration of Helsinki. The requirement for informed consent from individual subjects was waived, as we used de-identified secondary data.2 (m2). Obesity was defined as a BMI ≥ 25 (kg/m2), according to the World Health Organization recommendation for the Asia–Pacific region [Study data consisted of medical information based on a self-administered questionnaire and anthropometric and laboratory measurements as previously described . In briec region . Based oAll subjects fasted for at least 12 h prior to blood sampling; aspartate aminotransferase (AST), alanine aminotransferase (ALT) total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, glucose, HbA1c, and high-sensitivity C-reactive protein (CRP) levels were measured. All tests were performed using standard laboratory methods. We used a previously described method to assess the visceral fat area (VFA) on abdominal fat computed tomography (CT) images . The indHepatic ultrasonography  was performed to diagnose fatty liver disease by experienced radiologists who were unaware of the clinical information of the subjects . Fatty lAll colonoscopic examinations were performed by using a video colonoscopy system  by board-certified gastroenterologists who had performed more than 5000 colonoscopies ,28. For p value of less than 0.05 in the univariate analysis, those with clinical importance were subjected to multivariate analyses. All statistical analyses were performed using SPSS 22.0 , and p values < 0.05 were considered statistically significant.Data are presented as the mean ± standard deviation for normally distributed continuous variables and as proportions for categorical variables. Student’s t-test and analysis of variance were used to analyze continuous variables, and the differences between nominal variables were compared with the chi-square test. A logistic regression analysis was utilized to analyze the association between NAFLD or MAFLD and colorectal adenomas after adjusting for potential confounders. Among variables with a p < 0.001). The prevalence of colorectal adenoma was significantly higher in both NAFLD and MAFLD patients than in those without NAFLD/MAFLD. Baseline characteristics stratified by sex are provided in Among the 3441 subjects, the mean age was 52.4 years, and the male proportion was 61.9%. The prevalence rate of colorectal adenoma was 29.4%. The baseline characteristics of the study participants according to NAFLD or MAFLD are shown in p < 0.001). After adjusting for age, sex, BMI, diabetes, hypertension, smoking, triglyceride level, HDL cholesterol level, and VFA, the statistical significance of NAFLD disappeared . Moreover, diabetes and smoking showed significant associations with an increased risk of colorectal adenoma ; however, there was no significant association between MAFLD and colorectal adenoma in the multivariate analysis .We investigated the association between colorectal adenomas and NAFLD/MAFLD. In the univariate model, the presence of NAFLD showed a significant association with colorectal adenoma. NAFLD was associated with a 45% increased risk of colorectal adenomas , while diabetes and smoking showed significant associations with an increased risk of colorectal adenoma in men in the multivariate analysis . MAFLD was independently associated with the risk of colorectal adenoma in women .When we performed stratified analysis according to sex, NAFLD was independently associated with the risk of colorectal adenoma in women . However, there was no statistical significance in the increased risk of the NAFLD-with-obesity group compared with the no-NAFLD group. When we compared the MAFLD group with the no-fatty liver disease group, there was no significant difference in the risk for colorectal adenoma. Moreover, when we further divided the MAFLD group into three categories according to BMI and diabetes, the proportion of colorectal adenoma was the highest in the MAFLD-with-diabetes group; however, there was no significant difference regarding the risk of colorectal adenoma in the multivariate analysis, as displayed in Since diabetes and obesity are both well-known confounders and significant risk factors for colorectal adenoma, we evaluated the relationship between colorectal adenoma and NAFLD with diabetes or obesity. After adjusting for age, sex, smoking, triglyceride level, HDL cholesterol level, hypertension, VFA, and BMI, the NAFLD-with-diabetes group had a significantly increased risk for colorectal adenoma compared with the no-NAFLD group  during colonoscopy vary widely among endoscopists and are therefore an important factor to consider when comparing adenoma prevalence between groups . The oveIn this study, there was no difference in the risk of colorectal adenomas in those with either NAFLD or MAFLD among the total subjects. In contrast to our results, a recent study performed in Japan reported that nonobese MAFLD was the most important factor associated with the presence of colorectal adenomas, with an increased OR of 3.19 . The strNAFLD is a sexually dimorphic disease with regard to epidemiological and clinical features, including liver-related outcomes . To analp < 0.05) in this study, which supports this explanation.The sex-specific association of colorectal adenoma and NAFLD/MAFLD in our study may possibly be related to sexual dimorphism in NAFLD. Women who are overweight or obese before menopause have a lower risk of hepatic steatosis and have a higher hepatic insulin sensitivity than men, which might contribute to a more beneficial cardiometabolic effect in women. Since this effect seems to be related to estrogen concentrations and alterations in adipose tissue distribution, these processes might change with menopause, progressing adiposity, and worsening hepatic insulin resistance . In a reWaist circumference, obesity, lipid profiles, glucose, and hypertension are known factors that play roles in the process of colorectal adenoma . Among tTo our knowledge, this is the largest study to have examined the relationship between MAFLD and colorectal adenomas. In addition, we evaluated multiple metabolic confounding factors, including VFA, by utilizing CT. However, this study has several limitations. First, due to its observational study design, cautious interpretation is required. Second, although liver biopsy is considered to be the gold standard for the diagnosis of NAFLD, it was assessed only by ultrasonography in this study. Ultrasonography has limitations, including interpersonal variability, inaccuracy in patients with low liver fat contents, and the inability to quantify fibrosis . HoweverIn conclusion, we demonstrated that NAFLD and MAFLD were significantly associated with colorectal adenoma in women. NAFLD and MAFLD with advanced fibrosis showed an increased risk of colorectal adenoma. Therefore, clinicians should be aware of the increased risk of colorectal adenoma in NAFLD patients, especially for women. Further experimental observations that could be conducted to define genetic and sex differences in populations with different eating habits are needed.


Background: Identifying and treating hearing loss can help improve communication skills, which often leads to improved quality of life. Many people do not seek medical treatment and, therefore, go undiagnosed for an extended period before realizing they have hearing loss. This study presents a self-administered, low-cost, smartphone-based hearing test application (HearTest) to quantify the pure-tone hearing thresholds of a user. The HearTest application can be used with commercially available smartphone devices and an earphone with the mentioned specification. Methods: Air-conduction-based pure-tone audiometry for the smartphone application was designed and implemented to detect hearing thresholds using a traditional “10 dB down and 5 dB up” approach. Employed smartphone-earphone combination was calibrated with respect to a GSI-61 audiometer and insert earphone ER-3A to maintain clinical standards with the help of subjective testing on 20 normal-hearing (NH) participants. Results: Further subjective testing on 14 participants with NH and retesting on five participants showed that HearTest achieves high-accuracy audiogram within clinically acceptable limits (≤10 dB HL mean difference) when compared with the reference clinical audiometer. Hardware challenges and limitations in air-conduction-based hearing tests through smartphones and ways to improve their accuracy and reliability are discussed. Conclusion: The proposed smartphone application provides a simple, affordable, and reliable means for people to learn more about their hearing health without needing access to a formal clinical facility. Hearing loss affects 470 million people in the world and it is expected to double by the year 2050 . UntreatMany advanced adaptive signal processing algorithms have been developed that implement distinct parts of the hearing aid processing pipeline in modern smartphones in real time. The combination of such novel algorithms developed for monaural and binaural hearing applications and the smartphone provide an immensely powerful and cost-effective platform to assist people with hearing disorders ,7,8,9,10Most people with moderate-to-profound hearing impairments live in low- and middle-income countries . ConductProgress in smartphone technology offers a way to build an application for air-conduction-based hearing tests. The advantages of the smartphone-based solution are portability, self-assistance, low cost, and it being less time consuming compared to the formal clinical facilities. Smartphone-based applications can have high reliability, sensitivity, and specificity towards identifying potential hearing loss ,15,16. SThe study of  revealedThe purpose of this study is to provide an accurate, low-cost, and widely accessible smartphone-based application (HearTest) for quantifying an individual’s hearing ability. HearTest performs pure-tone audiometry (PTA) and plots the test results on an audiogram. In this study, we have used the commercially available iPhone XR smartphone and Sennheiser CX3.00 earphones for calibration and testing purposes. The selected smartphone-earphone combination was calibrated with respect to GSI-61 audiometer and insert earphones (ER-3A), maintaining clinical standards. As part of the calibration, the initial assumption about reference equivalent threshold sound pressure level (RETSPL) values was based on the findings of Ho et al. in . SubjectUnlike other smartphone-based screening applications, the proposed smartphone application can quantify the hearing threshold levels within clinically accepted standards for NH people.We propose a novel subjective test-based approach to calibrate a smartphone-earphone combination with respect to the reference audiometer.In PTA, we use continuous tones that are different from regular short interval tones. The advantages of using continuous tones are that they minimize harmonic distortions and do not require the attention of the user.Compared with previous works related to smartphone-based hearing test applications, the major contributions of this paper are as follows:In the remainder of this text, to minimize the redundancy we define the following terms. By the HearTest application, it is implied that it is used with the combination of the iPhone (8/10/XR) and the Sennheiser CX300 earphone device. A reference audiometer refers to the standard clinical GSI-61 audiometer used with insert earphones (ER-3A). The rest of the paper is organized as follows: Subjective tests conducted during this study can be divided into the following four batches based on the HearTest build version . Application in pre-calibration stage is named “HearTest v1” and the post-calibration stage is “HearTest v2”.Subjective test batch 1 (B1): For the first version of the application, a total of 20 participants self-identified as having NH ranging from ages 19 to 28 years with mean and standard deviation as Subjective test batch 2 (B2): A total of 14 participants also self-identified as having NH ranging from ages 20 to 30 years with mean and standard deviation as Subjective test batch 3 (B3): five participants from B1 were again called for retesting with the HearTest v2.Subjects were recruited through a university volunteer research program. All participants were fluent English speakers and had no other reported handicapping conditions. Human subjects’ approval was received from Institutional Review Board of the University of Texas at Dallas, protocol number: 14-72.During each subjective test, the participant first went through the standard clinical pure-tone audiometry followed by the HearTest application. Both the tests were carried out via air conduction and were performed in an isolated sound booth. For the clinical audiometry, the GSI-61 audiometer and ER-3A insert earphone with specified The standard modified Hughson Westlake approach of down 10 dB, up 5 dB commonly used in audiometric testing was used for quantifying the hearing loss thresholds. Most audiometers have attenuators that are calibrated in 5 dB steps, and more sophisticated models also provide testing in 1 dB, 2 dB, or some other step size that can be manually adjusted. The range of intensities that can be tested with audiometers usually goes from −10 to 120 dB HL. The proposed HearTest application has an upper limit on maximum dB HL that can be tested. It is limited by the maximum output level of the smartphone. HearTest can analyze the hearing loss ranging from −10 to 80 dB HL (except the maximum limit for 0.25 kHz is 75 dB HL).The application tested six frequencies, 0.25, 0.5, 1, 2, 4, and 8 kHz, for both ears. The test began by presenting the test tone at 30 dB HL. If there was no response from the subject, then the intensity was increased in 10 dB steps until the subject confirmed the test tone to be of sufficient intensity. Once the response was obtained, the test tone was lowered by 10 dB until no response and then was increased in steps of 5 dB. The level that could be heard first after three reversals was assumed to be the hearing threshold level. The responses were recorded on the local memory of the device. Test tones were played continuously so that harmonic distortions were minimum and the subject’s attention did not become an issue . When thThe HearTest application started by asking the user for identification and showed instructions about the test procedure. It asked the user to turn the phone volume to maximum and sit in a quiet location before proceeding towards the test. The graphical user interface of the application is presented in After the successful completion of the hearing test for both ears, the application moved to the pure-tone audiogram page, as shown in The results of hearing tests are quantified as the hearing level (HL) in decibels for each test frequency. HL is for a given ear at a specified frequency using a transducer, measured with an audiometer calibrated to reference equivalent threshold level (RETSPL) for air conduction ,32. For For example, standard RETSPLs for pure tone when used with calibrated audiometer and insert earphones (ER-3A) are available in ANSI S3.6-2010 and are shown in Before building the application, RETSPLs were unknown for selected device combinations, we started with using the best available estimate for RETSPLs. Ho et al. presented RETSPLs for EarPods (MB770G) when used with Apple iPad mini 1 . We usedp-value greater than 10%. Spearman’s correlation was calculated as data are not normally distributed and might contain outliers. We used this disagreement between the reference audiometer and HearTest v1 to correct the RETSPL values. Due to the linear relation between hearing thresholds and RETSPLs as per  was carried with respect to the clinical audiometer (GSI-61 with insert earphones ER-3A) through subjective testing on 20 NH participants. Evaluation of the calibrated version of the application with 14 NH participants showed a maximum 4.6 dB HL of mean absolute difference when compared with the reference audiometer, which is significantly lower than the clinically accepted standard (10 dB HL). Retesting of five NH participants before and after calibration supported the validity of the calibration. Relatively low variation of maximum output level among devices of the same model (Apple) suggested that it is feasible to carry out the hearing test using the same calibrated RETSPL values until earphones of the specified impedance and sensitivity are used. Future steps on validation of the application through subjective testing on people with HI and challenges due to “crossover” phenomena were discussed.