EFEMP1 contributes to light-dependent ocular growth in zebrafish

  1. Jiaheng Xie
  2. Bang V. Bui
  3. Patrick T. Goodbourn
  4. Patricia R. Jusuf

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Myopia (short-sightedness) is the most common ocular disorder. It generally develops after over-exposure to aberrant visual environments, disrupting emmetropization mechanisms that should match eye growth with optical power. A pre-screening of strongly associated myopia-risk genes identified through human genome-wide association studies implicates efemp1 in myopia development, but how this gene impacts ocular growth remains unclear. Here, we modify efemp1 expression specifically in the retina of zebrafish. We found that under normal lighting, efemp1 mutants developed axial myopia, enlarged eyes, reduced spatial vision and altered retinal function. However, under myopia-inducing dark-rearing, compared to control fish, mutants remained emmetropic and showed changes in retinal function. Efemp1 modification changed the expression of efemp1 , egr1 , tgfb1a , vegfab and rbp3 genes in the eye, and changes the inner retinal distributions of myopia-associated EFEMP1, TIMP2 and MMP2 proteins. Efemp1 modification also impacted dark-rearing-induced responses of vegfab and wnt2b genes and above-mentioned myopia-associated proteins. Together, we provided robust evidence that light-dependent ocular growth is regulated by efemp1 .

Summary

This study shows that retina-specific modification of efemp1 expression in zebrafish results in myopic eye, and impacts responses to myopia-inducing dark-rearing in eye growth, retinal function, and myopia-associated molecular expression and distribution, implicating light-dependent regulation of efemp1 in ocular development.

Article activity feed

  1. Version published to 10.1101/2023.12.18.572096v2 on bioRxiv
  2. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The manuscript by Xie et al investigates the role of efemp1 in mediating ocular growth. Efemp1, a secreted extracellular matrix glycoprotein, was previously identified as a myopia-risk gene in human GWAS studies. Given that myopia is linked to aberrant eye shape, the authors investigated whether and how this gene mediates eye growth. Using a CRISPR based approach in zebrafish the authors knocked out efemp1 specifically in the retina and established that a myopic eye results. They went further and investigated visual function in these mutant fish using the optomotor response and electroretinograms. As dark-rearing in many animal models has been linked to the induction of myopia, the authors examined the effects of a dark-rearing regimen in efemp1 mutants and found surprisingly that they did not show signs of myopia. Lastly, the expression and distribution of several myopia-associated genes was investigated in the retina of efemp1 mutants and following dark-rearing.

    1. The starting point for this study was the generation of a "retina-specific knockout mutant of the efemp1 gene". However, evidence for a 'successful' knockout at the protein level is missing.

    We have clarified the exact nature of our efemp1*2C-Cas9 model further. The mutants have mosaic genetic modification that do not simply lead to gene deletion (knockout). We have reworded throughout the manuscript to avoid statement indicating the efemp1 *2C-Cas9 fish as a knockout model and instead used “genetic modification” or “genetic disruption”, etc:

    “This gene editing system led to mosaic retinal mutations; each Cas9-expressing retinal cell that were driven by the rx2 promoter would perform its own CRISPR gene editing process, and as a result, even within an individual retina, there were different types of indels (e.g., loss- or gain-of-function mutations, or milder mutations that may cause mislocalization) in different cells.” (Line 104–108).

    For the same reason, it is very challenging to show such a mosaic genetic editing in the protein level. First of all, we were not able to find commercial anti-EFEMP1 antibody for zebrafish that targets specifically the editing sites in our fish model. This means that mutated *efemp1 *DNAs that were transcribed and translated would produce mutant EFEMP1 protein that might still be recognized by an anti-EFEMP1 antibody, although their dysfunction might manifest as altered distribution and thus abnormal ocular development.

    On the other hand, in this study we used a headloop PCR technique, a sensitive genotyping approach that specifically suppresses amplification of wild-type but not mutated *efemp1 *DNA to show that there were genetic modifications in our mutants. However, likely due to the patchy distribution of Cas9-expressing retinal cells (Fig 1A′) and the non-uniform nature of gene editing, our genotyping results showed weak mutant bands (Fig 1C–D), implicating low editing rates. The fact that only a proportion of mutations would result in loss of the protein would make it difficult to distinguish the gene editing in the retina via immunostaining or western blot.

    We have added following in the Results section to indicate the difficulties in showing genetic modification at the protein level for the efemp12C-Cas9 model:

    “On the other hand, due to the mosaic nature of the gene editing, the patchiness of Cas9-expressing retinal cells (Fig 1A′) and the potentially low editing rate, as well as the unavailability of commercial anti-EFEMP1* antibodies that targets specifically the CRISPR editing sites, efemp1 modification in our mutant model at the protein level is challenging to show.*” (Line 125–128)

    Immunostaining for Efemp1 in sections of the entire retina from control and mutant fish would have helped here. It is only in Figure 7 B, C that portions of the inner retina from control and efemp1 2c-Cas9 fish are shown with Efemp1 immunostaining. Control and mutant retinae show slight relative differences in Efemp1 fluorescence levels which are difficult to reconcile with a knock-out scenario.

    As mentioned above, our model is not simply a knockout but a combination of a range of indels that may produce mutant proteins. At least some of them are therefore still likely to bind with the anti-EFEMP1 antibody used in the present study; the antibody does not bind to EFEMP1 regions corresponding to sgRNAs target sites on zebrafish efemp1 DNA. We have added this detail in the Methods to clarify.

    “Noting that the anti-EFEMP1 antibody does not bind to EFEMP1 regions corresponding to sgRNAs target sites on zebrafish efemp1 DNA, thus mutant proteins (if any) may still be labeled by the antibody.” (Line 790–792)

    Therefore, it makes sense that our result showed differences in relative EFEMP1 fluorescence between groups across the inner retina rather than complete loss of EFEMP1 immunostaining in mutant retinas.

    resumably this phenotype is a result of the mosaic expression of Cas9 (GFP) shown in Fig 1? Can the authors explain the reason for this mosaicism?

    We believe that the “mosaic expression of Cas9” the reviewer mentioned is the “patchy distribution of Cas9-expressing retinal cells” as we mentioned in the above response. Yes this is also partially the reason why mutant retinas still present EFEMP1 immunostaining. The patchy (or mosaic) Cas9 expression in the retina of our mutant model can be because we use the Gal4/UAS system to drive the 2C-Cas9 gene editing system. Mosaic expression has long been noticed as a drawback of the Gal4/UAS system. We have modified the manuscript to explain the mosaic Cas9 expression in the mutant retina:

    “The patchiness of Cas9 expression in the mutant retina may attribute to the Gal4/UAS system (Halpern et al., 2008).” (Line 103–104)

    Given this mosaic expression would one expect Efemp1 immunoreactive areas intermingled with areas devoid of Efemp1 in the mutant retina?

    This happens only in cells that CRIPSR eliminates production of EFEMP1, but due to patchy Cas9 expression and perhaps only a little proportion of Cas9-positive cells will lose EFEMP1 protein, our immunostaining did not show apparent intermingling. Importantly, it is worth noting that as our explanation above, anti-EFEMP1 antibody may be able to bind with mutant EFEMP1 proteins and thus EFEMP1 immunostaining will still present in retinal cells with successful gene editing.

    Further, do deficits in the various functional assays the authors perform correlate with the degree of mosaicism?

    We appreciate the reviewer’s interesting idea. As primary goal of the present study is to determine whether retinal-specific *efemp1 *modification has any effect on ocular refraction, we aimed to use fish with as more Cas9-expressing cells as possible for functional analysis, and thus fish used were not expected to have discernible difference in degree of Cas9 expression mosaicism. Therefore, it is not known that whether there is a correlation between ocular deficits and Cas9 expression mosaicism. We thank the reviewer’s suggestion and will bear this idea in mind for future experimental design.

    In the same vein, in Figure 2 the authors refer to variation in GFP levels in the efemp12c-Cas9. It is not clear whether the authors mean levels of GFP in individual cells or numbers of GFP+ cells. Presumably the latter. Could the authors clarify?

    We have added details in the Methods of the manuscript to clarify:

    *“Post-hoc retinal histology indicated that intensity of eGFP fluorescence is corresponding to eGFP positive cell number; fish with higher eGFP fluorescence level had more eGFP positive cells.” *(Line 723–725)

    In my opinion understanding and characterizing the efemp12c-Cas9 fish thoroughly is key to interpreting the phenotypes the authors show subsequently.

    We agree with the reviewer. Due to the characteristics of our 2C-Cas9 model mentioned above, headloop PCR, which is highly sensitive for determining occurrence of gene mutations regardless indel types, is so far the most practical approach for us to provide evidence of successful gene editing. Because there was limited means to show gene modification in the protein level for our mutant model (as mentioned above), we instead provided functional verification of gene modification using OMR. We showed that functionally our 2C-Cas9 model have comparable phenotype with efemp1-knockdown zebrafish that have robust gene disruption induced by morpholino. Overall, with this evidence we believe that there were efemp1 modification in our fish model. Given no other manipulations, the phenotypes are presumably due to the mosaic mutations generated here. We would speculate (though have no data to show this) that a more even and complete knockout of Efemp1 throughout all of the retinal neurons would increase the size of the phenotypic changes seen even more. It was important for us to target the eye to assess the role in the local emmetropisation processes rather than mixing it with possible other CNS defects confounding the phenotype. We were excited to be able to observe quantifiable phenotypes even with such a mosaic randomized mutation model shown here and believe it gives more strength to the role of Efemp1.

    Reviewer #1 (Significance (Required)):

    The wide range of assays the authors perform to assess visual deficits is commendable. Such a comprehensive approach ranging from anatomical, behavioral and electrophysiological assays is poised to identify changes that could otherwise be overlooked. Given the increasing use of zebrafish as models of ocular diseases, this study provides a solid roadmap of the types of analysis possible. This work should be interesting to researchers in the field of myopia research and to basic vision researchers interested in using the zebrafish as a model organism.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary: In this study, the authors used the zebrafish model to study efemp1, a gene that was previously found to be associated with myopia. They used CRISPR-Cas9 to create specific efemp1 knockout in the retina in a mosaic manner. They used a few histological and physiological techniques to evaluate the resulting mutant and found that the efemp1 mutants developed symptoms that are consistent with myopia. The authors further quantified the expression of a few potential target genes in the eye that are potentially implicated in myopia phenotype. The authors also evaluated the differential phenotype of the efemp1 mutant grown in different light conditions that might contribute to myopia.

    Major comments:

    Overall, the authors have provided convincing evidence of the phenotype created by their efemp1 perturbation. Their experiments were thoroughly done and extensively analyzed. They even discussed some potential shortcomings of their study. Their study is a nice first step towards a better understanding of the efemp1 gene function in ocular growth and in myopia. All my comments below should be addressed by clarifications and discussions and not by any new experiments or projects.

    Minor comments:

    1. Elaborate the rationale for choosing efemp1 from the original GWAS study for zebrafish investigation. The authors only mentioned that this gene is among the highest in the rank and its role in myopia is not clear. However, there are quite a few other genes in the GWAS study that were ranked as high, if not higher than efemp1.

    We thank the reviewer for the suggestion. Firstly, in a previous study, we used the high-throughput zebrafish optomotor response assay coupled with morpholino gene knockdown to screen top-ranked myopia-risk genes from the GWAS study. To use zebrafish as a model, additionally we took into account several other factors including existence of zebrafish orthologues, gene expression in the eye, association of ocular phenotypes with risk genes shown in previous zebrafish studies, fatality of gene depletion and availability of characterized morpholinos to prioritize GWAS-associated risk genes for screening. With significant reduction of OMR responses in efemp1 morphants, *efemp1 *was selected as a gene of interest for investigation. As our pre-screen is currently an unpublished study, we were not showing the data in the manuscript, but we are happy to show relevant results to the reviewer if requested. To clarify the selection of this gene, we have added a brief statement in the Introduction:

    “*Our previous study (unpublished) screening GWAS-associated myopia-risk genes with high-throughput optomotor response measurement and morpholino gene knockdown indicated that knockdown of efemp1 in larval zebrafish reduced spatial-frequency tuning function, making efemp1 a candidate gene worth *for further investigation for myopia development.” (Line 53–57)

    On the other hand, in the Introduction of our manuscript, we indeed had covered that in humans, *efemp1 *disruptions, with either gain- or loss-of functions, would lead to visual disease, such as Malattia Leventinese, doyne honeycomb retinal dystrophy, juvenile-onset open-angle glaucoma, or high myopia. These also implicated the importance in understanding the role of *efemp1 *in ocular development.

    Elaborate the rationale for choosing retina as the target tissue of efemp1 knockout, especially when the original GWAS study indicated the expression of EFEMP1 is in cornea, RPE, and sclera, but not in retinal cells.

    Firstly, *efemp1 *is expressed in the retina as shown by our immunostaining in zebrafish and in situ hybridization in mouse in a previous study (PMID: 26162006). We have modified the manuscript to clarify this point:

    “EFEMP1 is a secreted extracellular matrix glycoprotein widely expressed throughout the human body, especially in elastic fiber-rich tissues, for examples, the brain, lung, kidney and eye including the retina (Livingstone et al., 2020; Mackay et al., 2015).” (Line 51–53)

    In future studies it will be interesting to perform similar somatic efemp1 manipulation in other ocular tissues to examine whether this gene has tissue-dependent functions for ocular growth. Nonetheless, our results demonstrated that at the very least retinal *efemp1 *is involved in ocular development.

    Secondly, the rx2 gene is indeed also expressed in the RPE in zebrafish (PMID: 11180949), meaning that there were also RPE cells expressing Cas9 driven by rx2. We have added this detailed to the manuscript:

    “In this transgenic zebrafish line, Tg(rx2:Gal4) is expressed specifically in the retina and the RPE (Chuang and Raymond, 2001), due to the retina-specific retinal homeobox gene 2 (rx2) promoter.” (Line 95–97)

    Importantly, as myopia generally develops due to dysregulated gene-environment interactions, modification of *efemp1 *specifically in the light-sensing retina allowed us to investigate the interaction of *efemp1 *with visual environment. We have added this point to the manuscript:

    “In order to investigate the role of the efemp1 gene and its interaction with visual environment, we first generated a zebrafish line with efemp1 modification specifically in the retina (efemp12C-Cas9; Fig 1A), the light-sensing tissue in the eye, using a 2C-Cas9 somatic CRISPR gene editing system (Di Donato et al., 2016).” (Line 92–95)

    Discuss possible ways of modifying efemp1 gene in the retina that would be more uniform and would not create mosaicism and/or heterogenous mutations that can complicate downstream characterizations and interpretations as the authors currently experienced.

    We appreciate the reviewer’s suggestion. One possible way of generating uniform tissue-specific gene modification is to use the Cre-loxP recombination system. We have modified the Discussion of manuscript as per reviewer’s suggestion:

    “To avoid such heterogeneous tissue-specific gene editing, the Cre-LoxP system is an option­: using tissue-specific driven Cre recombination to delete LoxP flanked exons of the target gene.” (Line 482–486)

    • Added to discussion –

    The authors should elaborate further on the effect of the mosaicism and heterogenous mutations on efemp1, a presumably excreted protein, on regulating the ocular growth.

    We appreciate the reviewer’s interesting point of view. However, it is very difficult to identify a regionalized effect of mosaicism and heterogenous mutations of efemp1 on ocular growth even with dissected eyes. It is likely that distribution of Cas9-expressing cells was mosaic but still overall even across the retina. Perhaps in other models that allow controlled regional efemp1 manipulation in the eye, for sample, using gene promoters that present dorsal to ventral gradient, comparisons between modified and unmodified regions in the same eye will help to unravel whether efemp1 regulates eye growth only around the location where it was produced.

    How did the downstream genes they studied affect by the messing up of the extracellular Efemp1? Is it through altering the Egf signal transduction?

    Throughout the Discussion we have tried to cover how efemp1 disruption affect myopia-associated genes where it is possible by linking our results with literature. However, there were not enough details from the literature showing direct pathways between *efemp1 *and the tested myopia-risk genes. These will be interesting topics for further investigation. To our knowledge, there is no evidence that myopia-associated genes we analyzed in the study are transduced by Egf signaling.

    If possible, discuss the original SNP that was associated with efemp1 and the potential mechanisms through which the SNP affects human EFEMP1; Then, discuss how the study of zebrafish efemp1 mutant can aid our understanding of the human's SNP.

    Unfortunately, this information is not available. In the meta-analysis our work is based on Efemp1 ranked highly based on biological and statistical evidence. In figure 5 of Tedje et al., 2018, we can see Efemp1 in the first place. Where available, the annotation (light blue column) would indicate whether the variant was found in exonic, UTR or transcribing RNA. Nothing was identified for Efemp1 – which could mean that it is expressed in regulatory sequencing further away.

    Typo: Page 15, Line 299: Loss of this gene "promotes".

    Thanks to the reviewer, we have corrected the typo.

    Reviewer #2 (Significance (Required)):

    This study is an interesting and potentially significant addition to the ophthalmology field, as it conducted an initial characterization of a candidate gene for myopia in zebrafish and observed a relevant phenotype after the gene knockout. Colleagues in the myopia field will find the results interesting. In addition, colleagues in the zebrafish field will find the in-depth characterizations and tools used in the paper very informative.

    I have conducted research in the human genetics of ophthalmology, gene expression analysis, zebrafish eye development and diseases. I believe my background allows me to effectively appreciate and evaluate the findings of this manuscript.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary The authors use a retinal-specific promoter to target zebrafish efemp1 for inactivation to study its effects on the eye. Their use of the DiDonato/del Bene 2C-Cas9 system is a good method to target only cells that express a specific promoter i.e. rx2. Following this (mosaic and transient) targeting of efemp1, the authors describe enlarged eyes and myopia development, as well as reduced spatial visual sensitivity and altered retinal function by ERG analysis. Furthermore, expression levels of egr1, tgfb1a, vegfb, and rbp3 are altered, as well as Timp2 and Mmp2 proteins. Finally, dark-rearing of efemp1 mutant fish is reported to lead to emmetropization, rather than myopia.

    Major comments

    1. The data presented by the authors are interesting, and likely due to efemp1 disruption in the eye. However, the authors should clarify or explain several points, and improve on experimental rigor. Figure 1 C, D- PCRs are not convincing for loss of efemp1. The authors should consider PCR reactions that would show deletion driven by both CRISPRs, or an RFLP reaction based on conventional PCR that would show differences if individual CRISPRs were effective.

    Our zebrafish model is not simply a complete knockout model (please see response to reviewer 1’s comment 1 for details). In our model, even in a retina, there will be different indels in cells that expresses Cas9, including gain- or loss-of-function mutations, or mutations that do not even influence its function. In some cases, even with CRISPR cutting, DNA will recover to be wildtype. Thus, even with FACS to sort for Cas9+ (GFP+) cells, it is not possible to provide evidence for such gene modification using conventional PCR, because as long as there is a unmutated target sequence there will be PCR production. Because of this, headloop PCR as a well-established, highly sensitive approach is specifically suitable for our case.

    There needs to be better evidence that efemp1 is being edited (e.g. Western blot, or qPCR).

    As described in our response to reviewer 1’s comment 1, due the way *efemp1 *gene was modified in the retina in our model and the unavailability of suitable commercial antibodies, western blot is currently not an option for us. For qPCR, theoretically it is a way to show genetic modification at the transcriptional level, if combined with FACS from dissected eyes and sgRNA target sites specific primers. However, in reality it is not very practical to perform. First of all, even in our model with more Cas9+ cells, due to the patchy expression, the number of these cells are in fact low in a retina. This means that the number of fish to get enough cells for RNA isolation would be much higher, likely to be hundreds of fish. Moreover, in each clutch the number of fish with higher Cas9+ cell number is generally low, estimated to be only ~5%. Overall, this indicates that a large number of fish are required to even just get one sample for such an experiment. With evidence from headloop PCR and visual phenotype verification (OMR; Fig 1E–H and Fig S1), we believe it is certain that efemp1 gene has been modified. As mentioned also, the ability to identify quantifiable phenotypic differences in this model despite the mosaic Cas9 activity and random indels in different cells is highly suggestive of a full knockout of Efemp1 in the eye causing an even larger phenotype.

    The data in Figure 7 are not convincing that EFEMP1 protein levels are substantially reduced in mutants.

    This is expected. Please see response to reviewer 1’s comment 2.

    Why are efemp12C-Cas9 eyes smaller with normal lighting? (Figure S2)

    Fig S2 showed that efemp1*2C-Cas9 fish have smaller eye size than control fish only at 2 weeks of age. As shown by our survival data (Fig 2C), fish with more severe gene modification (implicated by more GFP+ cells, GFP+++ fish) are possibly died by 4 weeks of age, likely due to severe deficits in visually driven predation and subsequently nutrition deficiency. These fish thus gradually develop smaller size of the body including the eye with age, compared to control fish. Therefore, it makes senses that overall mutant fish have smaller eyes at 2 weeks of age but as GFP+++ fish die by 4 weeks, the group averaged eye size returned to a level similar to control fish. The fish survived are likely the ones that have mild mutations, which allow them to remain some levels of vision for feeding and develop without discernibly smaller eye size. Because there was variability of eye size in zebrafish caused by either development or gene manipulation, we used a relative calculation (ratio of retinal radius to lens radius) as a myopia index for comparison.

    The clustering of datapoints in Figure 2B, 4B, overlaps extensively between control and mutant, and it is not easy to be sure that the high significance scores (***) are accurate.

    We thank the reviewer for pointing out this concern. Though data points overlapped to some levels, in general difference between group means were apparent and the range that they deviate (i.e., mean ± SEM) were barely overlap. We realise it was difficult to see the SEM error bars, as they were so close to the mean, that they were hard to distinguish. We have adjusted our figures for clearer visualization of the error bars. Hopefully this will better show how far apart the data are as reflected by the significance scores.

    The authors should consider discussing whether loss of efemp1 is developmental only, or sustained. rx2 is likely to be switched off after development, and retinal cells that arise after the rx2:Gal4 ceases to be active will have a normal quotient of efemp1.

    Genetic modification in our mutant model is sustained. It is true that *rx2 *is only transiently expressed during early development, but once gDNA in a cell was modified by a CRISPR editing event driven by the 2C-Cas9 system, it remains throughout cell divisions (the same mutation would be copied during DNA synthesis) and cell lifetime. In addition, it has been showed that in adult teleost activation of *rx2 *in retinal stem cells in the retinal ciliary marginal zone determines its fate to form retinal neurons (PMID: 25908840). Therefore, in new neurons derived from retinal stem cells in the adult zebrafish retina, there is expression of rx2 to drive the 2C-Cas9 system for genetic modification. We have added relevant details to the Result section:

    “Despite the mosaicism, the mutations resulted from the 2C-Cas9 system in retinal cells is expected to be sustained. Also, in adult teleost, activation of rx2 in retinal stem cells in the retinal ciliary marginal zone determines its fate to form retinal neurons (Reinhardt et al., 2015). This suggested that in new neurons derived from retinal stem cells in the adult zebrafish retina, there may be expression of rx2 to drive the 2C-Cas9 system for genetic modification.” (Line 108–116)

    The authors should also consider a more detailed discussion of the mechanism mediated by/through efemp1 that alters retinal function and expression of other genes.

    We appreciate the reviewer’s suggestion. It is possible to add more detailed discussion to the manuscript for potential mechanistic links, but ultimately such content would be highly speculative and may lead to over-interpretation of the data. Moreover, a comprehensive overview of detailed mechanisms of how *efemp1 *may alter retinal function and expression of relevant genes will require space and significantly lengthen the manuscript, with however only minimal improvement. Therefore, we believe it is reasonable to only touch the most relevant as we did for the manuscript.

    Finally, since a full mouse knockout of efemp1 exists (Daniel et al, 2020), it is not clear why a retinal-specific zebrafish model would give better insight into the phenotype.

    There are several advantages of our 2C-Cas9 zebrafish model. Firstly, with a retinal specific modification of the *efemp1 *gene, we are able to rule out systematic effect. Essentially our focus is the role of *efemp1 *in specifically ocular development. Secondly, with their smaller size, rapid development, high reproductivity, and ease of genetic and environmental manipulations, zebrafish allow us to perform large-scale high-throughput investigation with different genetic and environmental combinations. Furthermore, by changing the promoter that drives Gal4 expression in our model, we can target precisely different retinal neuron subtypes to characterize which and how different visual circuits are involved.

    Minor comments

    "Myopia is the most common ocular disorder" is overly broad and needs qualifiers.

    We appreciate the reviewer’s rigorousness. However, myopia is in fact the most common ocular disorder around the world. We have mentioned in the Introduction that “Myopia (short-sightedness) is now the most common visual disorder, and is predicted to impact approximately half of the world population by 2050 (Holden et al., 2016).” Therefore, we believe a qualifier is appropriate.

    Line 36 - what ocular changes cannot be easily managed?

    We thank the reviewer’s suggestion. We have modified the Introduction manuscript to add some examples:

    “Although considered manageable with optical correction, the development of high levels of myopia (or pathological myopia) brings with it ocular changes that promote eye diseases that cannot be easily managed (glaucoma, cataract, myopic maculopathy, etc.) (Hayashi et al., 2010; Ikuno, 2017; Marcus et al., 2011).” (Line 34–37)

    Why does loss of retinal efemp1 cause reduced OMR response? Unlikely to be refractive error at this stage.

    We have modified the Discussion as per the reviewer’s concern:

    “We noticed that although 5 dpf efemp12C-Cas9 fish overall were not myopic relative to efemp1+/+ fish (Fig 2B), they showed reduced spatial-frequency tuning function (Fig 1E–H). This phenotype, if not due to refractive error, can be a result of altered visual processing, as aberrant extracellular matrix caused by efemp1 disruption may lead to dysfunctional synapses (Dityatev and Schachner, 2006).” (Line 491–494)

    Which Timp2 (Timp2a or Timp2b) is visualized in Figure 7?

    Thanks to the reviewer for raising this point. We have added relevant details to the Methods:

    “The anti-TIMP2 antibody was developed based on human TIMP2. In a previous study this antibody was showed to label for zebrafish TIMP2a (Zhang et al., 2003). As similarity of zebrafish TIMP2b to human TIMP2 is much lower than that of zebrafish TIMP2a (60.55% vs. 71.23%), labelling of zebrafish TIM2b is less likely. Yet, we are not able to completely rule out this possibility due to lack of information of the exact immunogen.” (Line 790–794)

    Why is the inner retina studied for altered protein expression, but not the rest of the eye? Myopia is primarily driven by growth of the outer retinal/sclera.

    The reason why we focused on the inner retina is that in our study, prominent expression differences of our proteins of interest between groups were mainly noticed on the inner but not outer retina. We agree that the outer retina is a key driver for visually regulated ocular growth, yet the inner retina also plays a crucial role. There is abundant evidence that the inner retina is involved in development of ocular refraction. For examples, Cx36, Egr1 and dopamine pathways in the inner retina have been reported to be associated with regulation of ocular refraction (PMID: 10412059; PMID: 28602573; PMID: 25052990; PMID: 32547367). We believe it is reasonable to focus on the inner retina, were we observed robust quantifiable expression for the tested proteins in our case.

    Reviewer #3 (Significance (Required)):

    General assessment: This study uses retinal-specific inactivation of efemp1 with a clever methodology to study its effects on the eye. However, the necessity of these experiments is not well explained, as a full mouse knockout line exists. • Advance: There are some interesting observations about gene expression following efemp1 inactivation, and useful experiments that look at the combination of genetics with environmental conditions on refractive error. This builds on studies by the Hulleman group on efemp1's role in the eye by adding functional information. • Audience: This will be of interest to both basic researchers and clinicians who study genetic influencers of the eye.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary

    The authors use a retinal-specific promoter to target zebrafish efemp1 for inactivation to study its effects on the eye. Their use of the DiDonato/del Bene 2C-Cas9 system is a good method to target only cells that express a specific promoter i.e. rx2. Following this (mosaic and transient) targeting of efemp1, the authors describe enlarged eyes and myopia development, as well as reduced spatial visual sensitivity and altered retinal function by ERG analysis. Furthermore, expression levels of egr1, tgfb1a, vegfb, and rbp3 are altered, as well as Timp2 and Mmp2 proteins. Finally, dark-rearing of efemp1 mutant fish is reported to lead to emmetropization, rather than myopia.

    Major comments

    The data presented by the authors are interesting, and likely due to efemp1 disruption in the eye. However, the authors should clarify or explain several points, and improve on experimental rigor. Figure 1 C, D- PCRs are not convincing for loss of efemp1. The authors should consider PCR reactions that would show deletion driven by both CRISPRs, or an RFLP reaction based on conventional PCR that would show differences if individual CRISPRs were effective. There needs to be better evidence that efemp1 is being edited (e.g. Western blot, or qPCR). The data in Figure 7 are not convincing that EFEMP1 protein levels are substantially reduced in mutants. Why are efemp12C-Cas9 eyes smaller with normal lighting? (Figure S2) The clustering of datapoints in Figure 2B, 4B, overlaps extensively between control and mutant, and it is not easy to be sure that the high significance scores (***) are accurate. The authors should consider discussing whether loss of efemp1 is developmental only, or sustained. rx2 is likely to be switched off after development, and retinal cells that arise after the rx2:Gal4 ceases to be active will have a normal quotient of efemp1. The authors should also consider a more detailed discussion of the mechanism mediated by/through efemp1 that alters retinal function and expression of other genes. Finally, since a full mouse knockout of efemp1 exists (Daniel et al, 2020), it is not clear why a retinal-specific zebrafish model would give better insight into the phenotype.

    Minor comments

    "Myopia is the most common ocular disorder" is overly broad and needs qualifiers. Line 36 - what ocular changes cannot be easily managed? Why does loss of retinal efemp1 cause reduced OMR response? Unlikely to be refractive error at this stage. Which Timp2 (Timp2a or Timp2b) is visualized in Figure 7? Why is the inner retina studied for altered protein expression, but not the rest of the eye? Myopia is primarily driven by growth of the outer retinal/sclera.

    Significance

    • General assessment: This study uses retinal-specific inactivation of efemp1 with a clever methodology to study its effects on the eye. However, the necessity of these experiments is not well explained, as a full mouse knockout line exists.
    • Advance: There are some interesting observations about gene expression following efemp1 inactivation, and useful experiments that look at the combination of genetics with environmental conditions on refractive error. This builds on studies by the Hulleman group on efemp1's role in the eye by adding functional information.
    • Audience: This will be of interest to both basic researchers and clinicians who study genetic influencers of the eye.
  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this study, the authors used the zebrafish model to study efemp1, a gene that was previously found to be associated with myopia. They used CRISPR-Cas9 to create specific efemp1 knockout in the retina in a mosaic manner. They used a few histological and physiological techniques to evaluate the resulting mutant and found that the efemp1 mutants developed symptoms that are consistent with myopia. The authors further quantified the expression of a few potential target genes in the eye that are potentially implicated in myopia phenotype. The authors also evaluated the differential phenotype of the efemp1 mutant grown in different light conditions that might contribute to myopia.

    Major comments:

    Overall, the authors have provided convincing evidence of the phenotype created by their efemp1 perturbation. Their experiments were thoroughly done and extensively analyzed. They even discussed some potential shortcomings of their study. Their study is a nice first step towards a better understanding of the efemp1 gene function in ocular growth and in myopia. All my comments below should be addressed by clarifications and discussions and not by any new experiments or projects.

    Minor comments:

    • Elaborate the rationale for choosing efemp1 from the original GWAS study for zebrafish investigation. The authors only mentioned that this gene is among the highest in the rank and its role in myopia is not clear. However, there are quite a few other genes in the GWAS study that were ranked as high, if not higher than efemp1.
    • Elaborate the rationale for choosing retina as the target tissue of efemp1 knockout, especially when the original GWAS study indicated the expression of EFEMP1 is in cornea, RPE, and sclera, but not in retinal cells.
    • Discuss possible ways of modifying efemp1 gene in the retina that would be more uniform and would not create mosaicism and/or heterogenous mutations that can complicate downstream characterizations and interpretations as the authors currently experienced.
    • The authors should elaborate further on the effect of the mosaicism and heterogenous mutations on efemp1, a presumably excreted protein, on regulating the ocular growth. How did the downstream genes they studied affect by the messing up of the extracellular Efemp1? Is it through altering the Egf signal transduction?
    • If possible, discuss the original SNP that was associated with efemp1 and the potential mechanisms through which the SNP affects human EFEMP1; Then, discuss how the study of zebrafish efemp1 mutant can aid our understanding of the human's SNP.
    • Typo: Page 15, Line 299: Loss of this gene "promotes".

    Significance

    This study is an interesting and potentially significant addition to the ophthalmology field, as it conducted an initial characterization of a candidate gene for myopia in zebrafish and observed a relevant phenotype after the gene knockout. Colleagues in the myopia field will find the results interesting. In addition, colleagues in the zebrafish field will find the in-depth characterizations and tools used in the paper very informative.

    I have conducted research in the human genetics of ophthalmology, gene expression analysis, zebrafish eye development and diseases. I believe my background allows me to effectively appreciate and evaluate the findings of this manuscript.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Xie et al investigates the role of efemp1 in mediating ocular growth. Efemp1, a secreted extracellular matrix glycoprotein, was previously identified as a myopia-risk gene in human GWAS studies. Given that myopia is linked to aberrant eye shape, the authors investigated whether and how this gene mediates eye growth. Using a CRISPR based approach in zebrafish the authors knocked out efemp1 specifically in the retina and established that a myopic eye results. They went further and investigated visual function in these mutant fish using the optomotor response and electroretinograms. As dark-rearing in many animal models has been linked to the induction of myopia, the authors examined the effects of a dark-rearing regimen in efemp1 mutants and found surprisingly that they did not show signs of myopia. Lastly, the expression and distribution of several myopia-associated genes was investigated in the retina of efemp1 mutants and following dark-rearing.

    The starting point for this study was the generation of a "retina-specific knockout mutant of the efemp1 gene". However, evidence for a 'successful' knockout at the protein level is missing. Immunostaining for Efemp1 in sections of the entire retina from control and mutant fish would have helped here. It is only in Figure 7 B, C that portions of the inner retina from control and efemp12c-Cas9 fish are shown with Efemp1 immunostaining. Control and mutant retinae show slight relative differences in Efemp1 fluorescence levels which are difficult to reconcile with a knock-out scenario. Presumably this phenotype is a result of the mosaic expression of Cas9 (GFP) shown in Fig 1? Can the authors explain the reason for this mosaicism? Given this mosaic expression would one expect Efemp1 immunoreactive areas intermingled with areas devoid of Efemp1 in the mutant retina? Further, do deficits in the various functional assays the authors perform correlate with the degree of mosaicism? In the same vein, in Figure 2 the authors refer to variation in GFP levels in the efemp12c-Cas9. It is not clear whether the authors mean levels of GFP in individual cells or numbers of GFP+ cells. Presumably the latter. Could the authors clarify? In my opinion understanding and characterizing the efemp12c-Cas9 fish thoroughly is key to interpreting the phenotypes the authors show subsequently.

    Significance

    The wide range of assays the authors perform to assess visual deficits is commendable. Such a comprehensive approach ranging from anatomical, behavioral and electrophysiological assays is poised to identify changes that could otherwise be overlooked. Given the increasing use of zebrafish as models of ocular diseases, this study provides a solid roadmap of the types of analysis possible. This work should be interesting to researchers in the field of myopia research and to basic vision researchers interested in using the zebrafish as a model organism.

  6. Version published to 10.1101/2023.12.18.572096v1 on bioRxiv