Genetic and pharmacologic alterations of claudin9 levels suffice to induce functional and mature inner hair cells

Reviewer #1 (Public review):

The focus of this manuscript was to investigate the role of Cldn9 in the development of the mammalian cochlea. The main rationale of the study is the fact that cochlear hair cells do not regenerate, so when damaged they are lost forever, causing irreparable hearing loss. The authors have attempted to address this problem by inducing the ectopic production of additional hair cells and test whether they acquire the morphological and functional characteristics of native hair cells. They show that downregulation of Cldn9 using a well-established genetic manipulation of transgenic mice led to the production of extra numerary inner hair cells, which were able to survive for several months. By performing a large battery of experiments, the authors were able to determine that the native and ectopic inner hair cells have comparable morphological and physiological characteristics. There are several conclusions highlighted by the authors in different parts of the manuscript, including the key role of Cldn9 in coordinating embryonic and postnatal development, the differentiation of supporting cells into inner hair cells, and the possible use of Cldn9 to induce inner hair cell differentiation following deafness induced by hair cell loss.

Comments on revised version:

The authors have addressed the following points raised during the first submission: statistical analysis and wave 1 analysis. However, very little was done to address the other key aspects of my report, which are essential for the interpretation of the results. As mentioned in my previous report, some aspects of the work are not justified by the current data and will require either a tone-down of the claims or further experiments.

For example, one puzzling finding that is not addressed in the manuscript is the lack of functional benefit from these additional inner hair cells. In fact, it appears to be detrimental based on the increased ABR thresholds and EP. So, it is not clear to this reviewer the advantage of this approach.

It is not clear what direct evidence there is, apart from some immunostaining, indicating that the ectopic inner hair cells derive from the supporting cells. This part would benefit from a more careful consideration and maybe an attempt at a more direct experimental approach. Alternatively, the text should be modified accordingly.

One point that should be made clear throughout the manuscript is that the ectopic inner hair cells are generated in a cochlea that is undergoing normal maturation. Thus, there is no guarantee that modulating the expression levels of Cldn9 in a deaf mouse lacking hair cells would produce the same result as that shown in this study. This point should be at least discussed.

Reviewer #3 (Public review):

The study by Chen et al reports an interesting and previously unknown phenomenon of generation of supernumerary inner hair cells (IHCs) in response to downregulation of Cldn9 during embryonic or postnatal development. The authors developed an inducible doxycycline (dox)-tet-OFF-Cldn9 transgenic mice to regulate expression levels of Cldn9 and show that downregulation of Cldn9 resulted in additional, although incomplete row of IHCs immediately adjacent to the original IHC row. These induced extra IHCs had similar well-developed hair bundles, able to mechanotransduce and were innervated by auditory neurons, resembling wild-type IHCs. In addition, the authors knock down Cldn9 postnatally using shRNA injections in P1-7 mice with similar induction of extranumerary IHCs next to the original row of IHCs. The conclusions of this paper are mostly well supported by the data. However, some data analyses are limited, and some important controls are not shown.
The data from this study are important and promising for future gene therapy applications. The generation of extra IHCs postnatally using downregulation of Cldn9 by shRNA could potentially be used as a replacement of IHCs lost after noise-induced trauma, ototoxic agents, or other environmental trauma. However, it is not clear if downregulation of CLDN9 in adult mice would lead to extranumerary IHCs. On the other hand, the replacement of lost inner hair cells due to various genetic mutations by inducing supernumerary mutant IHCs with the same abnormalities would not be reasonable.

The authors show that postnatally generated ectopic IHCs are viable and mechanotransducive, but the hearing function of the mice with ectopic hair cells is not improved. However, the ectopic hair cells seems to be generated from supporting cell trans-differentiation, and the intricate mosaic of the organ of Corti is altered (the extra row of IHCs seems to be positioned immediately adjacent to the original IHC row), which could by itself lead to hearing issues. It is not clear if the newly formed unusual junctions between the ectopic and original IHCs are sufficiently tight to prevent leakage of the endolymph to the basolateral surface of IHCs. Also, it is not clear if the other organ of Corti tight junctions could lose their tightness due to the downregulation of Cldn9, which could over time affect the endocochlear potential and hearing abilities as shown by this study.

Overall, the manuscript could be of interest to scientists working in the inner ear development and regeneration field, and to the hearing researchers in general and perhaps developmental biologists and cell biologists interested in tight junction proteins and their function.

Strength

The methodologies used are solid and convincing. There is a great potential for practical use of these valuable findings and new knowledge on IHC developmental regulation by Cldn9 expression.

Weakness

Some of the data in this study would benefit from showing corresponding negative controls and higher-resolution images of CLDN9 localization, which the authors chose not to show in the revised manuscript. Importantly, CLDN9 immunofluorescence staining data look different from previously published observations and show cytoplasmic staining of supporting cells only and did not show the staining of tight junctions between the OHCs and supporting cells as well as between the IHCs and supporting cells as reported previously (Kitajiri et al., 2004; Nakano et al., 2009, Ramzan et al., 2021). The organ of Corti schematics showing CLDN9 expression reflects the authors' immunostaining data but is unusual considering that CLDN9 localizes to the tight junctions of the reticular lamina as was shown by immuno-EM in this study and described in previous publications (Kitajiri et al., 2004; Nakano et al., 2009, Ramzan et al., 2021). However, the authors did not provide an explanation for these discrepancies in the Discussion of the manuscript.

Also, more detailed investigations would in some instances clarify the data. For example, it is not clear if the downregulation of Cldn9 affects the other genes known to participate in cell fate determination, and why downregulation of Cldn9 expression resulted in production of extranumerary inner hair cells only and not the other cell types, like OHCs, for example.

Reviewer #4 (Public review):

The work by Yingying Chen, Jeong Han Lee, and co-authors summarizes the morphological and functional outcomes of Cldn9 loss in the inner ear, particularly in the organ of Corti. While the study does not provide mechanistic insights into how the developmental loss of Cldn9 leads to ectopic hair cell formation, the phenomenon itself is curious. The work primarily focuses on a detailed characterization of the ectopic hair cells, which is well done. Despite the lack of mechanistic insights, the study will be of interest to the inner ear field if several major issues with the manuscript are addressed.

(1) The title, "Genetic and pharmacologic alterations of claudin9 levels suffice to induce functional and mature inner hair cells," is misleading. First, both manipulations (knockout and knockdown) are genetic, and no pharmacology is involved. Second, both manipulations are carried out during the embryonic and neonatal periods, and there is no evidence of mature hair cell regeneration in this study. The title should be revised to reflect this. A more accurate title could be: "Developmental loss of Cldn9 results in functional ectopic inner hair cells that persist through adulthood."
(2) Contact-mediated lateral inhibition in hair cell fate determination is one of the most well-studied phenomena in the inner ear field, and numerous groups have shown that it is mediated by Notch signaling. This must be added to the introduction.
(3) A large body of literature has demonstrated that Notch inhibition alone is not sufficient to regenerate hair cells in adult mice. Therefore, if the loss of claudins disrupts Notch signaling-the proposed mechanisms in the discussion - it is unlikely to be a viable therapeutic strategy for hair cell regeneration in the adult ear. Furthermore, no hair cell ablation experiments were conducted to demonstrate what could be considered true regeneration. These speculative statements should be removed or revised accordingly.
(4) Cldn9 is a tight junction protein and should localize to the membrane. Yet, the data presented show what appears to be diffuse cytoplasmic staining, which is concerning.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

The focus of this manuscript was to investigate the role of Cldn9 in the development of the mammalian cochlea. The main rationale of the study is the fact that cochlear hair cells do not regenerate, so when damaged they are lost forever, causing irreparable hearing loss. The authors have attempted to address this problem by inducing the ectopic production of additional hair cells and testing whether they acquire the morphological and functional characteristics of native hair cells. They show that downregulation of Cldn9 using a well-established genetic manipulation of transgenic mice led to the production of extra numerary inner hair cells, which were able to survive for several months. By performing a large battery of experiments, the authors were able to determine that the native and ectopic inner hair cells have comparable morphological and physiological characteristics. There are several conclusions highlighted by the authors in different parts of the manuscript, including the key role of Cldn9 in coordinating embryonic and postnatal development, the differentiation of supporting cells into inner hair cells, and the possible use of Cldn9 to induce inner hair cell differentiation following deafness induced by hair cell loss.

Strengths:

Several of the conclusions in this study are well supported by the experimental work.

Weaknesses:

Some aspects of the data and its interpretation needs better explanation and requires further investigation.

(1) The Results section is the most difficult part to read and understand. It contains a very limited, and in some places confusing and repetitive, description of the data. Statistical analysis is missing for some of the key data (e.g., ABRs), and in some places the text contradicts the data presented in the figures (e.g., Figure 8). I am sure carefully revising the text would clarify some of these issues.

We thank the reviewer for the suggestion. We revised parts of the results section and added the statistical analysis to the ABRs and DPOAE (lines 151-159; Page 29, lines 846-880).

(2) One puzzling finding that is not addressed in the manuscript is the lack of functional benefit from these additional inner hair cells. In fact, it appears to be detrimental based on the increased ABR thresholds. Maybe it would be useful to analyze the wave 1 characteristics.

We thank the reviewer for the suggestion. We added the wave 1 characteristics as S8.

(3) It is not clear what direct evidence there is, apart from some immunostaining, indicating that the ectopic inner hair cells derive from the supporting cells. This part would benefit from a more careful consideration and maybe an attempt at a more direct experimental approach.

We thank the reviewer for the suggestion. We intend to investigate the origin of the ectopic inner hair cells using (for example, a qRT-PCR, sm FISH, etc.) in our future study.

(4) One point that should be made clear throughout the manuscript is that the ectopic inner hair cells are generated in a cochlea that is undergoing normal maturation. Thus, there is no guarantee that modulating the expression levels of Cldn9 in a deaf mouse lacking hair cells would produce the same result as that shown in this study. My guess is that it probably won't, but I am sure this could be tested (maybe in the future) using the excellent experimental approach applied in this study.

That is a great point. We will explore it in our future experiments.

Reviewer #2 (Public Review):

Summary:

The generation of functional extranumerary inner hair cells (IHCs) in postnatal mice, particularly with virus-mediated knockdown of Cldn9 mRNA expression in the neonatal cochlear duct, is an important observation. It is significant because not many studies exist that report molecular manipulations of the neonatal organ of Corti that result in the generation of new hair cells that remain functional and appear to be intact for an extended time, here more than one year. Overall, this is a carefully conducted study; the observations are clear, and the methods are solid. Two independent methods for reducing the expression of Cldn9 mRNA were used: a conditional transgenic model and AAV-mediated knockdown with shRNA. The lack of a functional explanation of how the reduced expression of Cldn9 specifically leads to the formation of extranumerary IHCs leaves open questions. For example, it is not clear whether there is indeed a fate change happening and whether Cldn9 reduction affects developmental processes. The discussion of how Cldn9 reduction potentially affects Notch signaling, without hard evidence, is handwaving.

Strengths:

It is a very interesting observation and somewhat unexpected in its specificity for inner hair cells. Using two different approaches to manipulate Cldn9 expression provides a strong experimental foundation. The study is conducted quantitatively and with care.

Weaknesses:

The lack of mechanistic insight results in an open-ended story where at least the potential interaction of Cldn9 reduction with known and well-characterized signaling pathway components should have been investigated. This missed opportunity limits the scope of the study and should be addressed: How does Cldn9 downregulation affect the expression levels of other known genes linked to hair cell production and cell fate decisions? Quantitative RT-PCR works well for the authors, and comparing the expression of Notch or other known pathway components could provide mechanistic insight.

We thank the reviewer for the suggestion. We did quantitative RT-PCR to compare the expression of Notch or other known pathway components in our future work. Besides, we used smFISH with ccnd1 probe and cdkn1b probe to detect cyclin D1 and cyclin-dependent kinase inhibitor 1B (p27) separately in the mouse cochlea. GAPDH was selected as a reference gene. The quantification results showed no significant difference between Cldn9^+/T mice and Cldn9^+/+ mice at P2, P7, and P14.

It is unclear how P21 inner hair cells were identified for the patch-clamp experiments shown in Fig 4E-H. This is a challenging endeavor without the possibility of using specific markers.

We did not have a specific marker for IHCs. However, one with experience in hair bundle morphology and knowledge of their location in the epithelia can identify IHCs from the upright microscope.

Please also address the numerous minor points outlined below; it will improve the paper's readability.

Thanks. Please find the point-to-point answers below.

Please include page numbers and line numbers in a revised manuscript.

We include page numbers and line numbers in a revised manuscript.

Reviewer #3 (Public Review):

This important study by Chen et al help in advancing our knowledge about the regulation of inner hair cell (IHC) development and revealed the role of Cldn9 in IHC embryonic and postnatal induction by transdifferentiation from the supporting cells. The authors developed an inducible doxycycline (dox)-tet-OFF-Cldn9 transgenic mice to regulate expression levels of Cldn9 and show that downregulation of Cldn9 resulted in additional, although incomplete row of IHCs immediately adjacent to the original IHC row. These induced extra IHCs had similar well developed hair bundles, able to mechanotransduce and were innervated by auditory neurons resembling wild-type IHCs. In addition, the authors knock down Cldn9 postnatally using shRNA injections in P1-7 mice with similar induction of extranumerary IHC next to the original row of IHCs. The conclusions of this paper are mostly well supported by the data, but some data analysis needed to be clarified and some crucial controls should be provided to improve the confidence in the presented results. There is a great potential for practical use of these valuable findings and new knowledge on IHC developmental regulation to design Cldn9 gene therapy in the future.

The described by Chen et al mechanisms of extra hair cell generation by suppression of the tight junction protein Cldn9 expression level are very interesting and previously unknown. In particular, the generation of extra IHCs postnatally using downregulation of Cldn9 by shRNA could potentially be very useful as a replacement of HCs lost after noise-induced trauma, ototoxic agents, or other environmental trauma. On the other hand, the replacement of lost hair cells due to various genetic mutations by inducing a supernumerary IHCs with the same abnormalities would not be reasonable.

The authors show that postnatally generated ectopic IHCs are viable and mechanotransducive, but it would be nice to show the maturation steps of ectopic IHC during this postnatal period. For example, stereocilia bundles of the ectopic hair cells should mature later than the original IHCs. A few days after viral delivery of shRNA, you should be able to observe immature IHC bundles that unequivocally will define newly generated IHCs. Unfortunately, the authors show only examples of already mature ectopic IHCs at P21 and in 5-6 weeks old mice and at relatively low resolution. Also, during maturation, IHCs usually have transient axo-somatic synapses that are not present in mature IHCs. It would be great to see if, in 5-6 weeks old mouse, the ectopic IHCs still have axo-somatic synapses or not, and if the majority of the ectopic IHCs have innervation. Some of the data in this study would benefit from showing corresponding controls and some - from higher resolution imaging.

We appreciate the reviewer's suggestion. The objective of the paper is to report the phenomenon and present the coarse features of the Cldn9-mediated induced ectopic hair cells. The systematic details are for future studies, which are ongoing and out of the current scope.

In the mammalian cochlea, each HC is separated from the next by intervening supporting cells, forming an invariant and alternating mosaic along the cochlea's length. Cochlear supporting cells in some conditions can divide and trans-differentiate into HCs, serving as a potential resource for HC differentiation, using transcription and other developmental signaling factors.

However, when ectopic hair cells are generated from supporting cell trans-differentiation, the intricate mosaic of the organ of Corti is altered, which could by itself lead to hearing issues. In case of downregulation of Cldn9, the extra row of IHCs seems to be positioned immediately adjacent to the original IHC row. It is not clear if the newly formed unusual junctions between the ectopic and original IHCs are sufficiently tight to prevent leakage of the endolymph to the basolateral surface of IHCs. Also, it is not clear if the other organ of Corti tight junctions could lose their tightness due to the downregulation of Cldn9, which could over time affect the endocochlear potential as shown by this study and hearing abilities.

There was a slightly increased ABR threshold (5 dB -15 dB) (Fig. 4A) and a decrease in the magnitude of the EP and the rise in the K⁺ concentration in the endolymph and perilymph of Cldn9+/T mice compared to from age-matched littermates (S10) indicated there might be a compromised epithelium tight junction. The downregulation of Cldn9 affected the endocochlear potential and hearing abilities ((Fig. 4A, S10) after 2m, suggesting an age-dependent effect. The effective downregulation of Cldn9 would require proper titration of Cldn9 levels to induce extra hair cells with intact epithelial integrity; work may require additional studies.

Importantly, CLDN9 immunofluorescence staining data that show cytoplasmic staining of supporting cells should be revisited and the organ of Corti schematics showing CLDN9 expression should be corrected, considering that CLDN9 localizes to the tight junctions of the reticular lamina as was shown by immunoEM in this study and described in previous publications (Kitajiri et al., 2004; Nakano et al., 2009, Ramzan et al., 2021). While the current version of the manuscript will interest scientists working in the inner ear development and regeneration field, it could be more valuable to hearing researchers outside this immediate field and perhaps developmental biologists and cell biologists after proper revision.

We appreciate the reviewer's comments. We were concerned about the observation, but the results were consistent. Indeed, that was the motivation for performing the immunoEM (S3). A follow-up report may address it further.

Recommendations for the authors:

Reviewer #2 (Recommendations For The Authors):

Please address the points I made about the presentation (word choice, inconsistencies in labeling, etc). It ultimately helps a reader to understand and to follow your logic. This is an important observation.

We corrected the inconsistencies in labeling and addressed the points you suggested.

Making the extra effort to investigate a possible interaction between Cldn9 and Notch signaling would substantially increase the significance of the work.

Thanks for the suggestions. We will explore it in our future work.

Minor points:

Some sentences would benefit from revision:

- The abstract argues that hearing loss is incurable because mammalian hair cells are terminally differentiated (3rd sentence). This is not accurate.

Mammalian HCs are terminally differentiated by birth, making HC loss challenging to replace.

- The second sentence of the second paragraph of the introduction, "Cochlear SCs can divide and trans-differentiate into HCs, serving as a potential resource for HC differentiation, using transcription and developmental signaling factors (White et al., 2006)," should be referenced in the context of the animal's age. This feature of supporting cells is transient and only observed in neonatal mice. The following sentences in the same paragraph would also benefit from being placed into the same context when appropriate.

We thank the reviewer for the suggestion. These sentences have been corrected.

- Introduction: "But functional features of the newly developed HC are circumspect." The authors probably meant "circumspect," but is this the appropriate word? Also, please use the plural of HC = HCs.

The sentence has been corrected to “but the functional features of the newly developed HCs are circumspect”.

- Introduction: Isn't an essential function of tight junctions in the organ of Corti the separation of fluid-filled spaces? Perhaps additional functions of tight junction proteins are unclear, but at least this one function appears clear.

We thank the reviewer for the suggestion. We added the “additional” before the “function” in this sentence.

- Introduction: "using shRNA injection in postnatal (P) days (P1-7) mice." This is a rather vague statement that could be better defined. Perhaps mention that the injections targeted the round window and that an AAV-based method was used. Also, it is not clear from the methods whether the injection needle pierced the round window. Please clarify. Likewise, the methods state that these experiments were conducted in P1-P15 mice, but the main text says P1-P7. Later, in the results section and in the figure legend for Fig 7, the mice are between P1-P7 and P14; the figure itself is labeled with P1 and P14. However, data is presented (Fig 6) for injections at P2, P4, P7, and P14. In the text referring to Fig 6B in the results section, it is stated, "By contrast, the P14-21 inner ear transfected with Cldn9-shRNA produced no detectable increase..." Only data for P2, P4, P7, and P14 injections are presented. These are minor issues, but please check the inconsistencies because they make it difficult to follow.

We corrected this sentence to “Analogous additional putative IHCs differentiation was observed when Cldn9-shRNA was injected through the round window to postnatal (P) days (P2-7, and P14) mice…”. The label in Fig 7A has been changed to P2-7, and the text referring to Fig 6B in the result section has been changed to “the P14 inner ear transfected with Cldn9-shRNA produced no detectable increase...".

- Last statement of the Introduction: "making Cldn9 a viable target for generating transformed IHCs." It is not clear what transformed IHCs are.

We replaced the transformed with supernumerary.

- To understand the Southern Blot analysis in Fig 1E, the location of BstAPI and BamHI restriction sites and the probe need to be illustrated in Fig 1D.

The restriction sites BstAPI, (Bst), and BamHI (Bam) are indicated (Fig. 1D).

- Please define the purple arrows and arrowheads in Fig 1D. What do the different colors for the backbone mean? I see red and green, but also orange and yellow in the floxed allele. In Fig 1F, is "Knock-in" synonymous with homozygote? Would it be clearer to use the nomenclature Cldn9(T/T), Cldn9(T/+), and Cldn9(+/+), which is used later in the text?

We have made the changes as requested.

- Results, first paragraph: "Results of RT-PCR..." This refers to quantitative RT-PCR; please add the word "quantitative."

Thanks. We added “quantitative” to the sentence.

- Results and Fig S1. Is the strong upregulation of Cldn9 mRNA (S1A) also reflected in stronger Cldn9 immunoreactivity?

Yes, the strong upregulation of Cldn9 mRNA showed higher cldn9 immunoreactivity.

- Results, Fig 1. Please add a schematic drawing showing all elements of the inducible gene expression cassette in the final transgenic allele, and please illustrate how the system works. This helps the reader to understand the strong Cldn9 mRNA upregulation in Cldn9(T/T) mice, where expression is likely driven by the CMV promoter and reciprocally, in the presence of doxycycline, the suppression of transcription by binding of the tTA-dox protein to the TRE elements of the modified CMV promoter. Is this a correct assumption?

Yes, this is a correct assumption

- Results, about Fig S3. Why is it important to investigate Cldn6 and ILDR1 levels in the context of Cldn9 downregulation? Also, that is meant with "no comparative differences in others?". If a potential compensatory effect is suspected, why are the authors not systematically characterizing the expression of other tight junction proteins with quantitative RT-PCR? The results shown in S3 are anecdotal, without proper quantification, and lack context.

The goal is to examine the potential compensatory changes in other TJ proteins. It was not to examine all possible TJ proteins localized in the inner ear.

Results, section headed with "Downregulation of..." First sentence. Fig. 2A-C à Fig. 2A-E.

Thanks. We corrected the sentence “5-week-old mice Cldn9^+/T cochleae displayed a notable row of ectopic HCs (Fig. 2A-C).” to “5-week-old mice Cldn9^+/T cochleae displayed a notable row of ectopic HCs (Fig. 2A-E).”

The same section: "were negatively labeled with anti-prestin antibody." Consider "were not labeled with antibody to prestin." Likewise, a few sentences below, please consider rephrasing "the ectopic HCs ... reacted positively to otoferlin antibodies". Also, "...expressed multiple CtBP2 labeling..." - this reads like an incomplete sentence.

Thanks for the suggestions. We have corrected the three sentences mentioned.

The phrase "putative ectopic" lacks clarity because "putative" could refer to "ectopic" (like an adverb). Consider swapping the two words and writing "ectopic putative IHCs" or simply "ectopic IHCs."

Thanks for the suggestions. We replaced the “putative ectopic IHCs” with “ectopic IHCs” in all contexts.

Please use more precise figure labels when referring to a specific figure panel. For example, "Additionally, the ectopic HCs show IHC bundle features (Fig. 2)," - Bundles are shown in Fig 2D and Fig 2E. Please check all instances where a full figure is mentioned, but the specific reference is to a panel of the figure. Another example, "... using quantitative RT-PCR (S7)..." would be more specific if Fig S7A is referred to.

Thanks for the suggestions. We checked all instances and corrected the labels. Thanks!

"IHC counts at different ages (P2-P21) and the cochlear frequency segments (4-32 kHz) demonstrate..."- the figure shows data for 8 kHz and 32 kHz; please revise: "segments (8 kHz and 32 kHz) demonstrate."

This sentence has been revised based on your suggestion. Thanks!

Please add a legend to Fig. 3C (like the one shown in Fig. 2F).

Thanks for the reminder. The legend for Fig. 3C was modified.

Fig 4A and Fig 4B. It is impossible to distinguish the open/closed circles and the many lines. Please consider a different format or an extended supplemental figure. Also, drawing a line connection between the 32 kHz and click data points in 4A is inappropriate.

Instead of the open/closed circles, the dashed line means Cldn9^+/+ mice, and solid lines represent Cldn9^+/T mice. We added the line labels. The line connecting between 32 kHz and click data points was removed.

Fig 4, legend. Please define BHB and BHC levels.

BHB and BHC are defined.

The paragraph "Synaptic features of PE IHCs match original IHCs" is confusing because it states the following: "The synapses between the IHCs and auditory neurons at the apical, middle, and basal cochlear locations from 5-week-old Cldn9+/+ and Cldn9+/T mice show substantial differences." The meaning of the heading, therefore, does not match what is ultimately shown and discussed.

We have changed the title to “Synaptic features of ectopic IHCs and original IHCs”.

Moreover, no actual features of synapses are investigated; CtBP2/Homer pairs were used to identify afferent synapses, which this reviewer would argue provides a reasonable estimate of the number of synapses where pre- and post-synaptic markers are detected in close vicinity. It would be helpful to describe the method for counting juxtaposed CtBP2 and Homer-labeled puncta with more detail.

The method section now includes more information about the synapse count, which this reviewer would argue provides a reasonable estimate of the number of synapses where pre- and post-synaptic markers are detected in close proximity.

The final concluding sentence of the section also suggests that synaptic transmission from PE IHCs might be compromised because significant differences in synapse numbers were identified. It would be important to mention this.

Thanks for the reminder. We added this information to the final concluding sentence.

Fig. 5C, 5D; legend. Is "co-expressed" the right word choice? Consider "colocalized" or "juxtaposed".

The "co-expressed" has been replaced with "colocalized".

Voltage-clamp recordings of P21 inner hair cell mechanoelectrical transduction currents. This reviewer cannot identify a previous publication describing the details of this method on P21 cochlear inner hair cells; this seems like an excellent methodological advance.

Yes, we can record data from older mice. Thanks for pointing it out.

"Transfection in vivo of Cldn9 shRNA," the P14-21 inner ear transfected with Cldn9-shRNA." Plus, additional use of the word "transfection." Transfection generally means the introduction of plain nucleic acid into cells. The word refers to methods that do not use viruses. In contrast, "transduction" is the term used for virus-mediated gene transfer. The authors used AAVs. Please correct for appropriate scientific terminology.

Thanks for the clarification. This information has been corrected accordingly.

"A slight decline in the amplitude of the EP and a substantial rise in perilymph K+ was detected in 8-month-old Cldn9+/T (S7)." Probably Fig. S8A,B is meant.

Yes, it referred to Fig. S8 A, B. We corrected it in the result section. Thanks!

Heading "Discussions" -> "Discussion"

The focus of the second part of the discussion on potential interactions between Cldn9 suppression and known signaling pathways is essential. The logic that is presented with respect to Notch signaling, however, is not clear and misleading. For example, it is not obvious what is meant by "Cldn9 subserves the signaling catalyst to activate NICD cascades" and whether this statement is supported by any published data.

The statement was a suggestion and has been qualified with a “may” clause (line 299).

The authors might consider discussing whether the observed effect caused by Cldn9 elimination is a specific role of the Cldn9 protein itself or is an epiphenomenon resulting from cytomechanical changes in the developing and maturing organ of Corti. This would add a potential Notch-independent component for a possible interpretation of the observations.

We state lines 302-304 “Alternatively, Cldn9 levels disruption may alter the mechanical properties of the developing and maturing organ of Corti that may trigger ectopic IHC differentiation, an epiphenomenon independent of the Notch signaling“.

Methods:

"Deletion of the selection marker in the tTA cassette by crossing the F1 mouse with the embryonic Cre line (B6.129S4-Meox2tm1(cre)Sor/J)." This sentence seems to be incomplete.

Thanks for pointing it out. This sentence has been rewritten.

"Images were captured under a confocal microscope." Consider writing "with a confocal microscope".

This sentence has been corrected. Thanks!

RNA extraction and... How many mice were used per experiment? 10-15 or just 10?

The mice number for the RNA extraction is between 10 and 15. Thanks

Reviewer #3 (Recommendations For The Authors):

Below are my suggestions, questions, and criticisms.

(1) The red outline on Fig1A schematic does not correspond to the previously published expression pattern of CLDN9 in the organ of Corti reticular lamina tight junctions (Kitajiri et al, 2004, Nakano et al., 2009, Ramzan et al., 2021). Also, there are no tight junctions all around the pillar cells. The tight junctions are restricted to the sites of tight attachments between two cells. The immunofluorescence staining using CLDN9 antibody looks rather cytoplasmic (Fig 1 and Fig S1) than associated with the tight junctions as it was shown by immunoEM data here and reported previously (Kitajiri et al, 2004; Nakano et al, 2009; Ramzan et al, 2021). Please correct the schematic and explain your data.

We have redrawn the diagram (Fig. 7).

(2) The CLDN9 staining in Figure 1, B and C, highlights the cytoplasm of the supporting cells, and hair cells devoid of the staining. From the images in Fig. S1C, it also looks like CLDN9 is present only in supporting cells and not in hair cells? How would the authors reconcile their data with Cldn9 expression data from the gEAR database and Ramzan et al.'s 2021 RNAscope data? Please provide the validation of the antibody used in this study.

We recognize the reviewer’s concern but RNA and protein levels are not always in parallel.

(3) Figure 1D. The dash lines from the targeting vector to the wt allele seem to indicate a recombination event. Please do not show the recombination event, instead just show what part of the targeting vector was incorporated to replace wt Cldn9. There is no description in the figure 1 legend what purple arrows and arrowheads mean and what yellow and orange line segments in the floxed allele schematic indicate. Please also show where the BstAPI and BamHI restriction enzyme sites are.

We have provided supplement Fig 1., and have noted the BstAPI and BamHI restriction enzyme sites in Fig. 1D.

(4) What does the organ of Corti that has 40-to-55-fold increase in Cldn9 mRNA expression looks like before dox treatment? Any abnormalities at all? How is CLDN9 protein localization looks in the Cldn9+/T untreated mice? Do they have normal number of IHCs? Cldn9+/T untreated mice should be used as another control at least in Figure S1. What does the organ of Corti that has a 40-to-55-fold increase in Cldn9 mRNA expression look like before dox treatment? Are there any abnormalities at all?

The untreated Cldn9^+/T mice can grow normally but are not fertile. So, we used a very low concentration of dox water (0.1 mg/ml) instead of normal water to keep the breeding pairs. The protein level increased in the Cldn9^+/T mice compared with Cldn9^+/+mice. With 0.1 mg/ml dox water, they also showed ectopic IHCs.

(5) It is interesting that decline of 0.4-0.6-fold in mRNA level leads to about 8-fold decrease in protein level based on your immunoEM data on tight junctions of IHC with supporting cells. Do you observe the same effect in OHC-SC tight junctions, or the decrease was observed selectively around IHCs?

The reviewer is alluding to matching RNA and protein levels. It appears that for Clnd9 one cannot expect a closely matched relationship.

(6) The quality of the immunoEM data is great, but a control of secondary antibody alone staining in wt and Cldn9+/T dox treated should be shown and compared to the Cldn9+/T treated sample.

We thank the reviewer for raising the issue. Secondary antibodies are used as a control in all immunoEMs in the laboratory. We opted not to show negative results.

(7) The authors observed a decrease in Cldn6 expression albeit not quantitative in response to Cldn9 downregulation. How were the immunofluorescence signals compared and evaluated? Please provide a detailed description of the method used. Did the authors used the same image acquisition parameters? Was the Cldn9 and Cldn6 immunostaining done using same protocol with the same aliquot and dilution of the secondary antibodies, etc.? The staining for CLDN6 seems to be concentrated in the cytoplasm of supporting cells, and not in the tight junctions, similar to CLDN9 immunoreactivity shown in Fig. S1C and to the ILDR1 pattern of staining in Fig. S3. How can the authors explain this? How were the antibodies validated?

The Cldn9 and Cldn6 immunostaining were done using the same protocol with the same aliquot and dilution of the secondary antibodies.

(8) CLDN14 is also expressed in the organ of Corti tight junctions. What happened to this TJ protein during CLDN9 downregulation?

We detected Cldn14 with immunostaining in the Cldn9+/T mice and Cldn9+/+ mice fed with 0.25 mg/ml dox water, and the results showed increased expression of Cldn14 in Cldn9+/T mice. Detail alterations of other TJ proteins have been reserved for future studies.

(9) When supernumerary IHCs were observed in Cldn9+/T mice, have the authors noticed a corresponding decrease in supporting cells surrounding IHCs? Quantification of the IHCs supporting cells would be useful. Do the ectopic IHCs have apical tight junctions with original IHCs or they are surrounded by supporting cells?

We quantified the SCs around the IHCs but did not detect significant differences among the groups.

(10) The authors indicated that viable PE IHCs were observed in 15 months old Cldn9+/T dox treated mice. How stereocilia bundles look in these ectopic hair cells? Are they preserved similar to the original IHCs or degenerated? It is hard to see this in Fig 3, phalloidin panel. High-resolution SEM would show this better.

For the remaining ectopic IHCs in 15 months, we did not detect apparent differences in hair bundles compared with the original IHCs.

(11) Interestingly, the authors indicate that the highest number of the ectopic IHCs were developed in the apical turn and the higher elevation of ABR threshold was also observed at low frequencies end. This may indicate that extra IHCs do not help hearing function.

The extra IHCs showed along the whole cochlea, even though it is more obvious in the apical turn. The declined hearing may have resulted from the leakage of the endolymph K+ to the perilymph and EP decline.

(12) No age-matched wt control is shown for decreased expression of Cldn9 after shRNA injection at P2 (Fig. 6A).

As indicated earlier, we opted to state but did not show negative results.

(13) Figure 6C. The better- quality SEM images showing a longer stretch of IHCs are needed to convince readers that there are ectopic IHCs that are well preserved in 5-6 weeks old mice in all cochlear turns after GFP-Cldn9 shRNA treatment at P2-P7.

In S4, we showed that there are ectopic IHCs along the cochlear axis.

(14) Do scrambled shRNA control samples had some ectopic IHCs? This control is missing in Fig.6D.

No scrambled shRNA controls did not show ectopic IHCs. We have stated it.

(15) Figure 7B, lower schematic. There are no known continuous tight junctions and CLDN9 expression around the OHCs and IHCs. CLDN9 is known to be concentrated at the reticular lamina tight junctions which separate the endolymph from perilymph. Please, correct all schematics accordingly.

We have made the changes as requested.

Minor comments:

(1) Page 1, Abstract. I would not say "making HC loss incurable" since recent gene therapy results show some advances in this direction. Please rephrase more accurately.

We have made the changes as requested.

(2) Page 4, Results, line 5; please rephrase "PCR of tail tissue samples performed genotyping."

It has been corrected to “The genotyping was performed by the PCR with the tail tissue.”

(3) Fig. 1 legend, panel B, replace "showing IHC stained myosin7a" with "showing IHC stained by myosin7a". Also, in the same sentence, "phalloidin, actin (green) antibodies," Phalloidin is not an antibody; please change this.

Thanks. We have corrected this information.

(4) Fig 2C, IHC label obscures the view of IHCs, please move this label out and use an arrow to point to IHCs.

We have made the changes as requested.

(5) Figure 4, title. Replace "currents elicited original" with "current elicited from original".

This sentence has been corrected. Thanks.

(6) Figure 4, panel A. It is hard to see the open symbols on the graph. Are they associated with the dash lines? Please make them more visible or indicate what dash lines are. "ABR threshold for (n=12)" should be "ABR threshold for Cldn9+/+(n=12)"?

Yes, they are associated with the dash lines. We added the labels for the solid lines and dash lines. "ABR threshold for (n=12)" was corrected to "ABR threshold for Cldn9+/+(n=12)."

(7) Figure 4, legend. "Within each wt and heterozygote mice, there was no significant shift...". Do you mean within each group of mice? Also "Mean DPOAE threshold for 2-8 mos (n=9) was tested,..." Do you mean (n=9) for each group or what group?

Yes, "Within each wt and heterozygote mice, there was no significant shift..." has been revised. The number of mice in each group for the DPOAE test was clarified in the Fig. 4B legend. Thanks.

(8) Please label the X axis in Figure 4D.

The X-axis has been labeled (Time (s))

(9) Figure 4 B, do the colors of the lines indicate the same age groups as in Fig 4A? Do the dash lines associate with open symbols? Please state this clearly in the figure's legend.

Yes. We added this information in Fig. 4B legend.

(10) Figure 4D. Please label the X axis of the fluorescence intensity graph.

The X-axis has been labeled (Time (s))

(11) Figure 4G, legend. Replace "(mean +std)" with "(mean +SD)" for consistency here and in Figure 5 legend.

Thanks. We replaced "(mean +std)" with "(mean +SD) in the legend of Fig. 4G and Fig.5 and Fig.6.

(12) Figure 5B, legend. Replace "makers" with "markers".

Thanks. This information was corrected.

(13) Figure 6A, legend. There is no downregulation of Cldn9 by shRNA shown in "S5". Do the authors mean Figure S7? Please, correct "S5" to "Fig. S7".

This information was corrected. Thanks.

(14) Figure 6A, legend. There is no reduced CLDN9 protein expression shown in Fig. 1C. Do the authors mean Fig. 6A, third panel? Please correct the phrase "reduced protein expression (Fig. 1C) is shown in the 3rd Panel (Cldn9, red)" accordingly, and do not capitalize "p" in the "3rd Panel".

This information was corrected. Thanks (line 917-918).

(15) Also there, replace "The right Panel shows two rows of IHCs (marked HC marker, Myo7a (cyan), and the merged photomicrograph" with "The right panel shows the merged image with two rows of IHCs stained with HC marker Myo7a (cyan) and the expression of Ad-GFP-mCldn9 shRNA (green) in the adjacent row of supporting cells". Please indicate in what cells Ad-GFP-mCldn9 shRNA (green) is expressed. It looks like only one row of supporting cells has this green signal.

This information was corrected.

(16) Figure 6B, legend. Replace "Examples of photomicrographs of sections of the whole-mount cochlea of P2, P4, P7, and P14 Cldn9 shRNA injected mice" with "Examples of phalloidin stained whole-mount organ of Corti samples from cochleae of the wild-type mice injected at P2, P4, P7 and P14 with Cldn9 shRNA"

This sentence has been modified based on your suggestions. Thanks!

(17) Replace "action labeling" with "actin labeled."

Thanks! The "action labeling" has been replaced with "actin labeled." Line 924

(18) Figure 6C. Insert "C" before SEM images description in the legend. The authors stated that SEM images of "5-6-wks-old mice" are shown. Please indicate the exact age of mice shown on each image and at what age these mice received the virus injection.

Thanks! The “C” has been added. We have noted that the SEM images are from 5-week-old mice" in the legend, and the virus was injected at P2.

(19) Figure 6D, legend. Last sentence: move "are significantly different" and insert this between "IHCs" and "at P2 apex".

This information was corrected.

(20) Figure S7, legend. Replace "(sram)" with "(scram)" as in the figure itself. Also, Indicate the age of samples at the harvesting time for imaging and the age at injection of Cldn9 shRNA.

"(sram)" has been replaced with "(scram)". The age of samples at the harvesting time for imaging and the age at injection of Cldn9 shRNA are indicated.

(21) Figure S8. Replace "4 mos-old" and "8 mos-old" with "4 months-old" and "8 months-old" everywhere in the legend and in the figure labels.

We have made the changes as suggested.

(22) Page 8, 5th lane from the bottom. Change "EP and K+ concentration endolymph" to "EP and K+ concentration of the endolymph".

It has been corrected. Thanks.

(23) Page 8, next to the last sentence before the Discussion. Wrong figure number, please replace "(S7)" with "Fig. S8".

It has been corrected. Thanks.