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

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 a careful revision of the text would clarify some of these issues.

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.

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.

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.

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 is working well for the authors, and a comparison of the expression of Notch or other known pathway components could provide mechanistic insight.

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.

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

Please 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.
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.
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 be of interest to scientists working in the inner ear development and regeneration field, it could be more valuable to the hearing researchers outside this immediate field and perhaps developmental biologists and cell biologists after proper revision.