Jag1 represses Notch activation in lateral supporting cells and inhibits an outer hair cell fate in the medial compartment of the developing cochlea

  1. Sandra de Haan
  2. Agustin A. Corbat
  3. Christopher R. Cederroth
  4. Lisa G. Autrum
  5. Simona Hankeova
  6. Elizabeth C. Driver
  7. Barbara Canlon
  8. Matthew W. Kelley
  9. Emma R. Andersson

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Abstract

Notch signaling regulates both inner and middle ear morphogenesis and establishes a strict pattern of sensory cells in the organ of Corti in the mammalian cochlea. Patients with Alagille syndrome have impaired Notch signaling (∼94% with JAG1 mutations) resulting in sensorineural and conductive hearing loss. Here, we investigate the function of Jag1-mediated Notch activation in cochlear patterning and signaling using the Jag1 “Nodder” ( Jag1 Ndr/Ndr ) mouse model of Alagille syndrome. Jag1 Ndr/Ndr mice exhibited severe vestibular and auditory deficits and a dose-dependent increase in ectopic inner hair cells and a reduction in outer hair cells. Single cell RNA sequencing of the organ of Corti demonstrated a global dysregulation of genes associated with inner ear development and deafness. Analysis of individual cell types indicated a novel role for Jag1 in repressing Notch activation in lateral supporting cells and revealed a function for Jag1 in gene regulation and development of outer hair cells. Additionally, “outer hair cell-like” SLC26A5 (Prestin) positive cells were present in the medial compartment and pillar cell region of Jag1 Ndr/Ndr mice and exhibited location-dependent expression of the inner hair cell fate-regulator Tbx2 , revisiting the potency of Tbx2 in driving inner hair cell commitment in “outer hair cell-like” cells in the Jag1-mutant IHC compartment. Our data reveals a novel role for Jag1 in repressing Notch activation in lateral supporting cells and highlights involvement for Notch signaling in inner versus outer hair cell specification and positioning.

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    Response and revision plan

    Manuscript number: RC- 2024-02380

    Corresponding author(s): Emma R Andersson

    1. General Statements

    We sincerely appreciate the thorough and positive review provided by all reviewers. Their comments have provided valuable suggestions to improve and enhance clarity of our study on the role of Jag1-mediated Notch signaling in cochlear development, and its implications for Alagille syndrome. Furthermore, their feedback has underscored the significance of our study in elucidating patterning and hearing deficits, and its relevance for therapeutic considerations*. *

    2. Description of the planned revisions

    Comment from BioRxiv

    In addition to comments from appointed reviewers, Jaime García-Añoveros emailed us with a comment on our BioRxiv preprint. Professor García-Añoveros was interested in our finding thatTbx2 is expressed in OHC-like cells (Fig5), because his lab has shown that Tbx2 is an inner hair cell determinant (García-Añoveros et al., 2022). Fig 5 shows quantifications of Tbx2 RNAscope punctae in sections, showing that Tbx2 is expressed in Jag1Ndr/Ndr outer hair cell-like cells, in the inner hair cell compartment, at similar levels to that expressed by the extra inner hair cells also present in *Jag1Ndr/Ndr *mice. He suggested we perform RNAscope for Tbx2 on wholemount cochlear preparations, to confirm the Fig 5 data from cross sections. While we are confident of our quantifications, which were based on optical slice sections Reviewer comments

    We have already implemented some of the reviewer suggestions, as detailed under point 3, and the list below is therefore discontinuously numbered.

    Reviewer 1

    *Comments regarding quality of images: the picture quality for Figure 4b is low, especially for F-actin staining. Please enhance the intensity. (check image). Fig. 1g, poor quality. The WT cochlea looks severely disorganized. (replace image) *

    Response

    Figure 4b and Fig1g images will be improved or replaced. We plan a more extensive analysis of the adult phenotype, to also address comment #1 from Reviewer 2 (described below in response to Reviewer 2, #1).

    Reviewer 2

    Fig1g shows a very abnormal cross section through the cochlear duct. There are no clearly visible Deiters' cells. Is this the case? Loss of outer hair cell function should only increase thresholds about 40dB, and there are increased thresholds reported here of 60+, despite remaining outer hair cells. This could be accounted for by the conduction defects, but also, there may be defects in the adult ear not observed earlier. Is there any inner hair cell loss? Deiter cell loss? Are inner and outer hair cell stereocilia normal? These may account for the severe hearing loss.

    Response

    To further characterize the adult cochlear phenotype, we will quantify the number of IHCs, OHCs and SCs with immunohistological staining of cryosections from adult Jag1Ndr/Ndr mice, and address in the Discussion section how this phenotype relates to the observed hearing loss. Additionally, we plan to analyze ABR wave-I characteristics of existing recordings to further study auditory nerve fiber responses and IHC function.

    We have added a discussion of the relative contribution of middle and inner ear defects to the overall hearing loss in the Discussion section (lines 385-399), to also address comment #3 from reviewer 1 (below in section 3 "revisions that have been already incorporated in transferred manuscript").

    Reviewer 2

    *What is the rationale for reporting differences in the p-value that are not significant at the adjusted p-value? Since these are whole genome analysis it is only appropriate to report significance by adjusted p-values. *

    *One of the novel aspects of this study is the finding that Notch components are upregulated in the Jag1Ndr/Ndr mutants (although some of these results are not significant at the adjusted p value). Given the potential significance that these results would indicate (including c-inhibition), it would be important to confirm upregulation of key Notch components in situ using RNA-scope or immunohistochemistry. *

    Response

    We agree that multiple hypothesis testing should be corrected for (with adjusted p values), which we have done in all analyses. However, we considered it relevant to report enriched or depleted genes that reached a meaningful fold difference and p-value threshold, even though the adjusted p-value threshold was not met. Our hope was that this would provide transparency and allow for consideration of the different sample sizes (different abundance of specific cell types), allowing the reader to explore the data. For further transparency, a distinction in labelling of significant adj. p-values and p-values was previously made in the original manuscript.

    We thank the reviewer for pointing out that the Notch target gene upregulation is an interesting and novel finding. We will perform RNAscope experiments to validate the upregulation of Notch components and target genes at P5, including *Jag1, Jag2, Hes5, Nrarp, Tns1 *and Cxcl12. Quantification of the RNA scope signal will also provide an alternative approach to testing whether the enrichment/upregulation of Notch target genes is statistically significant.

    __Reviewer 1 __

    Text and figure comments: Scale bar missing in Figure1b and Figure1h.* Please mention the scale bar presented mm in the figure legends for Figure 2; Figure 3; SFigure 6.*

    Response

    Scale bar information will be added to the specified figures.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer 1

    Developmentally hair cells develop from the base to the apex starting from the IHC to OHC. The observation of the changes in HC pattern indicates the impact of Notch in timing and maturation status of HC differentiation. Likely by the time when OHCs are supposed to be developed, which is dictated by the suppression of IHC and the activation of OHC signals, due to the dysregulation of Jag1, the IHC signaling cannot be sufficiently suppressed, whereas the OHC signaling cannot be sufficiently activated. This has a positional effect as further it is from the IHCs, more mature OHC can develop. Could the authors dig deeper into the scRNAseq data to see if they can isolate the profile of extra IHCs in the JagNdr/Ndr mouse, to see if they can detect the expression of some OHC genes albeit at much lower levels?

    Response

    There were no significant gene expression differences between Jag1Ndr/Ndr and Jag1+/+ IHCs. As we expect the Jag1Ndr/Ndr IHC pool to contain similar numbers of de facto IHCs and ectopic IHCs, failure to detect any differences suggests that the ectopic IHCs are transcriptionally similar to de facto IHCs. To further address the ectopic IHC signature, we subsetted, renormalized and reclustered the Jag1Ndr/Ndr and Jag1+/+ IHCs. No Jag1Ndr/Ndr-specific clusters were identified in this analysis (new Supplementary Fig 4c). In addition, we analysed the expression of IHC- and OHC-specific markers to assess the faithfulness of Jag1Ndr/Ndr IHCs and OHCs. As reported in our original manuscript, Jag1Ndr/Ndr OHCs expressed lower levels of OHC markers. However, Jag1Ndr/Ndr IHCs were indistinguishable from *Jag1+/+ * IHCs (new Supplementary Fig 4b). These new analyses also address comment #2 by Reviewer 2 (see below).

    As the reviewer pointed out that development of HCs occurs from base to apex, we have added a quantification of apex and base regions of the P5 phenotype to Sfig5 and described this data in the Results section (lines 230-231).

    Reviewer 1

    It is difficult to dissect the contribution of middle ear malformation and inner ear defects to hearing loss in Alagille syndrome with the current model. For the development of any therapy, the two main factors have to be analyzed separately. One option is to generate an inner ear-specific JagNdr/Ndr model to bypass the middle ear issue, which can be evaluated for potential therapy. This part should be discussed.

    Response

    We agree that the relative contribution of middle and inner ear defects to hearing loss in a Jag1-compromised setting cannot be assessed with Jag1Ndr/Ndr mice. Generation of an inner-ear specific Jag1 Nodder model to bypass middle ear defects and address the relative contribution of middle and inner ear defects, would be technically challenging/impossible since the Nodder mouse model carries a single missense mutation in Jag1 and must be carefully maintained on a mixed genetic background to fully recapitulate Alagille syndrome. However, previous elegant work from other groups has dissected the function of Jag1 in supporting cells and neural crest, and how defects in each of these systems contribute to hearing loss. We therefore now comprehensively discuss this work by others (lines 385-399).

    Reviewer 1

    *In Figure 1, the author mentioned the major defects found in the vestibular system. Is there any difference in the vestibular system at the cellular level? Some evidence will be informative. *

    Jag1Ndr/Ndr mice completely lack the posterior semicircular canal, which explains the head nodding behavior observed in our model, since the posterior semicircular canal detects head-tilting towards the shoulders. We have no data on the hair cells located in the saccule or utricle. Since the paper focusses on patterning and hearing, rather than balance, we consider further analysis of the vestibular system at cellular level outside of the scope of our paper.

    Reviewer 2

    From the UMAP plot in Fig 2b, it seems that the scRNA-seq data did not reveal any change in cell identities in the Jag1Ndr/Ndr ears. This result is not really discussed in the results or discussion-particularly why the OHC-like cells, extra IHCs, and absent Hensen's cells are not revealed in this analysis.

    Response

    In our scRNAseq dataset we were unable to identify, with certainty, an OHC-like population. After subsetting HCs, we did observe an additional OHC population exclusive to homozygous animals. However, after RNAscope validation, this population might have arisen from contamination with PCs. IHCs were transcriptionally similar between wildtype and homozygous animals, and we were unable to identify the ectopic IHCs. We additionally reported fewer to almost absent HeCs in the homozygous dataset. This data has been shown in the Results section (Fig2b) and in Supplementary Table 8 (number of cells per cell type) and has been discussed in the Discussion section. To further address the lack of separation of IHCs and ectopic IHCs, and failure to identify OHC-like cells, we have added additional panels assessing IHCs and OHC gene expression to SFigure4. This also addressed comments #2 addressed by Reviewer 1 (see above).

    Reviewer 2

    *It is difficult to know which cells are extra (+1), including inner hair cells. Since scRNAseq did not reveal a different gene signature for these 'extra' cells, it is more appropriate to just count them all together. *

    Response

    We have merged the quantification of IHCs and +1 IHCs to total IHCs in Fig4c. Separate original quantification of IHCs and +1 IHCs is reported in SFigure5, since the data presented in this way reflect a doubling of the IHC row.

    Reviewer 2

    Additionally, a previous report has suggested that JAG1 mediates cis-inhibition in the medial region of the cochlea. The data presented here do not show an upregulation of Notch signaling in the medial supporting cells, suggesting this is not the case. This should be discussed.

    Response

    It is indeed interesting to note that, although with comparable sample size for medial and lateral populations, upregulation of Notch activation is restricted to lateral SCs, and not, despite previous indications (Basch et al., 2016), observed in medial SC populations. We have discussed the possibility for cis-inhibition to a greater extent in the Discussion section (lines 310-311).

    Reviewer 2 and Reviewer 3

    *Pg 9 Discussion: The sentence: "The JAG1NDR missense mutant is expressed in vivo, and traffics normally, but does not bind or activate NOTCH1", is somewhat misleading because it suggests this allele has no function. Based on the milder ear phenotype to null alleles as well as survival suggests that this allele is hypomorphic. This should be clarified and discussed. *

    The authors should provide a more detailed description of the Nodder mice (the nature of the mutation and how it may effect Notch1 and Notch2 receptor activation) in the introduction.

    Response

    We now introduce the *Nodder *mouse model (Hansson et al., 2010) and signaling defects to a greater extent in the Introduction section (lines 66-68).

    Reviewer 2

    Pg 5 third paragraph, "Differential gene expression analysis identified 40 up- and 42-downregulated genes in Jag1Ndr/Ndr versus Jag1+/+ IPhCs, with pathway dysregulation similar to the pseudobulk analyses (Fig3c, Supp.Table 5,6)"-should be 40 downregulated and 42 upregulated. Similarly: Pg 6 second paragraph: Differential gene expression analysis identified 1 up and* 42-downregulated genes in Jag1Ndr/Ndr DCs versus Jag1+/+ DCs-should be 1 down and 42 up. *

    Response

    Thank you for catching our accidental inversion here. The text has been corrected accordingly.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Reviewer 1

    To study how Jag1 insufficiency affects the development, the authors included the JagNdr/Ndr mouse model. To fully understand the characteristics of the Nodder mouse model, it's necessary to include the direct age-dependent comparison of the Jag1 level (by qPCR/and or Western blot) between Jag1+/+ v.s. from JagNdr/Ndr in Figure 1 at some selected stages to correlate the Jag1 insufficiency with the "Nodder" model. A spatial expression comparison of Jag1 between Jag1+/+ v.s. from JagNdr/Ndr from different the main age groups should be included in SFigure 2, together with Notch target genes.

    The JAG1 Nodder mutation results in a hypomorphic ligand that is unable to bind and activate the Notch1 receptor (Hansson et al., 2010). The ligand itself, however, is still expressed, and its protein expression can even be upregulated in vivo (Hansson et al., 2010). Therefore, performing quantitative expression analysis of JAG1 expression (by qPCR or immunohistochemistry) would not provide insights into the levels of JAG1 activity. Instead, we show that there is decreased Notch target gene expression at the prosensory domain stage, as a proxy for Notch activation levels (SFigure2*. *A more detailed introduction of the model is provided in the Introduction (lines66-68), to also address a comment from Reviewer 2, #7 and Reviewer 3 comment #2.

    Reviewer 3

    The mutant form of Jagged1 in Nodder mice is trafficked to the cell surface, and while this mutant form of Jagged1 is incapable of activating the Notch1 receptor it may interact with "new" proteins, gaining new functions. My recommendation to the authors is to determine whether similar defects occur in conditional Jag1 knockout mice (increased Notch signaling in lateral supporting cells and presence of ectopic outer-hair cell like cells). The ability to disrupt Jag1 function at different stages of development may also help to determine why Jag1 deficiency renders some outer hair cells insensitive to Tbx2. If this is not possible due to time constrains, I would recommend a more in-depth discussion of the limitations of using Nodder mice.

    Jag1 conditional knockout at various stages, has not been reported to result in ectopic OHC-like cells (Brooker et al., 2006; Chrysostomou et al., 2020; Gilels et al., 2022). However, two other Jag1 missense mutants display atypical hair cells in the IHC compartment, which could be the OHC-like cells we report here (Kiernan et al., 2001; Tsai et al., 2001). Taken together, these data would suggest that Jag1 loss of function in supporting cells is not sufficient to result in OHC-like cells, but that constitutive Jag1 insufficiency can drive OHC-like cell formation. We now cite these data and discuss possible interpretations, as suggested (lines 324-331).

    References

    Basch, M. L., Brown, R. M., Jen, H.-I., Semerci, F., Depreux, F., Edlund, R. K., Zhang, H., Norton, C. R., Gridley, T., Cole, S. E., Doetzlhofer, A., Maletic-Savatic, M., Segil, N., & Groves, A. K. (2016). Fine-tuning of Notch signaling sets the boundary of the organ of Corti and establishes sensory cell fates. ELife, 5, 841-850. https://doi.org/10.7554/eLife.19921

    Brooker, R., Hozumi, K., & Lewis, J. (2006). Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear. Development, 133(7), 1277-1286. https://doi.org/10.1242/dev.02284

    Chrysostomou, E., Zhou, L., Darcy, Y. L., Graves, K. A., Doetzlhofer, A., & Cox, B. C. (2020). The notch ligand jagged1 is required for the formation, maintenance, and survival of Hensen's cells in the mouse cochlea. Journal of Neuroscience, 40(49). https://doi.org/10.1523/JNEUROSCI.1192-20.2020

    García-Añoveros, J., Clancy, J. C., Foo, C. Z., García-Gómez, I., Zhou, Y., Homma, K., Cheatham, M. A., & Duggan, A. (2022). Tbx2 is a master regulator of inner versus outer hair cell differentiation. Nature, 605(7909). https://doi.org/10.1038/s41586-022-04668-3

    Gilels, F. A., Wang, J., Bullen, A., White, P. M., & Kiernan, A. E. (2022). Deletion of the Notch ligand Jagged1 during cochlear maturation leads to inner hair cell defects and hearing loss. Cell Death and Disease, 13(11). https://doi.org/10.1038/s41419-022-05380-w

    Hansson, E. M., Lanner, F., Das, D., Mutvei, A., Marklund, U., Ericson, J., Farnebo, F., Stumm, G., Stenmark, H., Andersson, E. R., & Lendahl, U. (2010). Control of Notch-ligand endocytosis by ligand-receptor interaction. Journal of Cell Science, 123(Pt 17), 2931-2942. https://doi.org/10.1242/jcs.073239

    Kiernan, A. E., Ahituv, N., Fuchs, H., Balling, R., Avraham, K. B., Steel, K. P., & Hrabé de Angelis, M. (2001). The Notch ligand Jagged1 is required for inner ear sensory development. Proceedings of the National Academy of Sciences of the United States of America, 98(7), 3873-3878. https://doi.org/10.1073/pnas.071496998

    Tsai, H., Hardisty, R. E., Rhodes, C., Kiernan, A. E., Roby, P., Tymowska-Lalanne, Z., Mburu, P., Rastan, S., Hunter, A. J., Brown, S. D. M., & Steel, K. P. (2001). The mouse slalom mutant demonstrates a role for Jagged1 in neuroepithelial patterning in the organ of Corti. Hum Mol Genet, 10(5), 507-512. https://doi.org/10.1093/hmg/10.5.507

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Patients with Alagille syndrome have impaired Notch signaling (~94% with JAG1 mutations) resulting in sensorineural and conductive hearing loss. To gain a better understanding of the genesis of these functional defects, the authors conduct a detailed examination of a mouse model of Alagille syndrome, called Nodder mice (Jag1 Ndr/Ndr). Consistent with previously reported phenotypes for Jag1 mutant mice, the authors observed severe vestibular and auditory defects that were accompanied by semicircular canal abnormalities, and defects in the patterning of the auditory sensory epithelium (medial boundary defect causing duplication of inner hair cells and inner phalangeal cells and reduction in outer hair cells and lateral supporting cells). What makes this study stand out from previous studies is the elegant use of single cell RNA-sequencing technology. Their scRNA_seq data suggest that 1) Jag1 lowers Notch signaling in lateral supporting cells and 2) Jag1 regulates the gene expression of outer hair cells. Further marker analysis revealed ectopic outer hair cell-like cells in the medial compartment and pillar cell region of Jag1 Ndr/Ndr mice. Surprisingly the outer hair cell-like cells closest to inner hair cells expressed Tbx2. Previous studies have shown that ectopic activation of Tbx2 is sufficient to convert outer hair cells into inner hair cells, suggesting that Jag1 deficiency may render these outer hair cells insensitive to Tbx2.

    Overall, this is a very well-designed and executed study. The main conclusions of the study are well supported by the presented data and both data and methods are presented in a clear and detailed manner. I have only few suggestions for improvement:

    Major comment:

    The mutant form of Jagged1 in Nodder mice is trafficked to the cell surface, and while this mutant form of Jagged1 is incapable of activating the Notch1 receptor it may interact with "new" proteins, gaining new functions. My recommendation to the authors is to determine whether similar defects occur in conditional Jag1 knockout mice (increased Notch signaling in lateral supporting cells and presence of ectopic outer-hair cell like cells). The ability to disrupt Jag1 function at different stages of development may also help to determine why Jag1 deficiency renders some outer hair cells insensitive to Tbx2. If this is not possible due to time constrains I would recommend a more in-depth discussion of the limitations of using Nodder mice.

    Minor comment:

    The authors should provide a more detailed description of the Nodder mice (the nature of the mutation and how it may effect Notch1 and Notch2 receptor activation) in the introduction.

    Significance

    Strength of the study: The establishment and in-depth characterization of a Jag1 homozygous mutant mouse model (Nodder mice) to study the effects of Alagille syndrome on the auditory and vestibular system. Another strength is the characterization of the cell-type specific effects of Jag1 mutations using single cell transcriptomics.

    Limitation of the study: It is unclear if the missense mutation in Jag1 that is present in Nodder mice causes "only" a loss of function. It is possible that a mutant form of Jag1 protein gains new functions (see also major comment).

    Advance: This study demonstrates a novel role for the Notch ligand Jag1 in repressing Notch activation in lateral supporting cells and uncovers an involvement for Jag1-activated Notch signaling in inner versus outer hair cell specification and positioning.

    Audience: This study will be of interest for both clinical and basic scientist as it provides novel insights into how Alagille syndrome effects the auditory system and novel mechanistic insights into the complex and cell-type specific role Notch signaling.

    My field of expertise is: inner ear/ cochlea development, Notch signaling, cell fate specification and differentiation of cochlear supporting cells.

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    Referee #2

    Evidence, reproducibility and clarity

    Alagille's syndrome is a developmental disorder in which the vast majority of patients have heterozygote mutations in the gene for the Notch ligand Jagged1. Patients with Alagille's have developmental defects in the heart, liver, eye and ear. This manuscript describes a hypomorphic new allele of Jag1 (Ndr mutation), in which homozygotes animals survive, allowing analysis of the postnatal and adult inner ear. The authors show that Jag1Ndr/Ndr mutants demonstrate hearing loss, vestibular defects, and patterning defects in the cochlea. Similar to previous studies of Jag1 in the ear, the authors find a decrease of outer hair cells, an increase of inner hair cells, and misplaced outer hair cells in the inner hair cell region. The authors perform scRNA-seq and analyze transcriptional effects in different cell populations. Interestingly, the authors present data that shows that the Notch pathway is upregulated in supporting cells at postnatal day 5, suggesting Jag1 may be playing an inhibitory role during postnatal maturation. The authors also show intriguing expression data regarding the OHC-like cells in the OHC region. Overall the study is well-performed and provides new information regarding the hair cell and supporting cell patterning defects caused by a reduction of Jag1-Notch signaling. However, most of the histological analysis was performed during development, and thus the origin of the severe hearing loss is not completely clear.

    Comments

    Major:

    Fig1g shows a very abnormal cross section through the cochlear duct. There are no clearly visible Deiters' cells. Is this the case? Loss of outer hair cell function should only increase thresholds about 40dB, and there are increased thresholds reported here of 60+, despite remaining outer hair cells. This could be accounted for by the conduction defects, but also, there may be defects in the adult ear not observed earlier. Is there any inner hair cell loss? Deiter cell loss? Are inner and outer hair cell stereocilia normal? These may account for the severe hearing loss.

    From the UMAP plot in Fig 2b, it seems that the scRNA-seq data did not reveal any change in cell identities in the Jag1Ndr/Ndr ears. This result is not really discussed in the results or discussion-particularly why the OHC-like cells, extra IHCs, and absent Hensen's cells are not revealed in this analysis.

    It is difficult to know which cells are extra (+1), including inner hair cells and inner phalangeal cells. Since scRNAseq did not reveal a different gene signature for these 'extra' cells, it is more appropriate to just count them all together.

    What is the rationale for reporting differences in the p-value that are not significant at the adjusted p-value? Since these are whole genome analysis it is only appropriate to report significance by adjusted p-values.

    One of the novel aspects of this study is the finding that Notch components are upregulated in the Jag1Ndr/Ndr mutants (although some of these results are not significant at the adjusted p value). Given the potential significance that these results would indicate (including c-inhibition), it would be important to confirm upregulation of key Notch components in situ using RNA-scope or immunohistochemistry.

    Additionally, a previous report has suggested that JAG1 mediates cis-inhibition in the medial region of the cochlea. The data presented here do not show an upregulation of Notch signaling in the medial supporting cells, suggesting this is not the case. This should be discussed.

    Pg 9 Discussion: The sentence: "The JAG1NDR missense mutant is expressed in vivo, and traffics normally, but does not bind or activate NOTCH1", is somewhat misleading because it suggests this allele has no function. Based on the milder ear phenotype to null alleles as well as survival suggests that this allele is hypomorphic. This should be clarified and discussed.

    Minor:

    Pg 5 third paragraph, "Differential gene expression analysis identified 40 up- and 42-downregulated genes in Jag1Ndr/Ndr versus Jag1+/+ IPhCs, with pathway dysregulation similar to the pseudobulk analyses (Fig3c, Supp.Table 5,6)"-should be 40 downregulated and 42 upregulated.

    Similarly: Pg 6 second paragraph: Differential gene expression analysis identified 1 up and 42-downregulated genes in Jag1Ndr/Ndr DCs versus Jag1+/+ DCs-should be 1 down and 42 up

    Significance

    This study corroborates and extends previous studies of the role of JAG1 in the inner ear. The role of JAG1 in cochlear development is not well understood compared to other Notch ligands, because it is expressed in the supporting cells and not the hair cells. The single cell RNAseq analysis presented here sheds new light on how JAG1 may be functioning postnatally. In addition, because this is a novel allele of JAG1 in which the homozygotes survive, we can further understand how the phenotype may affect hearing and balance in Alagille syndrome patients. This study will be interesting to those who study developmental biology, Notch signaling and the inner ear.

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    Referee #1

    Evidence, reproducibility and clarity

    Notch signaling regulates inner and middle ear morphogenesis and establishes a strict pattern of sensory cells in the organ of Corti in the mammalian cochlea. In the paper, the authors investigate the function of Jag1-mediated Notch activation in cochlear patterning and signaling using a novel Jag1 "Nodder" (Jag1Ndr/Ndr) mouse model of Alagille syndrome. In the transgenic mouse model, they found that the mice exhibited severe vestibular and auditory defects including an increase in ectopic inner hair cells, and a reduction in outer hair cells. By single-cell RNA study of the organ of Corti, the authors demonstrated a global dysregulation of genes associated with inner ear development and deafness. They observed that the role of Tbx2 in IHC specification is likely influenced by Notch signaling. This was a well-designed study with quality data that provided valuable information on the effect of dysregulated Jag1 on human Alagille syndrome. Given the recent success of a gene therapy clinical trial for human genetic hearing loss, this study helps to answer some key questions related to hearing, which should be of significance guiding future development of potential therapy.

    To study how Jag1 insufficiency affects the development, the authors included the JagNdr/Ndr mouse model. To fully understand the characteristics of the Nodder mouse model, it's necessary to include the direct age-dependent comparison of the Jag1 level (by qPCR/and or Western blot) between Jag1+/+ v.s. from JagNdr/Ndr in Figure 1 at some selected stages to correlate the Jag1 insufficiency with the "Nodder" model. A spatial expression comparison of Jag1 between Jag1+/+ v.s. from JagNdr/Ndr from different the main age groups should be included in SFigure 2, together with Notch target genes.

    Developmentally hair cells develop from the base to the apex starting from the IHC to OHC. The observation of the changes in HC pattern indicates the impact of Notch in timing and maturation status of HC differentiation. Likely by the time when OHCs are supposed to be developed, which is dictated by the suppression of IHC and the activation of OHC signals, due to the dysregulation of Jag1, the IHC signaling cannot be sufficiently suppressed, whereas the OHC signaling cannot be sufficiently activated. This has a positional effect as further it is from the IHCs, more mature OHC can develop. Could the authors dig deeper into the scRNAseq data to see if they can isolate the profile of extra IHCs in the JagNdr/Ndr mouse, to see if they can detect the expression of some OHC genes albeit at much lower levels?

    It is difficult to dissect the contribution of middle ear malformation and inner ear defects to hearing loss in Alagille syndrome with the current model. For the development of any therapy, the two main factors have to be analyzed separately. One option is to generate an inner ear-specific JagNdr/Ndr model to bypass the middle ear issue, which can be evaluated for potential therapy. This part should be discussed.

    In Figure 1, the author mentioned the major defects found in the vestibular system. Is there any difference in the vestibular system at the cellular level? Some evidence will be informative.

    Scale bar missing in Figure1b and Figure1h.

    The picture quality for Figure 4b is low, especially for F-actin staining. Please enhance the intensity.

    Please mention the scale bar presented m in the figure legends for Figure 2; Figure 3; SFigure 6.

    Fig. 1g, poor quality. The WT cochlea looks severely disorganized.

    Significance

    Notch signaling regulates inner and middle ear morphogenesis and establishes a strict pattern of sensory cells in the organ of Corti in the mammalian cochlea. In the paper, the authors investigate the function of Jag1-mediated Notch activation in cochlear patterning and signaling using a novel Jag1 "Nodder" (Jag1Ndr/Ndr) mouse model of Alagille syndrome. In the transgenic mouse model, they found that the mice exhibited severe vestibular and auditory defects including an increase in ectopic inner hair cells, and a reduction in outer hair cells. By single-cell RNA study of the organ of Corti, the authors demonstrated a global dysregulation of genes associated with inner ear development and deafness. They observed that the role of Tbx2 in IHC specification is likely influenced by Notch signaling. This was a well-designed study with quality data that provided valuable information on the effect of dysregulated Jag1 on human Alagille syndrome. Given the recent success of a gene therapy clinical trial for human genetic hearing loss, this study helps to answer some key questions related to hearing, which should be of significance guiding future development of potential therapy.

  5. Version published to 10.1101/2024.02.02.577075v1 on bioRxiv