Pleiotropic function of Dlx5/6 in coordinating the development of mammalian vocal and auditory organs

  1. Frida Sánchez-Garrido
  2. Victoria Bouzerand
  3. Markéta Kaiser
  4. Chloé Chaumeton
  5. Anastasia Fontaine
  6. Tomáš Zikmund
  7. Jozef Kaiser
  8. Églantine Heude

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Abstract

Acoustic communication, essential for mammalian social interactions, involves both effector (vocal) and receptor (auditory) organs that present a wide variety of morphologies between species. The molecular mechanisms supporting the harmonious diversification of effector and receptor systems along with the evolution of species-specific acoustic communication are still poorly understood. It is conceivable that common genetic pathways determine the parallel morphogenesis of vocal and auditory systems. Here, we investigated this question by generating mutant mice with targeted invalidation of Dlx5/6 genes in the otic placode and cephalic neural crest cells, contributing to the formation of the ear and vocal tract components. We show that Dlx5/6 inactivation provokes simultaneous defects of the outer, middle and inner ear and of the jaw, pharynx and larynx musculoskeletal systems. Our findings support the notion that Dlx5/6 genes played a pleiotropic role in the co-adaptation of vocal and auditory systems in mammals.

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    Reply to the reviewers

    Manuscript number: RC-2024-02835

    Corresponding author: Eglantine, Heude

    RESPONSE TO REVIEWERS

    Minor changes include:

    - concerns of Reviewer #1 with additional work to complete a new Supp. Fig. 3.

    - some text modifications to avoid ambiguity and misunderstanding, and to address the concerns of Reviewers #1 and #2.

    - addition of three references to develop some aspects of the discussion following the suggestions from Reviewer #3.

    Reviewer #1

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

    This manuscript investigates the role of Dlx5/6 in development of the ear and vocal tract components. They generate a new conditional mouse knockout of Dlx5/6 using a Sox10 driver, which deletes functional forms of these factors in the otic placode (the primordium of the entire inner ear) and in neural crest cells, which contribute to the middle and outer ear, the otic capsule, as well as the craniofacial skeleton and cartilage including that of the larynx, and tendons.

    The authors describe the ear and craniofacial phenotypes in these mutants using marker analysis on sections, some whole mounts and microCT imaging. They confirm previously described ear and craniofacial phenotypes, and add additional information on tongue, thyroid and hyoid cartilages, and associated mesoderm-derived muscles. Finally, they assess innervation finding subtle changes innervations patterns.

    They conclude that Dlx5/6 are required for normal development of auditory and vocal structures and suggest that these factors coordinate both systems and that this is important for their co-evolution.

    The experiments presented are straightforward phenotypic analysis, which is well presented with high quality imaging. However, it is not clear how well the conclusions are supported by the data because

    i) The number of animals analysed and showing the reported phenotypes is not clearly stated; some figure legends list some n numbers, but it is sometimes ambiguous whether they refer to controls or experimental specimen. In other cases, n=2 is too low to draw firm conclusions.

    We thank the reviewer for highlighting this point and for giving us the opportunity to clarify it.

    The number of specimens mentioned correspond to the number of mutants analyzed versus the equivalent number of controls. For most figures, at least 3 mutants and 3 corresponding controls have thus been studied independently, unless for the microCT analysis (n=2 each condition) that however complete histological data on sections (n=6 each condition) presented at equivalent stage. Our data showing highly penetrant and reproducible results, the n numbers appear significant in support of our conclusions. To avoid ambiguity, we have systematically completed the n numbers with the mention 'each condition' in the figure legends.

    ii) The nature of the controls is not stated, so we cannot know what is compared. Are controls WT, heterozygous for Dlx5 and -6, single KOs, single hets?

    The lack of clarity in methods and controls does not allow others to reproduce the findings easily.

    To address this concern, we have now added the genotype of controls in the Materials and Methods section. For all experiments, the controls of mutant specimens were from the same littermate. We included as control genotypes the specimens heterozygous for the Cre and/or flox or homozygous for the *flox *only (genotypes equivalent to breeders), which we had previously validated as showing a normal phenotype compared to non-genetically modified 'wild-type' specimens. This observation is consistent with what had been described previously in constitutive inactivated Dlx5/6 mouse models, for which the development of heterozygous animals was unaffected by the mutation (e.g. Heude et al. 2010 PNAS).

    Based on the fairly limited data presented here, the authors make some far-reaching suggestions that are not supported by experimental findings. For example, they propose that their study points to "co-adaptation of the effector and receptor organs during acoustic communication diversification in land vertebrates" and that Dlx5/6 play a role in this process. This idea appears to be the main motivation for the current study, however, there is little evidence to support such conclusions.

    Likewise, they suggest that a "common Sox10-Dlx5/6-BMP signaling axis coordinates the morphogenesis of both CNCC and otic placode derivatives for the proper formation of the vocal and auditory complex". There is no evidence provided that Sox10 controls any of the other genes and functional experiment related to Sox10 are not carried out, while the Dlx5/6-BMP link has been established previously.

    Overall, the authors need to improve numbers, experimental information and controls, and given the descriptive nature of the manuscript they should refrain from wide-ranging conclusions.

    We regret that Reviewer #1 took our discussion points as conclusions, which was not our purpose. Rather, Reviewer #3 stated that 'The authors are careful in the way they present the evidence, and only go beyond their data to add a few 'speculative' paragraphs at the end of the Discussion' (see below). We believe that the Discussion section is the appropriate space to formulate hypotheses based on experimental results, mainly while speculating on evolutionary aspects, that cannot be technically tested but are still conceptually of interest.

    Concerning the Sox10-Dlx5/6-BMP axis, we agree with Reviewer #1 that we do not provide evidences that this common genetic program regulates the formation of vocal and auditory systems. We have added additional nuance to the text to avoid ambiguity, and we expect that our propositions will inspire future research on the issues raised.

    The analysis could also be strengthened by focusing on the novel aspects, providing a developmental time series as to when phenotypes are first observed combined with marker analysis e.g. looking at different compartments of the otic vesicle, cell types etc., and providing higher magnification images to describe the phenotypes (e.g. those in figure 1) will strengthen the paper and might provide some novelty.

    Reviewer #1 (Significance (Required)):

    The authors present a new conditional knock out line for Dlx5/6. The major limitation of the study is that the data presented largely confirm what has already been published including the regulation of BMP pathway components and non-cell autonomous effects on muscle. As far as I am aware, the only new additions to the literature are the analysis of the cartilages and tongue.

    The phenotypic analysis is minimal, there is no developmental time series and no other functional experiments to address some of the suggestions made by the authors.

    My expertise is in ear formation, and I am aware of the craniofacial literature.

    The novelty of our study is based on the generation of a new conditional mouse model to address the pleiotropic role of Dlx5/6 in coordinating the development of both the vocal tract and auditory components, an aspect that has never been considered before.

    To this end, we have combined a variety of high-resolution imaging techniques to achieve an unprecedented analysis of critical markers of placode, neural crest and mesoderm derivatives (including a neural crest lineage analysis). We initially selected key early embryonic (E10.5-E11.5), fetal (E14.5) and perinatal (E16.5-E17.5) stages, but we agree with Reviewer #1 that an additional stage is relevant in our context to complete this developmental time series. We have now added in a new Supp. Fig. 3 including the analysis of a late embryonic stage (E12.5). In particular, our results show that primary chondrogenic condensations are affected at the level of the vocal tract and auditory compartments. Furthermore, our data reveal that the differentiation defects of the masticatory and tongue musculature occur at the transition between early (E11.5) and late (E12.5) embryonic stages. We are grateful to Reviewer #1 for his comments, which allowed us to present these new and interesting results.

    Reviewer #2

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

    In this manuscript, Sanchez-Garrido and colleagues analyze the consequences of Dlx5/6 deletion on cranial neural crest- and otic placode-derived structures development. The authors show that Dlx5/6 inactivation causes broad defects of the outer, middle and inner ear as well as malformations of the jaw, pharynx and larynx musculoskeletal systems. The authors conclude that Dlx5/6 play an important role in the regulation of vocal and auditory systems development in mammals.

    Reviewer #2 (Significance (Required)):

    The work is well-presented and illustrated, with appropriate description of methods. The role of this gene family in regulating craniofacial structures is not completely novel. The analysis is largely descriptive providing little mechanistic insights. The authors propose a possible link between Dlx5/6 activity and BMP signaling as the culprit for the defects observed in the mutants, however this is not supported experimentally. The work is viewed as too preliminary at this stage.

    We are appreciative of the feedback from Reviewer #2. Our aim was to gain insight into the genetic basis of the coordinated development of the vocal tract and hearing complex. As mentioned above for Reviewer #1, we have added modifications to the discussion to nuance and develop our propositions. We hope that our study will help to further develop some of the aspects we have explored.

    Reviewer #3

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

    This is a very good paper presenting results examining the role of DLX5/6 in the formation of ear and vocal tract structures by means of mutant mice. I believe the evidence offered in this paper in favor of such a role is strong and well presented.

    Reviewer #3 (Significance (Required)):

    I think this is a very good study, contributing to our knowledge of the action of DLX5/6, giving us insights into the genes' role in ear and vocal tract structures, critical for vocalization and the pairing of auditory and oral capacities. The authors are careful in the way they present the evidence, and only go beyond their data to add a few 'speculative' paragraphs at the end of the Discussion.

    I would encourage the authors to consider making mention of Gokhman et al's (2021) work, where genes similarly impacting craniofacial and oral capacities are discussed: https://www.nature.com/articles/s41467-020-15020-6

    I would also have liked the authors to cite work on GLI3 and larynx formation (given that both GLI3 and HAND2 control DLX5 and 6) and play an important role in Neural Crest-related processes: https://elifesciences.org/articles/77055 / https://elifesciences.org/articles/56450

    In this context, the authors mention work on the role of Dlx5/6 in GABAergic neurons. Do the authors believe there to be a connection (mediated by the SHH pathway) between the ganglionic eminence and neural crest development, or are these two findings only convergent at the level of the phenotype?

    We are grateful to Reviewer #3 for his positive assessment of our study and for his very relevant suggestions of works to consider in the context of our research. This has allowed us to complete and develop exciting points in our discussion, which we hope will stimulate further lines of research regarding the diversification of acoustic communication abilities during mammalian evolution, including the emergence of speech in humans.

    To answer the reviewer's question, we do not believe that there is a link between the ganglionic eminences and neural crest derivatives, which develop independently in two embryonic compartments under the regulation of distinct genetic programs. However, the observation that Dlx5/6 expression in the brain regulates socialization and vocalization behaviors in adult mice suggest that the genes have a broader pleiotropic role in acoustic communication, both during development and in the adult at the cognitive level.

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    Referee #3

    Evidence, reproducibility and clarity

    This is a very good paper presenting results examining the role of DLX5/6 in the formation of ear and vocal tract structures by means of mutant mice. I believe the evidence offered in this paper in favor of such a role is strong and well presented.

    Significance

    I think this is a very good study, contributing to our knowledge of the action of DLX5/6, giving us insights into the genes' role in ear and vocal tract structures, critical for vocalization and the pairing of auditory and oral capacities. The authors are careful in the way they present the evidence, and only go beyond their data to add a few 'speculative' paragraphs at the end of the Discussion.

    I would encourage the authors to consider making mention of Gokhman et al's (2021) work, where genes similarly impacting craniofacial and oral capacities are discussed. https://www.nature.com/articles/s41467-020-15020-6

    I would also have liked the authors to cite work on GLI3 and larynx formation (given that both GLI3 and HAND2 control DLX5 and 6) and play an important role in Neural Crest-related processes: https://elifesciences.org/articles/77055 https://elifesciences.org/articles/56450 In this context, the authors mention work on the role of Dlx5/6 in GABAergic neurons. Do the authors believe there to be a connection (mediated by the SHH pathway) between the ganglionic eminence and neural crest development, or are these two findings only convergent at the level of the phenotype?

  3. Review Commons

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

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Sanchez-Garrido and colleagues analyze the consequences of Dlx5/6 deletion on cranial neural crest- and otic placode-derived structures development. The authors show that Dlx5/6 inactivation causes broad defects of the outer, middle and inner ear as well as malformations of the jaw, pharynx and larynx musculoskeletal systems. The authors conclude that Dlx5/6 play an important role in the regulation of vocal and auditory systems development in mammals.

    Significance

    The work is well-presented and illustrated, with appropriate description of methods. The role of this gene family in regulating craniofacial structures is not completely novel. The analysis is largely descriptive providing little mechanistic insights. The authors propose a possible link between Dlx5/6 activity and BMP signaling as the culprit for the defects observed in the mutants, however this is not supported experimentally. The work is viewed as too preliminary at this stage.

  4. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    This manuscript investigates the role of Dlx5/6 in development of the ear and vocal tract components. They generate a new conditional mouse knockout of Dlx5/6 using a Sox10 driver, which deletes functional forms of these factors in the otic placode (the primordium of the entire inner ear) and in neural crest cells, which contribute to the middle and outer ear, the otic capsule, as well as the craniofacial skeleton and cartilage including that of the larynx, and tendons.

    The authors describe the ear and craniofacial phenotypes in these mutants using marker analysis on sections, some whole mounts and CT imaging. They confirm previously described ear and craniofacial phenotypes, and add additional information on tongue, thyroid and hyoid cartilages, and associated mesoderm-derived muscles. Finally, they assess innervation finding subtle changes innervation patterns.

    They conclude that Dlx5/6 are required for normal development of auditory and vocal structures and suggest that these factors coordinate both systems and that this is important for their co-evolution. The experiments presented are straightforward phenotypic analysis, which is well presented with high quality imaging. However, it is not clear how well the conclusions are supported by the data because

    i) The number of animals analysed and showing the reported phenotypes is not clearly stated; some figure legends list some n numbers, but it is sometimes ambiguous whether they refer to controls or experimental specimen. In other cases, n=2 is too low to draw firm conclusions.

    ii) The nature of the controls is not stated, so we cannot know what is compared. Are controls WT, heterozygous for Dlx5 and -6, single KOs, single hets?

    The lack of clarity in methods and controls does not allow others to reproduce the findings easily. Based on the fairly limited data presented here, the authors make some far-reaching suggestions that are not supported by experimental findings. For example, they propose that their study points to "co-adaptation of the effector and receptor organs during acoustic communication diversification in land vertebrates" and that Dlx5/6 play a role in this process. This idea appears to be the main motivation for the current study, however, there is little evidence to support such conclusions. Likewise, they suggest that a "common Sox10-Dlx5/6-BMP signaling axis coordinates the morphogenesis of both CNCC and otic placode derivatives for the proper formation of the vocal and auditory complex". There is no evidence provided that Sox10 controls any of the other genes and functional experiment related to Sox10 are not carried out, while the Dlx5/6-BMP link has been established previously. Overall, the authors need to improve numbers, experimental information and controls, and given the descriptive nature of the manuscript they should refrain from wide-ranging conclusions. The analysis could also be strengthened by focusing on the novel aspects, providing a developmental time series as to when phenotypes are first observed combined with marker analysis e.g. looking at different compartments of the otic vesicle, cell types etc., and providing higher magnification images to describe the phenotypes (e.g. those in figure 1) will strengthen the paper and might provide some novelty.

    Significance

    The authors present a new conditional knock out line for Dlx5/6. The major limitation of the study is that the data presented largely confirm what has already been published including the regulation of BMP pathway components and non-cell autonomous effects on muscle. As far as I am aware, the only new additions to the literature are the analysis of the cartilages and tongue. The phenotypic analysis is minimal, there is no developmental time series and no other functional experiments to address some of the suggestions made by the authors.

    My expertise is in ear formation, and I am aware of the craniofacial literature.

  5. Version published to 10.1101/2024.12.13.628362v1 on bioRxiv