Activity regulates a cell type-specific mitochondrial phenotype in zebrafish lateral line hair cells

  1. Andrea McQuate
  2. Sharmon Knecht
  3. David W Raible

  • Curated by eLife

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    Mutations in mitochondrial genes can lead to deafness but the mitochondrial biology of sensory hair cells is not well understood. In this study, high-resolution imaging of mitochondrial development in sensory hair cells of normal and mutant zebrafish lateral line systems was described. The authors provide evidence that the formation of the mitochondrial architecture requires normal hair cell activity. This paper is of potential interest to researchers interested in metabolic homeostasis and sensory hair cell biology.

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Abstract

Hair cells of the inner ear are particularly sensitive to changes in mitochondria, the subcellular organelles necessary for energy production in all eukaryotic cells. There are over 30 mitochondrial deafness genes, and mitochondria are implicated in hair cell death following noise exposure, aminoglycoside antibiotic exposure, as well as in age-related hearing loss. However, little is known about the basic aspects of hair cell mitochondrial biology. Using hair cells from the zebrafish lateral line as a model and serial block-face scanning electron microscopy, we have quantifiably characterized a unique hair cell mitochondrial phenotype that includes (1) a high mitochondrial volume and (2) specific mitochondrial architecture: multiple small mitochondria apically, and a reticular mitochondrial network basally. This phenotype develops gradually over the lifetime of the hair cell. Disrupting this mitochondrial phenotype with a mutation in opa1 impacts mitochondrial health and function. While hair cell activity is not required for the high mitochondrial volume, it shapes the mitochondrial architecture, with mechanotransduction necessary for all patterning, and synaptic transmission necessary for the development of mitochondrial networks. These results demonstrate the high degree to which hair cells regulate their mitochondria for optimal physiology and provide new insights into mitochondrial deafness.

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  1. Version published to 10.7554/elife.80468 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    In this manuscript, McQuate et al. use serial block face SEM to provide a high resolution, 3D analysis of mitochondrial structure in hair cells and surrounding supporting cells of the zebrafish lateral line. They first demonstrate that hair cells have a higher mitochondrial volume as compared to supporting cells, which likely reflects the high metabolic load of these sensory cells. Their deeper analysis of mitochondrial morphology in hair cells reveals that the base of the hair cell - near the presynapse is dominated by a large, networked mitochondrion, while the apex of the cell is dominated by many small mitochondria. By examining hair cells at different stages of development, the authors show that specialized features of hair cell mitochondria are gradually established over the course of development. Finally, by examining hair cells in mutants that lack mechanosensation or presynaptic calcium responses, McQuate et al. reveal that cellular activity contributes to the development of appropriate mitochondrial morphology and localization within hair cells. This dataset, which will be made publicly available, is an immense resource to the community and will facilitate the generation of novel hypotheses about hair cell mitochondrial function in health and disease.

    Strengths:

    1. The painstaking acquisition and analysis of hair cell EM data in a genetically tractable system that is easily accessible for in vivo functional experiments to address hypotheses that emerge from this work.
    1. The use of multiple datasets and analysis methods to cross-validate results.
    1. The thoughtful, careful analysis of the data highlights the richness of the dataset.
    1. The use of both wild-type and mutant animals substantially adds to the manuscript, providing significantly more insight than wild-type data alone.

    Weaknesses:

    1. The manuscript could more strongly highlight the utility of this dataset and facilitate its future use by providing a summary table that lists each sample together with salient details.
    1. The authors examine an opa-1 mutant with altered mitochondrial fission (which consequently has changes in mitochondrial morphology and organization) to suggest that aberrant mitochondrial architecture negatively impacts mitochondrial function. However, mitochondrial fusion is thought to be critical for mitochondrial health beyond just altered architecture. Because fusion has other roles, it is difficult to use this manipulation to conclude that it is simply disruptions in mitochondrial architecture that alters function.
    1. Although the work of acquiring and reconstructing EM data is labor-intensive, ideally, multiple fish would be examined for each genotype. Readers should take into consideration that one of the mutant datasets is derived from just one animal.

    We thank Reviewer 1 for pointing out the “painstaking acquisition” that went into this study, the “thoughtful, careful analysis,” and the “richness of the dataset.” We believe we have addressed the aforementioned weaknesses.

    Reviewer #2 (Public Review):

    Sensory hair cells have high metabolic demands and rely on mitochondria to provide energy as well as regulate homeostatic levels of intracellular calcium. Using high-resolution serial block face SEM, the authors examined the influences of both developmental age and hair cell activity on hair cell mitochondrial morphology. They show that hair cell mitochondria develop a regionally specific architecture, with the highest volume mitochondria localized to the basolateral presynaptic region of hair cells. Data obtained from mutants lacking either mechanotransduction or presynaptic calcium influx provide evidence that hair cell activity shapes regional mitochondrial morphology. These observed specializations in mitochondrial morphology may play an important role in mitochondrial function, as mutants showing disrupted hair cell mitochondrial architecture showed depolarized mitochondrial potentials and impaired evoked mitochondrial calcium influx.

    This work provides novel and intriguing evidence that mechanotransduction and presynaptic calcium influx play important roles in shaping subcellular mitochondrial morphology in sensory hair cells. Yet there was a lack of consistency in the analysis and presentation of the data which made it difficult to contextualize and interpret the results. This study would be greatly strengthened by i) consistent definitions for hair cell maturation, ii) comparable data analysis of cav1.3a mutant and cdh23 mutant mitochondrial morphologies, and iii) more detailed descriptions and interpretations of the UMAP analysis.

    We thank Reviewer #2 for thinking the work is “novel and intriguing”. We have addressed the weaknesses raised.

    Reviewer #3 (Public Review):

    McQuate et al have succeeded in reconstructing 3D images of mitochondria and discovered unique structural features of mitochondria in zebrafish hair cells. Compared to the other cell types, such as central and peripheral support cells, Hair cells have many elongated and connected mitochondria and they seem to be involved in hair cell and ribbon synapses development. These findings will contribute to understanding the mechanisms for mitochondrial network regulation.

    Using the SBFSEM technique, the authors provide clear 3D images of hair cells and the technique improves the resolution of the image to understand the structural parameters of not only mitochondria but also ribbon synapses compared to typical fluorescent imaging. These results are very attractive and have the high potential to broadly apply to 3D imaging of any type of organelles, cells, and tissues. On the other hand, however, the authors provide the data from a small sample size, and the functional experiments to make a conclusion are lacking. Some missing representative images and the nonunified methods of grouping for the analysis make the reviewer concerned.

    We thank the Reviewer for thinking the results are “very attractive and have the high potential to broadly apply to 3D imaging of any type or organelles, cell, and tissues.” We agree. We have addressed the weaknesses raised

  3. eLife

    eLife assessment

    Mutations in mitochondrial genes can lead to deafness but the mitochondrial biology of sensory hair cells is not well understood. In this study, high-resolution imaging of mitochondrial development in sensory hair cells of normal and mutant zebrafish lateral line systems was described. The authors provide evidence that the formation of the mitochondrial architecture requires normal hair cell activity. This paper is of potential interest to researchers interested in metabolic homeostasis and sensory hair cell biology.

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, McQuate et al. use serial block face SEM to provide a high resolution, 3D analysis of mitochondrial structure in hair cells and surrounding supporting cells of the zebrafish lateral line. They first demonstrate that hair cells have a higher mitochondrial volume as compared to supporting cells, which likely reflects the high metabolic load of these sensory cells. Their deeper analysis of mitochondrial morphology in hair cells reveals that the base of the hair cell - near the presynapse is dominated by a large, networked mitochondrion, while the apex of the cell is dominated by many small mitochondria. By examining hair cells at different stages of development, the authors show that specialized features of hair cell mitochondria are gradually established over the course of development. Finally, by examining hair cells in mutants that lack mechanosensation or presynaptic calcium responses, McQuate et al. reveal that cellular activity contributes to the development of appropriate mitochondrial morphology and localization within hair cells. This dataset, which will be made publicly available, is an immense resource to the community and will facilitate the generation of novel hypotheses about hair cell mitochondrial function in health and disease.

    Strengths:
    1. The painstaking acquisition and analysis of hair cell EM data in a genetically tractable system that is easily accessible for in vivo functional experiments to address hypotheses that emerge from this work.
    2. The use of multiple datasets and analysis methods to cross-validate results.
    3. The thoughtful, careful analysis of the data highlights the richness of the dataset.
    4. The use of both wild-type and mutant animals substantially adds to the manuscript, providing significantly more insight than wild-type data alone.

    Weaknesses:
    1. The manuscript could more strongly highlight the utility of this dataset and facilitate its future use by providing a summary table that lists each sample together with salient details.
    2. The authors examine an opa-1 mutant with altered mitochondrial fission (which consequently has changes in mitochondrial morphology and organization) to suggest that aberrant mitochondrial architecture negatively impacts mitochondrial function. However, mitochondrial fusion is thought to be critical for mitochondrial health beyond just altered architecture. Because fusion has other roles, it is difficult to use this manipulation to conclude that it is simply disruptions in mitochondrial architecture that alters function.

    3. Although the work of acquiring and reconstructing EM data is labor-intensive, ideally, multiple fish would be examined for each genotype. Readers should take into consideration that one of the mutant datasets is derived from just one animal.

  5. eLife

    Reviewer #2 (Public Review):

    Sensory hair cells have high metabolic demands and rely on mitochondria to provide energy as well as regulate homeostatic levels of intracellular calcium. Using high-resolution serial block face SEM, the authors examined the influences of both developmental age and hair cell activity on hair cell mitochondrial morphology. They show that hair cell mitochondria develop a regionally specific architecture, with the highest volume mitochondria localized to the basolateral presynaptic region of hair cells. Data obtained from mutants lacking either mechanotransduction or presynaptic calcium influx provide evidence that hair cell activity shapes regional mitochondrial morphology. These observed specializations in mitochondrial morphology may play an important role in mitochondrial function, as mutants showing disrupted hair cell mitochondrial architecture showed depolarized mitochondrial potentials and impaired evoked mitochondrial calcium influx.

    This work provides novel and intriguing evidence that mechanotransduction and presynaptic calcium influx play important roles in shaping subcellular mitochondrial morphology in sensory hair cells. Yet there was a lack of consistency in the analysis and presentation of the data which made it difficult to contextualize and interpret the results. This study would be greatly strengthened by i) consistent definitions for hair cell maturation, ii) comparable data analysis of cav1.3a mutant and cdh23 mutant mitochondrial morphologies, and iii) more detailed descriptions and interpretations of the UMAP analysis.

  6. eLife

    Reviewer #3 (Public Review):

    McQuate et al have succeeded in reconstructing 3D images of mitochondria and discovered unique structural features of mitochondria in zebrafish hair cells. Compared to the other cell types, such as central and peripheral support cells, Hair cells have many elongated and connected mitochondria and they seem to be involved in hair cell and ribbon synapses development. These findings will contribute to understanding the mechanisms for mitochondrial network regulation.

    Using the SBFSEM technique, the authors provide clear 3D images of hair cells and the technique improves the resolution of the image to understand the structural parameters of not only mitochondria but also ribbon synapses compared to typical fluorescent imaging. These results are very attractive and have the high potential to broadly apply to 3D imaging of any type of organelles, cells, and tissues. On the other hand, however, the authors provide the data from a small sample size, and the functional experiments to make a conclusion are lacking. Some missing representative images and the nonunified methods of grouping for the analysis make the reviewer concerned.

  7. Version published to 10.1101/2022.06.17.496604v1 on bioRxiv