Live imaging of hair bundle polarity acquisition demonstrates a critical timeline for transcription factor Emx2

  1. Yosuke Tona
  2. Doris K Wu  Is a corresponding author
  1. NIDCD, NIH, United States

Abstract

The asymmetric hair bundle on top of hair cells (HCs), comprises a kinocilium and stereocilia staircase, dictates HC's directional sensitivity. The mother centriole (MC) forms the base of the kinocilium, where stereocilia are subsequently built next to it. Previously we showed that transcription factor Emx2 reverses hair bundle orientation and its expression in the mouse vestibular utricle is restricted, resulting in two regions of opposite bundle orientation (Jiang et al, 2017). Here, we investigated establishment of opposite bundle orientation in embryonic utricles by live-imaging GFP-labeled centrioles in HCs. The daughter centriole invariably migrated ahead of the MC from the center to their respective peripheral locations in HCs. Comparing HCs between utricular regions, centriole trajectories were similar but they migrated towards opposite directions, suggesting that Emx2 pre-patterned HCs prior to centriole migration. Ectopic Emx2, however, reversed centriole trajectory within hours during a critical time-window when centriole trajectory was responsive to Emx2.

Data availability

The following figures contain the source data files. Figure 2 (source data 1), Figure 2 supplement 2 (source data 1-3), Figure 2 supplement 3 (source data 1-4), Figure 2 supplement 4 (source data 1-2), Figure 3 (source data 1-2), figure 3 supplement 2 (source data 1), Figure 4 (source data 1-2), Figure 5 (source data 1), Figure 6 (source data 1-2), Figure 7 (source data 1), Figure 7 supplement 1 (source data 1), Figure 8 (source data 1-2), Figure 9 (source data 1).

Article and author information

Author details

  1. Yosuke Tona

    Section on Sensory Cell Regeneration and Development, Laboratory of Molecular Biology, NIDCD, NIH, Bethesda, United States
    Competing interests
    No competing interests declared.
  2. Doris K Wu

    Section on Sensory Cell Regeneration and Development, Laboratory of Molecular Biology, NIDCD, NIH, Bethesda, United States
    For correspondence
    wud@nidcd.nih.gov
    Competing interests
    Doris K Wu, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1400-3558

Funding

National Institutes of Health (1ZIADC000021)

  • Yosuke Tona

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal experiments were conducted according to NIH guidelines and under the approved Animal Care Protocol of NIDCD/NIH (#1212-17).

Reviewing Editor

  1. Tanya T Whitfield, University of Sheffield, United Kingdom

Publication history

  1. Received: May 28, 2020
  2. Accepted: September 17, 2020
  3. Accepted Manuscript published: September 23, 2020 (version 1)
  4. Version of Record published: October 5, 2020 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 1,184
    Page views
  • 155
    Downloads
  • 4
    Citations

Article citation count generated by polling the highest count across the following sources: PubMed Central, Crossref, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Yosuke Tona
  2. Doris K Wu
(2020)
Live imaging of hair bundle polarity acquisition demonstrates a critical timeline for transcription factor Emx2
eLife 9:e59282.
https://doi.org/10.7554/eLife.59282

Further reading

    1. Cell Biology
    2. Neuroscience
    Yu Wang, Meghan Lee Arnold ... Barth D Grant
    Research Article Updated

    Caenorhabditis elegans neurons under stress can produce giant vesicles, several microns in diameter, called exophers. Current models suggest that exophers are neuroprotective, providing a mechanism for stressed neurons to eject toxic protein aggregates and organelles. However, little is known of the fate of the exopher once it leaves the neuron. We found that exophers produced by mechanosensory neurons in C. elegans are engulfed by surrounding hypodermal skin cells and are then broken up into numerous smaller vesicles that acquire hypodermal phagosome maturation markers, with vesicular contents gradually degraded by hypodermal lysosomes. Consistent with the hypodermis acting as an exopher phagocyte, we found that exopher removal requires hypodermal actin and Arp2/3, and the hypodermal plasma membrane adjacent to newly formed exophers accumulates dynamic F-actin during budding. Efficient fission of engulfed exopher-phagosomes to produce smaller vesicles and degrade their contents requires phagosome maturation factors SAND-1/Mon1, GTPase RAB-35, the CNT-1 ARF-GAP, and microtubule motor-associated GTPase ARL-8, suggesting a close coupling of phagosome fission and phagosome maturation. Lysosome activity was required to degrade exopher contents in the hypodermis but not for exopher-phagosome resolution into smaller vesicles. Importantly, we found that GTPase ARF-6 and effector SEC-10/exocyst activity in the hypodermis, along with the CED-1 phagocytic receptor, is required for efficient production of exophers by the neuron. Our results indicate that the neuron requires specific interaction with the phagocyte for an efficient exopher response, a mechanistic feature potentially conserved with mammalian exophergenesis, and similar to neuronal pruning by phagocytic glia that influences neurodegenerative disease.

    1. Cell Biology
    2. Genetics and Genomics
    Mi Huang, Daniel Coral ... Sebastian Kalamajski
    Research Article Updated

    Genetic variation at the MTIF3 (Mitochondrial Translational Initiation Factor 3) locus has been robustly associated with obesity in humans, but the functional basis behind this association is not known. Here, we applied luciferase reporter assay to map potential functional variants in the haplotype block tagged by rs1885988 and used CRISPR-Cas9 to edit the potential functional variants to confirm the regulatory effects on MTIF3 expression. We further conducted functional studies on MTIF3-deficient differentiated human white adipocyte cell line (hWAs-iCas9), generated through inducible expression of CRISPR-Cas9 combined with delivery of synthetic MTIF3-targeting guide RNA. We demonstrate that rs67785913-centered DNA fragment (in LD with rs1885988, r2 > 0.8) enhances transcription in a luciferase reporter assay, and CRISPR-Cas9-edited rs67785913 CTCT cells show significantly higher MTIF3 expression than rs67785913 CT cells. Perturbed MTIF3 expression led to reduced mitochondrial respiration and endogenous fatty acid oxidation, as well as altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed mitochondrial OXPHOS complex assembly. Furthermore, after glucose restriction, the MTIF3 knockout cells retained more triglycerides than control cells. This study demonstrates an adipocyte function-specific role of MTIF3, which originates in the maintenance of mitochondrial function, providing potential explanations for why MTIF3 genetic variation at rs67785913 is associated with body corpulence and response to weight loss interventions.