1. Cell Biology
  2. Developmental Biology

Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure

  1. Ian A Swinburne
  2. Kishore R Mosaliganti
  3. Srigokul Upadhyayula
  4. Tsung-Li Liu
  5. David G C Hildebrand
  6. Tony Y -C Tsai
  7. Anzhi Chen
  8. Ebaa Al-Obeidi
  9. Anna K Fass
  10. Samir Malhotra
  11. Florian Engert
  12. Jeff W Lichtman
  13. Tomas Kirchhausen
  14. Eric Betzig
  15. Sean G Megason  Is a corresponding author
  1. Harvard Medical School, United States
  2. Boston Children’s Hospital, United States
  3. Janelia Research Campus, Howard Hughes Medical Institute, United States
  4. Harvard University, United States
https://doi.org/10.7554/eLife.37131
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Cite this article
  1. Ian A Swinburne
  2. Kishore R Mosaliganti
  3. Srigokul Upadhyayula
  4. Tsung-Li Liu
  5. David G C Hildebrand
  6. Tony Y -C Tsai
  7. Anzhi Chen
  8. Ebaa Al-Obeidi
  9. Anna K Fass
  10. Samir Malhotra
  11. Florian Engert
  12. Jeff W Lichtman
  13. Tomas Kirchhausen
  14. Eric Betzig
  15. Sean G Megason
(2018)
Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure
eLife 7:e37131.
https://doi.org/10.7554/eLife.37131
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  3. Download .RIS
7 figures, 14 videos, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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ES lumen slowly inflates and rapidly deflates every 0.3–4.5 hr.

(A) Illustration of the adult human inner ear showing cochlea, semicircular canals (SCCs), and endolymphatic duct and sac (ES, red arrowhead) and their organization of tissue (green), endolymph …

https://doi.org/10.7554/eLife.37131.003
Figure 1—figure supplement 1
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Early ES development and additional examples of wild-type ES inflation and deflation.

(A) ES morphogenesis begins at 36-hr post fertilization (hpf) as an evagination in the dorsal epithelial wall of the otic vesicle (green arrowhead points to nascent ES, see also Video 1). Scale bar …

https://doi.org/10.7554/eLife.37131.004
Figure 2
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Hydrostatic pressure transmits endolymph through duct to inflate the ES.

(A) Illustration of larval zebrafish highlighting sagittal plane of image acquisition (blue square). (B) Time points of individual sagittal slices of raw data from 3D time course (endolymph labeled …

https://doi.org/10.7554/eLife.37131.008
Figure 3 with 1 supplement
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Lmx1bb is necessary for development of the ES’s ability to form breaks in its diffusion barrier and deflate.

(A) Lateral view of wild-type and lmx1bbjj410/jj410 mutant ears imaged by bright-field microscopy at 80 hpf, asterisk labels greatly enlarged mutant ES. Scale bar, 100 μm. (B) Slices from 3D …

https://doi.org/10.7554/eLife.37131.011
Figure 3—figure supplement 1
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Inflation of additional mutant ES.

Quantification of segmented ES volumes (primary axis, green) and leak in fluorescence (secondary axis, magenta) from an additional time-lapse of an lmx1bbjj410/jj410 mutant (see Video 6).

https://doi.org/10.7554/eLife.37131.012
Figure 4
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Lamellar protrusions at the tip of the ES exist in open and closed configurations.

(A) Select images from serial-section scanning electron microscopy of a 5.5 dpf zebrafish’s right inner ear. Dorsal is up, lateral left, medial right, ventral down, anterior top, and posterior …

https://doi.org/10.7554/eLife.37131.015
Figure 5
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AO-LLSM reveals dynamics of ES cells.

(A) Illustration of AO-LLSM mounting strategy for imaging ES using volcano mount. (B) Representative LLSM images without adaptive optics (AO), with AO, and with AO followed by deconvolution. (C) …

https://doi.org/10.7554/eLife.37131.017
Figure 6
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Basal lamellae are dynamic.

(A) 3D rendering of AO-LLSM data, three sequential time points 30 s apart (membrane citrine depicted in grey, Video 10). Bright patches on surface move (red, blue, green arrows). (B) Consecutive 3D …

https://doi.org/10.7554/eLife.37131.020
Figure 7
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Basal lamellae open prior to deflation.

(A,C) 3D renderings of segmented cells and ES lumen. (A) Dorsal view. (B) Raw data of membrane citrine AO-LLSM data spanning same time range as in (A). First and last time point are overlaid with …

https://doi.org/10.7554/eLife.37131.024

Videos

Video 1
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Early ES development.

Video begins with schematic of experimental set-up and context of the presented field of view. Then, an annotated time point is presented of the upcoming video. The presented video is of a sagittal …

https://doi.org/10.7554/eLife.37131.005
Video 2
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Wild-type ES inflates and deflates.

Video begins with an illustration depicting the context of the presented field of view, which is a sagittal slice encompassing the developing ES from a 4D time course. Then, an annotated time point …

https://doi.org/10.7554/eLife.37131.006
Video 3
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Wild-type ES inflates and deflates.

A video of two time courses, sagittal slices from 4D datasets, quantified in Figure 1—figure supplement 1B (left) and Figure 1—figure supplement 1C (right). Fluorescence from membrane citrine, shown …

https://doi.org/10.7554/eLife.37131.007
Video 4
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Endolymph periodically inflates ES and then released into periotic space.

Time course of otic vesicle injected with 3 kDa dextran-Texas red at 55 hpf. Panels are transverse volumes of same time course. Left, labeled endolymph presented in yellow. Right, labeled endolymph …

https://doi.org/10.7554/eLife.37131.009
Video 5
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Endolymph released into periotic space followed by perilymph leak-in.

Time course of otic vesicle injected with 3 kDa dextran-Texas red at 55 hpf, and perilymph labeled with 10 kDa dextran- Alexa Fluor 488. First three panels are transverse volumes while the fourth is …

https://doi.org/10.7554/eLife.37131.010
Video 6
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Mutant ES over-inflates.

Video of sagittal slice from 4D dataset of lmx1bbjj410/jj410 mutant- quantified in Figure 3D. Fluorescence from membrane citrine shown in green. Perilymph highlighted with fluorescence from 3 kDa …

https://doi.org/10.7554/eLife.37131.013
Video 7
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Mutant ES over-inflates.

Video of sagittal slice from 4D dataset of lmx1bbjj410/jj410 mutant- quantified in Figure 3—figure supplement 1. Fluorescence from membrane citrine shown in green. Perilymph highlighted with …

https://doi.org/10.7554/eLife.37131.014
Video 8
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Serial-section electron micrographs of wild-type ES at 5.5 dpf.

Sections are 60 nm thick and color overlays highlight cells with lamellar barriers or basal lamellae. Second half explores organization of cells in space using cell segmentations.

https://doi.org/10.7554/eLife.37131.016
Video 9
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Slab view of ES time course acquired with lattice light-sheet microscopy with adaptive optics.

15 sequential slices (300 nm slice spacing) were combined as a maximum intensity projections (MIP) to make a 4.5 μm slab. 7 sequential 4.5 μm slabs were tiled to consolidate the presentation of a …

https://doi.org/10.7554/eLife.37131.018
Video 10
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3D rendering of tracked and segmented cells and ES lumen.

An anterior view on the left and dorsal view on the right. Segmented ES lumen is colored magenta. All other objects are ES cells. Labeled cubes indicate body axes. Same time course as Video 9.

https://doi.org/10.7554/eLife.37131.019
Video 11
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3D rendering of membrane citrine signal.

The video begins with an annotated time point from the 3D rendering of signal from an AO-LLSM time course. A yellow dotted line highlights the rendered ES, green arrows point to basal lamellae, and …

https://doi.org/10.7554/eLife.37131.021
Video 12
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Slab view of myosin-GFP during ES time course.

Seven sequential slices (600 nm slice spacing) were combined as a maximum intensity projections (MIP) to make a 4.2 μm slabs. 4 sequential 4.2 μm slabs, starting with the distal tip of the ES on the …

https://doi.org/10.7554/eLife.37131.022
Video 13
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Representative heat shock dnRac1 time course.

Embryos were heat shocked at 55 hpf. At 57 hpf, α-bungarotoxin protein and 3 kDa dextran-Texas red were injected into the hearts. Time course began at 58 hpf, membrane citrine is green, perilymph is …

https://doi.org/10.7554/eLife.37131.023
Video 14
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Endolymph time course at high time resolution reveal sites of release.

Four embryos with 3 kDa dextran-Texas red injected into the otic vesicle. First three are 3-D rendered volumes from a dorsal view. The fourth fish is 3-D rendered from a transverse view (as in Videos…

https://doi.org/10.7554/eLife.37131.025

Tables

Key resources table
Reagent type (species)
or resource		DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Danio rerio)ABZIRC, Eugene, ORZFIN ID: ZDB-GENO-960809–7
Strain, strain background (Danio rerio)lmx1bb mutant, ale uchu (jj410 allele)ZIRC, Eugene, OR, PMID: 17574823jj410; ZFIN ID: ZDB-ALT-070426–3Schibler and Malicki, 2007
Strain, strain background (Danio rerio)Tg(actb2:mem-citrine-citrine)hm30Megason lab, PMID: 25303534hm30; ZFIN ID: ZDB-ALT-150209–1Xiong et al., 2014
Strain, strain background (Danio rerio)Tg(actb2:mem-citrine)/(actb2:Hsa.H2b-tdTomato)hm32Megason lab, PMID: 27535432hm32; ZFIN ID:
ZDB-ALT-161213–1		Aguet et al., 2016
Strain, strain background (Danio rerio)Tg(actb2:mem-citrine)/(actb2:Hsa.H2b-tdTomato)hm33Megason lab, PMID: 27535432hm33; ZFIN ID: ZDB-ALT-161213–2Aguet et al., 2016
Strain, strain background (Danio rerio)Tg(−5.0lmx1bb:d2eEGFP)mw10gift from Brian Link's lab, PMID: 19500562mw10; ZFIN ID: ZDB-ALT-091218–2McMahon et al., 2009
Strain, strain background (Danio rerio)Tg(actb2:mem-mcherry2)hm29Megason lab, PMID: 23622240hm29; ZFIN ID: ZDB-ALT-130625–1Xiong et al., 2013
Strain, strain background (Danio rerio)Tg(hsp70:rac1_T17N-p2a-mem-cherry2)hm35Megason lab, rac1 mutant plasmid gift from Raz lab, this paperhm35Kardash et al., 2010
Strain, strain background (Danio rerio)Tg(elavl3:GCaMP5G)a4598gift from Alexander Schier's lab, PMID: 23524393a4598; ZFIN ID: ZDB-ALT-130924–1Ahrens et al., 2013
Strain, strain background (Danio rerio)Tg(actb2:myl12.1-EGFP)e2212gift from C.P. Heisenberg's lab, PMID: 25535919e2212; ZFIN ID: ZDB-ALT-130108–2Compagnon et al., 2014
Strain, strain background (Danio rerio)Tg(actb2:mCherry-Hsa.UTRN)e119gift from C.P. Heisenberg's lab, PMID: 25535919e119; ZFIN ID: ZDB-ALT-151029–2Compagnon et al., 2014
Antibodymouse anti ZO-1Thermo Fisher Scientific, Waltham, MAZO1-1A12
Antibodyrabbit anti collagen IIAbcam, Cambridge, United Kingdomab209865
Antibodyrabbit anti lamininSigma-Aldrich, St. Louis, MOL9393
Recombinant DNA reagentpet-28b-Cas9-Hisgift from Alexander Schier's lab, PMID: 24873830addgene id: 47327Gagnon et al., 2014
Recombinant DNA reagentpmtb-t7-alpha-bungarotoxinMegason lab, PMID: 26244658addgene id: 69542Swinburne et al., 2015
Sequence-based reagentfoxi in situ probes,Danio rerioPCRtemplate + T7 reaction (Sigma)RefSeq:NM_181735Thisse and Thisse, 2014
Sequence-based reagentbmp4 in situ probes, Danio rerioPCRtemplate + T7 reaction (Sigma)RefSeq:NM_131342Thisse and Thisse, 2014
Sequence-based reagentlmx1bb sgRNA (exon 2), Danio rerioannealedoligos + SP6 reaction (NEB)GenBank:CR376762Gagnon et al., 2014
Sequence-based reagentlmx1bb sgRNA (exon 3),Danio rerioannealed oligos + SP6 reaction (NEB)GenBank:CR376762Gagnon et al., 2014
Peptide, recombinant proteincas9 proteinMegason labpurification scheme fromGagnon et al., 2014
Peptide, recombinant proteinalpha-bungarotoxinTocris Bioscience (Bristol, United Kingdom)Tocris catalog number 2133Swinburne et al., 2015
Commercial assay or kitmMessage mMachine T7 ULTRA kitThermo Fisher Scientific, Waltham, MAAM1345
Chemical compound, drugDextran, Texas Red, 3000 MWThermo Fisher Scientific, Waltham, MAD-3329
Chemical compound, drugDextran, Alexa Fluor 488, 10000 MWThermo Fisher Scientific, Waltham, MAD-22913
Chemical compound, drugtricaine methanosulfateSigma-Aldrich, St. Louis, MOE10521
Chemical compound, drugnonenyl succinic anhydrideElectron Microscopy Sciences, Hatfield, PA19050
Chemical compound, drugDMP-30Electron Microscopy Sciences, Hatfield, PA13600
Chemical compound, drug1,2,7,8-diepoxyoctane (97%)Sigma-Aldrich, St. Louis, MO139564
Chemical compound, drugSorensen's Phosphate BufferElectron Microscopy Sciences, Hatfield, PA11600–10
Chemical compound, drugglutaraldehyde, EM gradeElectron Microscopy Sciences, Hatfield, PA16220
Chemical compound, drugparaformaldehydeElectron Microscopy Sciences, Hatfield, PA15710
Chemical compound, drugpotassium ferricyanideSigma-Aldrich, St. Louis, MO702587
Chemical compound, drugosmium tetroxideElectron Microscopy Sciences, Hatfield, PA19140
Chemical compound, druguranyl acetateElectron Microscopy Sciences, Hatfield, PA22400
Chemical compound, drugmaleic acidSigma-Aldrich, St. Louis, MOm5757
Chemical compound, drugacetronitrileElectron Microscopy Sciences, Hatfield, PA10020
Chemical compound, drugTaab 812 ResinMarivac Ltd., Nova Scotia, Canada
Software, algorithmMovingROIExtract, convertFormathttps://github.com/krm15/AO-LLSM
Software, algorithmConvertToMegacapture, GoFigure2ContoursToMesheshttps://github.com/krm15/GF2Exchange
Software, algorithmItk-snapwww.itksnap.org
PMID: 16545965		Yushkevich et al., 2006
Software, algorithmFluorenderwww.sci.utah.edu/software/fluorender.html
PMID:23584131		Wan et al., 2012
Software, algorithmHandBrakehttps://handbrake.fr/
Software, algorithmLabVIEWNational Instruments
Software, algorithmMATLAB (R2014A)www.mathworks.com
Software, algorithmParaViewwww.paraview.org
Software, algorithmGoFigure2Xiong et al., 2013
Software, algorithmFIJI (imagJ)www.fiji.sc
PMID: 22743772		Schindelin et al., 2012
Software, algorithmcellPreprocess, multiscalePlanarityAndVoting3D, DistanceFromMask, resample, MorphologicalErosionOnLabelImageFilter, SizeThreshold, MembraneSegmentation, MembraneSegmentationWithMarkersImageFilter, CellSegmentationStatisticshttps://github.com/krm15/ACME/tree/MultithreadLookup
Software, algorithmZen softwarehttp://www.zeiss.com/microscopy/us/products/microscope-software/zen-lite.html
OtherVolcano mould,‘frosted extreme detail’https://www.shapeways.com/, this paperVolcano400
OtherFemtoJet 4xEppendorf, Hamburg, Germany5253000017
OtherNanojectDrummond Scientific, Broomall, PA3-000-204
OtherTungsten wireSigma-Aldrich, St. Louis, MO267554–9.5G

Additional files

Transparent reporting form
https://doi.org/10.7554/eLife.37131.026

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