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

  1. Ian A Swinburne  Is a corresponding author
  2. Kishore R Mosaliganti  Is a corresponding author
  3. Srigokul Upadhyayula  Is a corresponding author
  4. Tsung-Li Liu  Is a corresponding author
  5. David G C Hildebrand
  6. Tony Y -C Tsai
  7. Anzhi Chen  Is a corresponding author
  8. Ebaa Al-Obeidi  Is a corresponding author
  9. Anna K Fass
  10. Samir Malhotra  Is a corresponding author
  11. Florian Engert  Is a corresponding author
  12. Jeff W Lichtman  Is a corresponding author
  13. Tomas Kirchausen  Is a corresponding author
  14. Eric Betzig  Is a corresponding author
  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
7 figures, 14 videos, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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 (beige), perilymph (magenta), and bone (black). Illustrations of the adult and larval zebrafish inner ear showing ES (indicated with red arrowheads, see also Figure 1—figure supplement 1 and Video 1 for how the zebrafish ES first forms). (B) Micrograph of larval zebrafish, sagittal view. (B’) In situ of foxi1 highlights position of ES (red arrowhead), n = 12. (C) Illustration of imaging setup. (D) Slices and select time points from 3D confocal time course showing a single inflation and deflation event from a live zebrafish embryo. Cell membranes (green) are labeled using ubiquitous membrane citrine transgenes. Perilymph (magenta) is labeled with 3 kDa dextran-Texas red. ES identified with dotted blue outline and yellow arrow. Lumen of inflated ES identified with dashed white outline in 64:36 panel. (E) Corresponding 3D meshes of the segmented ES lumen volume. (F) Quantification of segmented ES volumes (primary axis, green) and leak in fluorescence (secondary axis, magenta) over multiple cycles (see also Figure 1—figure supplement 1B,C and Videos 23). (G) Histogram of times between peak inflation volumes, compiled from eight different time courses and 54 inflations. Scale bars 100 μm for (B) and 10 μm for (D,E).

https://doi.org/10.7554/eLife.37131.003
Figure 1—figure supplement 1
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 10 μm. (B–C) Quantification of segmented ES volumes (primary axis, green) and leak in fluorescence (secondary axis, magenta) over multiple cycles. (B) Quantification of segmented ES volumes (primary axis, green) and leak in fluorescence (secondary axis, magenta) over multiple cycles (see left panel of Video 3). (C) Additional time-lapse analysis (see right panel of Video 3).

https://doi.org/10.7554/eLife.37131.004
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 yellow by single dye injection into otic vesicle, membrane citrine in cyan). (C) Illustration of larval zebrafish highlighting transverse perspective (blue box) for rendered volumes of ES. (D) Time points from time course of raw data rendered in 3D transverse view (endolymph in yellow, ES tissue outlined with dashed line) showing endolymph flowing through duct to ES and then out to perilymph (blue arrow). (E) Illustration of strategy for laser ablating otic vesicle cells with point-scanning 2-photon laser to ablate 2–3 targeted cells. (F) Slices from 4D confocal time course after laser ablation showing ES deflation. ES lumen is outlined with a white dashed line in (F), n = 4. (G) Time points from time course rendered in 3D transverse view (endolymph in yellow, perilymph in magenta, ES tissue outlined with dashed line), n = 8. Blue arrows indicate endolymph expulsion or perilymph leak in events (D,G). All scale bars 10 μm.

https://doi.org/10.7554/eLife.37131.008
Figure 3 with 1 supplement
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 confocal time course of an lmx1bb transcriptional reporter (cyan, Tg(lmx1bb:egfp)mw10/mw10; yellow, Tg(actb2:mem-mcherry2)hm29), n = 3. (C) Slices and select time points from 3D confocal time course of lmx1bbjj410/jj410 mutant embryos. Membrane (green) from ubiquitous membrane citrine transgenes. Perilymph (magenta) from 3 kDa dextran-Texas red, n = 4. (D) Quantification of segmented ES volumes (primary axis, green) and leak-in fluorescence (secondary axis, magenta) from lmx1bbjj410/jj410 time course in (C) (see also Figure 3—figure supplement 1 and Videos 56). (E) 3D transverse view (endolymph in yellow) from timelapse showing endolymph in dilated mutant ES, outlined with dashed blue line, n = 2. (F) Small regions with thin membranes (asterisks) form in the inflated ES of wild-type but not lmx1bb mutants. (G) Quantification of minimum epithelial thickness versus inflated ES volume in mutant (plotted in red, n = 9) and wild-type (plotted in black, n = 14). Compiled from 65 to 80 hpf embryos. (H) Uneven labeling from Tg(lmx1bb:egfp) reveals thin basal processes (white arrow). (I) Wild-type ES examples with sparsely labeled cells: membrane-labeled citrine (green) in a membrane-labeled cherry background (magenta), white arrows indicate lamellar projections, n = 15. (J) lmx1bbjj410/jj410 mutant ES examples with sparsely labeled cells: membrane cherry (magenta) in a membrane citrine background (green), n = 9. (K) Cartoon schematic of apico-basal organization of ES. (L) Supporting whole-mount immuno-stains for basal and apical markers (collagen and ZO-1, both magenta) in a membrane-labeled citrine background (green), n = 16, 15, 6, 32, 21, and 12 for (i-vi). Scale bars in (B-L), 10 μm.

https://doi.org/10.7554/eLife.37131.011
Figure 3—figure supplement 1
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
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 bottom of the series z-stack. Cells forming lamellar barriers were labeled (color overlays, consistent across panels) to highlight connectivity of lamellae. Presented slices are a subset from the series, each separated by 960 nm (Video 8). The lumen of the ES is labeled and outlined with a white dashed line in third panel; perilymph (peri.). (B) Lamellae interdigitate and can form tongue-in-groove structures (inset). (C) Lamellae can interweave. (D) Example of lamellar junctions in an open configuration. (E) Cells in endolymphatic duct have basal lamellae, with the presented duct connecting with ES in panel A. (F) 3D rendering of ES segmentation from serial micrographs shown in panel (A) and Video 8. Black dotted-outline encompasses area of closed, endolymph-filled lamellae. Black mesh highlights areas of membrane overlap between lamellae that are spread open. (G) Electron-dense tight junctions (yellow arrows) present in cells that also have spread basal protrusions. An opening in the apical junctions creates a path from the duct to the basal protrusions (magenta arrow, slices in three panels 1.2 μm apart). (H, H’) lmx1bbjj410/jj410 embryos maintain apical junctions between ES cells (yellow arrows). (I) Mutant ES cells lack basal protrusions (red arrow). Scale bar in (A) is 1000 nm, (F) is 5 μm, and all other scale bars are 500 nm. (B–G) Serial section electron microscopy, n = 2, additional transmission EM, n = 3. (H–I) Serial section EM of mutant, n = 1, transmission EM, n = 3.

https://doi.org/10.7554/eLife.37131.015
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) Three orthogonal views of ACME membrane reconstruction. (D) Three orthogonal views of raw fluorescence signal overlaid with cell segmentations and ES lumen segmentation (magenta), n = 4. (E) 3D rendering of segmented cells and ES lumen (magenta). (F) Volume measurements of segmented ES lumen, imaged every 30 s for over 3 hr (Videos 810). Blue arrowheads point to time points presented in G and I. (G) Top, dorsal perspective displaying 3D renderings of segmented cells. Bottom, maximum intensity projections (MIP) of 4.5 μm slab through tip of ES shows raw data of the ES for the same time points. (H) Secondary axis presents green cell’s thickness versus time (green cell in (G)). Again, primary axis is volume of ES lumen for comparison. (I) Top, 3D renderings of just green cell from (G) and magenta lumen highlight stretching of cell, dorsal-medial perspective. Bottom, centered cross-sectional view of raw data overlaid with green cell’s and ES lumen’s segmentations for the same time points. (J) Secondary axis is plot of grey cell’s thickness (grey in (G)). (K) Top, 3D renderings of only grey cell and magenta lumen. Bottom, centered cross-section view of raw data overlaid with grey cell’s and ES lumen’s segmentations for same time points. (L) Scatter plot of results from Spearman correlation test of cell thickness trajectories and ES lumen volume trajectories for individual inflation and deflation intervals that are monotonic (example intervals bracketed in H and J). Green region highlights significant correlation (p-value<10−3) between cells thinning during inflation or thickening during deflation. Green arrowhead points to test result for bracketed interval in H. Grey region highlights instances where there is no significant correlation between the trajectory of cell thickness and lumen volume. Grey arrowhead points to test result for bracketed interval in J (grey arrowhead). Magenta region highlights significant correlation between cells thinning during deflation or thickening during inflation. Magenta arrowhead points to test result for bracketed interval in J (magenta arrowhead). Y-axis was capped at 10 so that all values greater than or equal to 10 plot as 10, n = 99. All scale bars 10 μm.

https://doi.org/10.7554/eLife.37131.017
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 renderings overlaid as red (71:18:00), blue (71:18:30), and then green (71:19:00). Immobile regions remain grey, while regions of displacement are red, blue, and green. Arrows point to moving lamellae. (C) Additional example of moving lamellae, again visualized by overlaying consecutive images in red (70:08:30), blue (70:09:00), and then green (70:09:30). Arrows point to moving lamellae. (D) Top, time points of 3D rendered segmentations spanning 30 min. Below, 4.5 μm MIP slabs of the raw data for the same time points. For both views, arrow points to same region where lumen segmentation is slowly exposed as thin lamellar region expands. (E) Top, time points of 3D rendered segmentations spanning 2 min. Below, 4.5 μm MIP slabs of the raw data for the same time points. For both views, arrow points to same region where lumen segmentation rapidly inflates as thin lamellar region expands. (F) Consecutive 3D renderings, same as (E), overlaid as red (71:46:00), blue (71:46:30), and then green (71:47:00). Arrows point to rapidly inflating lamellae. (G) Consecutive 3D renderings, overlaid as red (70:48:30), blue (70:49:00), and then green (71:49:30). Arrows point to rapidly inflating lamellae, same region as (F). (H) 4.2 μm MIP slabs of a time point from a time-lapse of wild-type embryos expressing myosin GFP (top) mCherry-utrophin (middle), and merged (myosin, green, utrophin, magenta, see Video 12), n = 4. (I) 4.2 μm MIP slabs of a time point from a time-lapse of lmx1bb crispant embryos expressing myosin GFP (see Video 12), n = 2. (J) Dilated mutant ES collapses following puncture of the otic vesicle with a tungsten needle, n = 5. All scale bars 10 μm.

https://doi.org/10.7554/eLife.37131.020
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 segmentations for cells and lumen (magenta) neighboring the region of valve opening. In (A–D) arrows point to site of lamellae separating, dotted outlines indicate field of views in B and D, en. indicates endolymph lumen, peri. indicates perilymph. (C) Anterior view of another instance of lamellae separating. (D) Raw data spanning time range of (C). (E) Dorsal view of endolymph volume within ES lumen and time points before and after two release events. Middle and right panels show x-y and y-z ortho-planes of the isolated release sites, highlighted with yellow arrows (See Video 14), n = 8 (F) Illustrated pressure relief mechanism.

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

Videos

Video 1
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 slice from a 4D time course of early ES development (white arrow points to ES in introduction). ES morphogenesis begins at 36 hr post fertilization (hpf) as an evagination in the dorsal-anterior-lateral epithelial wall of the otic vesicle. Fluorescence from membrane citrine, shown in grey. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.005
Video 2
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 is presented of the upcoming video, a green arrow points to the ES, a dotted line outlines the ES lumen, the otic vesicle is labeled ventral to the ES, and the perilymph surrounds the ES structure, labeled in magenta. Video of sagittal slice from 4D dataset, quantified in Figure 1F. Fluorescence from membrane citrine, shown in green. Perilymph highlighted with fluorescence from 3 kDa dextran-Texas red, shown in magenta. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.006
Video 3
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 in green. Perilymph highlighted with fluorescence from 3 kDa dextran-Texas red, shown in magenta. Scale bars are 10 μm.

https://doi.org/10.7554/eLife.37131.007
Video 4
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 in yellow, membrane citrine in cyan. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.009
Video 5
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 a dorsal view of the same time course. Labeled endolymph presented in yellow, perilymph in magenta. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.010
Video 6
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 dextran-Texas red, shown in magenta. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.013
Video 7
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 fluorescence from 3 kDa dextran-Texas red, shown in magenta. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.014
Video 8
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
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 complete 3D time course. The membrane citrine signal is green and the 3 kDa dextran-Texas red perilymph highlighter is magenta. Lower right panel is an annotated reference, with the basal surface of the ES labeled and outlined with a dotted yellow line, the apical interface enclosing endolymph outlined with a dotted blue line, the endolymph within the ES lumen indicated with a blue arrow, exposed basal lamellae indicated with a green arrow, and the perilymph labeled with magenta text. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.018
Video 10
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
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 the surrounding space is labeled as perilymph with magenta text. Dorsal view of ES, membrane citrine signal rendered in 3D. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.021
Video 12
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 left, were tiled to consolidate the presentation of a complete 3D time course. On top is the time lapse of a wild-type embryo expression myosin-GFP. Below is an lmx1bb crispant expressing myosin-GFP. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.022
Video 13
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 magenta. Scale bar is 10 μm.

https://doi.org/10.7554/eLife.37131.023
Video 14
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 4 and 5). Scale bar is 10 μm.

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

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  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 Kirchausen
  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