Author response: Lamellar projections in the endolymphatic sac act as a relief valve to regulate inner ear pressure (2018)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The inner ear is a fluid-filled closed-epithelial structure whose function requires maintenance of an internal hydrostatic pressure and fluid composition. The endolymphatic sac (ES) is a dead-end epithelial tube connected to the inner ear whose function is unclear. ES defects can cause distended ear tissue, a pathology often seen in hearing and balance disorders. Using live imaging of zebrafish larvae, we reveal that the ES undergoes cycles of slow pressure-driven inflation followed by rapid deflation. Absence of these cycles in lmx1bb mutants leads to distended ear tissue. Using serial-section electron microscopy and adaptive optics lattice light-sheet microscopy, we find a pressure relief valve in the ES comprised of partially separated apical junctions and dynamic overlapping basal lamellae that separate under pressure to release fluid. We propose that this lmx1-dependent pressure relief valve is required to maintain fluid homeostasis in the inner ear and other fluid-filled cavities. https://doi.org/10.7554/eLife.37131.001 eLife digest The most internal part of the human ear, the inner ear, is essential for us to hear and have a sense of balance. It is formed by a complex series of connected cavities filled by a liquid. When sound waves and changes in the position of the body make this liquid move, specialized 'hair' cells can detect these subtle movements; neurons then relay this information to the brain where it is decoded and interpreted. For the inner ear to work properly, the body needs to finely regulate the pressure created by the liquid inside the cavities. For example, people with unstable pressure in their ears can experience deafness or problems with balance. A structure known as the endolymphatic sac, which is a balloon-like chamber connected to the rest of the inner ear by a thin tube, helps with this regulation. However, scientists are still unsure about how exactly the sac performs its role. One problem is that the inner ear is difficult to study because it is encased in one of the densest bones in the body. Many other animals also have inner ears, from fish to birds and mammals. Here, Swinburne et al. examine the inner ear of zebrafish embryos because, in this fish, the ear starts working before the bones around it form; the structure is therefore accessible for injections and microscopy. Experiments show that when the pressure in the inner ear rises, the endolymphatic sac slowly fills up with the ear liquid, and then it rapidly deflates. Fish with mutations that stop the sac from deflating have overinflated sacs, which is a symptom also found in certain patients with hearing and balance disorders. Looking into the details of these inflation-deflation cycles, Swinburne et al. found that the cells that form the sac have gaps between them, unlike a normal sheet of cells. A flap covers these gaps to keep the liquid in, but under pressure, the flap opens and the liquid can escape. These results show that the endolymphatic sac works as a pressure relief valve for the inner ear. Ultimately, understanding how pressure is regulated in the ear could help patients with inner ear disorders. It could also serve as a template to investigate how eyes, kidneys and the brain, which all have liquid-filled cavities, control their internal pressure. https://doi.org/10.7554/eLife.37131.002 Introduction Understanding the mechanisms by which organs use water-filled cavities to compartmentalize biochemical and biophysical environments is a fundamental problem. Because water is nearly incompressible, several organs harness and cope with water as an object that transmits force. Hydrostatic pressure inflates the eye during development (Coulombre, 1956). Later, unstable ocular pressure from reduced production of aqueous humor or reduced drainage can lead to blindness, as occurs in hypertonic maculopathy or glaucoma (Costa and Arcieri, 2007; Leske, 1983). Hydrostatic pressure appears to drive expansion of brain ventricles during development (Desmond, 1985; Desmond and Levitan, 2002; Lowery and Sive, 2005). Later, unstable hydrostatic pressure in brain ventricles is correlated with hydrocephaly and mental disorders (Hardan et al., 2001; Kurokawa et al., 2000). Hydrostatic pressure inflates and controls the size of the developing ear (Abbas and Whitfield, 2009; Hoijman et al., 2015; Mosaliganti et al., unpublished). Later, unstable pressure in the ear can cause deafness and balance disorders like Pendred syndrome and Ménière's disease (Belal and Antunez, 1980; Schuknecht and Gulya, 1983). This theme of harnessing hydrostatic pressure for normal development and controlling pressure for healthy physiology raises the question of how tissues regulate pressure. Tissue structures identified as important for pressure control include Schlemm's canal in the eye, arachnoid granules and the choroid plexus in the brain, and the endolymphatic duct and sac in the ear (Johnstone et al., 2011; Kimura and Schuknecht, 1965; Naito, 1950; Orešković et al., 2017; Pollay, 2010; Symss and Oi, 2013; Tripathi, 1974). A cavity's pressure could be managed via mechanisms involving molecular pores, molecular transporters, and the physical behavior of the tissue. Observing the mechanisms by which these tissue barriers control pressure has been limited by a range of obstacles such as optical accessibility and uncertainty in both the time- and length-scale on which they function. As such, not much is known about how these tissues manage fluctuating pressures because they have not been observed in vivo. The inner ear is a prominent example of an organ whose tissue form determines its physiology. It is filled with endolymph, the composition of which differs from other fluids such as plasma, perilymph, and cerebral spinal fluid in that it contains high potassium, low sodium, and high electric potential (Lang et al., 2007). This endolymph composition is necessary to drive ion currents into hair cells to convert fluid movement, caused by either the head's acceleration or by sound, into biochemical signals that underlie balance and hearing. The ear's endolymphatic duct connects the endolymph in the semicircular canals, cochlea, and other chambers to the endolymphatic sac (ES, Figure 1A,B). An ES-like structure is present in basal vertebrates, including lamprey and hagfish (Hammond and Whitfield, 2006), suggesting it plays an ancient role in inner ear function. The epithelium of the ES, as well as that of the rest of the ear, has an apical surface facing the internal endolymph and a basal surface facing the external perilymph (Figure 1A). Most of the adult ear is enclosed in a bony labyrinth within the temporal bone and perilymph is located in the space between the epithelium and bone (Figure 1A). In adults, the endolymphatic duct and sac are partially encased within the temporal bone such that the distal end of the endolymphatic sac protrudes out of the temporal bone into the cranial cavity (Figure 1A). Excessive hydrostatic pressure can tear the inner ear's epithelium, disrupting its electric potential (a pathology called endolymphatic hydrops). In contrast, low potassium or reduced endolymph production can lower hydrostatic pressure within the ear to the point where the ear chambers may collapse (Lang et al., 2007). Early work showed that ES ablation causes hydrostatic pressure to rise and the epithelium to tear (Kimura and Schuknecht, 1965; Naito, 1950), suggesting that the ES may maintain endolymph homeostasis. Recent work indicated that an explanted mouse ES gradually loses endolymph (Honda et al., 2017). While many molecular pores and transporters are expressed in the ES, it is unclear why it is organized as a dead-end tube and how it might reduce endolymph volume. Figure 1 with 1 supplement see all Download asset Open asset 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 2–3). (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 The ES has not been studied much in zebrafish because it has not always been clear that zebrafish possess this structure or at what time it forms during development (Haddon and Lewis, 1996; Hammond and Whitfield, 2006). In situ hybridization images at 52 hr post fertilization (hpf) showing expression of ES markers, foxi1 and bmp4, restricted to an ES-like structure positioned on the dorsal side of the otic vesicle are the strongest and developmentally earliest evidence for zebrafish having an ES (Figure 1B') (Abbas and Whitfield, 2009; Geng et al., 2013). However, its formation and physiological function remain unknown. Here, we present a detailed characterization of the ES in zebrafish performed using confocal microscopy, serial-section electron microscopy, adaptive optics lattice light-sheet microscopy, and a genetic mutant that together demonstrate that the ES contains a physical relief valve for regulating inner ear fluid pressure through regulated transepithelial fluid release. Results The endolymphatic sac exhibits cycles of inflation and deflation We first established an imaging system to identify the developmental origins of the zebrafish ES. Extended time-lapse imaging required long-term immobilization with α-bungarotoxin that permits normal development without the reduction in ear growth caused by long-term treatment with tricaine, bright fluorescent transgenic fish with contrast from a membrane-localized fluorescent protein to minimize bleaching and photoxicity, and mounting in a submerged agarose canyon that permits positioning of the zebrafish ear close to the coverslip (400 μm wide, walls of canyon secure the yolk and head, Figure 1C) (Swinburne et al., 2015). The zebrafish ear develops from a collection of epithelial cells that form a closed fluid-filled ellipsoidal structure called the otic vesicle (Whitfield, 2015). We saw that ES morphogenesis begins at 36 hpf as an evagination in the dorsal epithelial wall of the otic vesicle (Figure 1—figure supplement 1A, Video 1, Figure 1A–B, and ES identity confirmed by expression of foxi1, Figure 1B'). Between 36 and 60 hpf, the ES grows and elongates. These findings established that the zebrafish ES is optically accessible during embryonic and larval stages and that ES morphogenesis begins at 36 hpf. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 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 The zebrafish ear starts to sense body acceleration for the nervous system between 60 and 72 hpf, as demonstrated by the onset of the vestibulo-ocular reflex (Mo et al., 2010). We found that the ES begins to exhibit a physical behavior during the same time window. We observed that the lumen of the ES remains closed until 60 hpf, but between 60 and 65 hpf it begins cycles of slowly inflating and rapidly deflating (sagittal slices, Figure 1D, lumen volumes, Figure 1E, Figure 1—figure supplement 1, and Videos 2–3). Three-dimensional (3D) measurements showed that the ES lumen volume changes 5–20-fold through the course of each cycle (Figure 1E,F, and Figure 1—figure supplement 1B–C). The period between peak ES volumes exhibited a broad distribution (0.3–4.5 hr) with an average of 1.6 ± 0.8 hr (mean ± SD, histogram compiled from eight time courses and 54 peaks, Figure 1G). These initial observations suggested several potential causes of the inflation-deflation cycles of the ES including: a response to organ-wide increases in fluid pressure within the otic vesicle, a local tissue behavior wherein the ES inflates with fluid from the perilymph, or a local tissue behavior wherein cells in the ES periodically coordinate their movements to expand the ES volume by sucking fluid from the otic vesicle. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 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 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 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 Loss of the epithelial barrier is sufficient for ES deflation To assess whether ES inflations occur by transmission of endolymph pressure between the otic vesicle and the ES, we performed a single injection of a small volume of solution containing 3 kDa fluorescent dextran into the otic vesicle and followed its movement during inflation and deflation. We found that the duct is open and that endolymph flows from the otic vesicle into the ES during inflation (Figure 2A–D, Video 4). Additionally, upon deflation, endolymph rapidly leaked out of the ES and into the perilymphatic space (75:22 panel, Figure 2D). To determine whether pressure in the otic vesicle is transmitted to the ES for its inflation, we laser-ablated 2–3 cells within the wall of the otic vesicle distant from the ES at 64 hpf (Figure 2E). Shortly after ablation, the ES deflated and completely collapsed within 20 min (Figure 2F). These results indicate that fluid pressure in the otic vesicle inflates the ES volume, the ES tissue has elastic material properties, and a loss of epithelial integrity is sufficient for ES deflation. Figure 2 Download asset Open asset 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 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg 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 A core function of epithelial sheets is to act as a barrier that can prevent passive movement of molecules between an organ's interior and exterior. Deflations could be driven by local breaks in the epithelial barrier combined with elastic tissue collapse or by an alternative mechanism such as cellular reabsorption of endolymph. To distinguish between these mechanisms, we used dye injections into the heart to label the perilymph that surrounds the otic vesicle and ES (Figure 1D). Quantification of fluorescence within the lumen of the ES revealed that perilymph dye begins to leak into the ES lumen at the onset of each deflation (Figure 1F, visible leakage in Figure 1D, Videos 2–3). In the example shown deflation coincided with an influx of fluorescence from the perilymph into the lumen of the ES in each of the nine major inflation-deflation cycles. This coincident timing suggests that both deflation and dye leakage were due to transient breaks in the epithelial barrier, an interpretation that is consistent with the rapid release of endolymph to deflation (Figure Video 4). The that perilymph the deflating ES may the of a elastic might be to of the of and the of movement of perilymph by to limited and the of contrast for we could not alternative mechanisms such as from rapid or cellular To determine whether endolymph occurs at the same time as perilymph we labeled the two fluids with different and their with temporal (Figure Video We found a behavior deflation in which endolymph leaked out perilymph leaked in perilymph out with endolymph followed by deflation It is difficult to the and movement of fluid with the alternative interpretation that rapid or cellular the ES. A mechanism for this observed behavior a structure that driven by a pressure that rapidly opens during deflation to for by followed by influx by before Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Endolymph released into periotic space followed by perilymph Time course of otic vesicle injected with 3 kDa dextran-Texas red at 55 hpf, and perilymph labeled with 10 kDa are transverse volumes the is a dorsal view of the same time course. endolymph presented in yellow, perilymph in magenta. Scale bar is 10 μm. is essential for ES deflation The of the ES inflation and deflation cycles suggests might be To identify that to the of this physiology and to reveal in which it can we a mutant caused by a stop in the lmx1bb et al., that exhibited an ES. We found that the ES in lmx1bb mutants times the inflated ES it visible at hpf by microscopy (Figure To determine lmx1bb is expressed at an place and time for a to be an ES we a transgenic driven by the lmx1bb et al., expressed in ES cells at hpf, before the first ES inflation Figure consistent with lmx1bb expression for development of the of the ES to release pressure. in the development of the otic vesicle, lmx1bb is expressed in of the semicircular canals and of the inner ear that also exhibit development in the mutant et al., and 2007). is for ES development on of the otic and are many mutants with or defects that not have ES et al., 1996; et al., imaging and perilymph in mutant embryos revealed that the ES lumen (Figure Figure supplement 1, and Videos As in the we quantified the of perilymph into the ES lumen (secondary of Figure 3D and Figure supplement 1A). In the we observed perilymph the ES. Additionally, we mutants where the endolymph labeled and not leakage out of the distended ES epithelium (Figure These findings that the epithelial barrier remains in the mutant ES. Figure 3 with 1 supplement see all Download asset Open asset is necessary for development of the to form breaks in its barrier and (A) view of and mutant ears by microscopy at hpf, mutant ES. Scale 100 μm. (B) Slices from 3D confocal time course of an lmx1bb yellow, n = (C) Slices and select time points from 3D confocal time course of mutant (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 fluorescence (secondary axis, magenta) from time course in (C) (see also Figure supplement 1 and Videos (E) 3D transverse view (endolymph in from showing endolymph in mutant ES, outlined with dashed blue n = (F) with thin membranes form in the inflated ES of but not lmx1bb (G) Quantification of epithelial inflated ES volume in mutant in red, n = and in n = from 65 to hpf from thin basal (white arrow). Wild-type ES with labeled citrine (green) in a (magenta), white arrows indicate n = mutant ES with labeled membrane (magenta) in a membrane citrine (green), n = schematic of organization of ES. for basal and apical and both magenta) in a citrine (green), n = and for Scale bars in 10 μm. Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg ES Video of sagittal slice from 4D of quantified in Figure 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. Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg ES Video of sagittal slice from 4D of quantified in Figure supplement 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. barriers to form an ES relief valve The of ES deflation in the lmx1bb mutant suggests that a may be by the dorsal ES tissue between and lmx1bb mutants using confocal microscopy and membrane-localized fluorescent we observed that the ES tissue much in embryos during peak inflation in the in mutants inflated to a volume indicated by Figure data in compiled from 65 to hpf inflation In this often as a thin sheet ± as seen in the ES or the lmx1bb mutants in which two and were observed ± the n = is n = with a one of 4 the of the revealed thin that the basal side of ES cells (white Figure of cells in the confirmed the of thin basal in the ES, as well as in their organization (Figure in the mutant revealed the of thin basal in the ES (Figure To determine the organization of the ES tissue, we for basal (Figure and We found that the basal surface of the ES tissue the perilymph the otic vesicle (Figure We could not a clear in the of between and the work suggested that may be a of in the et al., and be necessary to determine whether or other are expressed in the ES of We also of the apical and found that it the ES tissue that