Direct comparison of hair-bundle development and function by confocal and electron microscopy
To examine the function of kinocilia during development, we utilized the zebrafish posterior lateral-line organ, which is an auxiliary sensory organ present in fish and amphibians, specialized for local water-flow detection. The lateral line consists of superficial clusters of hair cells called neuromasts, arranged along the length of the animal (, ). Hair cells of the lateral-line organ are born in pairs, and sister hair cells adopt opposing polarities along the anterior-posterior (A-P) axis of the fish (López-Schier et al., 2004
; Seiler et al., 2005
) (). The morphological and planar polarity of a given hair bundle is determined by the asymmetric localization of the actin-rich stereocilia relative to the kinocilium (, S1A and S1A’
Direct comparison of hair-bundle development using transgenic β-actin-GFP, FM 1-43 and SEM
Functional polarity of mature and immature zebrafish hair cells
For our study, we first developed a basic framework for characterizing the various stages of development in terms of morphology and function. To accomplish this in vivo
, we generated transgenic fish expressing β-actin-GFP
under control of a hair-cell specific myo6b
promoter and used confocal microscopy to image actin-GFP in hair bundles (). These animals were then assessed for mechanosensitivity with FM 1-43, a vital dye that labels hair cells with functional mechanotransduction channels (Gale et al., 2001
) (). Finally, ultrastructural features of the same hair bundles were examined using SEM ().
The results of in vivo
imaging followed sequentially by SEM revealed multiple stages of hair-bundle development within each neuromast. We therefore categorized stages of hair-bundle development. Hair bundles were defined as mature or immature, and immature hair bundles were further sub-classified as early, intermediate or late (, ). Actin localization showed that early hair bundles had a thin and uniform circumferential actin ring at the hair-cell surface. High-resolution SEM analysis revealed that these early bundles had 1 or 2 rows of stereocilia at a height roughly equal to the kinocilium (, ). Interestingly, early sister hair cells had correct planar polarity relative to each other and to the A-P axis of the fish (, Figure S1C
). Any minor misalignments in early bundle were comparable to those observed in mature bundles (Figure S1C
). At the intermediate stage, actin localization reflected an established morphological polarity and SEM showed that on average, three rows of stereocilia were present. In these intermediate bundles, the height of stereocilia was double that of early bundles, and the kinocilium was twice the height of the tallest stereocilia (, ). Late stage hair bundles showed a further increase in height, had 3 to 4 rows of stereocilia, and a kinocilium which was at least five times the height of the tallest stereocilia (, ). Only late stage hair bundles consistently labeled with FM 1-43 and had detectable tip links in SEM images (). This is in agreement with previous studies correlating FM 1-43 label with the presence of tip links and the onset of mechanosensitivity in mouse and chick hair cells (Géléoc and Holt, 2003
; Si et al., 2003
). Finally, mature hair bundles had between 4 to 5 rows of stereocilia, a bundle height of approximately 1.5 μm, and a kinocilial height of 14 μm (, ). This coordinated approach provided a fundamental reference for the development of hair-bundle morphology and the onset of mechanosensitive function that we applied to subsequent analyses.
Hair-bundle morphological development
Loss of kinocilia does not disrupt hair-bundle development
Upon establishing methods to track and categorize the stages of hair-bundle development, we then examined the role of kinocilia in the onset of mechanosensitivity. For this analysis, we characterized ovl/ift88
mutants that lack normal kinocilia (Tsujikawa and Malicki, 2004
). Using SEM and immunohistochemistry we confirmed that in ift88tz288b
mutants kinocilia were severely stunted (mature kinocilia length: 14.42 μm in wildtype, n = 25; 0.29 μm in ift88tz288b
, n = 11; example: ). We occasionally observed slightly elongated (> 5 μm) kinocilia in younger larvae (2 days post fertilization (dpf)) but never kinocilia that were mature in length (data not shown). This is consistent with previous reports using a maternal-zygotic ift88
mutant that suggest in younger larvae there is residual ift88
maternal RNA in ift88tz288b
mutants (Huang and Schier, 2009
). Importantly, SEM images of ift88tz288b
hair bundles showed that while kinocilia were severely stunted in both immature and mature hair bundles, hair-bundle development was not affected (). In contrast to a study of auditory hair cells in mice, loss of Ift88 and kinocilia did not result in hair-bundle planar polarity defects () (Jones et al., 2008
). Normal planar polarity indicates that either the kinocilium is not required to establish the coordinated organization of zebrafish hair bundles, or that in ift88tz288b
hair bundles, although severely stunted, kinocilia are still able to fulfill this role.
ovl/ift88 mutant hair cells lack kinocilia yet develop normally
In addition to unaltered hair-bundle development, we observed that at 2 dpf, a stage when each neuromast contains hair cells at multiple developmental stages — early, intermediate, late and mature —ift88tz288b mutant neuromasts had a similar number of hair cells compared to wildtype neuromasts (). Furthermore, we found that in ift88tz288b mutant hair cells, FM 1-43 label progressed on a similar scale in developing hair cells as in wildtype larvae (), suggesting that a functional, developmental comparison was valid.
The onset of mechanosensitivity precedes FM 1-43 label and requires kinocilia
Normal labeling of mutant hair cells with FM 1-43 suggested that mechanotransduction was unaffected in ift88tz288b
larvae. In many species, labeling with FM 1-43 is an accepted method to assess hair-cell activity, however, using a second method for assessing physiological responses in parallel is more comprehensive. Previous studies of hair-cell mechanosensitivity in zebrafish have relied on measurements of extracellular microphonic potentials (Trapani and Nicolson, 2011
). Such recordings represent the sum response of all hair cells in a sensory patch. Because each neuromast contains hair cells at different stages (), microphonic potentials are not suitable for determining the onset of mechanotransduction. We therefore developed a method to measure hair-cell activity at the resolution of single cells. For these experiments we generated transgenic fish expressing cameleon D3cpv (Palmer et al., 2006
), a genetically encoded FRET (fluorescence resonance energy transfer)-based calcium indicator, in hair cells ().
Using intact transgenic larvae expressing cameleon, we first characterized the responses of wildtype hair cells to confirm that FM 1-43 labeling correlated with mechanosensitivity. For this analysis, we examined neuromasts at 5 dpf and 2 dpf, to compare populations of mature and immature hair cells respectively (). When stimulated along the A-P axis using a fluid-jet, hair cells displayed robust mechanically-evoked calcium responses. Subsequently, when we labeled cells with FM 1-43, we confirmed that the majority of cells at 5 dpf and 2 dpf that labeled with FM 1-43 were mechanosensitive, as defined by a rise in intracellular calcium levels (5 dpf: 94% n = 128 cells; 2 dpf: 84% n = 44 cells).
When we extended our analysis to the immature, FM 1-43 unlabeled cells at 2 dpf, we made a striking observation. In contrast to previous studies, we reproducibly detected mechanosensitive calcium responses in cells that did not label with FM 1-43 (, cell 1; : 67% n = 39 cells). It should be noted that the responses of FM 1-43 unlabeled cells were significantly smaller than responses of FM 1-43 labeled cells (). Although smaller, mechanosensitive responses in FM 1-43 unlabeled cells were reliably detected over a wide range of fluid-jet pressures, using numerous stimulus paradigms and also using a piezo-driven glass fiber to deflect hair bundles (Figures S2E, S2H, and S2I
). Previous studies have shown that hair cells with incompletely assembled, improperly tensioned, or closed channels could be labeled by deflecting hair bundles in the presence of FM 1-43 (Lelli et al., 2009
). However, we found that neither application of additional FM 1-43, nor sustained deflection in the presence of FM 1-43 could label immature FM 1-43 unlabeled cells that were mechanosensitive (Figures S4A-S4A’” and S4B-S4B’”
). These observations suggest that despite their lack of FM 1-43 label, immature responses are robust and immature cells possess functional mechanosensitive channels.
Because the kinocilium is the first apical structures to appear in developing hair cells, one obvious question is whether the kinocilium is required for mechanosensitive responses in immature cells that do not label with FM 1-43. When we examined mechanically-evoked calcium responses in ift88tz288b mutants using our transgenic line expressing cameleon we found that similar to wildtype cells, in ift88tz288b mutants, the majority of FM 1-43 labeled cells were mechanosensitive (: 71% n = 55 cells). Of the ift88tz288b cells that labeled with FM 1-43, the magnitude of the responses was significantly reduced (). However, in contrast to wildtype larvae, we observed that ift88tz288b cells without FM 1-43 label were rarely mechanosensitive (: 6% n = 35 cells). Relative to wildtype, the reduction in response size and loss of a subset of immature responses suggests that either full-length kinocilia are required for transmitting the forces of mechanical stimuli to the bundle or that kinocilia are intrinsically involved in mechanosensitivity during development.
Morphological polarity does not accurately predict functional polarity in immature hair cells
To parse out the exact role of the kinocilia in the acquisition of mechanosensitivity, we characterized developing hair-cell responses in more detail. At 2 dpf, hair cells are positioned in a highly stereotyped fashion within each neuromast, along a line perpendicular to the axis of mechanosensitivity (). Sister cells are paired across this line, and are fated to have opposing polarities () (López-Schier et al., 2004
). In this arrangement, when mature, all cells initially present on the left side of the neuromast will respond to posterior stimuli, and those on the right side will respond to anterior stimuli.
In line with this polarized arrangement, in mature cells at 2 dpf, deflection in the positive direction (towards the kinocilium) was accompanied by a rise in intracellular calcium levels (, cell 2), whereas deflection in the negative direction (away from the kinocilium) resulted in a decrease in intracellular calcium, (, cell 2). When stained with phalloidin, the observed functional polarity of each mature cell at 2 dpf correlated with the morphological planar polarity of its hair bundle (, cell 2). Similar results were obtained at 5 dpf when the majority of neuromast hair cells are mature ().
In contrast to mature cells, we observed that the functional polarity of FM 1-43 unlabeled cells was often completely reversed — FM 1-43 unlabeled, immature cells frequently showed an increase in intracellular calcium when stimulated in the negative direction (70% derived from early and intermediate FM 1-43 unlabeled cells; , cell 1). Further, in FM 1-43 labeled cells that were still immature, we observed a large subset that responded bi-directionally (increase in intracellular calcium to both negative and positive deflections) (45% derived from intermediate and late FM 1-43 labeled cells; cell 3). Inappropriate functional polarity was surprising since our SEM analysis of hair-bundle ultrastructure revealed that developing bundles had correct planar polarity, which is thought to be predictive of correct functional polarity ( and S1C
Because correct hair-bundle polarity did not correlate with functional polarity in developing cells, it was unclear whether functional polarity was restricted to the A-P axis as in mature cells. To test A-P axis restriction, we examined responses to stimuli oriented at 45° or 90° relative to the A-P axis (Figures S1D-S1G
). We observed that in all cells, off-axis responses were substantially reduced (Figure S1D
). Residual off-axis responses are likely due to the fact that many hair bundles, even mature ones, are not perfectly aligned along the A-P axis (Figures S1A-S1C
). Importantly, the reduction in off-axis response was independent of developmental stage and FM 1-43 label indicating that even the youngest developing hair cells are directionally restricted along the A-P axis (Figures S1D-S1G
We also performed several controls to determine if the atypical functional polarity in immature hair cells was robust and reproducible. We found that similar results were observed for a range of fluid-jet intensities and using a piezo-driven glass fiber to deflect hair bundles (Figures S2F and S2G
). These results indicate that immature functional polarity is robust in response to stimulation with either glass fiber or fluid jet, and the signal generated correlates with the strength of the stimulus.
Functional polarity reverses during hair-bundle development; reversed responses require kinocilia
After confirming the reproducibility of immature hair-cell responses, we next examined whether (1) the patterns of immature functional polarity we observed in wildtype larvae correlated with specific stages of hair-bundle development and (2) if kinocilia were required for atypical functional polarity at specific stages of development.
To test for a correlation between developmental stage and type of response, we first analyzed the functional polarity of individual developing hair cells at 2 dpf, and then determined the developmental stage of each cell based on kinocilial length, FM 1-43 label, and localization of actin-rich bundles (). Using this approach, we identified a complete reversal of functional polarity during hair-bundle development (, ). Immature early cells predominantly responded to negative deflections, and did not label with FM 1-43 (; : 75% negative, 11% positive, 15% no response, n = 47). The functional polarity of intermediate cells was more heterogeneous (; : 27% negative, 24% bi-directional, 29% positive, 20% no response, n = 51), and nearly half of the cells labeled faintly with FM 1-43. Intermediate cells that responded to positive or bi-directional deflections labeled more consistently with FM 1-43 (). Finally, we observed that all immature cells with late hair bundles were labeled strongly by FM 1-43 and responded to only positive or bi-directional deflections (; : 49% bi-directional, 46% positive, 5% no response, n = 39). In morphologically mature hair bundles analyzed at 2 dpf, functional polarity was almost exclusively restricted to the positive direction (; : 4% bi-directional, 94% positive, 2% no response, n = 51). Collectively, these data indicate that developmental changes in hair bundle morphology strongly correlate with changes in functional polarity.
Upon establishing a developmental trend in functional polarity, we next sought to confirm that the loss of mechanosensitivity in FM 1-43 unlabeled ift88tz288b
cells was due to a loss at a specific developmental stage rather than affecting all stages of development. On the whole, we found that compared to wildtype larvae, in ift88tz288b
mutants, the percentage of mechanosensitive cells at 2 dpf was decreased (: wild type 89% n = 188, ift88 tz288b
32% n = 72). In wildtype larvae, half of these mechanosensitive cells labeled with FM 1-43 and responded exclusively to positive deflections (: wild type 47% n = 167). In ift88tz288b
mutants, the vast majority of mechanosensitive cells labeled with FM 1-43 and responded exclusively to positive deflections (: ift88tz288b
91% n = 23). These results are consistent with a loss of mechanosensitivity restricted to hair cells that did not label with FM 1-43. Further, in ift88tz288b
mutants, we observed a near absence of mechanosensitive cells at early and intermediate stages of development (; : wild type 83% n = 98, ift88 tz288b
11% n = 36). At these stages, responses to negative deflections were largely abolished in ift88tz288b
mutants (; : wildtype 50%, ift88tz288b
6%). In addition, at intermediate and late stages we observed no bi-directional responses (; : wildtype 34% n = 90, ift88tz288b
0% n = 33). The occasional responses to negative deflections observed in ift88tz288b
mutants are likely due to residual ift88
maternal RNA and the sporadic presence of kinocilia at 2 dpf in these animals (Huang and Schier, 2009
). Based on these results, we conclude that kinocilia are not simply acting to couple forces to stereocilia but rather are specifically required for the reversed functional polarity seen in early and intermediate FM 1-43 unlabeled cells and at later stages for the reversed response component of bi-directional responses. Moreover, these data confirm that kinocilia are required for mechanosensitivity during the initial stages of hair-cell development.
Timescale of functional polarity reversal
Though it was clear that kinocilia were required for the reversed functional polarity in immature hair cells, the time scale underlying these functional changes during development was unresolved. To understand the temporal basis of these functional changes, we took advantage of the rapid development of zebrafish hair bundles (bundles develop from early to mature in 14-16 hours) and conducted long-term in vivo imaging by making hourly measurements of functional polarity in developing cells for 5 hrs. These experiments identified two transitions in functional polarity during hair cell development ().
Development of mature functional polarity: first reversal, then refinement
The first transition we observed was in immature early cells. Most of these cells remained responsive only to deflections in the negative direction for the entire 5 hr time period (16/19). However, 3/19 showed a marked functional polarity reversal, transitioning from the negative to the positive direction (Transition 1: ). This transition was relatively slow — it occurred over several hours, and once it occurred, these cells did not revert back to exclusively reversed functional polarity. During this time window we observed no gross changes in cell movements that could account for a reversal in functional polarity. The functional reversal we observe is distinct from previous studies that find sister lateral-line hair cells often rotate after cytokinesis (Wibowo et al., 2011
). Post-cytokinetic rotation occurs much earlier during development of lateral line hair-cells, prior to when hair cells insert into the apical epithelium and form hair bundles (data not shown).
The second type of transition we observed was in bi-directionally responding cells. While only 6/77 cells responded bi-directionally at the initiation of imaging, 8 additional cells responded bi-directionally at least once over the subsequent 5 hr imaging period. Further, in less than 1 hr, bi-directionally responding cells could change their functional polarity and respond to positive deflections, or vice versa, a phenomenon we observed in 12/14 bi-directionally responding cells. By increasing our image acquisition rate, we found that 5 minutes was sufficient for a cell to convert from bi-directional to positive functional polarity, or vice versa (Transition 2: ), indicating that at this later developmental stage, cells oscillate rapidly between bi-directional and positive uni-directional functional polarity.
Structural correlates of kinocilial-based mechanosensation
Although we observed a developmental progression in the acquisition of functional polarity, it was unclear how immature, kinocilial-based mechanosensation was functionally distinct from traditional stereocilial-based mechanotransduction. To investigate the structural correlates of kinocilial-based mechanosensation, we first recorded mechanically-evoked calcium responses in developing hair cells and then used SEM to examine hair bundle ultrastructure of these same cells ().
Correlative calcium imaging and SEM reveals a change in link architecture during development
For these sequential imaging experiments, we examined 6 early, 15 intermediate and 9 late hair bundles. All cells with early bundles responded to negative deflections (). SEM imaging demonstrated that half of the early cells had one complete row of stereocilia and half had bundles with two nearly complete rows (example of a single complete row: ; example of 2 rows: ; n = 3 with 1 row, n = 3 with 2 rows). In contrast to early cells, cells which responded bi-directionally (example: ; n = 3 intermediate, 2 late), or only to positive deflections (example: ; n = 4 intermediate, 7 late), had bundles with detectable tip links. These data demonstrate a correlation between tip link formation and the acquisition of mature functional polarity.
Our sequential calcium imaging and SEM experiments revealed a structural feature that was common to all stages of development: kinocilial links. On average we observed two kinocilial links radiating from the kinocilia to the tip of each stereocilium ( and S4
). Linkages were horizontally oriented or slightly oblique with either an upward or downward angle ( and S4
). This link architecture is quite different than the arrangement observed in the mouse, chick and bullfrog, where dozens of lateral kinocilial links form a dense network connecting the kinocilium to adjacent stereocilia (Goodyear et al., 2005
; Hillman, 1969
; Tsuprun et al., 2004
Because the frequency and location of kinocilial links in zebrafish hair cells appeared more similar to tip links, we reasoned that kinocilial links, via deflection of the kinocilia, might function to gate mechanosensitive channels in immature bundles lacking tip links. Consistent with this hypothesis, ift88tz288b hair bundles, which lack mature kinocilia and atypical immature responses, also lack kinocilial links (). The absence of atypical responses in ift88tz288b hair cells and the similarities between zebrafish tip and kinocilial links suggest that kinocilia utilize kinocilial links for their mechanosensitive function.
Kinocilial-based mechanosensitivity is pharmacologically similar to mature, tip link-based transduction and requires kinocilial link components Cdh23 and Pcdh15
To further substantiate a role for kinocilial links in kinocilial-based mechanosensation, we examined zebrafish with null mutations in Cdh23 or Pcdh15, components of tip and kinocilial links. We found that similar to mature hair cells at 5 dpf (Figure S4E
), Cdh23 and Pcdh15 were both required for mechanosensitive responses at 2 dpf in hair cells at all stages (: cdh23
n = 81, pcdh15
n = 86). In addition, we observed that transient treatment of wildtype hair bundles with BAPTA, which breaks tip and kinocilial links (Assad et al., 1991
; Goodyear and Richardson, 2003
) also eliminated all responses (). Along with our SEM observations, these results indicate that kinocilial links are part of the gating component required for kinocilial-based mechanosensation in developing hair cells.
Nascent hair cells require similar linkage components and channel as mature cells
Because Cdh23 and Pcdh15 mediate the atypical responses in early cells, we hypothesized that other components of the transduction machinery may be required for reversed functional polarity. To determine if immature, kinocilial-based responses were mediated by a channel similar to that in mature hair cells, we treated wildtype hair cells with the known mechanotransduction channel blockers dihydrostreptomycin (DHS), amiloride, and curare (Farris et al., 2004
). Similar to mature cells at 5 dpf (Figure S4D
), each of these channel blockers significantly reduced the response magnitude in all hair cells at 2 dpf (: naïve 51.5%, DHS 10.3%, amiloride 0%, curare 3.9%). While the DHS block was less complete at 2 dpf than at 5 dpf, the degree of inhibition at 2 dpf was independent of FM 1-43 labeling (Figure S4C
), suggesting that partial DHS insensitivity may be a general feature of immature hair cells, as previously proposed (Santos et al., 2006
). These results suggest that in comparison to tip links, kinocilial-based mechanosensation may utilize a pharmacologically similar mechanosensitive channel.