(A) Top down view of neuromast hair bundles labeled with Tg(myo6b:β-actin-GFP). Three stages of immature hair-bundle development can be observed: early, intermediate and late. The unlabeled region on either the caudal or rostral side of each bundle is the insertion point of the kinocilium.
(A’) Arrows indicate planar polarity and functional polarity of hair bundles in (A). Dashed arrows indicate predicted planar polarity.
(B) SEM image of a neuromast at 3 dpf; asterisk denotes the many tall kinocilia. (C) SEM image of a mature hair bundle.
(D, E and F) Confocal images of hair bundles expressing β-actin-GFP. White dashed arrows indicate hair bundles of respective stage examined.
(D’, E’ and F’) White dashed lines outline cell bodies after FM 1-43 label of hair bundle examined in (D, E and F).
(D”, E” and F”) SEM images of hair bundles in (D, E and F).
All images were taken at 3 dpf. k = kinocilium, white arrowheads denote kinocilial links, solid white arrows denote tip links. Anterior is left, dorsal is up. Scale bars: 2.5 μm (A, B, D, E and F), 500 nm (C), 5 μm (D’, E’ and F’), 1 μm (D”, E” and F”). See also Figure S1.

(A and B) SEM image of an ift88^tz288b immature (A) and mature (B) hair bundle. (C and D) Tubulin and actin stain of bundles at 5 dpf in wildtype (C) and ift88^tz288b (D).
(E) The average number of hair cells per neuromast at 2 dpf in wildtype and ift88^tz288b neuromasts (n > 13 neuromasts).
(F) Proportion of FM 1-43 labeled hair cells in ift88^tz288b and wildtype neuromasts at 2 dpf (n > 13 neuromasts).
(G) The average FM 1-43 intensity per neuromast at 2 dpf (n > 13 neuromasts). An unpaired Student's t-test was used to compare number of hair cells per neuromast in (E and G). A Chi squared Fisher exact test was used to compare proportion of FM 1-43 labeled hair cells in (F). k = kinocilium. Scale bars: 500 nm (A and B), 5 μm (C and D). Error bars are s.e.m.

(A) A 5 dpf neuromast expressing cameleon. Circles represent cells analyzed and are color coded and correspond to data in (A’-C).
(A’ and A”) FM 1-43 and phalloidin stain of the neuromast in (A). Two anterior-polarized hair bundles (A1 and A2) and two posterior-polarized hair bundles (P1 and P2) are indicated.
(B and C) Response of four individual hair cells highlighted in (A) to the same anterior (B) and posterior (C) stimulus.
(D, D’ and D”) Same layout as (A-A”) except analysis is at 2 dpf. Hair cell stages are as follows: cell 1: early, cell 2: mature, cell 3: intermediate. (D) A neuromast expressing cameleon. (D’) FM 1-43 strongly labeled a pair of mature cells (cell 2 and sister), faintly labeled a pair of intermediate cells (cell 3 and sister), yet did not label a pair of early hair cells (cell 1 and sister). (D”) Phalloidin label of the neuromast in (D-D’).
(E and F) Mechanically-evoked calcium responses corresponding to cells 1-3. Negative and positive deflections are indicated with schematics. Kinocilium is denoted as the tall, gray structure. Cell 1 responded to only negative deflections. Cell 2 responded to positive deflections. Cell 3 responded to both negative and positive deflections.
(G) Hair-cell responses (black circles) from wildtype (n = 13 neuromasts) and ift88^tz288b (n = 21 neuromasts) at 2 dpf. This same data was broken down into FM 1-43 labeled hair cells (gray circles) and FM 1-43 unlabeled hair cells (open gray circles).
(H) Proportion of negative, bi-directional and positive responses at 2 dpf (n = 188 wildtype and 72 ift88^tz288b cells).
Errors bars are s.em. Scale bars: 5 μm. For stimulus controls, see also Figure S2.

(A) Overall time scale and functional polarity associated with each transition. Early immature cells first go through Transition 1: reversal. During this period, immature cells reverse their functional polarity from the negative to positive direction. Later immature cells enter Transition 2: refinement. At this stage functional polarity is in rapid flux between bi-directional and positive deflections. Once mature, cells respond to only positive deflections.
(B) Deflection in the positive direction for cells in Transition 1 (T1), or Transition 2 (T2), respectively.
(C) Deflection in the negative direction for the same cells as in (B).

(A-F) Calcium responses were measured in response to positive and negative hair bundles deflections from cells at each stage of bundle development.
(A’-F’) SEM images of the hair bundles from hair cells represented in (A-F)
All images were taken at 3 dpf, arrowheads indicate kinocilial links, arrows indicate tip links, scale bar is 500 nm. Inset is 2X magnification. For additional link examples see also Figure S3.

(A) All mutant cdh23 and pcdh15 cells at 2 dpf did not respond to stimulation. Treatment of wildtype hair cells with BAPTA also eliminated all responses (P < 0.001, n > 7 neuromasts).
(B) The mechanotransduction channel blockers amiloride, curare and DHS significantly blocked responses at 2 dpf (P < 0.001, n > 5 neuromasts).
(C) Diagram of link asymmetry in hair bundles, and the role of tip links versus kinocilial links in mediating mechanosensitivity in the positive and negative direction, respectively.
Circles indicate potential location for mechanosensitive channels that mediate these responses. An unpaired and paired Student's t-tests were used in (A) and (B) respectively. Error bars are s.e.m. See also Figure S4.