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Afferent neurons of the zebrafish lateral line are strict selectors of hair-cell orientation.

Faucherre A, Pujol-Martí J, Kawakami K, López-Schier H - PLoS ONE (2009)

Bottom Line: Each neuron forms synapses with hair cells of identical orientation to divide the neuromast into functional planar-polarity compartments.We also show that afferent neurons are strict selectors of polarity that can re-establish synapses with identically oriented targets during hair-cell regeneration.Our results provide the anatomical bases for the physiological models of signal-polarity resolution by the lateral line.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Sensory Cell Biology & Organogenesis, Centre de Regulació Genòmica, Doctor Aiguader, Barcelona, Spain.

ABSTRACT
Hair cells in the inner ear display a characteristic polarization of their apical stereocilia across the plane of the sensory epithelium. This planar orientation allows coherent transduction of mechanical stimuli because the axis of morphological polarity of the stereocilia corresponds to the direction of excitability of the hair cells. Neuromasts of the lateral line in fishes and amphibians form two intermingled populations of hair cells oriented at 180 degrees relative to each other, however, creating a stimulus-polarity ambiguity. Therefore, it is unknown how these animals resolve the vectorial component of a mechanical stimulus. Using genetic mosaics and live imaging in transgenic zebrafish to visualize hair cells and neurons at single-cell resolution, we show that lateral-line afferents can recognize the planar polarization of hair cells. Each neuron forms synapses with hair cells of identical orientation to divide the neuromast into functional planar-polarity compartments. We also show that afferent neurons are strict selectors of polarity that can re-establish synapses with identically oriented targets during hair-cell regeneration. Our results provide the anatomical bases for the physiological models of signal-polarity resolution by the lateral line.

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Related in: MedlinePlus

Afferent neurons innervate hair cells of identical polarity.Top views of neuromasts from HuC∶mem-TdTomato injected ET4 fish are depicted. (A–D) maximal projections (A,C) and phalloidin staining (B,D) of neuromasts innervated by an afferent neuron that exclusively innervates hair cells of the same polarity (A–B) and by an afferent neuron that also projects neurites to hair cells of the opposite polarity (C–D). Asterisks indicate the innervated hair cells. Double asterisks point the innervated hair cells of the opposite polarities than the hair cells marked with single asterisks. (E–L) Maximal projection (E–K) and phalloidin staining (L) of the same neuromast. White arrows indicate a non-stable neurite contacting hair cells of opposite polarity (double asterisks) compared to stable synapses (asterisks). Scale bars: 10 µm (A,C, E and K) and 5 µm (B, D and L).
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pone-0004477-g003: Afferent neurons innervate hair cells of identical polarity.Top views of neuromasts from HuC∶mem-TdTomato injected ET4 fish are depicted. (A–D) maximal projections (A,C) and phalloidin staining (B,D) of neuromasts innervated by an afferent neuron that exclusively innervates hair cells of the same polarity (A–B) and by an afferent neuron that also projects neurites to hair cells of the opposite polarity (C–D). Asterisks indicate the innervated hair cells. Double asterisks point the innervated hair cells of the opposite polarities than the hair cells marked with single asterisks. (E–L) Maximal projection (E–K) and phalloidin staining (L) of the same neuromast. White arrows indicate a non-stable neurite contacting hair cells of opposite polarity (double asterisks) compared to stable synapses (asterisks). Scale bars: 10 µm (A,C, E and K) and 5 µm (B, D and L).

Mentions: Our previous observations predict that each neuron innervates only a subset of the constituent hair cells of each lateral-line organ. Because neuromasts harbor two populations of hair cells of opposite polarities, we asked whether there was a relationship between neuronal contacts and the orientation of hair cells. Hair cells in the zebrafish lateral line can be visualized in vivo by virtue of specific GFP expression in the SqET4 or brn3c∶GFP transgenic lines [33]–[35]. To determine the contacts of afferent neurons with hair cells of a particular orientation, we performed triple labeling of 7 dpf SqET4 larvæ with fluorescent phalloidin to reveal stereocilia in blue and scatter-labeled afferents with mem-TdTomato (Figure 3 A–L). We performed three-dimensional live imaging analyses in a large number of specimens (N = 25), and found that afferent neurons formed two types of neurites. One type was thin along its entire length, whereas the second type had bulged termini. Thin neurites were long and very motile, extending and retracting very rapidly (Supplementary Movie S1). These neurites often did not contact any hair cell (Figure 2 C). When they established contacts with a hair cell, however, these were short-lived (Figure 3 E–J). In contrast, bulged neurites were shorter, less motile and established stable associations with hair cells (Figure 3 E–K). We found no direct relationship between thin neurites and hair cells of any orientation (Supplementary Movie S1). However, in 12 of 14 cases we observed that bulged neurites associated exclusively with hair cells of identical polarity (Figure 3 A–D and K–L) (Table 1) (Supplementary Movie S2). In the two exceptional cases (4 and 11 of Table 1), a neuron contacted hair cells of both polarities. However, the rare neurites that contacted one or two hair cells of the opposite polarity did not show a prominent bulging at their ends (Supplementary Movie S2). Interestingly, we documented an instance when a bulged neurite with a stable contact with one hair cell (Figure 3 E) projected an extension to contact a second target (arrowhead Figure 3 G). The second contact in this case is with an opposite-polarized hair cell (Figure 3 L). Over time the second contact destabilized for the bulged neurite to retain a stable contact with a single hair cell (Figure 3 K). This analysis highlights the essential need to perform long-time lapse sequence at high temporal resolution to define the dynamics of contacts between hair cells and neurons. Our observations suggest that only bulged neurites form synapses with hair cells. We further corroborated this conclusion by the establishment of an imaging method using DiASP to directly reveal synaptic communication between hair cells of a given polarity and the afferent neurons [11]. When applied in the medium, DiASP enters hair cells by passing directly through mechanically gated transduction channels on the stereocilia and subsequently permeates to the afferent neurons (Figure 1 R–S). DiASP entry into hair cells is blocked when the mechanotransducing channels are closed. To mechanically open transduction channels in hair cells of a selected orientation and close those of the opposite, we applied a fluid stream directed at the neuromasts along the anteroposterior axis of the fish (Supplementary Figure S1). After a brief period of directed water jet to deflected stereocilia, we applied DiASP to the stream. In contrast to the normal loading to all transducing hair cells in the neuromast, fluid jet application of DiASP labeled hair cells whose stereocilia were deflected positively, whereas the cells whose stereocilia were deflected negatively were not labeled (Figure 4 A–D). We reasoned that if each neuron synapses with hair cells of both orientations, DiASP should permeate to all neurons irrespectively of the orientation of the fluid jet. However, when we applied a polarized fluid jet to HGn39D specimens, we observed that DiASP did not label all fibers, suggesting that the unlabelled neurons did not establish synaptic contacts with hair cells of both polarities (Figure 4 E–H). This result is consistent with our previous observations, which together support the conclusion that each afferent neuron forms stable synaptic connections with hair cells of only one orientation.


Afferent neurons of the zebrafish lateral line are strict selectors of hair-cell orientation.

Faucherre A, Pujol-Martí J, Kawakami K, López-Schier H - PLoS ONE (2009)

Afferent neurons innervate hair cells of identical polarity.Top views of neuromasts from HuC∶mem-TdTomato injected ET4 fish are depicted. (A–D) maximal projections (A,C) and phalloidin staining (B,D) of neuromasts innervated by an afferent neuron that exclusively innervates hair cells of the same polarity (A–B) and by an afferent neuron that also projects neurites to hair cells of the opposite polarity (C–D). Asterisks indicate the innervated hair cells. Double asterisks point the innervated hair cells of the opposite polarities than the hair cells marked with single asterisks. (E–L) Maximal projection (E–K) and phalloidin staining (L) of the same neuromast. White arrows indicate a non-stable neurite contacting hair cells of opposite polarity (double asterisks) compared to stable synapses (asterisks). Scale bars: 10 µm (A,C, E and K) and 5 µm (B, D and L).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637426&req=5

pone-0004477-g003: Afferent neurons innervate hair cells of identical polarity.Top views of neuromasts from HuC∶mem-TdTomato injected ET4 fish are depicted. (A–D) maximal projections (A,C) and phalloidin staining (B,D) of neuromasts innervated by an afferent neuron that exclusively innervates hair cells of the same polarity (A–B) and by an afferent neuron that also projects neurites to hair cells of the opposite polarity (C–D). Asterisks indicate the innervated hair cells. Double asterisks point the innervated hair cells of the opposite polarities than the hair cells marked with single asterisks. (E–L) Maximal projection (E–K) and phalloidin staining (L) of the same neuromast. White arrows indicate a non-stable neurite contacting hair cells of opposite polarity (double asterisks) compared to stable synapses (asterisks). Scale bars: 10 µm (A,C, E and K) and 5 µm (B, D and L).
Mentions: Our previous observations predict that each neuron innervates only a subset of the constituent hair cells of each lateral-line organ. Because neuromasts harbor two populations of hair cells of opposite polarities, we asked whether there was a relationship between neuronal contacts and the orientation of hair cells. Hair cells in the zebrafish lateral line can be visualized in vivo by virtue of specific GFP expression in the SqET4 or brn3c∶GFP transgenic lines [33]–[35]. To determine the contacts of afferent neurons with hair cells of a particular orientation, we performed triple labeling of 7 dpf SqET4 larvæ with fluorescent phalloidin to reveal stereocilia in blue and scatter-labeled afferents with mem-TdTomato (Figure 3 A–L). We performed three-dimensional live imaging analyses in a large number of specimens (N = 25), and found that afferent neurons formed two types of neurites. One type was thin along its entire length, whereas the second type had bulged termini. Thin neurites were long and very motile, extending and retracting very rapidly (Supplementary Movie S1). These neurites often did not contact any hair cell (Figure 2 C). When they established contacts with a hair cell, however, these were short-lived (Figure 3 E–J). In contrast, bulged neurites were shorter, less motile and established stable associations with hair cells (Figure 3 E–K). We found no direct relationship between thin neurites and hair cells of any orientation (Supplementary Movie S1). However, in 12 of 14 cases we observed that bulged neurites associated exclusively with hair cells of identical polarity (Figure 3 A–D and K–L) (Table 1) (Supplementary Movie S2). In the two exceptional cases (4 and 11 of Table 1), a neuron contacted hair cells of both polarities. However, the rare neurites that contacted one or two hair cells of the opposite polarity did not show a prominent bulging at their ends (Supplementary Movie S2). Interestingly, we documented an instance when a bulged neurite with a stable contact with one hair cell (Figure 3 E) projected an extension to contact a second target (arrowhead Figure 3 G). The second contact in this case is with an opposite-polarized hair cell (Figure 3 L). Over time the second contact destabilized for the bulged neurite to retain a stable contact with a single hair cell (Figure 3 K). This analysis highlights the essential need to perform long-time lapse sequence at high temporal resolution to define the dynamics of contacts between hair cells and neurons. Our observations suggest that only bulged neurites form synapses with hair cells. We further corroborated this conclusion by the establishment of an imaging method using DiASP to directly reveal synaptic communication between hair cells of a given polarity and the afferent neurons [11]. When applied in the medium, DiASP enters hair cells by passing directly through mechanically gated transduction channels on the stereocilia and subsequently permeates to the afferent neurons (Figure 1 R–S). DiASP entry into hair cells is blocked when the mechanotransducing channels are closed. To mechanically open transduction channels in hair cells of a selected orientation and close those of the opposite, we applied a fluid stream directed at the neuromasts along the anteroposterior axis of the fish (Supplementary Figure S1). After a brief period of directed water jet to deflected stereocilia, we applied DiASP to the stream. In contrast to the normal loading to all transducing hair cells in the neuromast, fluid jet application of DiASP labeled hair cells whose stereocilia were deflected positively, whereas the cells whose stereocilia were deflected negatively were not labeled (Figure 4 A–D). We reasoned that if each neuron synapses with hair cells of both orientations, DiASP should permeate to all neurons irrespectively of the orientation of the fluid jet. However, when we applied a polarized fluid jet to HGn39D specimens, we observed that DiASP did not label all fibers, suggesting that the unlabelled neurons did not establish synaptic contacts with hair cells of both polarities (Figure 4 E–H). This result is consistent with our previous observations, which together support the conclusion that each afferent neuron forms stable synaptic connections with hair cells of only one orientation.

Bottom Line: Each neuron forms synapses with hair cells of identical orientation to divide the neuromast into functional planar-polarity compartments.We also show that afferent neurons are strict selectors of polarity that can re-establish synapses with identically oriented targets during hair-cell regeneration.Our results provide the anatomical bases for the physiological models of signal-polarity resolution by the lateral line.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Sensory Cell Biology & Organogenesis, Centre de Regulació Genòmica, Doctor Aiguader, Barcelona, Spain.

ABSTRACT
Hair cells in the inner ear display a characteristic polarization of their apical stereocilia across the plane of the sensory epithelium. This planar orientation allows coherent transduction of mechanical stimuli because the axis of morphological polarity of the stereocilia corresponds to the direction of excitability of the hair cells. Neuromasts of the lateral line in fishes and amphibians form two intermingled populations of hair cells oriented at 180 degrees relative to each other, however, creating a stimulus-polarity ambiguity. Therefore, it is unknown how these animals resolve the vectorial component of a mechanical stimulus. Using genetic mosaics and live imaging in transgenic zebrafish to visualize hair cells and neurons at single-cell resolution, we show that lateral-line afferents can recognize the planar polarization of hair cells. Each neuron forms synapses with hair cells of identical orientation to divide the neuromast into functional planar-polarity compartments. We also show that afferent neurons are strict selectors of polarity that can re-establish synapses with identically oriented targets during hair-cell regeneration. Our results provide the anatomical bases for the physiological models of signal-polarity resolution by the lateral line.

Show MeSH
Related in: MedlinePlus