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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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The HuC promoter drives the expression of mem-TdTomato in the afferent neurons.Maximal projections of HGn39D fish injected with HuC∶mem-TdTomato are depicted. (A) HuC promoter drives the expression of mem-TdTomato in the soma of the afferent neurons (HB: hinbrain, gP: Posterior lateral-line ganglion). (B–C) mem-TdTomato is expressed in GFP-positive neurons of HGn39D fish at the level of the defasciculated fibers (B) and of the dendrites beneath the neuromast (C). (D) mem-TdTomato is expressed in a single neurons. (E–G) Injection of HuC∶mem-TdTomato allows the mosaic expression of red neurons. A single red neuron (E) can be observed in HGn39D (F). (G) Overlay of HGn39D GFP and mem-TdTomato. Scale bars: 50 µm (A) and 10 µm (B–G).
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pone-0004477-g002: The HuC promoter drives the expression of mem-TdTomato in the afferent neurons.Maximal projections of HGn39D fish injected with HuC∶mem-TdTomato are depicted. (A) HuC promoter drives the expression of mem-TdTomato in the soma of the afferent neurons (HB: hinbrain, gP: Posterior lateral-line ganglion). (B–C) mem-TdTomato is expressed in GFP-positive neurons of HGn39D fish at the level of the defasciculated fibers (B) and of the dendrites beneath the neuromast (C). (D) mem-TdTomato is expressed in a single neurons. (E–G) Injection of HuC∶mem-TdTomato allows the mosaic expression of red neurons. A single red neuron (E) can be observed in HGn39D (F). (G) Overlay of HGn39D GFP and mem-TdTomato. Scale bars: 50 µm (A) and 10 µm (B–G).

Mentions: We observed that two or more afferent fibers de-fasciculate from the main nerve towards each neuromast in HGn39D (Figure 1 D, N, Q). The tight fasciculation of the axons does not allow their individual resolution at the level of the main nerve (Figure 1 D). Thus, it is possible that neurons project bifurcated fibres towards each neuromast. However, we speculated that since the afferent neurons outnumber neuromasts by a factor of two, more than one neuron could innervate each neuromast. To test this hypothesis, we took advantage of the fact that plasmid DNA injected into eggs is often expressed in a variegated manner. We generated larvæ expressing a membrane-localized red-fluorescent protein in individual afferent neurons, by injecting a cDNA coding for mem-TdTomato under the transcriptional control of the neuronal HuC promoter. Mem-TdTomato decorated the entire neuron, including the somata (Figure 2 A). In the background of the HGn39D transgenic line, this strategy allowed us to resolve individual neurons along their entire length (Figure 2 A–D), and also the thin neurites that form the dendritic arbors beneath the neuromasts (Figure 2 C–G). All the lateralis neurons that expressed mem-TdTomato in HGn39D were also positive for GFP and showed identical dynamics when compared to non-expressors. This indicates that HuC directs expression in afferents and that mem-TdTomato does not affect their behavior. We selected specimens expressing mem-TdTomato in single neurons for detailed analysis by confocal microscopy to find single fibers in 100% of the cases (N = 42) (Figure 2 D), indicating that neurons do not project branched fibers towards any individual neuromast. Together, these results show that each neuromast is contacted by more than one afferent neuron.


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)

The HuC promoter drives the expression of mem-TdTomato in the afferent neurons.Maximal projections of HGn39D fish injected with HuC∶mem-TdTomato are depicted. (A) HuC promoter drives the expression of mem-TdTomato in the soma of the afferent neurons (HB: hinbrain, gP: Posterior lateral-line ganglion). (B–C) mem-TdTomato is expressed in GFP-positive neurons of HGn39D fish at the level of the defasciculated fibers (B) and of the dendrites beneath the neuromast (C). (D) mem-TdTomato is expressed in a single neurons. (E–G) Injection of HuC∶mem-TdTomato allows the mosaic expression of red neurons. A single red neuron (E) can be observed in HGn39D (F). (G) Overlay of HGn39D GFP and mem-TdTomato. Scale bars: 50 µm (A) and 10 µm (B–G).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2637426&req=5

pone-0004477-g002: The HuC promoter drives the expression of mem-TdTomato in the afferent neurons.Maximal projections of HGn39D fish injected with HuC∶mem-TdTomato are depicted. (A) HuC promoter drives the expression of mem-TdTomato in the soma of the afferent neurons (HB: hinbrain, gP: Posterior lateral-line ganglion). (B–C) mem-TdTomato is expressed in GFP-positive neurons of HGn39D fish at the level of the defasciculated fibers (B) and of the dendrites beneath the neuromast (C). (D) mem-TdTomato is expressed in a single neurons. (E–G) Injection of HuC∶mem-TdTomato allows the mosaic expression of red neurons. A single red neuron (E) can be observed in HGn39D (F). (G) Overlay of HGn39D GFP and mem-TdTomato. Scale bars: 50 µm (A) and 10 µm (B–G).
Mentions: We observed that two or more afferent fibers de-fasciculate from the main nerve towards each neuromast in HGn39D (Figure 1 D, N, Q). The tight fasciculation of the axons does not allow their individual resolution at the level of the main nerve (Figure 1 D). Thus, it is possible that neurons project bifurcated fibres towards each neuromast. However, we speculated that since the afferent neurons outnumber neuromasts by a factor of two, more than one neuron could innervate each neuromast. To test this hypothesis, we took advantage of the fact that plasmid DNA injected into eggs is often expressed in a variegated manner. We generated larvæ expressing a membrane-localized red-fluorescent protein in individual afferent neurons, by injecting a cDNA coding for mem-TdTomato under the transcriptional control of the neuronal HuC promoter. Mem-TdTomato decorated the entire neuron, including the somata (Figure 2 A). In the background of the HGn39D transgenic line, this strategy allowed us to resolve individual neurons along their entire length (Figure 2 A–D), and also the thin neurites that form the dendritic arbors beneath the neuromasts (Figure 2 C–G). All the lateralis neurons that expressed mem-TdTomato in HGn39D were also positive for GFP and showed identical dynamics when compared to non-expressors. This indicates that HuC directs expression in afferents and that mem-TdTomato does not affect their behavior. We selected specimens expressing mem-TdTomato in single neurons for detailed analysis by confocal microscopy to find single fibers in 100% of the cases (N = 42) (Figure 2 D), indicating that neurons do not project branched fibers towards any individual neuromast. Together, these results show that each neuromast is contacted by more than one afferent neuron.

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