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Phoenix is required for mechanosensory hair cell regeneration in the zebrafish lateral line.

Behra M, Bradsher J, Sougrat R, Gallardo V, Allende ML, Burgess SM - PLoS Genet. (2009)

Bottom Line: We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts.The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells.Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.

View Article: PubMed Central - PubMed

Affiliation: National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.

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The development of the neuromasts in the lateral line is normal in the phoenix mutant larvae.(A) Semi-thin sections showing wild-type (top panels) and mutant neuromasts (bottom panels) in 5dpf (left panels) and 9dpf (right panels) larvae. (B) Camera lucida drawing for each section, highlighting the hair cells nuclei (red) and their cilia (green) and the supporting cells nuclei (blue). (C) Ultra-thin sections viewed by Electron Microscopy (EM) of a wild-type (top panel) and a mutant (lower panel) neuromast in 10dpf larvae. The hair cells stain darker than the supporting cells. (D) Camera lucida drawings of the EM sections, highlighting the hair cell nuclei (red), cell bodies (dark red), and cilia (green). The nuclei of the supporting cells are highlighted (blue). One apoptotic body was visible in (yellow). – 5 microns in (A) and 1 micron in (C).
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pgen-1000455-g001: The development of the neuromasts in the lateral line is normal in the phoenix mutant larvae.(A) Semi-thin sections showing wild-type (top panels) and mutant neuromasts (bottom panels) in 5dpf (left panels) and 9dpf (right panels) larvae. (B) Camera lucida drawing for each section, highlighting the hair cells nuclei (red) and their cilia (green) and the supporting cells nuclei (blue). (C) Ultra-thin sections viewed by Electron Microscopy (EM) of a wild-type (top panel) and a mutant (lower panel) neuromast in 10dpf larvae. The hair cells stain darker than the supporting cells. (D) Camera lucida drawings of the EM sections, highlighting the hair cell nuclei (red), cell bodies (dark red), and cilia (green). The nuclei of the supporting cells are highlighted (blue). One apoptotic body was visible in (yellow). – 5 microns in (A) and 1 micron in (C).

Mentions: We have utilized an assay to monitor hair cell regeneration in the lateral line of 5dpf zebrafish larvae, using transient exposure to copper sulphate (10mM) or neomycin (200mM) dissolved in the water. Our intention was to test for defective hair cell regeneration from a collection of mutant lines previously identified in a retroviral integration screen [45]. The major advantage of using retroviral constructs, over chemicals like ENU, as a mutagenic agent is the rapid identification of the mutated gene. This allows the spatio-temporal expression of the mutated genes to be compared to the observed phenotypes, facilitating selection of the mutants of interest. We reasoned that mutant larvae which develop a normal and functional lateral line and that exhibit expression of the mutated gene in neuromasts would be good candidates to test for defects in the regeneration of the hair cells. We found such a mutant line, which we called phoenix. Homozygotes displayed no behavioral defects (response to sound or mechanical stimulation was normal, data not shown) or visible morphological abnormalities until 5dpf, when the swim bladder failed to inflate. Later, around 7 to 8 dpf, mutants display necrosis in the liver, and death ensues at approximately 14 dpf. Because the gene was expressed in the lateral line neuromasts (see below), we performed a detailed assessment of this organ in 2 to 12 dpf larvae (Figure 1). Semi-thin sections through neuromasts (Figure 1A) of phoenix mutants (lower panels, 5dpf left panel and 9dpf right panel) were indistinguishable from wild-type larvae (upper panels). Camera lucida drawings (Figure 1B) of each section show the cilia (green) of hair cells and their nuclei (red) and the nuclei of the supporting cells (blue). Likewise, ultra-thin sections observed by electron microscopy (EM) (10dpf larvae shown in Figure 1C) did not present obvious differences between wild-type (top panel) and mutant neuromasts (lower panel). Camera lucida drawings (Figure 1D) show, like in Figure 1C, the cilia (green) of hair cells and their nuclei (red) and the nuclei of the supporting cells (blue). The cytoplasm of hair cells is outlined in dark red and an apoptotic body is shown in yellow in the lower panel. Note that apoptotic bodies, as the one present in the mutant, were found at the same rate in untreated wild-type and mutant sections, being probably products of the regular turnover of the supporting cells, which has been described previously [34]. We further observed wild-type and mutant neuromasts in three different transgenic backgrounds (Figure S1). As shown in live images, these lines express GFP in all the cells of the neuromast in the cldnB::GFP line [46] (Figure S1A), in the hair cells in the pou4f3::GFP line [47] (Figure S1B) or in a subset of supporting cells in the ET20::GFP line [48] (Figure S1C). Camera lucida drawings were added to each panel, for better illustration of the live images. To test the functionality of the hair cells in wild-type and phoenix mutant larvae, we used the well-described technique of monitoring the absorption of the vital dye FM1-43 [49]. We imaged live wild-type and mutant larvae in the cldnB::GFP line background (Figure S1A, GFP in green in first and third columns, FM1-43 in red in second and third columns. We added camera lucida drawings of the merged images in the fourth column). Again, we did not find a significant difference between the absorption of the dye in wild-type (top) and mutant (bottom) hair cells, nor did we see any significant observable structural differences. Therefore, although the gene is expressed in the lateral line, its initial development appears unaffected in mutant larvae.


Phoenix is required for mechanosensory hair cell regeneration in the zebrafish lateral line.

Behra M, Bradsher J, Sougrat R, Gallardo V, Allende ML, Burgess SM - PLoS Genet. (2009)

The development of the neuromasts in the lateral line is normal in the phoenix mutant larvae.(A) Semi-thin sections showing wild-type (top panels) and mutant neuromasts (bottom panels) in 5dpf (left panels) and 9dpf (right panels) larvae. (B) Camera lucida drawing for each section, highlighting the hair cells nuclei (red) and their cilia (green) and the supporting cells nuclei (blue). (C) Ultra-thin sections viewed by Electron Microscopy (EM) of a wild-type (top panel) and a mutant (lower panel) neuromast in 10dpf larvae. The hair cells stain darker than the supporting cells. (D) Camera lucida drawings of the EM sections, highlighting the hair cell nuclei (red), cell bodies (dark red), and cilia (green). The nuclei of the supporting cells are highlighted (blue). One apoptotic body was visible in (yellow). – 5 microns in (A) and 1 micron in (C).
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Related In: Results  -  Collection

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pgen-1000455-g001: The development of the neuromasts in the lateral line is normal in the phoenix mutant larvae.(A) Semi-thin sections showing wild-type (top panels) and mutant neuromasts (bottom panels) in 5dpf (left panels) and 9dpf (right panels) larvae. (B) Camera lucida drawing for each section, highlighting the hair cells nuclei (red) and their cilia (green) and the supporting cells nuclei (blue). (C) Ultra-thin sections viewed by Electron Microscopy (EM) of a wild-type (top panel) and a mutant (lower panel) neuromast in 10dpf larvae. The hair cells stain darker than the supporting cells. (D) Camera lucida drawings of the EM sections, highlighting the hair cell nuclei (red), cell bodies (dark red), and cilia (green). The nuclei of the supporting cells are highlighted (blue). One apoptotic body was visible in (yellow). – 5 microns in (A) and 1 micron in (C).
Mentions: We have utilized an assay to monitor hair cell regeneration in the lateral line of 5dpf zebrafish larvae, using transient exposure to copper sulphate (10mM) or neomycin (200mM) dissolved in the water. Our intention was to test for defective hair cell regeneration from a collection of mutant lines previously identified in a retroviral integration screen [45]. The major advantage of using retroviral constructs, over chemicals like ENU, as a mutagenic agent is the rapid identification of the mutated gene. This allows the spatio-temporal expression of the mutated genes to be compared to the observed phenotypes, facilitating selection of the mutants of interest. We reasoned that mutant larvae which develop a normal and functional lateral line and that exhibit expression of the mutated gene in neuromasts would be good candidates to test for defects in the regeneration of the hair cells. We found such a mutant line, which we called phoenix. Homozygotes displayed no behavioral defects (response to sound or mechanical stimulation was normal, data not shown) or visible morphological abnormalities until 5dpf, when the swim bladder failed to inflate. Later, around 7 to 8 dpf, mutants display necrosis in the liver, and death ensues at approximately 14 dpf. Because the gene was expressed in the lateral line neuromasts (see below), we performed a detailed assessment of this organ in 2 to 12 dpf larvae (Figure 1). Semi-thin sections through neuromasts (Figure 1A) of phoenix mutants (lower panels, 5dpf left panel and 9dpf right panel) were indistinguishable from wild-type larvae (upper panels). Camera lucida drawings (Figure 1B) of each section show the cilia (green) of hair cells and their nuclei (red) and the nuclei of the supporting cells (blue). Likewise, ultra-thin sections observed by electron microscopy (EM) (10dpf larvae shown in Figure 1C) did not present obvious differences between wild-type (top panel) and mutant neuromasts (lower panel). Camera lucida drawings (Figure 1D) show, like in Figure 1C, the cilia (green) of hair cells and their nuclei (red) and the nuclei of the supporting cells (blue). The cytoplasm of hair cells is outlined in dark red and an apoptotic body is shown in yellow in the lower panel. Note that apoptotic bodies, as the one present in the mutant, were found at the same rate in untreated wild-type and mutant sections, being probably products of the regular turnover of the supporting cells, which has been described previously [34]. We further observed wild-type and mutant neuromasts in three different transgenic backgrounds (Figure S1). As shown in live images, these lines express GFP in all the cells of the neuromast in the cldnB::GFP line [46] (Figure S1A), in the hair cells in the pou4f3::GFP line [47] (Figure S1B) or in a subset of supporting cells in the ET20::GFP line [48] (Figure S1C). Camera lucida drawings were added to each panel, for better illustration of the live images. To test the functionality of the hair cells in wild-type and phoenix mutant larvae, we used the well-described technique of monitoring the absorption of the vital dye FM1-43 [49]. We imaged live wild-type and mutant larvae in the cldnB::GFP line background (Figure S1A, GFP in green in first and third columns, FM1-43 in red in second and third columns. We added camera lucida drawings of the merged images in the fourth column). Again, we did not find a significant difference between the absorption of the dye in wild-type (top) and mutant (bottom) hair cells, nor did we see any significant observable structural differences. Therefore, although the gene is expressed in the lateral line, its initial development appears unaffected in mutant larvae.

Bottom Line: We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts.The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells.Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.

View Article: PubMed Central - PubMed

Affiliation: National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration.

Show MeSH
Related in: MedlinePlus