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Spinster homolog 2 (spns2) deficiency causes early onset progressive hearing loss.

Chen J, Ingham N, Kelly J, Jadeja S, Goulding D, Pass J, Mahajan VB, Tsang SH, Nijnik A, Jackson IJ, White JK, Forge A, Jagger D, Steel KP - PLoS Genet. (2014)

Bottom Line: The mechanism of action of Spns2 is still elusive in mammals.Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals.These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Wolfson Centre for Age-Related Diseases, King's College London, London, United Kingdom.

ABSTRACT
Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.

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Generation and manipulation of the knockout-first allele Spns2tm1a and progressive hearing loss measured by ABR.A, The promoter-driven cassette, including a splice acceptor site, an internal ribosome entry site (IRES) and a β-galactosidase reporter (lacZ), followed by a neomycin resistance marker expressed from an independent β-actin promoter, was inserted into intron2 of the Spns2 gene. FRT sites surround the inserted cassette and LoxP sites flank the critical exon (exon 3 of Spns2; http://www.knockoutmouse.org/martsearch/project/25171). The lacZ-tagged Spns2tm1b allele was generated by breeding Spns2tm1a-carrying mice to mice expressing Cre recombinase driven by the CMV promoter, to delete the floxed critical exon and the neomycin-containing promoter-driven selection cassette. Flp-mediated recombination removed the main inserted cassette to convert the knockout-first allele (Spns2tm1a) to Spns2tm1c, restoring Spns2 gene activity. Cre recombination driven by a suitable promoter such as the Sox10-Cre allele deleted the floxed exon 3 of the Spns2tm1c allele to generate a frameshift mutation (Spns2tm1d), triggering nonsense mediated decay of the transcript. B,C Spns2tm1a/tm1a mice showed progressive hearing impairment between 2 and 3 weeks of age. Raised mean ABR thresholds were detected as early as 2 weeks old in Spns2tm1a/tm1a mice (red, +/− SD), mainly from 24 kHz to 42 kHz with thresholds 30–40 dB higher than those of control mice (Spns2+/+, n = 10; Spns2+/tm1a, n = 16; Spns2tm1a/tm1a, n = 5). At 3 weeks old, hearing impairment became worse and all the frequencies were affected (Spns2+/+, n = 14; Spns2+/tm1a, n = 16; Spns2tm1a/tm1a, n = 13). Heterozygotes in blue; wildtypes in green; pale gray lines show thresholds of individual mutant mice. In C, the 2 week old mean mutant thresholds are plotted in pink with open circles for comparison with 3 week old data, indicating significant progression of the hearing loss (Kruskall-Wallis One-Way Analysis of Variance on Ranks H = 102.857, 17 degrees of freedom, p<0.001). D, Mean ABR thresholds of mutants (red) at 14 weeks old (n = 6, pale gray symbols represent individual mice) showed either very raised ABR thresholds or no response at all at the maximum sound level used (95 dB SPL). The green area shows the reference range for thresholds of wildtype mice of the same genetic background (n = 440), plotting the median and 2.5% to 97.5% percentiles. E,F, ABR thresholds of individual Spns2tm1a/tm1a and wildtype mice. Arrows at top indicate no response at the plotted maximum sound pressure level used. Heterozygous data are comparable to those of wildtypes (green) and not shown here. G, Mean thresholds of mutants at 2 weeks (red), 3 weeks (brown) and 14 weeks (gray) are shown plotted as the difference between mutant and wildtype (green) thresholds, showing an increasing difference between 2 and 3 weeks, partly due to increasing thresholds of mutants (see B and C) and partly due to continuing maturation of thresholds of wildtypes.
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pgen-1004688-g001: Generation and manipulation of the knockout-first allele Spns2tm1a and progressive hearing loss measured by ABR.A, The promoter-driven cassette, including a splice acceptor site, an internal ribosome entry site (IRES) and a β-galactosidase reporter (lacZ), followed by a neomycin resistance marker expressed from an independent β-actin promoter, was inserted into intron2 of the Spns2 gene. FRT sites surround the inserted cassette and LoxP sites flank the critical exon (exon 3 of Spns2; http://www.knockoutmouse.org/martsearch/project/25171). The lacZ-tagged Spns2tm1b allele was generated by breeding Spns2tm1a-carrying mice to mice expressing Cre recombinase driven by the CMV promoter, to delete the floxed critical exon and the neomycin-containing promoter-driven selection cassette. Flp-mediated recombination removed the main inserted cassette to convert the knockout-first allele (Spns2tm1a) to Spns2tm1c, restoring Spns2 gene activity. Cre recombination driven by a suitable promoter such as the Sox10-Cre allele deleted the floxed exon 3 of the Spns2tm1c allele to generate a frameshift mutation (Spns2tm1d), triggering nonsense mediated decay of the transcript. B,C Spns2tm1a/tm1a mice showed progressive hearing impairment between 2 and 3 weeks of age. Raised mean ABR thresholds were detected as early as 2 weeks old in Spns2tm1a/tm1a mice (red, +/− SD), mainly from 24 kHz to 42 kHz with thresholds 30–40 dB higher than those of control mice (Spns2+/+, n = 10; Spns2+/tm1a, n = 16; Spns2tm1a/tm1a, n = 5). At 3 weeks old, hearing impairment became worse and all the frequencies were affected (Spns2+/+, n = 14; Spns2+/tm1a, n = 16; Spns2tm1a/tm1a, n = 13). Heterozygotes in blue; wildtypes in green; pale gray lines show thresholds of individual mutant mice. In C, the 2 week old mean mutant thresholds are plotted in pink with open circles for comparison with 3 week old data, indicating significant progression of the hearing loss (Kruskall-Wallis One-Way Analysis of Variance on Ranks H = 102.857, 17 degrees of freedom, p<0.001). D, Mean ABR thresholds of mutants (red) at 14 weeks old (n = 6, pale gray symbols represent individual mice) showed either very raised ABR thresholds or no response at all at the maximum sound level used (95 dB SPL). The green area shows the reference range for thresholds of wildtype mice of the same genetic background (n = 440), plotting the median and 2.5% to 97.5% percentiles. E,F, ABR thresholds of individual Spns2tm1a/tm1a and wildtype mice. Arrows at top indicate no response at the plotted maximum sound pressure level used. Heterozygous data are comparable to those of wildtypes (green) and not shown here. G, Mean thresholds of mutants at 2 weeks (red), 3 weeks (brown) and 14 weeks (gray) are shown plotted as the difference between mutant and wildtype (green) thresholds, showing an increasing difference between 2 and 3 weeks, partly due to increasing thresholds of mutants (see B and C) and partly due to continuing maturation of thresholds of wildtypes.

Mentions: Spns2-deficient mice were initially discovered to be deaf during a large-scale screen of new mouse mutants carried out by the Sanger Institute's Mouse Genetics Project (MGP). The MGP uses the KOMP/EUCOMM resource of over 15,000 genes targeted in embryonic stem (ES) cells and aims to generate new mutants and screen them for a wide range of diseases and traits to reveal the function of 160 mutant genes each year [20]. Screening of hearing using the Auditory Brainstem Response (ABR) is part of the standardised battery of primary phenotypic tests and is carried out at 14 weeks of age [20]. Mutants generated from the KOMP/EUCOMM ES cell resource normally carry LoxP and Frt sites (Fig. 1A) engineered to facilitate further genetic manipulation to generate the conditional allele and then to knock out gene expression selectively [21].


Spinster homolog 2 (spns2) deficiency causes early onset progressive hearing loss.

Chen J, Ingham N, Kelly J, Jadeja S, Goulding D, Pass J, Mahajan VB, Tsang SH, Nijnik A, Jackson IJ, White JK, Forge A, Jagger D, Steel KP - PLoS Genet. (2014)

Generation and manipulation of the knockout-first allele Spns2tm1a and progressive hearing loss measured by ABR.A, The promoter-driven cassette, including a splice acceptor site, an internal ribosome entry site (IRES) and a β-galactosidase reporter (lacZ), followed by a neomycin resistance marker expressed from an independent β-actin promoter, was inserted into intron2 of the Spns2 gene. FRT sites surround the inserted cassette and LoxP sites flank the critical exon (exon 3 of Spns2; http://www.knockoutmouse.org/martsearch/project/25171). The lacZ-tagged Spns2tm1b allele was generated by breeding Spns2tm1a-carrying mice to mice expressing Cre recombinase driven by the CMV promoter, to delete the floxed critical exon and the neomycin-containing promoter-driven selection cassette. Flp-mediated recombination removed the main inserted cassette to convert the knockout-first allele (Spns2tm1a) to Spns2tm1c, restoring Spns2 gene activity. Cre recombination driven by a suitable promoter such as the Sox10-Cre allele deleted the floxed exon 3 of the Spns2tm1c allele to generate a frameshift mutation (Spns2tm1d), triggering nonsense mediated decay of the transcript. B,C Spns2tm1a/tm1a mice showed progressive hearing impairment between 2 and 3 weeks of age. Raised mean ABR thresholds were detected as early as 2 weeks old in Spns2tm1a/tm1a mice (red, +/− SD), mainly from 24 kHz to 42 kHz with thresholds 30–40 dB higher than those of control mice (Spns2+/+, n = 10; Spns2+/tm1a, n = 16; Spns2tm1a/tm1a, n = 5). At 3 weeks old, hearing impairment became worse and all the frequencies were affected (Spns2+/+, n = 14; Spns2+/tm1a, n = 16; Spns2tm1a/tm1a, n = 13). Heterozygotes in blue; wildtypes in green; pale gray lines show thresholds of individual mutant mice. In C, the 2 week old mean mutant thresholds are plotted in pink with open circles for comparison with 3 week old data, indicating significant progression of the hearing loss (Kruskall-Wallis One-Way Analysis of Variance on Ranks H = 102.857, 17 degrees of freedom, p<0.001). D, Mean ABR thresholds of mutants (red) at 14 weeks old (n = 6, pale gray symbols represent individual mice) showed either very raised ABR thresholds or no response at all at the maximum sound level used (95 dB SPL). The green area shows the reference range for thresholds of wildtype mice of the same genetic background (n = 440), plotting the median and 2.5% to 97.5% percentiles. E,F, ABR thresholds of individual Spns2tm1a/tm1a and wildtype mice. Arrows at top indicate no response at the plotted maximum sound pressure level used. Heterozygous data are comparable to those of wildtypes (green) and not shown here. G, Mean thresholds of mutants at 2 weeks (red), 3 weeks (brown) and 14 weeks (gray) are shown plotted as the difference between mutant and wildtype (green) thresholds, showing an increasing difference between 2 and 3 weeks, partly due to increasing thresholds of mutants (see B and C) and partly due to continuing maturation of thresholds of wildtypes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214598&req=5

pgen-1004688-g001: Generation and manipulation of the knockout-first allele Spns2tm1a and progressive hearing loss measured by ABR.A, The promoter-driven cassette, including a splice acceptor site, an internal ribosome entry site (IRES) and a β-galactosidase reporter (lacZ), followed by a neomycin resistance marker expressed from an independent β-actin promoter, was inserted into intron2 of the Spns2 gene. FRT sites surround the inserted cassette and LoxP sites flank the critical exon (exon 3 of Spns2; http://www.knockoutmouse.org/martsearch/project/25171). The lacZ-tagged Spns2tm1b allele was generated by breeding Spns2tm1a-carrying mice to mice expressing Cre recombinase driven by the CMV promoter, to delete the floxed critical exon and the neomycin-containing promoter-driven selection cassette. Flp-mediated recombination removed the main inserted cassette to convert the knockout-first allele (Spns2tm1a) to Spns2tm1c, restoring Spns2 gene activity. Cre recombination driven by a suitable promoter such as the Sox10-Cre allele deleted the floxed exon 3 of the Spns2tm1c allele to generate a frameshift mutation (Spns2tm1d), triggering nonsense mediated decay of the transcript. B,C Spns2tm1a/tm1a mice showed progressive hearing impairment between 2 and 3 weeks of age. Raised mean ABR thresholds were detected as early as 2 weeks old in Spns2tm1a/tm1a mice (red, +/− SD), mainly from 24 kHz to 42 kHz with thresholds 30–40 dB higher than those of control mice (Spns2+/+, n = 10; Spns2+/tm1a, n = 16; Spns2tm1a/tm1a, n = 5). At 3 weeks old, hearing impairment became worse and all the frequencies were affected (Spns2+/+, n = 14; Spns2+/tm1a, n = 16; Spns2tm1a/tm1a, n = 13). Heterozygotes in blue; wildtypes in green; pale gray lines show thresholds of individual mutant mice. In C, the 2 week old mean mutant thresholds are plotted in pink with open circles for comparison with 3 week old data, indicating significant progression of the hearing loss (Kruskall-Wallis One-Way Analysis of Variance on Ranks H = 102.857, 17 degrees of freedom, p<0.001). D, Mean ABR thresholds of mutants (red) at 14 weeks old (n = 6, pale gray symbols represent individual mice) showed either very raised ABR thresholds or no response at all at the maximum sound level used (95 dB SPL). The green area shows the reference range for thresholds of wildtype mice of the same genetic background (n = 440), plotting the median and 2.5% to 97.5% percentiles. E,F, ABR thresholds of individual Spns2tm1a/tm1a and wildtype mice. Arrows at top indicate no response at the plotted maximum sound pressure level used. Heterozygous data are comparable to those of wildtypes (green) and not shown here. G, Mean thresholds of mutants at 2 weeks (red), 3 weeks (brown) and 14 weeks (gray) are shown plotted as the difference between mutant and wildtype (green) thresholds, showing an increasing difference between 2 and 3 weeks, partly due to increasing thresholds of mutants (see B and C) and partly due to continuing maturation of thresholds of wildtypes.
Mentions: Spns2-deficient mice were initially discovered to be deaf during a large-scale screen of new mouse mutants carried out by the Sanger Institute's Mouse Genetics Project (MGP). The MGP uses the KOMP/EUCOMM resource of over 15,000 genes targeted in embryonic stem (ES) cells and aims to generate new mutants and screen them for a wide range of diseases and traits to reveal the function of 160 mutant genes each year [20]. Screening of hearing using the Auditory Brainstem Response (ABR) is part of the standardised battery of primary phenotypic tests and is carried out at 14 weeks of age [20]. Mutants generated from the KOMP/EUCOMM ES cell resource normally carry LoxP and Frt sites (Fig. 1A) engineered to facilitate further genetic manipulation to generate the conditional allele and then to knock out gene expression selectively [21].

Bottom Line: The mechanism of action of Spns2 is still elusive in mammals.Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals.These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Wolfson Centre for Age-Related Diseases, King's College London, London, United Kingdom.

ABSTRACT
Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.

Show MeSH
Related in: MedlinePlus