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Expression of cadherin 23 isoforms is not conserved: implications for a mouse model of Usher syndrome type 1D.

Lagziel A, Overlack N, Bernstein SL, Morell RJ, Wolfrum U, Friedman TB - Mol. Vis. (2009)

Bottom Line: The wild-type CDH23_V3 protein isoform, which has PDZ binding motifs but neither extracellular domains nor a transmembrane domain, localized exclusively to the outer plexiform layer of the retina containing photoreceptor cell synapses and to the synaptic region of auditory and vestibular hair cells.The time- and tissue-dependent expression patterns that we have shown for Cdh23 alternative transcripts suggest developmental roles and tissue-specific functions for the various transcripts.This species difference suggests that the mouse may not be a suitable model for studying the retinitis pigmentosa phenotype of human Usher syndrome type 1D.

View Article: PubMed Central - PubMed

Affiliation: Section on Human Genetics, Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850, USA.

ABSTRACT

Purpose: We compared cadherin 23 (Cdh23) mRNA and protein variants in the inner ear and retina of wild-type and mutant mice and primates to better understand the pleiotropic effects of Cdh23 mutations, and specifically to understand the absence of retinal degeneration in Cdh23 mutant mice.

Methods: Semiquantitative real-time PCR was used to compare the level of expression of Cdh23 alternative transcripts in the inner ear and retina of wild-type and homozygous Cdh23(v-6J) (waltzer) mice. Antibodies generated against CDH23 isoforms were used in immunohistochemistry, immunohistology, electron microscopy, and western blot analyses of mouse and primate inner ear and retina to study the distribution of these isoforms in various cellular compartments.

Results: Cdh23 mRNA alternative splice variants were temporally and spatially regulated in the inner ear and retina. In the mature mouse retina, CDH23 isoforms were broadly expressed in various cellular compartments of the photoreceptor layer. The wild-type CDH23_V3 protein isoform, which has PDZ binding motifs but neither extracellular domains nor a transmembrane domain, localized exclusively to the outer plexiform layer of the retina containing photoreceptor cell synapses and to the synaptic region of auditory and vestibular hair cells. The longest CDH23 protein isoform, CDH23_V1, appeared by western blotting to be the only one affected by the Cdh23(v-6J) mutation; it was expressed in the wild-type mouse inner ear, but not in the mouse retina. However, CDH23_V1 was detected in western blot analyses of monkey and human retinas.

Conclusions: The time- and tissue-dependent expression patterns that we have shown for Cdh23 alternative transcripts suggest developmental roles and tissue-specific functions for the various transcripts. Many of these isoforms continue to be expressed in waltzer mice. The longest CDH23 isoform (CDH23_V1), however, is not expressed in mutant mice and is necessary for normal inner ear function. The longest isoform is expressed in the retinas of primates, but not detected in the mouse retina. This species difference suggests that the mouse may not be a suitable model for studying the retinitis pigmentosa phenotype of human Usher syndrome type 1D.

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Validation of CDH23_V3 antisera. In A-C, arrows indicate untransfected cells; F-actin is stained in blue. A: A merged image showing HeLa cells 24 h after transfection with the expression vector GFP-Cdh23_v3a (see isoform designation Figure 1A). Cells were stained for CDH23_V3 with antibody TF648 (red) and F-actin antibody (blue). GFP fluorescence of transfected cells (green; B) overlapped with TF648 antibody staining (red; C) as revealed in the merged image (A). Scale bar equals 20 µm. D: A western blot was used to analyze protein extracts from HeLa cells transfected with GFP-Cdh23_v3a and untransfected controls. In the lysate of transfected cells a band of the expected size of roughly 50 kDa of the GFP-CDH23_V3a fusion protein (GFP approximately 27 kDa; CDH23_V3a approximately 26 kDa) was detected. E: Western blot of protein extracts from P60 mouse inner ear and retina revealed a band of roughly 26 kDa corresponding in size to CDH23_V3. β-actin was used as a loading control. Size standards are given in kDa.
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f2: Validation of CDH23_V3 antisera. In A-C, arrows indicate untransfected cells; F-actin is stained in blue. A: A merged image showing HeLa cells 24 h after transfection with the expression vector GFP-Cdh23_v3a (see isoform designation Figure 1A). Cells were stained for CDH23_V3 with antibody TF648 (red) and F-actin antibody (blue). GFP fluorescence of transfected cells (green; B) overlapped with TF648 antibody staining (red; C) as revealed in the merged image (A). Scale bar equals 20 µm. D: A western blot was used to analyze protein extracts from HeLa cells transfected with GFP-Cdh23_v3a and untransfected controls. In the lysate of transfected cells a band of the expected size of roughly 50 kDa of the GFP-CDH23_V3a fusion protein (GFP approximately 27 kDa; CDH23_V3a approximately 26 kDa) was detected. E: Western blot of protein extracts from P60 mouse inner ear and retina revealed a band of roughly 26 kDa corresponding in size to CDH23_V3. β-actin was used as a loading control. Size standards are given in kDa.

Mentions: Cdh23_v3a and Cdh23_v3b are short transcripts (708 and 600 nucleotides, respectively) initiated from a promoter in intron 65 and encode 235 and 199 amino acid proteins lacking ECs and transmembrane domains. CDH23_V3a/b expression was evaluated with antibodies TF648 and TF649, directed to the seven unique amino acids encoded at the N-terminus that differentiate this isoform from other CDH23 isoforms (Figure 1A). The antibodies were validated by transfecting a GFP-Cdh23_v3a (Figure 1A) expression vector into HeLa cells. Twenty-four hours after transfection the staining pattern obtained with the GFP-CDH23_V3a overlapped with the staining pattern obtained with antibody TF648. Nontransfected cells did not show antibody staining (Figure 2A-C).


Expression of cadherin 23 isoforms is not conserved: implications for a mouse model of Usher syndrome type 1D.

Lagziel A, Overlack N, Bernstein SL, Morell RJ, Wolfrum U, Friedman TB - Mol. Vis. (2009)

Validation of CDH23_V3 antisera. In A-C, arrows indicate untransfected cells; F-actin is stained in blue. A: A merged image showing HeLa cells 24 h after transfection with the expression vector GFP-Cdh23_v3a (see isoform designation Figure 1A). Cells were stained for CDH23_V3 with antibody TF648 (red) and F-actin antibody (blue). GFP fluorescence of transfected cells (green; B) overlapped with TF648 antibody staining (red; C) as revealed in the merged image (A). Scale bar equals 20 µm. D: A western blot was used to analyze protein extracts from HeLa cells transfected with GFP-Cdh23_v3a and untransfected controls. In the lysate of transfected cells a band of the expected size of roughly 50 kDa of the GFP-CDH23_V3a fusion protein (GFP approximately 27 kDa; CDH23_V3a approximately 26 kDa) was detected. E: Western blot of protein extracts from P60 mouse inner ear and retina revealed a band of roughly 26 kDa corresponding in size to CDH23_V3. β-actin was used as a loading control. Size standards are given in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743805&req=5

f2: Validation of CDH23_V3 antisera. In A-C, arrows indicate untransfected cells; F-actin is stained in blue. A: A merged image showing HeLa cells 24 h after transfection with the expression vector GFP-Cdh23_v3a (see isoform designation Figure 1A). Cells were stained for CDH23_V3 with antibody TF648 (red) and F-actin antibody (blue). GFP fluorescence of transfected cells (green; B) overlapped with TF648 antibody staining (red; C) as revealed in the merged image (A). Scale bar equals 20 µm. D: A western blot was used to analyze protein extracts from HeLa cells transfected with GFP-Cdh23_v3a and untransfected controls. In the lysate of transfected cells a band of the expected size of roughly 50 kDa of the GFP-CDH23_V3a fusion protein (GFP approximately 27 kDa; CDH23_V3a approximately 26 kDa) was detected. E: Western blot of protein extracts from P60 mouse inner ear and retina revealed a band of roughly 26 kDa corresponding in size to CDH23_V3. β-actin was used as a loading control. Size standards are given in kDa.
Mentions: Cdh23_v3a and Cdh23_v3b are short transcripts (708 and 600 nucleotides, respectively) initiated from a promoter in intron 65 and encode 235 and 199 amino acid proteins lacking ECs and transmembrane domains. CDH23_V3a/b expression was evaluated with antibodies TF648 and TF649, directed to the seven unique amino acids encoded at the N-terminus that differentiate this isoform from other CDH23 isoforms (Figure 1A). The antibodies were validated by transfecting a GFP-Cdh23_v3a (Figure 1A) expression vector into HeLa cells. Twenty-four hours after transfection the staining pattern obtained with the GFP-CDH23_V3a overlapped with the staining pattern obtained with antibody TF648. Nontransfected cells did not show antibody staining (Figure 2A-C).

Bottom Line: The wild-type CDH23_V3 protein isoform, which has PDZ binding motifs but neither extracellular domains nor a transmembrane domain, localized exclusively to the outer plexiform layer of the retina containing photoreceptor cell synapses and to the synaptic region of auditory and vestibular hair cells.The time- and tissue-dependent expression patterns that we have shown for Cdh23 alternative transcripts suggest developmental roles and tissue-specific functions for the various transcripts.This species difference suggests that the mouse may not be a suitable model for studying the retinitis pigmentosa phenotype of human Usher syndrome type 1D.

View Article: PubMed Central - PubMed

Affiliation: Section on Human Genetics, Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850, USA.

ABSTRACT

Purpose: We compared cadherin 23 (Cdh23) mRNA and protein variants in the inner ear and retina of wild-type and mutant mice and primates to better understand the pleiotropic effects of Cdh23 mutations, and specifically to understand the absence of retinal degeneration in Cdh23 mutant mice.

Methods: Semiquantitative real-time PCR was used to compare the level of expression of Cdh23 alternative transcripts in the inner ear and retina of wild-type and homozygous Cdh23(v-6J) (waltzer) mice. Antibodies generated against CDH23 isoforms were used in immunohistochemistry, immunohistology, electron microscopy, and western blot analyses of mouse and primate inner ear and retina to study the distribution of these isoforms in various cellular compartments.

Results: Cdh23 mRNA alternative splice variants were temporally and spatially regulated in the inner ear and retina. In the mature mouse retina, CDH23 isoforms were broadly expressed in various cellular compartments of the photoreceptor layer. The wild-type CDH23_V3 protein isoform, which has PDZ binding motifs but neither extracellular domains nor a transmembrane domain, localized exclusively to the outer plexiform layer of the retina containing photoreceptor cell synapses and to the synaptic region of auditory and vestibular hair cells. The longest CDH23 protein isoform, CDH23_V1, appeared by western blotting to be the only one affected by the Cdh23(v-6J) mutation; it was expressed in the wild-type mouse inner ear, but not in the mouse retina. However, CDH23_V1 was detected in western blot analyses of monkey and human retinas.

Conclusions: The time- and tissue-dependent expression patterns that we have shown for Cdh23 alternative transcripts suggest developmental roles and tissue-specific functions for the various transcripts. Many of these isoforms continue to be expressed in waltzer mice. The longest CDH23 isoform (CDH23_V1), however, is not expressed in mutant mice and is necessary for normal inner ear function. The longest isoform is expressed in the retinas of primates, but not detected in the mouse retina. This species difference suggests that the mouse may not be a suitable model for studying the retinitis pigmentosa phenotype of human Usher syndrome type 1D.

Show MeSH
Related in: MedlinePlus