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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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Primate and mouse CDH23 protein in inner ear and retina. A-C: western blot analyses of proteins separated on 3%–8% tris-acetate gels using the cytoplasmic domain antibody, TF7. A: Western analysis, using protein extracts from P0, and P90 wild-type and mutant mouse inner ear and retina. An approximately 350 kDa band corresponding to the largest CDH23 protein isoform was only detected in P0 mouse inner ear (arrow head). Faster migrating protein bands were present in wild-type and Cdh23v-6J and may represent lower molecular weight CDH23 protein isoforms (such as CDH23_V3). Tissue specific variation of these isoforms is better visualized in the western blot shown in panel B. B: Western analysis, using protein extracts from P4, and P60 wild-type mouse inner ear and retina. The high molecular weight band at roughly 350 kDa (arrowhead) was detected in the P4 inner ear protein sample. Traces of this band were also detected in the P60 inner ear protein sample. The faster migrating bands detected show variability in their appearance between young and adult tissue as well as variability between the inner ear and retina. C: Western analysis, using protein extracts from P0 mouse retina, human retina, and monkey retina. The high molecular weight band (arrow head A-C) detected in P0 wild-type mouse inner ear corresponds in size to the largest band detected in human and monkey retinas (arrowhead). β-actin was used as a loading control; size standards are given in kDa.
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f8: Primate and mouse CDH23 protein in inner ear and retina. A-C: western blot analyses of proteins separated on 3%–8% tris-acetate gels using the cytoplasmic domain antibody, TF7. A: Western analysis, using protein extracts from P0, and P90 wild-type and mutant mouse inner ear and retina. An approximately 350 kDa band corresponding to the largest CDH23 protein isoform was only detected in P0 mouse inner ear (arrow head). Faster migrating protein bands were present in wild-type and Cdh23v-6J and may represent lower molecular weight CDH23 protein isoforms (such as CDH23_V3). Tissue specific variation of these isoforms is better visualized in the western blot shown in panel B. B: Western analysis, using protein extracts from P4, and P60 wild-type mouse inner ear and retina. The high molecular weight band at roughly 350 kDa (arrowhead) was detected in the P4 inner ear protein sample. Traces of this band were also detected in the P60 inner ear protein sample. The faster migrating bands detected show variability in their appearance between young and adult tissue as well as variability between the inner ear and retina. C: Western analysis, using protein extracts from P0 mouse retina, human retina, and monkey retina. The high molecular weight band (arrow head A-C) detected in P0 wild-type mouse inner ear corresponds in size to the largest band detected in human and monkey retinas (arrowhead). β-actin was used as a loading control; size standards are given in kDa.

Mentions: We performed western blot analyses of mouse protein extracts from P0, P4, and adult wild-type, and P0 and adult mutant inner ear and retinas using TF7 antibody. Results were obtained from 3 to 4 independent protein extractions replicated 2 to 9 times. Representative western blots (Figure 8) revealed a large band of ~350 kDa corresponding in size to CDH23 longest protein isoform (CDH23_V1) only in P0 and P4 wild-type inner ear (Figure 8A-C). This large band was not detected in wild-type P0, P4, P60, or P90 retinas and was not detected in the waltzer Cdh23v-6J inner ear or retinal samples (Figure 8A-C). A trace of this high molecular cadherin 23 isoform was detected with antibody TF7 in the P60 inner ear sample (Figure 8B, lane 2), which was consistent with the reported reduction in cadherin 23 in the mature mouse inner ear [28,29,31,32]. Various small bands were also detected both in inner ear and retina that corresponded to the predicted sizes of CDH23_V2, roughly 120 kDa or CDH23_V3, approximately 26 kDa. Some variability in the appearance of the small bands was detected between inner ear or retina, the age of the mouse and the strain, either wild-type or mutant Cdh23v-6J. A high molecular weight band of roughly 350 kDa was detected in human and monkey retinas (Figure 8C) corresponding in size to the high molecular weight band detected in P0 and P4 inner ear (Figure 8A-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)

Primate and mouse CDH23 protein in inner ear and retina. A-C: western blot analyses of proteins separated on 3%–8% tris-acetate gels using the cytoplasmic domain antibody, TF7. A: Western analysis, using protein extracts from P0, and P90 wild-type and mutant mouse inner ear and retina. An approximately 350 kDa band corresponding to the largest CDH23 protein isoform was only detected in P0 mouse inner ear (arrow head). Faster migrating protein bands were present in wild-type and Cdh23v-6J and may represent lower molecular weight CDH23 protein isoforms (such as CDH23_V3). Tissue specific variation of these isoforms is better visualized in the western blot shown in panel B. B: Western analysis, using protein extracts from P4, and P60 wild-type mouse inner ear and retina. The high molecular weight band at roughly 350 kDa (arrowhead) was detected in the P4 inner ear protein sample. Traces of this band were also detected in the P60 inner ear protein sample. The faster migrating bands detected show variability in their appearance between young and adult tissue as well as variability between the inner ear and retina. C: Western analysis, using protein extracts from P0 mouse retina, human retina, and monkey retina. The high molecular weight band (arrow head A-C) detected in P0 wild-type mouse inner ear corresponds in size to the largest band detected in human and monkey retinas (arrowhead). β-actin was used as a loading control; size standards are given in kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2743805&req=5

f8: Primate and mouse CDH23 protein in inner ear and retina. A-C: western blot analyses of proteins separated on 3%–8% tris-acetate gels using the cytoplasmic domain antibody, TF7. A: Western analysis, using protein extracts from P0, and P90 wild-type and mutant mouse inner ear and retina. An approximately 350 kDa band corresponding to the largest CDH23 protein isoform was only detected in P0 mouse inner ear (arrow head). Faster migrating protein bands were present in wild-type and Cdh23v-6J and may represent lower molecular weight CDH23 protein isoforms (such as CDH23_V3). Tissue specific variation of these isoforms is better visualized in the western blot shown in panel B. B: Western analysis, using protein extracts from P4, and P60 wild-type mouse inner ear and retina. The high molecular weight band at roughly 350 kDa (arrowhead) was detected in the P4 inner ear protein sample. Traces of this band were also detected in the P60 inner ear protein sample. The faster migrating bands detected show variability in their appearance between young and adult tissue as well as variability between the inner ear and retina. C: Western analysis, using protein extracts from P0 mouse retina, human retina, and monkey retina. The high molecular weight band (arrow head A-C) detected in P0 wild-type mouse inner ear corresponds in size to the largest band detected in human and monkey retinas (arrowhead). β-actin was used as a loading control; size standards are given in kDa.
Mentions: We performed western blot analyses of mouse protein extracts from P0, P4, and adult wild-type, and P0 and adult mutant inner ear and retinas using TF7 antibody. Results were obtained from 3 to 4 independent protein extractions replicated 2 to 9 times. Representative western blots (Figure 8) revealed a large band of ~350 kDa corresponding in size to CDH23 longest protein isoform (CDH23_V1) only in P0 and P4 wild-type inner ear (Figure 8A-C). This large band was not detected in wild-type P0, P4, P60, or P90 retinas and was not detected in the waltzer Cdh23v-6J inner ear or retinal samples (Figure 8A-C). A trace of this high molecular cadherin 23 isoform was detected with antibody TF7 in the P60 inner ear sample (Figure 8B, lane 2), which was consistent with the reported reduction in cadherin 23 in the mature mouse inner ear [28,29,31,32]. Various small bands were also detected both in inner ear and retina that corresponded to the predicted sizes of CDH23_V2, roughly 120 kDa or CDH23_V3, approximately 26 kDa. Some variability in the appearance of the small bands was detected between inner ear or retina, the age of the mouse and the strain, either wild-type or mutant Cdh23v-6J. A high molecular weight band of roughly 350 kDa was detected in human and monkey retinas (Figure 8C) corresponding in size to the high molecular weight band detected in P0 and P4 inner ear (Figure 8A-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