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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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Different CDH23 protein isoforms localize to distinct cell layers of the retina. A, E, I, and M are differential interference contrast (DIC) images of longitudinal cryosections (B-D, F-H, J-L and N-P, respectively) through adult mouse retina showing photoreceptors cell layers. Indirect immunofluorescence labeling of CDH23 with the cytoplasmic domain antibody TF7 (B) revealed CDH23 in the ciliary region of photoreceptor cells, the ONL, and in the OPL. Double labeling with antibodies TF7 and centrin, a ciliary marker (C), revealed colocalization of CDH23 and centrin in the ciliary apparatus of the photoreceptor cells (D). F-H: Double labeling with antisera TF7 (F) and the synaptic protein PSD-95 with antisera PSD95 (G) revealed CDH23 colocalization with PSD-95 in the synaptic terminals in the OPL of photoreceptors cells (H). Double labeling with CDH23_V3 specific antibodies TF648 and TF649 (J, N) and PSD95 (K, O) revealed that CDH23_V3 was detected only in the OPL where it colocalized with PSD-95 (L, P). Scale bars represent 10 µm. Abbreviations: retinal pigment epithelium (RPE), outer segment (OS), inner segment (IS), outer nuclear layer (ONL), outer plexiform layer (OPL), and inner nuclear layer (INL).
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f3: Different CDH23 protein isoforms localize to distinct cell layers of the retina. A, E, I, and M are differential interference contrast (DIC) images of longitudinal cryosections (B-D, F-H, J-L and N-P, respectively) through adult mouse retina showing photoreceptors cell layers. Indirect immunofluorescence labeling of CDH23 with the cytoplasmic domain antibody TF7 (B) revealed CDH23 in the ciliary region of photoreceptor cells, the ONL, and in the OPL. Double labeling with antibodies TF7 and centrin, a ciliary marker (C), revealed colocalization of CDH23 and centrin in the ciliary apparatus of the photoreceptor cells (D). F-H: Double labeling with antisera TF7 (F) and the synaptic protein PSD-95 with antisera PSD95 (G) revealed CDH23 colocalization with PSD-95 in the synaptic terminals in the OPL of photoreceptors cells (H). Double labeling with CDH23_V3 specific antibodies TF648 and TF649 (J, N) and PSD95 (K, O) revealed that CDH23_V3 was detected only in the OPL where it colocalized with PSD-95 (L, P). Scale bars represent 10 µm. Abbreviations: retinal pigment epithelium (RPE), outer segment (OS), inner segment (IS), outer nuclear layer (ONL), outer plexiform layer (OPL), and inner nuclear layer (INL).

Mentions: The subcellular distribution of CDH23 in the retina was determined by analyzing cryosections through adult mice eyes by immunofluorescence with antibody TF7, which recognizes the CDH23 cytoplasmic domain, and therefore multiple Cdh23 isoforms (Figure 1A, lower panel). Cryosections of mouse retinas were double-labeled either with anti-centrin as a marker for the ciliary apparatus of photoreceptor cells [43], or anti- PSD95 as a marker for the pre- and post-synaptic terminals of photoreceptor cells located in the outer plexiform layer [50]. Double labeling with TF7 and anti-centrin (Figure 3A-D) revealed CDH23 in the ciliary region, the outer nuclear layer, and in the outer plexiform layer. Double-labeling with TF7 and anti-PSD95 (Figure 3E-H) confirmed localization of CDH23 in the outer plexiform layer. Higher resolution imaging of double-labeling immunofluorescence with TF7 and anti-centrin revealed partial colocalization of CDH23 protein isoforms and centrins in the basal bodies of the ciliary apparatus of photoreceptor cells (Figure 4). Pre-embedding high resolution immunoelectron microscopy, using antibody TF7, localized CDH23 in the basal body of the cilium and its adjacent centriole and confirmed CDH23 as a component of the ciliary apparatus (Figure 5) [42].


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)

Different CDH23 protein isoforms localize to distinct cell layers of the retina. A, E, I, and M are differential interference contrast (DIC) images of longitudinal cryosections (B-D, F-H, J-L and N-P, respectively) through adult mouse retina showing photoreceptors cell layers. Indirect immunofluorescence labeling of CDH23 with the cytoplasmic domain antibody TF7 (B) revealed CDH23 in the ciliary region of photoreceptor cells, the ONL, and in the OPL. Double labeling with antibodies TF7 and centrin, a ciliary marker (C), revealed colocalization of CDH23 and centrin in the ciliary apparatus of the photoreceptor cells (D). F-H: Double labeling with antisera TF7 (F) and the synaptic protein PSD-95 with antisera PSD95 (G) revealed CDH23 colocalization with PSD-95 in the synaptic terminals in the OPL of photoreceptors cells (H). Double labeling with CDH23_V3 specific antibodies TF648 and TF649 (J, N) and PSD95 (K, O) revealed that CDH23_V3 was detected only in the OPL where it colocalized with PSD-95 (L, P). Scale bars represent 10 µm. Abbreviations: retinal pigment epithelium (RPE), outer segment (OS), inner segment (IS), outer nuclear layer (ONL), outer plexiform layer (OPL), and inner nuclear layer (INL).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Different CDH23 protein isoforms localize to distinct cell layers of the retina. A, E, I, and M are differential interference contrast (DIC) images of longitudinal cryosections (B-D, F-H, J-L and N-P, respectively) through adult mouse retina showing photoreceptors cell layers. Indirect immunofluorescence labeling of CDH23 with the cytoplasmic domain antibody TF7 (B) revealed CDH23 in the ciliary region of photoreceptor cells, the ONL, and in the OPL. Double labeling with antibodies TF7 and centrin, a ciliary marker (C), revealed colocalization of CDH23 and centrin in the ciliary apparatus of the photoreceptor cells (D). F-H: Double labeling with antisera TF7 (F) and the synaptic protein PSD-95 with antisera PSD95 (G) revealed CDH23 colocalization with PSD-95 in the synaptic terminals in the OPL of photoreceptors cells (H). Double labeling with CDH23_V3 specific antibodies TF648 and TF649 (J, N) and PSD95 (K, O) revealed that CDH23_V3 was detected only in the OPL where it colocalized with PSD-95 (L, P). Scale bars represent 10 µm. Abbreviations: retinal pigment epithelium (RPE), outer segment (OS), inner segment (IS), outer nuclear layer (ONL), outer plexiform layer (OPL), and inner nuclear layer (INL).
Mentions: The subcellular distribution of CDH23 in the retina was determined by analyzing cryosections through adult mice eyes by immunofluorescence with antibody TF7, which recognizes the CDH23 cytoplasmic domain, and therefore multiple Cdh23 isoforms (Figure 1A, lower panel). Cryosections of mouse retinas were double-labeled either with anti-centrin as a marker for the ciliary apparatus of photoreceptor cells [43], or anti- PSD95 as a marker for the pre- and post-synaptic terminals of photoreceptor cells located in the outer plexiform layer [50]. Double labeling with TF7 and anti-centrin (Figure 3A-D) revealed CDH23 in the ciliary region, the outer nuclear layer, and in the outer plexiform layer. Double-labeling with TF7 and anti-PSD95 (Figure 3E-H) confirmed localization of CDH23 in the outer plexiform layer. Higher resolution imaging of double-labeling immunofluorescence with TF7 and anti-centrin revealed partial colocalization of CDH23 protein isoforms and centrins in the basal bodies of the ciliary apparatus of photoreceptor cells (Figure 4). Pre-embedding high resolution immunoelectron microscopy, using antibody TF7, localized CDH23 in the basal body of the cilium and its adjacent centriole and confirmed CDH23 as a component of the ciliary apparatus (Figure 5) [42].

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