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Gene profiling of postnatal Mfrprd6 mutant eyes reveals differential accumulation of Prss56, visual cycle and phototransduction mRNAs.

Soundararajan R, Won J, Stearns TM, Charette JR, Hicks WL, Collin GB, Naggert JK, Krebs MP, Nishina PM - PLoS ONE (2014)

Bottom Line: In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2).Levels of RPE65 were significantly decreased by 2.0-fold.In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, Maine, United States of America.

ABSTRACT
Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes are part of a regulatory network influencing postnatal posterior eye development.

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Cellular localization of Prss56 and Glul in B6 (C57BL/6J) and Mfrprd6/Mfrprd6 mice.(A) By in situ hybridization, in B6 (C57BL/6J) controls at P14, we observed Prss56 transcript in only very few cells of the inner nuclear layer (INL) of the retina (top panel), whereas in Mfrprd6/Mfrprd6 mutants, an intense staining of Prss56 transcript was observed in INL of the retina (bottom panel). (B) By 2-plex in situ hybridization, in B6 controls, we observed co-localization of Prss56 (red) and Glul (pseudo colored green) transcripts in only a few cell body of the (INL) of the retina (top panel), whereas in Mfrprd6/Mfrprd6, strong co-localization of Prss56 and Glul transcripts in the cell body of the INL of the retina was observed (bottom panel). (C) Glutamine synthetase (GS) staining of Müller cells. In both C57BL/6J (B6) and Mfrprd6/Mfrprd6 mice, Müller cells marked with glutamine synthetase showed a similar localization pattern (inset, top and bottom panels) as observed for Prss56 in situ hybridization staining, suggesting that Müller cells in the INL of retina express Prss56 transcripts at P14.
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pone-0110299-g006: Cellular localization of Prss56 and Glul in B6 (C57BL/6J) and Mfrprd6/Mfrprd6 mice.(A) By in situ hybridization, in B6 (C57BL/6J) controls at P14, we observed Prss56 transcript in only very few cells of the inner nuclear layer (INL) of the retina (top panel), whereas in Mfrprd6/Mfrprd6 mutants, an intense staining of Prss56 transcript was observed in INL of the retina (bottom panel). (B) By 2-plex in situ hybridization, in B6 controls, we observed co-localization of Prss56 (red) and Glul (pseudo colored green) transcripts in only a few cell body of the (INL) of the retina (top panel), whereas in Mfrprd6/Mfrprd6, strong co-localization of Prss56 and Glul transcripts in the cell body of the INL of the retina was observed (bottom panel). (C) Glutamine synthetase (GS) staining of Müller cells. In both C57BL/6J (B6) and Mfrprd6/Mfrprd6 mice, Müller cells marked with glutamine synthetase showed a similar localization pattern (inset, top and bottom panels) as observed for Prss56 in situ hybridization staining, suggesting that Müller cells in the INL of retina express Prss56 transcripts at P14.

Mentions: In the absence of an antibody that could reliably detect murine PRSS56, in situ hybridization was used to determine the cellular localization of the Prss56 transcript. In the no probe control, no Prss56 transcript was observed (data not shown). In the wild-type control at P14, confocal microscopy revealed a few cells in the retinal inner nuclear layer (INL) that showed expression of Prss56 transcript (Fig. 6A, upper panel). By contrast, in Mfrprd6 eyes at P14, intense specific staining of the transcript was observed in the INL (Fig. 6A, lower panel). This increased staining further validates both the microarray and qRT-PCR results of increased Prss56 transcripts in Mfrprd6 mutant eyes relative to controls.


Gene profiling of postnatal Mfrprd6 mutant eyes reveals differential accumulation of Prss56, visual cycle and phototransduction mRNAs.

Soundararajan R, Won J, Stearns TM, Charette JR, Hicks WL, Collin GB, Naggert JK, Krebs MP, Nishina PM - PLoS ONE (2014)

Cellular localization of Prss56 and Glul in B6 (C57BL/6J) and Mfrprd6/Mfrprd6 mice.(A) By in situ hybridization, in B6 (C57BL/6J) controls at P14, we observed Prss56 transcript in only very few cells of the inner nuclear layer (INL) of the retina (top panel), whereas in Mfrprd6/Mfrprd6 mutants, an intense staining of Prss56 transcript was observed in INL of the retina (bottom panel). (B) By 2-plex in situ hybridization, in B6 controls, we observed co-localization of Prss56 (red) and Glul (pseudo colored green) transcripts in only a few cell body of the (INL) of the retina (top panel), whereas in Mfrprd6/Mfrprd6, strong co-localization of Prss56 and Glul transcripts in the cell body of the INL of the retina was observed (bottom panel). (C) Glutamine synthetase (GS) staining of Müller cells. In both C57BL/6J (B6) and Mfrprd6/Mfrprd6 mice, Müller cells marked with glutamine synthetase showed a similar localization pattern (inset, top and bottom panels) as observed for Prss56 in situ hybridization staining, suggesting that Müller cells in the INL of retina express Prss56 transcripts at P14.
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pone-0110299-g006: Cellular localization of Prss56 and Glul in B6 (C57BL/6J) and Mfrprd6/Mfrprd6 mice.(A) By in situ hybridization, in B6 (C57BL/6J) controls at P14, we observed Prss56 transcript in only very few cells of the inner nuclear layer (INL) of the retina (top panel), whereas in Mfrprd6/Mfrprd6 mutants, an intense staining of Prss56 transcript was observed in INL of the retina (bottom panel). (B) By 2-plex in situ hybridization, in B6 controls, we observed co-localization of Prss56 (red) and Glul (pseudo colored green) transcripts in only a few cell body of the (INL) of the retina (top panel), whereas in Mfrprd6/Mfrprd6, strong co-localization of Prss56 and Glul transcripts in the cell body of the INL of the retina was observed (bottom panel). (C) Glutamine synthetase (GS) staining of Müller cells. In both C57BL/6J (B6) and Mfrprd6/Mfrprd6 mice, Müller cells marked with glutamine synthetase showed a similar localization pattern (inset, top and bottom panels) as observed for Prss56 in situ hybridization staining, suggesting that Müller cells in the INL of retina express Prss56 transcripts at P14.
Mentions: In the absence of an antibody that could reliably detect murine PRSS56, in situ hybridization was used to determine the cellular localization of the Prss56 transcript. In the no probe control, no Prss56 transcript was observed (data not shown). In the wild-type control at P14, confocal microscopy revealed a few cells in the retinal inner nuclear layer (INL) that showed expression of Prss56 transcript (Fig. 6A, upper panel). By contrast, in Mfrprd6 eyes at P14, intense specific staining of the transcript was observed in the INL (Fig. 6A, lower panel). This increased staining further validates both the microarray and qRT-PCR results of increased Prss56 transcripts in Mfrprd6 mutant eyes relative to controls.

Bottom Line: In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2).Levels of RPE65 were significantly decreased by 2.0-fold.In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, Maine, United States of America.

ABSTRACT
Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes are part of a regulatory network influencing postnatal posterior eye development.

Show MeSH
Related in: MedlinePlus