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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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Ingenuity pathway analysis identified Visual Cycle and Phototransduction pathways to be downregulated genes in homozygous Mfrprd6 mutant mice.(A) Genes in the visual cycle (RPE) and phototransduction pathway (rod photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (Rpe65, Lrat and Rgr), phototransduction pathway genes (Rgs9, Guca1b, Pde6a) and genes encoding structural components of the rod-cells (Rpgrip1 and Fscn2) that were validated by qRT-PCR. (B) Genes in the visual cycle (RPE) and phototransduction pathway (cone photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (Rpe65, Lrat and Rgr), Müller glia cell expressed gene (Rgr), phototransduction pathway genes (Rgs9 and Guca1b), and genes encoding structural components of the cone cells (RpGrip1 and Fscn2) that were validated by qRT-PCR. The molecules associated with the symbols are as depicted in the inset. The solid and dashed lines represent direct or indirect interactions, respectively, between the genes. The arrow indicates interaction between genes. A =  Activation, B = Binding, E = Expression (includes metabolism/synthesis for chemicals), I (Inhibition), PP (Protein-Protein binding), P (Phosphorylation/Dephosphorylation), RB (Regulation of binding), MB (Group/complex Membership).
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pone-0110299-g002: Ingenuity pathway analysis identified Visual Cycle and Phototransduction pathways to be downregulated genes in homozygous Mfrprd6 mutant mice.(A) Genes in the visual cycle (RPE) and phototransduction pathway (rod photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (Rpe65, Lrat and Rgr), phototransduction pathway genes (Rgs9, Guca1b, Pde6a) and genes encoding structural components of the rod-cells (Rpgrip1 and Fscn2) that were validated by qRT-PCR. (B) Genes in the visual cycle (RPE) and phototransduction pathway (cone photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (Rpe65, Lrat and Rgr), Müller glia cell expressed gene (Rgr), phototransduction pathway genes (Rgs9 and Guca1b), and genes encoding structural components of the cone cells (RpGrip1 and Fscn2) that were validated by qRT-PCR. The molecules associated with the symbols are as depicted in the inset. The solid and dashed lines represent direct or indirect interactions, respectively, between the genes. The arrow indicates interaction between genes. A =  Activation, B = Binding, E = Expression (includes metabolism/synthesis for chemicals), I (Inhibition), PP (Protein-Protein binding), P (Phosphorylation/Dephosphorylation), RB (Regulation of binding), MB (Group/complex Membership).

Mentions: To determine the molecular networks and biological pathways affected in Mfrprd6 mutant eye at the P14 time point, we examined the microarray data by Ingenuity Pathway Analysis (IPA). The top five canonical IPA pathways that were altered significantly in Mfrprd6 mutant eyes were B cell development, allograft rejection signaling, autoimmune thyroid disease signaling, phototransduction, cytotoxic T lymphocyte-mediated apoptosis of target cells, and visual cycle. The genes identified in these pathways and results of statistical tests are given in Table S1. Of particular interest are the phototransduction and visual cycle genes that were perturbed in Mfrprd6 mutant eyes. The Mfrprd6 allele, which is a loss of function mutation leading to the absence of MFRP protein from the RPE [8], is likely to have a direct effect on RPE cell function/maintenance. In accordance with this effect, visual cycle gene transcripts expressed in the RPE, including Rpe65 and Lrat, were decreased significantly in Mfrprd6 mice (Fig. 2, A, B). Transcripts of Rgr, which encode a visual cycle protein found in both RPE and Müller cells, were also significantly decreased (Fig. 2B). The Mfrprd6 mutation also affected retina-specific transcript levels, as evidenced by a significant relative fold change (RFC) of −1.2 to −2.0 in transcripts expressed in photoreceptor cells (Fig. 2, A, B). These included transcripts from genes specifically expressed in rod cells (Rho, Gnb1 and Gnb5; Fig. 2A), cone cells (Opn1sw, Gnat2, Gnb3 and Gnb5; Fig. 2B), or in both rods and cones (Rgs9, Rgs9bp, Prkaca, Pde6a, Pde6b, Guca1a and Guca1b; Fig. 2, A, B). Transcripts encoded by genes implicated in maintaining photoreceptor OS morphology, Fscn2 and Rpgrip1, were also significantly decreased in the Mfrprd6 mutant (Fig. 2, A, B), consistent with the early OS disorganization that is observed in this mutant.


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)

Ingenuity pathway analysis identified Visual Cycle and Phototransduction pathways to be downregulated genes in homozygous Mfrprd6 mutant mice.(A) Genes in the visual cycle (RPE) and phototransduction pathway (rod photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (Rpe65, Lrat and Rgr), phototransduction pathway genes (Rgs9, Guca1b, Pde6a) and genes encoding structural components of the rod-cells (Rpgrip1 and Fscn2) that were validated by qRT-PCR. (B) Genes in the visual cycle (RPE) and phototransduction pathway (cone photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (Rpe65, Lrat and Rgr), Müller glia cell expressed gene (Rgr), phototransduction pathway genes (Rgs9 and Guca1b), and genes encoding structural components of the cone cells (RpGrip1 and Fscn2) that were validated by qRT-PCR. The molecules associated with the symbols are as depicted in the inset. The solid and dashed lines represent direct or indirect interactions, respectively, between the genes. The arrow indicates interaction between genes. A =  Activation, B = Binding, E = Expression (includes metabolism/synthesis for chemicals), I (Inhibition), PP (Protein-Protein binding), P (Phosphorylation/Dephosphorylation), RB (Regulation of binding), MB (Group/complex Membership).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214712&req=5

pone-0110299-g002: Ingenuity pathway analysis identified Visual Cycle and Phototransduction pathways to be downregulated genes in homozygous Mfrprd6 mutant mice.(A) Genes in the visual cycle (RPE) and phototransduction pathway (rod photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (Rpe65, Lrat and Rgr), phototransduction pathway genes (Rgs9, Guca1b, Pde6a) and genes encoding structural components of the rod-cells (Rpgrip1 and Fscn2) that were validated by qRT-PCR. (B) Genes in the visual cycle (RPE) and phototransduction pathway (cone photoreceptors) are represented in this panel. The asterisk represents the visual cycle genes (Rpe65, Lrat and Rgr), Müller glia cell expressed gene (Rgr), phototransduction pathway genes (Rgs9 and Guca1b), and genes encoding structural components of the cone cells (RpGrip1 and Fscn2) that were validated by qRT-PCR. The molecules associated with the symbols are as depicted in the inset. The solid and dashed lines represent direct or indirect interactions, respectively, between the genes. The arrow indicates interaction between genes. A =  Activation, B = Binding, E = Expression (includes metabolism/synthesis for chemicals), I (Inhibition), PP (Protein-Protein binding), P (Phosphorylation/Dephosphorylation), RB (Regulation of binding), MB (Group/complex Membership).
Mentions: To determine the molecular networks and biological pathways affected in Mfrprd6 mutant eye at the P14 time point, we examined the microarray data by Ingenuity Pathway Analysis (IPA). The top five canonical IPA pathways that were altered significantly in Mfrprd6 mutant eyes were B cell development, allograft rejection signaling, autoimmune thyroid disease signaling, phototransduction, cytotoxic T lymphocyte-mediated apoptosis of target cells, and visual cycle. The genes identified in these pathways and results of statistical tests are given in Table S1. Of particular interest are the phototransduction and visual cycle genes that were perturbed in Mfrprd6 mutant eyes. The Mfrprd6 allele, which is a loss of function mutation leading to the absence of MFRP protein from the RPE [8], is likely to have a direct effect on RPE cell function/maintenance. In accordance with this effect, visual cycle gene transcripts expressed in the RPE, including Rpe65 and Lrat, were decreased significantly in Mfrprd6 mice (Fig. 2, A, B). Transcripts of Rgr, which encode a visual cycle protein found in both RPE and Müller cells, were also significantly decreased (Fig. 2B). The Mfrprd6 mutation also affected retina-specific transcript levels, as evidenced by a significant relative fold change (RFC) of −1.2 to −2.0 in transcripts expressed in photoreceptor cells (Fig. 2, A, B). These included transcripts from genes specifically expressed in rod cells (Rho, Gnb1 and Gnb5; Fig. 2A), cone cells (Opn1sw, Gnat2, Gnb3 and Gnb5; Fig. 2B), or in both rods and cones (Rgs9, Rgs9bp, Prkaca, Pde6a, Pde6b, Guca1a and Guca1b; Fig. 2, A, B). Transcripts encoded by genes implicated in maintaining photoreceptor OS morphology, Fscn2 and Rpgrip1, were also significantly decreased in the Mfrprd6 mutant (Fig. 2, A, B), consistent with the early OS disorganization that is observed in this mutant.

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