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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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qRT-PCR analysis of RPE and retinal- specific genes in homozygous Mfrprd6, Tulp1tvrm124 and Rpe65tvrm148 mutants.(A) In homozygous Mfrprd6 mutant mice, the transcripts in the visual cycle (Rpe65, Lrat and Rgr), phototransduction pathway (Rgs9, GuCa1b, Pde6a) and structural components of rods and cones (Fscn2 and RpGrip1) were significantly decreased relative to the wild-type control (B6/J), validating the microarray results. (B) qRT-PCR analysis in Tulp1tvrm124/Tulp1tvrm124 mutants at P14 revealed no significant change in any of the transcripts tested. (C) In Rpe65tvrm148/Rpe65tvrm148 mutants, there was only a significant increase in RpGrip1 from transcripts tested, relative to wild-type (B6/J) controls. The data are expressed as relative fold change (RFC) after normalizing to the wild-type control. RFC was calculated using ΔΔCT method after internal calibration to β-Actin control. Each value represents RFC ± S.E.M. * P<0.05 and ** P<0.001 relative to controls. N = 3–6 per group.
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pone-0110299-g003: qRT-PCR analysis of RPE and retinal- specific genes in homozygous Mfrprd6, Tulp1tvrm124 and Rpe65tvrm148 mutants.(A) In homozygous Mfrprd6 mutant mice, the transcripts in the visual cycle (Rpe65, Lrat and Rgr), phototransduction pathway (Rgs9, GuCa1b, Pde6a) and structural components of rods and cones (Fscn2 and RpGrip1) were significantly decreased relative to the wild-type control (B6/J), validating the microarray results. (B) qRT-PCR analysis in Tulp1tvrm124/Tulp1tvrm124 mutants at P14 revealed no significant change in any of the transcripts tested. (C) In Rpe65tvrm148/Rpe65tvrm148 mutants, there was only a significant increase in RpGrip1 from transcripts tested, relative to wild-type (B6/J) controls. The data are expressed as relative fold change (RFC) after normalizing to the wild-type control. RFC was calculated using ΔΔCT method after internal calibration to β-Actin control. Each value represents RFC ± S.E.M. * P<0.05 and ** P<0.001 relative to controls. N = 3–6 per group.

Mentions: To validate the microarray data, qRT-PCR analysis was performed on whole eyes from Mfrprd6 mutants and C57BL/6J mice. Transcripts that were significantly increased in the microarray analysis are listed in (Table 2). Transcripts that were significantly decreased in the microarray analysis (Table 3), including those encoding components of the visual cycle (Rpe65, Lrat, Rgr), phototransduction pathway (Rgs9, Pde6a, Guca1b), and involved in disc morphogenesis (Fscn2, Rpgrip1), were also reduced as determined by qRT-PCR (Fig. 3A). However, some of the changes in transcripts including Prph2 (−4.1795, q value  = 0.06), Optc (−2.395, q<0.05), Aqp5 (−2.197, q<0.05) (Table 3) were not validated by qRT-PCR. The failure to validate these transcripts was unlikely to be caused by assay limitations. Amplification primers were targeted to exon-exon junctions to amplify only processed RNA (Table 1); a PCR product of the correct size was verified; the PCR efficiency of the primers for genes of interest and calibrator was the same; CT values in control and Mfrprd6 mutant samples, indicated robust target amplification in both; and melting curve analysis confirmed the presence of a single amplified product. However, the lack of verification may be due the difference in samples used for the microarray and the qRT-PCR analyses, to the low abundance of some transcripts, or in some cases the differences were not significant (e.g. Prph2, which is highly expressed in the retina, had a FDR of 0.0605).


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)

qRT-PCR analysis of RPE and retinal- specific genes in homozygous Mfrprd6, Tulp1tvrm124 and Rpe65tvrm148 mutants.(A) In homozygous Mfrprd6 mutant mice, the transcripts in the visual cycle (Rpe65, Lrat and Rgr), phototransduction pathway (Rgs9, GuCa1b, Pde6a) and structural components of rods and cones (Fscn2 and RpGrip1) were significantly decreased relative to the wild-type control (B6/J), validating the microarray results. (B) qRT-PCR analysis in Tulp1tvrm124/Tulp1tvrm124 mutants at P14 revealed no significant change in any of the transcripts tested. (C) In Rpe65tvrm148/Rpe65tvrm148 mutants, there was only a significant increase in RpGrip1 from transcripts tested, relative to wild-type (B6/J) controls. The data are expressed as relative fold change (RFC) after normalizing to the wild-type control. RFC was calculated using ΔΔCT method after internal calibration to β-Actin control. Each value represents RFC ± S.E.M. * P<0.05 and ** P<0.001 relative to controls. N = 3–6 per group.
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pone-0110299-g003: qRT-PCR analysis of RPE and retinal- specific genes in homozygous Mfrprd6, Tulp1tvrm124 and Rpe65tvrm148 mutants.(A) In homozygous Mfrprd6 mutant mice, the transcripts in the visual cycle (Rpe65, Lrat and Rgr), phototransduction pathway (Rgs9, GuCa1b, Pde6a) and structural components of rods and cones (Fscn2 and RpGrip1) were significantly decreased relative to the wild-type control (B6/J), validating the microarray results. (B) qRT-PCR analysis in Tulp1tvrm124/Tulp1tvrm124 mutants at P14 revealed no significant change in any of the transcripts tested. (C) In Rpe65tvrm148/Rpe65tvrm148 mutants, there was only a significant increase in RpGrip1 from transcripts tested, relative to wild-type (B6/J) controls. The data are expressed as relative fold change (RFC) after normalizing to the wild-type control. RFC was calculated using ΔΔCT method after internal calibration to β-Actin control. Each value represents RFC ± S.E.M. * P<0.05 and ** P<0.001 relative to controls. N = 3–6 per group.
Mentions: To validate the microarray data, qRT-PCR analysis was performed on whole eyes from Mfrprd6 mutants and C57BL/6J mice. Transcripts that were significantly increased in the microarray analysis are listed in (Table 2). Transcripts that were significantly decreased in the microarray analysis (Table 3), including those encoding components of the visual cycle (Rpe65, Lrat, Rgr), phototransduction pathway (Rgs9, Pde6a, Guca1b), and involved in disc morphogenesis (Fscn2, Rpgrip1), were also reduced as determined by qRT-PCR (Fig. 3A). However, some of the changes in transcripts including Prph2 (−4.1795, q value  = 0.06), Optc (−2.395, q<0.05), Aqp5 (−2.197, q<0.05) (Table 3) were not validated by qRT-PCR. The failure to validate these transcripts was unlikely to be caused by assay limitations. Amplification primers were targeted to exon-exon junctions to amplify only processed RNA (Table 1); a PCR product of the correct size was verified; the PCR efficiency of the primers for genes of interest and calibrator was the same; CT values in control and Mfrprd6 mutant samples, indicated robust target amplification in both; and melting curve analysis confirmed the presence of a single amplified product. However, the lack of verification may be due the difference in samples used for the microarray and the qRT-PCR analyses, to the low abundance of some transcripts, or in some cases the differences were not significant (e.g. Prph2, which is highly expressed in the retina, had a FDR of 0.0605).

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