Limits...
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.

Show MeSH

Related in: MedlinePlus

Independent qRT-PCR validation of genes that were differentially expressed in Mfrprd6/Mfrprd6 mutants by array analysis.The upregulated gene Prss56 was evaluated at three different time points. (A) At P7, there was a 3.5-fold increase in Prss56 transcript and it increased to 14-fold by P14, followed by a further 70-fold increase in Prss56 transcript in Mfrprd6/Mfrprd6 mutants at P21. (B) At P14, when compared to the Mfrprd6/Mfrprd6 mutant, in Tulp1tvrm124/Tulp1tvrm124 mutants, there was no significant change in Prss56 transcript, whereas in Rpe65tvrm148/Rpe65tvrm148 mutants, there was a significant decrease. (C) Wildtype levels of Prss56 transcript revealed a significant decrease between P7 and P21 timpoints, whereas the decrease from P7 to P14 was not statistically significant. Data are expressed as relative fold change (RFC) in the Prss56 transcript after normalizing to the wild-type control (B6). RFC was calculated by the ΔΔCT method using β-actin as an internal calibrator. Each value represents RFC ± S.E.M. * P<0.05 and ** P<0.001 relative to controls. N = 3 per group.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214712&req=5

pone-0110299-g005: Independent qRT-PCR validation of genes that were differentially expressed in Mfrprd6/Mfrprd6 mutants by array analysis.The upregulated gene Prss56 was evaluated at three different time points. (A) At P7, there was a 3.5-fold increase in Prss56 transcript and it increased to 14-fold by P14, followed by a further 70-fold increase in Prss56 transcript in Mfrprd6/Mfrprd6 mutants at P21. (B) At P14, when compared to the Mfrprd6/Mfrprd6 mutant, in Tulp1tvrm124/Tulp1tvrm124 mutants, there was no significant change in Prss56 transcript, whereas in Rpe65tvrm148/Rpe65tvrm148 mutants, there was a significant decrease. (C) Wildtype levels of Prss56 transcript revealed a significant decrease between P7 and P21 timpoints, whereas the decrease from P7 to P14 was not statistically significant. Data are expressed as relative fold change (RFC) in the Prss56 transcript after normalizing to the wild-type control (B6). RFC was calculated by the ΔΔCT method using β-actin as an internal calibrator. Each value represents RFC ± S.E.M. * P<0.05 and ** P<0.001 relative to controls. N = 3 per group.

Mentions: PRSS56/Prss56 variants in human and mouse are associated with defects in ocular growth [17]–[19], [25], a process that is also affected by human MFRP mutations [1]–[6]. Therefore, we focused on characterizing Prss56 expression in greater detail. Microarray data indicated no difference in Prss56 transcript accumulation between Mfrprd6 mice and controls at P0, suggesting that the increase in Prss56 expression occurs during postnatal development of the Mfrprd6 eye. To assess the temporal variation of Prss56 expression in the postnatal period, qRT-PCR was performed at three different time points (Fig. 5A). At P7, there was a 3.5-fold increase in Prss56 transcript, which increased to 14-fold at P14 and 70-fold at P21 (Fig. 5A). Thus, the Mfrprd6 mutation causes a progressive accumulation of Prss56 transcript throughout postnatal development.


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)

Independent qRT-PCR validation of genes that were differentially expressed in Mfrprd6/Mfrprd6 mutants by array analysis.The upregulated gene Prss56 was evaluated at three different time points. (A) At P7, there was a 3.5-fold increase in Prss56 transcript and it increased to 14-fold by P14, followed by a further 70-fold increase in Prss56 transcript in Mfrprd6/Mfrprd6 mutants at P21. (B) At P14, when compared to the Mfrprd6/Mfrprd6 mutant, in Tulp1tvrm124/Tulp1tvrm124 mutants, there was no significant change in Prss56 transcript, whereas in Rpe65tvrm148/Rpe65tvrm148 mutants, there was a significant decrease. (C) Wildtype levels of Prss56 transcript revealed a significant decrease between P7 and P21 timpoints, whereas the decrease from P7 to P14 was not statistically significant. Data are expressed as relative fold change (RFC) in the Prss56 transcript after normalizing to the wild-type control (B6). RFC was calculated by the ΔΔCT method using β-actin as an internal calibrator. Each value represents RFC ± S.E.M. * P<0.05 and ** P<0.001 relative to controls. N = 3 per group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110299-g005: Independent qRT-PCR validation of genes that were differentially expressed in Mfrprd6/Mfrprd6 mutants by array analysis.The upregulated gene Prss56 was evaluated at three different time points. (A) At P7, there was a 3.5-fold increase in Prss56 transcript and it increased to 14-fold by P14, followed by a further 70-fold increase in Prss56 transcript in Mfrprd6/Mfrprd6 mutants at P21. (B) At P14, when compared to the Mfrprd6/Mfrprd6 mutant, in Tulp1tvrm124/Tulp1tvrm124 mutants, there was no significant change in Prss56 transcript, whereas in Rpe65tvrm148/Rpe65tvrm148 mutants, there was a significant decrease. (C) Wildtype levels of Prss56 transcript revealed a significant decrease between P7 and P21 timpoints, whereas the decrease from P7 to P14 was not statistically significant. Data are expressed as relative fold change (RFC) in the Prss56 transcript after normalizing to the wild-type control (B6). RFC was calculated by the ΔΔCT method using β-actin as an internal calibrator. Each value represents RFC ± S.E.M. * P<0.05 and ** P<0.001 relative to controls. N = 3 per group.
Mentions: PRSS56/Prss56 variants in human and mouse are associated with defects in ocular growth [17]–[19], [25], a process that is also affected by human MFRP mutations [1]–[6]. Therefore, we focused on characterizing Prss56 expression in greater detail. Microarray data indicated no difference in Prss56 transcript accumulation between Mfrprd6 mice and controls at P0, suggesting that the increase in Prss56 expression occurs during postnatal development of the Mfrprd6 eye. To assess the temporal variation of Prss56 expression in the postnatal period, qRT-PCR was performed at three different time points (Fig. 5A). At P7, there was a 3.5-fold increase in Prss56 transcript, which increased to 14-fold at P14 and 70-fold at P21 (Fig. 5A). Thus, the Mfrprd6 mutation causes a progressive accumulation of Prss56 transcript throughout postnatal development.

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