Work Description

Title: Dataset for "Myelin regulatory factor (Myrf) is a critical early regulator of retinal pigment epithelial development." Open Access Deposited

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Methodology
  • RNAscope in situ hybridization Heads were harvested from embryonic day 14.5 (e14.5) embryos, dissected eyes were harvested from postnatal day 0 (P0) – P21 pups, and optic nerves were harvested from P21 pups. For larger eyes (after P0), a hole was punctured through the cornea to allow penetration of the fixative. Samples were fixed in 4% buffered paraformaldehyde (0.1M NaPO4 pH7.3) for 0.5-4 hours at 22°C, dehydrated through increasing concentrations of ethanol up to 70%. The samples were then embedded in paraffin using the TissueTek VIP Model VIP5A-B1 (Sakura Finetek USA, Inc.) and the Shandon Histocentre 2 Model #64000012 embedding station (Thermo Fisher Scientific) and sectioned at 5-6m. For in situ hybridization, RNAscope was performed using the RNAscope Multiplex Fluorescent Detect V2 system (Advanced Cell Diagnostics [ACD], #323110). Briefly, paraffin was removed with two changes of Xylene and then washed in 100% ethanol (ETOH). Sections were treated with the hydrogen peroxide reagent for 10 minutes followed by two washes in distilled water. Target retrieval was performed using boiling 1 X Target Retrieval Reagent for 7 minutes, followed by washing in distilled water and 100% ETOH. After drying the slides, the sections were treated with Protease Plus in prewarmed humidity chamber for 25 minutes at 40°C then washed in distilled water. Prewarmed RNAscope probes were then applied, and slides were incubated in a humidity chamber at 40°C for 2 hours. The probes used were Mm-Myrf (ACD, 524061), Mm-Upk3b (ACD, 568561), Mm-Wfikkn2 (ACD, 531321), Mm-Id3 (ACD, 445881), Mm-Bmp2-E3 (ACD, 427341), and a negative control probe (ACD, 320871). After hybridization of the probe, sections were washed in 1 X Wash Buffer then incubated at 40°C with Amp1 reagent for 30 minutes, Amp2 reagent for 30 minutes, and Amp3 reagent for 15 minutes, with washes in 1 X Wash Buffer between each step. For signal development, the sections were then incubated in HRP-C1 for 15 minutes at 40°C, washed in 1 X Wash Buffer and then incubated with Cyanine 3, Opal-Tm 570 (Akoya Biosciences, FP1488001KT) fluorophore diluted 1:1500 in TSA plus buffer (ACD, 322809) for 30 minutes at 40°C. Sections were then washed in 1 X Wash Buffer then treated with HRP Blocker for 15 minutes at 40°C, stained with DAPI (Sigma, MBD0015) and mounted in ProLong Gold Antifade (Invitrogen, P36930).

  • Immunohistochemistry and OTX2/TUNEL staining Antibody staining was performed using established methods [28], with the antibody conditions can be found in Supplemental Table 5. All antibodies were incubated at 4°C overnight. Nuclei for all samples were stained with DAPI (Sigma, MBD0015) and mounted in ProLong Gold Antifade Mountant (Invitrogen, P36930). RPE flatmounts were done as described previously [28]. ERMN immunostaining was quantitated using ImageJ. The integrated density of ERMN signal was normalized to the integrated density of EZRN signal. The fold change was calculated between Rx>cre Myrffl/fl mutants and Myrffl/fl controls. The unpaired T Test was used to calculate the p value. Co-staining of the rabbit anti-OTX2 (Abcam, ab21990) and apoptotic cells using In Situ Cell Death Detection Kit, TMR red (Roche, 12156792910) was performed on e14.5 paraffin sections. OTX2 immunohistochemistry was performed as described above. Apoptotic cells were identified using the protocol from the In Situ Cell Death Detection Kit, TMR red. Sections pretreated with DNaseI were used as a positive control and sections without the Enzyme Solution were used as a negative control. Nuclei were stained with DAPI (Sigma, MBD0015) and mounted in ProLong Gold Antifade Mountant (Invitrogen, P36930). OTX2 positive, TUNEL positive, and DAPI positive cells were counted using Fiji, ImageJ software. A minimum of two slides were counted per sample. For the OTX2 total counts, the data is displayed as the total number of the OTX2 stained cells per total number of DAPI cells in the region counted. For the RPE apoptosis counts, the data is displayed as the total number of TUNEL positive cells per the total number of RPE cells, marked by OTX2. Each point on the graph represents an individual sample.
Description
  • MYRF is a gene that regulates the development and function of the retinal pigmented epithelium (RPE), which play an important role in maintaining photoreceptor structure and function. Mutations in patients have been implicated in eye size disorders, particularly causing a small, but structurally normal eye. We have utilized a molecular technique, single cell RNA sequencing, to investigate how loss of Myrf specifically in the RPE in a mouse model impacts downstream gene expression at three developmental timepoints and used this information to define the role of Myrf in development. Our work identified key cytoskeletal structural genes specific to the RPE, Ermn and Upk3b, and a gene important for the cell survival, Sox10, as critical targets of Myrf. In addition, we have identified and confirmed that the TGFbeta signaling pathway is dysregulated when Myrf is lost during development. This pathway is particularly relevant in RPE health and eye growth. Our electron microscopy and histologic analyses also confirm a defect in RPE structure and function. We place MYRF within a hierarchy of genes involved in RPE development and introduce novel candidate genes for further study as retinal degeneration and nanophthalmos candidate genes.
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Depositor
Contact information
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Funding agency
  • Other Funding Agency
Other Funding agency
  • Glaucoma Research Foundation

  • Bright Focus Foundation

  • Knights Templar Eye Foundation

  • National Eye Institute

  • Research to Prevent Blindness

  • E. Matilda Ziegler Foundation for the Blind
Keyword
Resource type
Last modified
  • 04/01/2025
Published
  • 04/01/2025
DOI
  • https://doi.org/10.7302/vna2-n507
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To Cite this Work:
Brinkmeier, M. L., Wang, S. Q., Pittman, H., Cheung, L. Y., Prasov, L. (2025). Dataset for "Myelin regulatory factor (Myrf) is a critical early regulator of retinal pigment epithelial development." [Data set], University of Michigan - Deep Blue Data. https://doi.org/10.7302/vna2-n507

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Files (Count: 11; Size: 8.97 GB)

Thumbnailthumbnail-column Title Original Upload Last Modified File Size Access Actions
Brinkmeieretal2025DeepBlue.txt 2025-03-31 2025-03-31 8.26 KB Open Access
Count_Wfikkn2.tar 2025-03-24 2025-03-24 599 MB Open Access
CountingTgfb.tar 2025-03-24 2025-03-24 829 MB Open Access
Counting_Bmp2.tar 2025-03-24 2025-03-24 1.65 GB Open Access
Counting_Id3.tar 2025-03-24 2025-03-24 22.8 MB Open Access
Ermn_Ezr_Counts.tar 2025-03-24 2025-03-24 60.5 MB Open Access
OTX2_Counts.tar 2025-03-24 2025-03-24 391 MB Open Access
Sox10Counts.tar 2025-03-24 2025-03-24 1.89 GB Open Access
TyrcreMyrf_Counts.tar 2025-03-24 2025-03-24 2.09 GB Open Access
Upk3b_Counts.tar 2025-03-24 2025-03-24 147 MB Open Access
pSMAD_Counts.tar 2025-03-24 2025-03-24 1.35 GB Open Access

Date: 24 March, 2025

Dataset Title: Myelin regulatory factor (Myrf) is a critical early regulator of retinal pigment epithelial development.

Dataset Contact: Lev Prasov [email protected]

Dataset Creators:
Name: Michelle L. Brinkmeier
Email: [email protected]
Institution: Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105
https://orcid.org/0000-0001-9505-4682

Name: Su Qing Wang
Email: [email protected]
Institution: Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105
ORCID:

Name: Hannah Pittman
Email: [email protected]
Institution: Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109
ORCID: https://orcid.org/0009-0004-4170-1898

Name: Leonard Y Cheung
Email:[email protected]
Institution: Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794
ORCID: https://orcid.org/0000-0002-0912-9594

Name: Lev Prasov
Email: [email protected]
Institution: Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105
ORCID: https://orcid.org/0000-0002-6635-1116

Funding: This work was supported with grants from the National Eye Institute (NEI, https://www.nei.nih.gov/, K08-EY032098, K12EY022299 to LP, ), the Bright Focus Foundation (https://www.brightfocus.org/, M2022011N to LP), the Research to Prevent Blindness Career Development Award (https://www.rpbusa.org/, to LP), the Knights Templar Eye Foundation Career Starter Award and Competitive Renewal (https://www.ktef.org/, to LP), the E. Matilda Ziegler Foundation for the Blind (http://emzfoundation.com/, to LP), and the Glaucoma Research Foundation (https://glaucoma.org/, to LP). The NEI P30 core grant (EY002687) helped support histology, imaging, and molecular biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Key Points:
- We use single cell RNA sequencing to compare transcripts expressed in the mouse eye cup across three developmental time points in an retinal pigmented epithelial (RPE) cell specific deletion of Myrf and controls
- We identified and confirmed alterations in genes involved in the ultrastructure of the RPE, in proliferation, and in TGFb signaling.

Research Overview:
MYRF is a gene that regulates the development and function of the retinal pigmented epithelium (RPE), which play an important role in maintaining photoreceptor structure and function. Mutations in patients have been implicated in eye size disorders, particularly causing a small, but structurally normal eye. We have utilized a molecular technique, single cell RNA sequencing, to investigate how loss of Myrf specifically in the RPE in a mouse model impacts downstream gene expression at three developmental timepoints and used this information to define the role of Myrf in development. Our work identified key cytoskeletal structural genes specific to the RPE, Ermn and Upk3b, and a gene important for the cell survival, Sox10, as critical targets of Myrf. In addition, we have identified and confirmed that the TGFbeta signaling pathway is dysregulated when Myrf is lost during development. This pathway is particularly relevant in RPE health and eye growth. Our electron microscopy and histologic analyses also confirm a defect in RPE structure and function. We place MYRF within a hierarchy of genes involved in RPE development and introduce novel candidate genes for further study as retinal degeneration and nanophthalmos candidate genes.

Methodology:
Transcripts for Upk3b, Wfikkn2, Id3, Bmp2, and Myrf were identified by RNAscope in situ hybridization.
Protein localization for ERMN, EZR, SOX10, TGFB2, pSMAD, and TMEM98 were identified by immunohistochemistry using fluorescent conjugated secondary antibodies.
Apoptotic cells in the RPE were detected using OTX2 immunostaining followed by TUNEL cell death staining.

Files contained here:
Images used for OTX2/TUNEL counting:
The labels on these files indicate the date of the experiment (06.15, eg), the age (e14), the genotype/age (Myrfflfl, eg), the sample number (10546-2, eg), and experiment (OTX2TUNEL, eg), the slide number, and the magnification. Merge means that the red, green, and DAPI channels were imaged and merged together.

Images from ERMN/EZR immunostaining:
The labels for these images give the mouse ear tag number (3419, 3420, 3603, 3608, 3420, or 3443), the slide number (S#), the age (P21), the genotype, and the antibodies.Merge means that the red, green, and DAPI channels were imaged and merged together.

Images from Upk3b RNAscope:
The important information in the labels for these images include the postnatal day 0 mouse number (P0-1, P0-16, P0-2, P0-3, P0-4, P0-5, and P0-6), the genotype, and the probe (Upk3b). Some images have RM which indicates the RxcreMyrf strain. Merge means that the red, green, and DAPI channels were imaged and merged together.

Images from Sox10 immunostaining:
The image labels from the SOX10 immunostaining experiment contain the age (e13, e15, or P3) the animal number (9818-3 or P3-2, eg), and the magnification (20x or 40x). Merge means that the red, green, and DAPI channels were imaged and merged together. Some of the images are labeled MelanASOX10, these were costained with the two antibodies, MelanA in red and SOX10 in green.

Images from Wfikkn2 RNAscope:
The important information in the image labels from the Wfikkn2 RNAscope experiment are the postnatal day 0 mouse number (P0-14, P0-16, P0-1, P0-2, P0-4, P06, P0-3, and P0-5), the genotype, and the magnification. Some images have RM which indicates the RxcreMyrf strain. Merge means that the red, green, and DAPI channels were imaged and merged together.

Images from Tgfb2 immunostaining:
Images from the TGFB2 immunostaining are labeled with the sample number (3744-1 or P0-8, eg), the genotype, and the magnification (20x or 40x). Merge means that the red, green, and DAPI channels were imaged and merged together.

Images Id3 RNAscope:
Images from the Id3 RNAscope are labeled with the sample number (3774-1 or P0-8, eg), the genotype, and the magnification. Some images have RM which indicates the RxcreMyrf strain. Merge means that the red, green, and DAPI channels were imaged and merged together.

Images pSMAD immunostaining:
Images from the pSMAD immunostaining are labeled with the sample number (3744-1 or P0-1, eg), the age (P0), the genotype, and the magnification (20x or 40x). Some images have RM which indicates the RxcreMyrf strain. Merge means that the red, green, and DAPI channels were imaged and merged together.

Images TyrcreMyrf Myrf RNAscope:
Images from the TyrcreMyrf Myrf RNAscope are labeled with TM to denote the TyrcreMyrf strain, the sample number (6279-2, eg), the age (e18),the genotype, MyrfRNAscope for the probe, and the magnification (20x or 40x). Merge means that the red, green, and DAPI channels were imaged and merged together.

Images TyrcreMyrf TMEM98 immunostaining:
Images from the TyrcreMyrf TMEM98 immunostaining are labeled with TM to denote the TyrcreMyrf strain, the date (31425, eg), the age (e18), the genotype, the sample number (7042-5, eg), TMEM98 for the antibody, and the magnification (20x or 40x). Merge means that the red, green, and DAPI channels were imaged and merged together.

Images Bmp2 RNAscope:
Images from the Bmp2 RNAscope experiment are labeled with RM for the Rxcre Myrf strain, the age (P0), the sample number (3744- or P0-12, eg), the genotype, the magnification, and if multiple eyes from one sample were used, they will appear as Eye 1 and Eye 2. Merge means that the red, green, and DAPI channels were imaged and merged together.

Related publication(s):
Brinkmeier et al. (2025), Myelin regulatory factor (Myrf) is a critical early regulator of retinal pigment epithelial development. Forthcoming.

Use and Access:
This data set is made available under a Attribution-NonCommercial 4.0 International (CC BY-NC 4.0).

To Cite Data:
Brinkmeier, M.L., Wang, S.Q., Pittman, H., Cheung, L.Y., Prasov, L. (2025). Myelin regulatory factor (Myrf) is a critical early regulator of retinal pigment epithelial development. [Data set]. University of Michigan - Deep Blue. https://deepblue.lib.umich.edu/data/concern/data_sets/tm70mw254/

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