Shtn1, a retinal ganglion cell (RGC) specific gene, regulates RGC neurite development: Insights from an RGC direct somatic cell reprogramming model
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1072052 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27833564_1.fastq.gz | Fastqsanger.gz | |||
| SRR27833564_2.fastq.gz | Fastqsanger.gz | |||
| SRR27833565_1.fastq.gz | Fastqsanger.gz | |||
| SRR27833565_2.fastq.gz | Fastqsanger.gz | |||
| SRR27833566_1.fastq.gz | Fastqsanger.gz |
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Project description:Retinal ganglion cells (RGCs) are the projection neurons of the retina. Proper development of the axon and dendrite of RGCs is the basis for these cells to function as projection neurons to deliver visual information to the brain. The purpose of this study was to investigate the function of a newly identified RGC-specific gene, Shtn1, in neurite development in RGCs. Using IF, we demonstrated that SHTN1 is distinctively expressed in RGCs during the period in which RGCs actively develop and adjust the connections of their neurites with upstream and downstream neurons. Using the iRGC system, we demonstrated that Shtn1 promotes the growth and complexity of neurites, and thus the electrophysiological maturation, of iRGCs. RNA-Seq analyses showed that Shtn1 may also regulate gene expression and neurogenesis in iRGCs. These findings improve our understanding of the molecular machinery governing RGC neurite development, and may help to optimize future RGC regeneration methods.
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Project description:Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse and Xenopus, coupled with unbiased genome-wide microarray profiling. Localized mRNA from the isolated growth cones of Xenopus laevis and Mus musculus retinal ganglion cells were subjected to microarray analysis
2010-11-23 | E-GEOD-25166 | biostudies-arrayexpress
Project description:Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse and Xenopus, coupled with unbiased genome-wide microarray profiling.
2010-11-23 | GSE25166 | GEO
Project description:The goal of this experiment was to identify markers and potential regulators of J-RGC development.
2018-04-09 | GSE102888 | GEO
Project description:The goal of this experiment was to investigate the molecular mechanism of how Set-beta regulates neurite growth. Set-beta’s subcellular localization is regulated by posttranslational modifications. We found that Set-beta suppresses neurite growth of purified postnatal rat retinal ganglion cell (RGC) primary neurons when it is overexpressed in the nucleus, whereas recruiting Set-beta to cellular membrane by fusing myr-tag to its N-terminus promotes neurite growth. Here, we transfected purified by immunopanning postnatal rat RGC with wild-type Set-beta which localizes to the nucleus, myr-Set-beta which is recruited to cellular membranes, and mCherry control, and analyzed with microarrays Set-beta’s subcellular localization-dependent effects on gene expression. We found that wild-type Set-β regulated expression of significantly more genes than myr-Set-β, consistent with wild-type Set-β’s nuclear localization and previously described roles in regulating transcription. These data reveal potential downstream gene effectors regulating neurite growth, and specific candidate genes could be validated and tested in future experiments.
2014-04-30 | GSE56951 | GEO
Project description:Genetic signatures of retinal ganglion cells revealed through single cell profiling [dissociated RGC]
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Project description:Subcellular expression profiling of the growth cones of retinal ganglion cells (RGC)
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Project description:The goal of this experiment was to investigate the molecular mechanism of how Set-beta regulates neurite growth. Set-betaM-bM-^@M-^Ys subcellular localization is regulated by posttranslational modifications. We found that Set-beta suppresses neurite growth of purified postnatal rat retinal ganglion cell (RGC) primary neurons when it is overexpressed in the nucleus, whereas recruiting Set-beta to cellular membrane by fusing myr-tag to its N-terminus promotes neurite growth. Here, we transfected purified by immunopanning postnatal rat RGC with wild-type Set-beta which localizes to the nucleus, myr-Set-beta which is recruited to cellular membranes, and mCherry control, and analyzed with microarrays Set-betaM-bM-^@M-^Ys subcellular localization-dependent effects on gene expression. We found that wild-type Set-M-NM-2 regulated expression of significantly more genes than myr-Set-M-NM-2, consistent with wild-type Set-M-NM-2M-bM-^@M-^Ys nuclear localization and previously described roles in regulating transcription. These data reveal potential downstream gene effectors regulating neurite growth, and specific candidate genes could be validated and tested in future experiments. Acutely purified by immunopanning postnatal day 4 (P4) retinal ganglion cells (RGCs) transfected with mCherry, wild-type Set-M-NM-2, and myr-Set-M-NM-2, and co-transfected with pMAX-GFP (Lonza) constructs, were plated at high density on uncoated glass Lab-Tek II chamber slides (Thermo Fisher Scientific). The following morning, after gentle trituration, GFP-positive cells were FACS-isolated at 37M-BM-0C in PBS with BD FACSAria I cell sorter at 20 psi using a 130 M-BM-5m nozzle; GFP-negative threshold was set by first processing untransfected RGCs. Cells were gated on a FSC-A versus SSC-A plot to exclude dead cells and/or debris, and aggregates were removed using single cell gating with FSC-H versus FSC-W and SSC-H versus SSC-W plots. Immediately after isolation, GFP-positive cells were diluted in pre-equilibrated growth medium, centrifuged 15 minutes at 80 g, resuspended in growth medium in a 48-well tissue culture plastic plate as above, and cultured 2 more days in growth medium, as above. The Quick-RNA MicroPrep kit (R1050, Zymo Research) was used for RNA extraction 3 days after transfection, according to the manufacturer's instructions. Total RNA were used as input for the whole transcriptome amplification Ovation Pico WTA System V2 kit (3302-12, NuGEN), the cDNA product was quantified with Nanodrop 8000 Spectrophotometer (Thermo Scientific) and its quality was examined with a Bioanalyzer 2100 using the Nano 6000 kit (Agilent). 5 M-BM-5g of cDNA product from each sample was used as input for fragmentation and labeling using the Encore Biotin Module kit (4200-12, NuGEN); the fragmentation product quality was tested as above. The labeled product was hybridized to whole genome GeneChip Rat Gene 2.0 ST Arrays (Affymetrix). The staining, washing and scanning of the arrays was carried out using a Fluidics 450 station, GeneChip Operating Software and GeneChip Scanner 3000 7G (Affymetrix). Image intensities were analyzed with Expression Console (Affymetrix) from CEL files for quality control using standard GeneChip Rat Gene 2.0 ST Array control probes (Affymetrix), and to determine the RMA expression values and for annotations. RMA expression values were normalized to the median for each sample.
2014-04-30 | E-GEOD-56951 | biostudies-arrayexpress
Project description:To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested at 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (p<0.05, FDR<1%) were quantified by SWATH-MS. Bioinformatics data analysis showed that there were 35 up-regulated and 24 down-regulated proteins in the temporal quadrant while 19 and 5 proteins were up-regulated and down-regulated, respectively, in the nasal quadrant respectively (n=4, p<0.05; fold change ≥1.4 fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants including CLU, GFAP, GNG5, IRF2BPL, L1CAM and CPLX1. Linear regression analysis indicated a strong association between the data obtained by SWATH-MS and MRM-MS (temporal, R2=0.97; nasal, R2=0.96). Gene ontology analysis reveals statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.
2021-09-07 | PXD026783 | Pride