{
"@context": "http://schema.org",
"@type": "ImageObject",
"@id": "https://doi.org/10.6084/m9.figshare.12457982.v16",
"url": "https://figshare.com/articles/figure/Id1_Id2_Id3_and_Id4_in_adult_mouse_brain_lateral_wall_V-SVZ_stem_cells/12457982/16",
"additionalType": "Figure",
"name": "Id1, Id2, Id3, and Id4 in adult mouse brain lateral wall V-SVZ neural stem cells",
"author": [
{
"name": "Hyung-Song Nam",
"givenName": "Hyung-Song",
"familyName": "Nam",
"@type": "Person",
"@id": "https://orcid.org/0000-0002-0429-5446"
},
{
"name": "Mario Capecchi",
"givenName": "Mario",
"familyName": "Capecchi",
"@type": "Person"
}
],
"description": "The R script in Nam and Capecchi, 2020 used to analyze the scRNA-seq dataset in Mizrak et al., 2019 (https://doi.org/10.1016/j.celrep.2018.12.044) was updated to Seurat 3. Results are similar to Seurat 2.
Clustering results of selected genes RNA, as well as tabulated results of AverageExpression command for selected genes RNA.
To validate the AverageExpression results, qRT-PCR measurements of several genes RNA in Morizur et al., 2018 (https://doi.org/10.1016/j.stemcr.2018.06.005) were utilized. These selected genes RNA levels were compared against the AverageExpression results. The trends were very similar, suggesting that normalized scRNA-seq datasets can also reveal the average RNA expression level of cell populations otherwise obtained by FACS sorting and qRT-PCR.
==============
A graph of the RNA levels of Id1, Id2, Id3, and Id4. The relative RNA abundance in the lateral wall V-SVZ qNSC's seems to be Id2 > Id3 > Id4 > Id1. How the RNA levels correlate to protein levels is unclear, although the Id1 protein in particular was measured directly by C-terminal fusion of the Venus yellow fluorescent protein in a knock-in mouse (https://doi.org/10.1016/j.stem.2009.08.017, https://doi.org/10.1016/j.ccr.2011.11.025, https://doi.org/10.1242/jcs.096198, https://doi.org/10.1016/j.stemcr.2014.09.012, https://doi.org/10.7554/elife.01197, https://doi.org/10.1016/j.devcel.2019.05.032).
A graph of the RNA levels of the Id genes and other marker genes in ependymal cells and the neurogenic lineage cells.
==============
Several marker genes RNA expression in clusters.
==============
Some flow cytometry data files from my PhD thesis work on Id1 in the V-SVZ, published at https://doi.org/10.1016/j.stem.2009.08.017. Some experiments with the Id1-VenusYFP mice (http://www.informatics.jax.org/allele/MGI:4366905). The figure pdf file shows the gates that were utilized to detect Id1-Venus+ cells from dissociated V-SVZ cells as well as Id1-Venus+ cells in cultured V-SVZ cells. The zip files contain the FCS data files. The file names were modified to be descriptive.
As described in the paper many years ago, there were neural Id1-Venus+ cells as well as endothelial Id1-Venus+ cells in the V-SVZ (mice >6 weeks of age). Culturing the V-SVZ cells in neurosphere-forming media with EGF and FGF-2 was a quick way to enrich for the neural lineage cells without FACS sorting, etc. In the cultures of neural lineage cells of the V-SVZ thus obtained, there were Id1-Venus+ and Id1-Venus- cells that could be discerned by flow cytometry. The Id1-Venus-high cells formed self-renewing neurospheres, etc as described in the paper.
The Id1-Venus fusion protein actually generated a very sensitive read-out of the endogenous Id1 protein. Why I think so is as follows. The Id1 protein was very difficult to detect in the brain tissue (i.e., no signal with conventional indirect immunofluorescence in both cell types). I could only visualize it after Tyramide Signal Amplification. The labeling efficiency with the Id1IRES-creERT2 allele was also somewhat low. Although other reasons are also possible, these suggested low protein expression level. Thus, there may not be so many copies of the Id1 protein in the cell. Yet, the Id1-Venus protein was detectable with flow cytometers. To reiterate, even though there aren't many copies of the Id1 protein, it could be visualized probably because of the VenusYFP's brightness.
It is noted here that even though the absolute level of the Id1 protein may be low, its level is highest in the stem/progenitor cells if one considers the relative levels along the neurogenic lineage.
==============
Two confocal images of YFP+ cells from Id1IRES-creERT2/+; ROSA26LSL-YFP/+ mice that were scored to be \"B1\" cells from Mirzadeh et al., 2008 (https://doi.org/10.1016/j.stem.2008.07.004). The whole mount immunfluorescence technique wasn't as optimized as it is now, so the images aren't so clear. However, what is clear is that these cells are different from the cells labeled by the Lrig1T2A-iCreERT2 allele. So how is it possible to get Lrig1 out of Id1high cells when they label cells with different morphologies? Although it wasn't apparent with the previous technique and the Id1IRES-creERT2 mouse, more recent results with another Id1 allele suggest that Id1 also labels the cells labeled by Lrig1 (unpublished preliminary observation). So, a working model is that there are at least two different types of stem cells: Id1 reveals both, whereas Lrig1 reveals just one subset. Additional work is being done.
",
"license": "https://creativecommons.org/licenses/by/4.0/legalcode",
"keywords": "60410 Neurogenetics, FOS: Biological sciences, FOS: Biological sciences, Neuroscience, Cell Biology",
"contentSize": "26098434 Bytes",
"dateCreated": "2020-10-18",
"datePublished": "2020",
"dateModified": "2020-12-08",
"predecessor_of": {
"@id": "https://doi.org/10.6084/m9.figshare.12457982",
"@type": "CreativeWork"
},
"schemaVersion": "http://datacite.org/schema/kernel-4",
"publisher": {
"@type": "Organization",
"name": "figshare"
},
"provider": {
"@type": "Organization",
"name": "datacite"
}
}