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"url": "https://figshare.com/articles/figure/Id1_Id2_Id3_and_Id4_in_adult_mouse_brain_lateral_wall_V-SVZ_stem_cells/12457982/72",
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"name": "Nam, Hyung-song",
"givenName": "Hyung-song",
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"name": "Benezra, Robert",
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{
"name": "Capecchi, Mario",
"givenName": "Mario",
"familyName": "Capecchi"
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"titles": [
{
"title": "Id1, Id2, Id3, and Id4 in adult mouse brain lateral wall V-SVZ neural stem cells"
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"subject": "60410 Neurogenetics",
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"subject": "Neuroscience"
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{
"subject": "Cell Biology"
},
{
"subject": "Developmental Biology"
}
],
"contributors": [],
"dates": [
{
"date": "2021-01-16",
"dateType": "Created"
},
{
"date": "2022-02-02",
"dateType": "Updated"
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"date": "2021",
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{
"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 result.
Selected marker genes RNA in clusters.
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.
==============
Some data from my PhD work on Id1 in the V-SVZ published at https://doi.org/10.1016/j.stem.2009.08.017. The work with the Id1 mice led to the discovery of Lrig1 in the V-SVZ.
----------------------
Production of the Id1-floxed allele mouse (http://www.informatics.jax.org/allele/MGI:4366910), the Id1-Venus allele mouse (http://www.informatics.jax.org/allele/MGI:4366905), the Id1-IRES-creERT2 allele mouse (http://www.informatics.jax.org/allele/MGI:4366863), and the ROSA26-StLa allele mouse (http://www.informatics.jax.org/allele/MGI:4366911).
There were two variants of the Id1 targeting vector. The first variant utilized a long 5' arm and a DTA cassette. The second variant utilized a shorter 5' arm and DTA and TK cassettes. The lengths of the homology arms in the first variant were derived from Yan et al., 1997 (https://doi.org/10.1128/mcb.17.12.7317). The actual genomic DNA was subcloned from a clone of RPCI-23 C57BL/6J mouse genome BAC library using recombineering.
I would put up the Sanger sequencing files of the targeting vectors up here if I could, but I'm not sure if I have the intellectual property rights to do that. Regardless, I have re-analyzed the Sanger sequencing results of the Id1 targeting vectors, and did not find any errors. (The material I did put up here after going through them again after more than ten years are (1) copied and pasted from my PhD thesis, (2) raw data from my PhD thesis work (Southern blots, PCR gels, restriction digests, and flow data), and (3) re-analysis of the data using modern software (flow data and sequence analyses for the knock-in allele targeting vectors and Southern blots).)
By the way, these targeting vectors were constructed on a high copy backbone. With the large genomic insert, the plasmid DNA of the final construct was very difficult to prepare because the E. coli harboring it grew very slowly, and the plasmid was prone to rearrangements by recombination. This sometimes happens - for example, see https://www.addgene.org/61580/ for comments from the Hongkui Zeng laboratory. To make it work, plasmid prep from every culture of the clone had to be checked for recombination by restriction digests before using. Preps of recombined plasmids had to be discarded. The low copy backbone plasmids are much easier to work with because they don't behave this way. For anybody still doing gene targeting, I would recommend them instead (see https://doi.org/10.6084/m9.figshare.12847649.v7 for links to Addgene).
----------------------
Flow cytometry data. The figure pdf 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 actual 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.
As an aside, the Id1-Venus fusion protein reporter actually generated a very sensitive read-out of the Id1 protein. These are why I think so. 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. Then, there may not be so many copies of the Id1-Venus protein in the cell. Yet, the Id1-Venus protein was detectable with flow cytometers.
----------------------
If the Discussion of the paper wasn't clear, 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.
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A confocal image from a coronal section of wildtype embryonic (~E12.5) spinal cord. Id1 (green), Nestin (red), and DAPI (blue).
----------------------
Two coronal section images from hippocampus of Id1IRES-creERT2/+; ROSA26StLa/+ mouse. 1 month after tamoxifen inductions. Non-endothelial X-gal+ cells in the dentate gyrus.
A confocal image from coronal section of hippocampus of Id1IRES-creERT2/+; ROSA26StLa/+ mouse. 1 month after tamoxifen inductions.Tau-b-gal (green), NeuN (red), and DAPI (blue).
----------------------
A confocal z-stack from coronal section of a wildtype mouse brain V-SVZ, >6 weeks old. A maximum projection image montage and a movie of the z-stack. Id1 (green). Mcm2 (red). DAPI (blue).
----------------------
A confocal z-stack from coronal section of a wildtype mouse brain V-SVZ, >6 weeks old. 48 hours after the cessation of Ara-C infusion (for 6 days, as described in Nam and Benezra, 2009). EdU was injected 30 minutes before perfusion. A maximum projection image montage and a movie of the z-stack. Id1 (green). EdU (red). DAPI (blue).
----------------------
Two confocal images of YFP+ cells from ventricular wall whole mount of 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).
==============
Previously, the whole mount immunfluorescence technique wasn't as optimized as it is now, so the images weren't so clear. However, putting the previous work in context with my more recent work, what is clear is that the cells labeled by the Id1IRES-creERT2 allele were different from the cells labeled by the Lrig1T2A-iCreERT2 allele (see https://doi.org/10.6084/m9.figshare.12731900.v6). 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, more recent results 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.
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