Hyung-song Nam, PhD thesis, 2009

https://doi.org/10.6084/m9.figshare.21920040

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posted on 2025-10-10, 19:56 authored by Hyung-song NamHyung-song Nam, Robert Benezra

As of year 2020, seemed to me like there was still some interest on Id genes. So, after more than ten years, I went through my 2009 PhD thesis work on Id1 again and put up some of the data here. Here are (1) minimally processed versions of images and figures presented in my PhD thesis and/or <a href="https://doi.org/10.1016/j.stem.2009.08.017" rel="noreferrer" target="_blank">Nam and Benezra, 2009</a> paper, (2) previously unpublished images and data, and (3) re-analyzed sequence data, restriction digest maps, and Southerns.• ID1 during developmentImmunofluorescence on neuronally differentiated mouse ES cell cultures: Green - ID1; Red - TUBB3.Immunofluorescence on a coronal section of wildtype mouse embryo spinal cord: Green - ID1; Red - NESTIN; Blue - DAPI.• ID1 in adultsMy first successful analysis of ID1 and ID3 expression in the adult brain. DAB immunohistochemistry.Epifluorescence images of immunofluorescence on coronal sections of a >6 week old wildtype mouse brain: Green - ID1; Red - KI-67 or MCM2; Blue - DAPI. Large files. Download then view. For macOS, I recommend a program <a href="https://github.com/jurplel/qView" target="_blank">qView</a>.Confocal images of immunofluorescence on coronal sections of a >6 week old wildtype mouse brain. (1) Green - ID1; Red - MCM2; Blue - DAPI. (2) Yellow - ID1; Magenta - ASCL1; Cyan - DAPI. (3) Yellow - ID1; Magenta - OLIG2; Cyan - DAPI. (4) Green - ID1; Red - EdU; Blue - DAPI. Ara-C infused for 6 days. Perfused 48 h after cessation of the infusion. 30 min before the perfusion, EdU was injected. The small green dots are probably non-specific protein precipitates in the block buffer. (5) Yellow - ID1; Magenta - GFAP. (6) Green - ID1; Red - S100-B; Blue - DAPI.• Evidence of in vivo neurogenesis from the Id1-expressing cellsGenetic inducible fate mapping set up.X-gal histochemistry on coronal sections. Striatal region. Uninduced Id1IRES-creERT2/+; Rosa26LSL-lacZ/+ mouse. Induced Id1IRES-creERT2/+; Rosa26StLa/+ mouse 3 days and 1 month after. Brightfield images. Large files.YFP+ cells from lateral ventricular wall whole mount of Id1IRES-creERT2/+; Rosa26LSL-YFP/+ mice scored to be "B1" cells from <a href="https://doi.org/10.1016/j.stem.2008.07.004" target="_blank">Mirzadeh et al., 2008</a>.GFP+ cells from whole mount of Id1IRES-creERT2/+; Tg(GFAP-LSL-GFP)/+ mouse lateral ventricular wall. Perhaps because of the GFP protein expression level from the GFAP promoter, the morphologies of the GFP+ cells were not revealed as well as with the Rosa26 reporters.Additional images.• ID1 in another neurogenic regionX-gal histochemistry on coronal sections. Hippocampus region. Induced Id1IRES-creERT2/+; Rosa26StLa/+ mouse 1 month after. Brightfield images. Large files. Note non-endothelial X-gal+ cells in the SGZ.X-gal stained sections above zoomed in.Confocal images of immunofluorescence on coronal sections. ID1 and various markers.Confocal image of immunofluorescence on coronal section. Induced Id1IRES-creERT2/+; Rosa26StLa/+ mouse brain 1 month after. Hippocampus region. Green - Tau-β-gal; Red - NeuN; Blue - DAPI.• Flow cytometric analysis of Id1-Venus mouseThe gates utilized to flow cytometrically detect the ID1-Venus+ cells from the V-SVZ.Tried wider gates for the ID1-Venus and GFAP detection. Could see more cells in the double-positive gate, but the larger cells were more autofluorescent.The first successful FACS sort of the ID1-Venus+ cells.• Additional experiments with the Id1Venus, Id1floxed, and other alleles• Supplementary notes on Id1 mice productionThe <a href="http://www.informatics.jax.org/allele/MGI:4366910" target="_blank">Id1-floxed allele mouse</a>, the <a href="http://www.informatics.jax.org/allele/MGI:4366905" target="_blank">Id1-Venus allele mouse</a>, the <a href="http://www.informatics.jax.org/allele/MGI:4366863" target="_blank">Id1-IRES-creERT2 allele mouse</a>, and the <a href="http://www.informatics.jax.org/allele/MGI:4366911" target="_blank">Rosa26-StLa allele mouse</a>.Dr. Jonathan Perk had previously generated and characterized the Southern blot probe for Id1 targeting before I joined the lab.I designed the targeting vectors for the alleles above and generated two variants of the Id1 targeting homology arms using recombineering. The first had a long 5' arm and a 3' DTA cassette. The homology arm lengths were from <a href="https://doi.org/10.1128/mcb.17.12.7317" target="_blank">Yan et al., 1997</a>. For the second, I shortened the 5' arm and added a TK cassette there in addition to the 3' DTA cassette. The genomic DNA was retrieved from a clone of the RPCI-23 C57BL/6J genomic DNA library into pKO2.1, pKO2.2, or pDTA-TK backbone plasmids (that I created using pKO II, PL253, and pGalK). Because these backbones had a high copy number origin of replication, the plasmid DNA of the final constructs with the large genomic DNA insert were initially very difficult to prepare in large scale. The E. coli harboring them grew very slowly, and the plasmids were prone to random rearrangements by recombination. Therefore, bacterial culture and antibiotic selection conditions were optimized. Resultant plasmid preps were screened for good clones, and recombined plasmid preps were discarded. The targeting vectors thus prepared for electroporations were verified by restriction enzyme digests and Sanger sequencing. In addition to the Sanger sequencing traces posted here, also see Addgene NGS sequences of (1) <a href="https://www.addgene.org/20966/" rel="noreferrer" target="_blank">ID1-Venus</a>, (2) <a href="https://www.addgene.org/188230/" rel="noreferrer" target="_blank">IRES-creERT2-FNF cassette</a>, and (3) <a href="https://www.addgene.org/182363/" rel="noreferrer" target="_blank">StLa reporter backbone (i.e., minus tauLacZ and Rosa26 homology arms) subsequently cloned miR-124 target sites</a>.The core facility at The Rockefeller University performed C57BL/6 mouse ES cell electroporations of the targeting vectors and initial Southern blots on the ES cell colonies (except for the Id1-IRES-creERT2 project). The core facility at the Sloan Kettering Institute performed blastocyst injections, chimera breeding, and Southern blots on genomic DNA from germline mice at the end of the process.An example of Id1Venus allele mouse breeding experiment. The idea is that because knock-out of both Id1 and Id3 is embryonic lethal, if mice that only have the Id1Venus allele (either as Id1V/V or Id1V/-) on Id3-/- genetic background are born (i.e., not embryonic lethal), then that can be interpreted as the Id1V allele can substitute for the Id1+ allele. In other words, if the Id1V allele were non-functional, then the Id1V/V; Id3-/- or Id1V/-; Id3-/- embryos would have died in utero because at least one copy of functional Id1 or Id3 is necessary for embryonic viability. But that was not observed, and Id1V/V; Id3-/- mice were born. So, the ID1-Venus fusion protein expressed from the Id1V allele is inferred to be functional. By the way, I noted <a href="http://www.informatics.jax.org/allele/MGI:4366905" target="_blank">here</a> initially that I had generated Id1V/-; Id3-/- mice. Unfortunately, that recollection from memory was incorrect. Review of the records indicated I had generated Id1V/V; Id3-/- mice instead as explained above. The note was corrected.✼In summary, in the lateral ventricular walls of mice older than 6 weeks, there were neural ID1-Venus+ cells as well as endothelial ID1-Venus+ cells. Culturing the lateral wall cells in neurosphere-forming media with FGF2 and EGF was a quick way to enrich for the neural lineage cells without FACS. In the cultures of the lateral wall neural lineage cells thus obtained, there were ID1-Venus+ and ID1-Venus- cells that could be discerned. The ID1-Venus-high cells FACS sorted from the cultures formed self-renewing neurospheres and so on as described in the paper.As an aside, the Venus yellow fluorescent protein was very bright, and it enabled a read-out of the ID1 protein that is present at a low level. These are why I thought the ID1 protein expression level is low. First, the ID1 protein was initially very difficult to detect in the brain tissue. Basically, with conventional indirect immunofluorescence, there was no detectable signal in the neural lineage cells and the endothelial cells. I could only visualize the ID1 protein with Tyramide Signal Amplification (TSA). Second, the labeling efficiency with the Id1IRES-creERT2 allele was also low. Although other interpretations are also possible, these suggested to me low protein expression level. If so, then there may indeed not be so many copies of the ID1 protein in wildtype mice cells and ID1-Venus protein in knock-in mice cells. Yet, the ID1-Venus was detectable albeit somewhat dim due to the expression level.Following that line of reasoning, if the title and discussion of the Nam and Benezra, 2009 paper were confusing, I note here that even though the absolute levels of the ID1 protein may be low, if one considers the relative levels along neurogenic lineage progression, it is high in the stem/progenitor cells. More specifically, the Id1 mRNA level seems to be highest in the activated stem cells, but it is almost as high in the quiescent stem cells (see <a href="https://figshare.com/ndownloader/files/47985901" rel="noreferrer" target="_blank">here</a>, clicking this link will download a file). As for the protein levels, I don't know of a good study that convincingly demonstrated a clear difference in the quiescent versus activated stem cells. There were studies done with BMP4- and FGF2+BMP4-treated cultured cells (I've done that too, see <a href="https://figshare.com/ndownloader/files/52291715" rel="noreferrer" target="_blank">here</a>, clicking this link will download a file), but are those cells the same as in vivo quiescent stem cells? I'm not sure. Nowadays, I suppose one can do flow cytometry of the Id1-Venus mouse cells stained with all the other markers that have been defined. Intracellular flow cytometry with the ID1 antibody could work too.By the way, some of the steps in the ID1 TSA immunostaining protocol in our paper (<a href="https://www.cell.com/cms/10.1016/j.stem.2009.08.017/attachment/ad03871a-80aa-432c-a6a2-ee0a73e3d1c9/mmc1.pdf" rel="noreferrer" target="_blank">link</a>) came from the MSKCC Molecular Cytology Core Facility (brain freezing, biotin-SAv-HRP amplification, etc) and some came from the Varmus lab via Dr. Rocio Sotillo (antigen retrieval in a steamer, etc). I think I stumbled upon the Tyramide-Alexa 488 reagent in an Invitrogen catalog.Moving on.