LRIG1 and CDK6 expression in the lateral ventricular wall cells

Some preliminary experiments done with the Lrig1^T2A-tdTomato allele and Cdk6^T2A-td-sfGFP allele mice in the year 2014.

But first, some previous data from my PhD work to help contextualize these results.

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Lrig1 and Cdk6 expression data from single cell RNA sequencing clusters (in vivo expression pattern).

Additional background information on Cdk6 expression in the brain.

Lrig1^T2A-tdTomato allele targeting vector map. The NGS sequence is available at https://www.addgene.org/134320/sequences/. One can verify that the homology arms map to Lrig1 using NCBI BLAST.

Notes on genotyping. Genotyping results.

The tdTomato fluorescence in brains from Lrig1^+/+, Lrig1^{T2A-tdTomato/+}, and Lrig1^{T2A-tdTomato/T2A-tdTomato} mice.

Adherent cultures of unfractionated lateral wall cells from an adult Lrig1^{T2A-tdTomato/+} mouse. The adherent culture technique is from Pollard et al., 2006. See below for the media I used. Early passage cells. Unambiguous RFP signal in the proliferating cells visible to the eyes. Why does an in vivo marker of quiescence label proliferating cells in vitro? More below.

Cdk6^T2A-td-sfGFP allele targeting vector map. The NGS sequence is available at https://www.addgene.org/134321/sequences/. One can verify that the homology arms map to Cdk6 using NCBI BLAST.

Cdk6^T2A-td-sfGFP mouse characterization. Additional images. Flow cytometry files.

By the way, the images from the Lrig1^T2A-tdTomato mice and Cdk6^T2A-td-sfGFP mice above show T2A clearly "works" because the ribosome-skipped tdTomato and td-sfGFP filled the entire cell as expected. The same T2A sequence above was also utilized in the Lrig1^T2A-iCreERT2 allele. The Lrig1^T2A-iCreERT2 allele vector NGS sequence is available at https://www.addgene.org/126651/sequences/. NCBI BLAST.

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The lateral ventricular walls were dissected from brains of single or double heterozygous adult Lrig1 and Cdk6 reporter mice. The dissected lateral walls were dissociated to single cells with papain, then analyzed with a custom FACSCanto flow cytometer as described in Nam and Capecchi, 2020. This showed clearly tdTomato+ and/or td-sfGFP+ cells from the reporter alleles (in vivo expression pattern). Much more fluorescence signal than the dimmer signal of the Id1^VenusYFP fusion protein allele (link).

The tdTomato and/or td-sfGFP signals from the Lrig1 and Cdk6 reporter alleles allowed for FACS sorting. The FACS sorted cells indeed showed tdTomato and/or td-sfGFP signals that could be visualized (images of tdTomato+ cells not shown).

One can appreciate the different populations with different LRIG1 and CDK6 expression levels. For example, there were prominent populations that were positive for LRIG1-T2A-tdTomato and negative for CDK6-T2A-td-sfGFP. Would one of these be the in vivo LRIG1+ neurogenic stem cells? At this point, I'm not 100% sure where the neurogenic stem cells are in the flow data. I would need to do some more experiments.

Regardless, when cultures were initiated from FACS sorted LRIG1-T2A-tdTomato-low/neg CDK6-T2A-td-sfGFP-high/int cells, they proliferated and formed large neurospheres in single cell or bulk cultures. Interestingly, the LRIG1-T2A-tdTomato signal in the cells was initially low/negative then increased during culture such that all cells became LRIG1-T2A-tdTomato-positive, similar to the image shown above of unfractionated cells adherent cultures.

Flow cytometric measurements of LRIG1-T2A-tdTomato and CDK6-T2A-td-sfGFP in adherent cultures from fractionated or unfractionated lateral wall cells of double heterozygous adult mouse brains (in vitro expression pattern). Early passage cells.

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So, how about the finding that the Lrig1^T2A-iCreERT2 mouse line doesn't initially label many proliferating cells in vivo after tamoxifen inductions? I'm not sure what that means with regards to Lrig1 expression in cultured NSPC that are proliferating (our observations described above, Jeong et al., 2020, and Marqués-Torrejón et al., 2021). Is this one of those things induced by tissue culture that changes the cells from their in vivo identity or something? Again, if Lrig1 is induced during tissue culture and highly expressed in proliferating cells in vitro when proliferating cells in vivo do not express high levels of Lrig1, what does that mean? I'm not sure. But data from multiple independent laboratories are concordant in that knocking it out in cultured cells or in vivo seems to have similar effects.

If I could speculate, I guess the FACS sorted cells were adapting to the culture media and so on, perhaps even changing in culture. The visible changes seemed to occur relatively fast, i.e., the LRIG1-T2A-tdTomato signal was up-regulated and the CDK6-T2A-td-sfGFP was down-regulated within days of culture. Are those all the changes that occur, i.e., the short-term gene expression changes? Or, are there long-term gene expression changes as well? Furthermore, do other things also change over a longer time scale in culture? Animal experiments are a lot more work and a lot more expensive to do, but, to me, seems like that's the best that can be done right now.

Finally, the FACS sorted ID1-Venus-high cells from adherent cultures of unfractionated adult Id1^V/V mouse brain lateral wall cells showed higher Lrig1 mRNA levels than the ID1-Venus-low cells. But doesn't the image above show all adherent NSPC's are LRIG1-T2A-tdTomato-positive? What is going on? (1) The Lrig1 levels in the cultured ID1-Venus-low cells might not be zero. (2) The Lrig1 levels in the cultured ID1-Venus-high cells might still be higher than in the cultured ID1-Venus-low cells. (3) Finally, because the tdTomato isn't fused to LRIG1 (unlike the ID1-Venus fusion protein, for example), and tdTomato has its own decay time, I suppose there is also a possibility that the tdTomato can perdure in the cultured ID1-Venus-low cells. For an example of perdurance, look at the images of the Cdk6^{T2A-td-sfGFP/+} mouse immunofluorescence here. Although the Cdk6 mRNA decreases significantly in the neuroblasts, the DCX+ neuroblasts still show a lot of td-sfGFP immunoreactivity.

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To generate these cultures, dissociated primary cells were first cultured in Ultra Low Binding plates (Corning). The resultant neurospheres were dissociated, and the cells were plated on regular plates (different sources) coated with laminin and poly-ornithine (Sigma).

Media:

DMEM/F-12 + GlutaMAX (GIbco)

20 mM HEPES (Gibco)

1x B27 minus Vitamin A (Gibco)

20 ng/ml mouse FGF2 (R & D, Peprotech, Gold Bio)

20 ng/ml mouse EGF (same sources)

2 µg/ml heparin (Sigma, tissue culture grade)

1x Primocin (InvivoGen, especially good for fungus)

Others often supplement with N2, insulin-transferrin-selenium, chick embryo extract, etc.

Incubated at 37 deg C and 7.5% CO2.

At the outset of my postdoctoral training work, I again tried as the base medium Neurobasal-A which I used during my PhD work and also Neurobasal. For whatever strange reason, the cells did not grow in those media. Instead, the cells grew in media with DMEM/F-12 as the base medium, so I just stuck with it without over-thinking it. At the time, I didn't try to figure out the differences in the media or the incubation condition because I thought it might take too much time to troubleshoot. Having compared my notes in January 2023, it could be because of the glutamine vs. GlutaMAX supplementation. During my PhD work, the Neurobasal-A medium was supplemented with both glutamine and GlutaMAX. Whereas, during my postdoctoral work, the Neurobasal-A medium was supplemented with GlutaMAX only.

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The Lrig1^T2A-tdTomato mice will be available from Jax (# 036457).

The Cdk6^T2A-td-sfGFP mice will be available from Jax (# 036458).

We refer interested investigators to Jax to obtain these mouse lines.