Flow cytometric analysis of LRIG1 protein in the lateral ventricular wall cells

Expanded presentation of the flow cytometry data included in the Nam and Capecchi, 2020 paper.

Lrig1 expression pattern from GENSAT and EUCOMM.

The Lrig1^T2A-tdTomato allele targeting vector was deposited for sharing at Addgene. The NGS sequence is available at https://www.addgene.org/134320/sequences/. NCBI BLAST.

Notes on PCR genotyping. Lrig1^T2A-tdTomato mouse line genotyping results.

Brains from Lrig1^+/+, Lrig1^{T2A-tdTomato/+}, and Lrig1^{T2A-tdTomato/T2A-tdTomato} mice. Note the tdTomato fluorescence in the cerebellum.

The Lrig1^T2A-iCreERT2 allele targeting vector was deposited for sharing at Addgene. The NGS sequence is available at https://www.addgene.org/126651/sequences/. NCBI BLAST.

A control immunostaining was done with a polyclonal goat anti-mouse LRIG1 antibody from R & D Systems (https://www.rndsystems.com/products/mouse-lrig1-antibody_af3688) on cerebellum sagittal section from a 1c2 Lrig1^{T2A-iCreERT2/+} mouse. The anti-LRIG1 signal in the cerebellum was consistent with the known expression pattern (see above). Furthermore, the anti-LRIG1 immunoreactivity signal seemed to co-localize with the anti-cre antibody immunoreactivity signal.

RFP-labeled cells in the cerebellum of the Lrig1^{T2A-iCreERT2/+}; Rosa26^Ai14/+ mouse (a magnified view of an image here).

Lateral ventricular wall slices from brains of adult mice (>12 weeks of age) were disociated to single cells. The cells were stained with the polyclonal goat anti-LRIG1 antibody. The anti-LRIG1 immunoreactivity signal was sensitive to the amount of papain used for dissociation. Thus, the amount of the papain was titrated down to where the tissue was still dissociated while retaining the anti-LRIG1 immunoreactivity. The antibody dilutions were also titrated. The primary antibody was visualized with biotinylated donkey anti-goat secondary and streptavidin Alexa 647. Although the signal was still faint, the biotin-streptavidin staining amplified it over the signal from the Alexa 488-conjugated anti-LRIG1 antibody. In "unstained" samples, the primary antibody staining was omitted.

The LRIG1-T2A-tdTomato-high cells expressed the highest levels of the LRIG1 protein, whereas the LRIG1-T2A-tdTomato-intermediate cells expressed lower levels of the LRIG1 protein. The LRIG1 protein level on the RFP-labeled cells in 2f1 Lrig1^{T2A-iCreERT2/+}; ROSA26^Ai14/+ mice was similar to the LRIG1 protein level on the LRIG1-T2A-tdTomato-intermediate cells.

At this point, I'm not 100% sure where the neurogenic stem cells are in the flow data (also see here). I would need to do some more experiments.

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As one can see above, using fluorescence microscopy, the RFP signal from the Lrig1^T2A-tdTomato allele was visible to the eyes from the cerebellum, but not from the ventricular wall (not shown). In other words, when I dissected out the brains from the Lrig1^{T2A-tdTomato/+} mice and looked at them with a fluorescence stereomicroscope, I saw red fluorescence signal in the cerebellum and the olfactory bulbs, but not in the ventricular wall. That would be consistent with a lower Lrig1 expression level in the ventricular wall, and thus a lower level of the tdTomato protein and fluorescence from it. Furthermore, the LRIG1 protein was undetectable in the ventricular wall using the aforementioned antibody in indirect immunofluorescence presumably also due to the lower expression level there. The sfGFP-iCre-ERT2 protein could not be detected as well. In other words, the same LRIG1 protein was expressed at different levels in different regions of the mouse brain. Thus, a more sensitive method, flow cytometry, was utilized to detect the LRIG1 protein in the ventricular wall cells.

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

The 2f1 Lrig1^T2A-iCreERT2 mice (069516-UCD) will be available from MMRRC.

We refer interested investigators to Jax or MMRRC to obtain the mouse lines.