LRIG1 expression in the mouse cerebellum

Images of GENSAT Tg(Lrig1-EGFP) BAC transgenic mouse brain and EUCOMM Lrig1^lacZ/+ knock-in mouse brain show that Lrig1 is highly expressed in the cerebellum (in an absolute rather than a relative sense by the way). Also see Figure 2 panels T and T' in Nam and Capecchi, 2020.

Map of Lrig1^T2A-tdTomato allele targeting vector. NGS sequence of the targeting vector (link). NCBI BLAST to verify that the targeting vector homology arms map to Lrig1 (link). Lrig1^T2A-tdTomato mice genotyping from intercross of heterozygotes at generation N19 of backcross to C57BL/6J. Brains from Lrig1^+/+, Lrig1^{T2A-tdTomato/+}, and Lrig1^{T2A-tdTomato/T2A-tdTomato} mice. Note the tdTomato fluorescence in the cerebellum.

Map of Lrig1^T2A-iCreERT2 allele targeting vector. NGS sequence of the targeting vector (link). NCBI BLAST (link).

The polyclonal goat anti-mouse LRIG1 antibody from R & D Systems (https://www.rndsystems.com/products/mouse-lrig1-antibody_af3688) utilized in Nam and Capecchi, 2020 for immunofluorescence and flow cytometry analyses was tested by immunostaining the 1c2 Lrig1^{T2A-iCreERT2/+} mouse cerebellum. The resulting anti-LRIG1 immunostaining signal was consistent with the GENSAT and EUCOMM images. Because the LRIG1 protein expression level was high in the cerebellum, an attempt was made to also visualize in the cerebellum the sfGFP-iCre-ER^T2 protein from the Lrig1^T2A-iCreERT2 allele. A rabbit monoclonal anti-cre antibody from Cell Signaling Tech (https://www.cellsignal.com/products/primary-antibodies/cre-recombinase-d7l7l-xp-rabbit-mab/15036) was tested to visualize the sfGFP-iCre-ER^T2 fusion protein. A faint signal was observed near the LRIG1+ region. However, I did not follow this up further with wildtype mouse control, etc, and the cerebellum immunostaining was not included in Nam and Capecchi, 2020.

RFP labeling in the cerebellum of the Lrig1^{T2A-iCreERT2/+}; Rosa26^Ai14/+ mice.

An image from Wang et al., 2018 (https://doi.org/10.1117/12.2285406) shows the RFP-labeled cells in the cerebellum of the Coffey Lrig1^creERT2/+; Rosa26^Ai14/+ mice live imaged by 3-photon microscopy (Chris Xu group at Cornell).

By the way, these indicate we correctly knocked in into Lrig1.

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In contrast to the cerebellum, the LRIG1 protein was undetectable in the ventricular wall using the same antibody in indirect immunofluorescence presumably due to lower expression level there. The sfGFP-iCre-ER^T2 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.

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 to a lesser extent in 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. However, the tdTomato signal in the ventricular wall cells was visible when analyzed with a flow cytometer, a much more sensitive instrument to detect the fluorescence.

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As an aside, because of the high Lrig1 expression level in the cerebellum, the cerebellum can be easily labeled with RFP using the Lrig1^T2A-iCreERT2 allele or the Coffey Lrig1^creERT2 allele (images shown here and more here). This is actually useful because the RFP in the cerebellum can serve as a proxy read-out for the tamoxifen induction efficiency elsewhere. That is, after I do the dissection of the tamoxifen-induced mouse brain for the ventricular wall whole mount, I can look with the fluorescence dissecting microscope at the cerebellum that remains. If the RFP labeling of the cerebellum is bright, that usually means the V-SVZ is labeled well with the RFP as well. Conversely, if the RFP labeling of the cerebellum is dim, then that usually means the tamoxifen induction failed for some unknown reason(s), and thus that sample can be de-prioritized for the analysis.

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

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