Genome editing-based refinement of GPCR visualization in mice using the oxytocin receptor as a model

Figures

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  Fig. 1. Epitope-tagging strategy to visualize GPCR in mouse models. (A) Schematic of the epitope-tagging strategy. In the absence of a suitable antibody for the protein of interest (POI), genetic introduction of an appropriate tag (shown as a yellow TAG) enables the detection with an anti-tag antibody. (B) Targeting strategy to insert an epitope-tag (shown as a yellow TAG) just upstream from the translational stop codon (TGA, shown in red) of the mouse Oxtr gene via CRISPR/Cas9. Solid bars indicate coding regions, and open bars indicate non-coding regions. PAM and guide sequence to recruit CRISPR components onto the Oxtr stop codon are depicted at the top. (C) Designs of three knock-in alleles for epitope-tagged Oxtr. Tag sequences (3xHA, smFP, or 3xALFA) are fused 3’ to the Oxtr coding sequence. The DNA sequences of three variants are available at Zenodo (https://zenodo.org/records/17446559) and DDBJ (Accession numbers: LC897328, LC897329, and LC897330), and the genomic DNA sequences for three knock-in lines are also supplied at Zenodo (https://zenodo.org/records/17446592) and DDBJ (Accession numbers: LC897331, LC897332, and LC897333).

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  Fig. 2. Structural prediction and functional validation of C-terminally tagged Oxtr. (A) Snake plot representation of mouse Oxtr obtained from GPCRdb (https://gpcrdb.org/), illustrating the topology of the seven transmembrane helices and intracellular/extracellular regions. (B) Structural models of Oxtr predicted by AlphaFold 3. Five independent models are overlaid to illustrate structural consistency in the transmembrane core and variability in flexible regions, particularly the relatively long C-terminal tail. (C–F) Predicted 3D structures of epitope-tagged Oxtr proteins in complex with Gq, generated by AlphaFold 3. (C) Oxtr-3xHA–Gq complex, (D) Oxtr-smFP–Gq complex, (E) overall structure of the smFP tag, and (F) Oxtr-3xALFA–Gq complex. Epitope-tagged Oxtr proteins are shown in gray, epitope tags in red, and the GFP scaffold in light green. The smFP (E) is designed by inserting a total of 10 HA epitopes at the N- and C-termini, as well as into an internal loop of the GFP scaffold10. The Gq heterotrimer subunits are depicted as Gαq (orange), Gβ (blue), and Gγ (green). Because the C-terminal tail of Oxtr is relatively long and likely to be conformationally flexible, the positioning of…

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  Fig. 3. smFP exhibits higher sensitivity than 3xHA or 3xALFA for Oxtr detection in the adult mouse brain. Oxtr-expressing cells are visualized in the brains (Bregma − 1.82 mm) of 7-week-old homozygous mice carrying (A–B) Oxtr-3xHA, (C–D) Oxtr-smFP, or (F–G) Oxtr-3xALFA. In the Oxtr-smFP mice (C–D), staining signals are markedly stronger compared with Oxtr-3xHA (A–B) and Oxtr-3xALFA mice (F–G), in regions previously reported to express Oxtr, including the primary somatosensory area (S1), hippocampal dentate gyrus (DG) and anterior CA2/CA3, paraventricular thalamus (PVT), posteroventral part of the medial amygdala (MePV), posteromedial cortical amygdaloid area (PMCo), and ventromedial hypothalamus (VMH). Panels (B), (D), and (G) show magnified views of the white-boxed region in S1, where individual neurons are more clearly visualized with a higher signal-to-noise ratio in the Oxtr-smFP mouse (D) than in Oxtr-3xHA (B) or Oxtr-3xALFA (G) mice. (E) Quantification of HA-stained neurons in the S1 corresponding to (B) and (D) from N = 6 mice (see Supplementary Fig. 2 for details). Data are mean ± SEM. Oxtr-smFP showed a significantly higher number of labeled neurons than Oxtr-3xHA, demonstrating…

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  Fig. 4. C-terminally fused 3xHA, smFP, and 3xALFA do not impair peripheral Oxtr function during lactation. Oxytocin is essential for milk ejection, and pups born to Oxtr knockout (KO) mothers reportedly die soon after birth due to impaired breastfeeding. Panels (A–C) show pups (postnatal day 3) from homozygous Oxtr-3xHA, Oxtr-smFP, and Oxtr-3xALFA mothers, respectively. As indicated by white ovals, their digestive tracts are filled with milk. Panels (D–F) show the corresponding mothers, whose mammary glands are well developed, with prominent nipples for nursing (white arrows). These observations indicate that the C-terminal tags do not interfere with peripheral oxytocin signaling, including its fundamental role in milk ejection.

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  Fig. 5. C-terminally fused 3xALFA tag impairs Oxtr trafficking to the cell membrane. Localization of C-terminally tagged Oxtr at the plasma membrane is examined in three cell types: cortical neurons (A–F), pancreatic islet cells (G–L), and mammary myoepithelial cells (M–R). In homozygous Oxtr-3xHA mice, 3xHA-tagged Oxtr exhibits uniform distribution along the cell membrane surrounding the cell body (A, B, G, H, M, N). In homozygous Oxtr-smFP mice, membrane signals are generally uniform in neurons (C, D) and mammary cells (O, P), although some heterogeneity is observed. In pancreatic cells (I, J), intracellularly invaginated signals occasionally appear (white arrows), suggesting that in certain cell types the smFP tag partially interferes with membrane trafficking. In contrast, in homozygous Oxtr-3xALFA mice, 3xALFA-tagged Oxtr shows pronounced intracellular retention in all three cell types (E, F, K, L, Q, R, white arrowheads), indicating severe impairment of receptor trafficking and membrane insertion.

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  Fig. 6. Oxtr-smFP enables detailed mapping of subcellular Oxtr localization in neurons. (A) 3xHA-tagged Oxtr-expressing neurons from P0 primary cultures (DIV7) are visualized using anti-HA antibody (magenta), showing co-localization with MAP2-positive dendrites (green). (A’) HA-positive Oxtr-expressing neurons are sparsely distributed among cultured cells. (a1–a4) Super-resolution images of the regions indicated by the white boxes in (A). (a1–a3) Although 3xHA-tagged Oxtr proteins (magenta) appear to be distributed along MAP2-positive dendrites (green), the signal is relatively diffuse and less clearly resolved due to higher background staining. (a4) Oxtr signals are also broadly distributed throughout the soma. (B) smFP-tagged Oxtr-expressing neurons from P0 primary cultures (DIV7). (B’) Compared with Oxtr-3xHA, HA-positive Oxtr-expressing neurons are more readily detected in Oxtr-smFP cultures, indicating higher detection sensitivity. (b1–b4) Super-resolution images of the regions indicated by the white boxes in (B). (b1–b3) smFP-tagged Oxtr proteins are observed as clearer punctate signals distributed along MAP2-positive dendrites, allowing Oxtr localization to be traced farther into…