Expression of Endogenous Epitope-Tagged GPR4 in the Mouse Brain

Area of Science:

• Neuroscience
• Molecular Biology
• Physiology

Background:

• GPR4 is a proton-sensing G protein-coupled receptor involved in vital physiological processes.
• Previous GPR4 expression studies relied on indirect methods, lacking direct protein visualization in the mouse brain.
• Understanding GPR4 localization is key to elucidating its role in central nervous system functions, particularly CO2/H+ modulation.

Purpose of the Study:

• To generate a GPR4-HA knock-in mouse model for direct visualization of GPR4 protein expression in the central nervous system.
• To precisely map GPR4 protein distribution within distinct brain regions.
• To correlate GPR4 protein localization with its known or suspected physiological functions.

Main Methods:

• CRISPR/Cas9 knock-in technology was employed to tag the endogenous GPR4 locus with a hemagglutinin (HA) epitope.
• Immunohistochemistry using a specific anti-HA antibody was performed on mouse brain tissue.
• GPR4 mRNA detection was used to complement protein expression data.

Main Results:

• GPR4 protein was detected in specific brain regions, including the retrotrapezoid nucleus (RTN), raphe nuclei, medial habenula, lateral septum, and thalamic nuclei.
• GPR4 expression was observed in diverse neuronal types (excitatory, inhibitory, aminergic), not limited by neurochemical identity.
• While GPR4 mRNA was present in brain vascular endothelium, protein immunoreactivity was absent in these cells.

Conclusions:

• The GPR4-HA mouse model enables direct visualization of GPR4 protein in the mouse brain for the first time.
• Identified GPR4 protein localization in the RTN supports its role in CO2-regulated breathing.
• This study provides a crucial resource for investigating GPR4's function in various CO2/H+-modulated brain processes.