Brain-wide mapping and synaptic localization of C1QL3 using a novel epitope-tagged knock-in mouse

Synapse formation and function are coordinated spatially and temporally by a host of synaptic proteins that regulate neuronal signaling, synapse specificity, and plasticity; many of which are implicated in neuropsychiatric disorders. Members of the C1q/TNF superfamily function as synaptic organizers, shaping synapse assembly and maintenance. Among them, C1QL3 plays a putative role in trans-synaptic adhesion and modulation of synaptic strength, but the lack of a reliable antibody to detect it has severely limited the ability to map its endogenous localization and study its biochemical properties. Here, we present a novel epitope-tagged knock-in mouse line (C1ql32HA), in which two hemagglutinin (HA) epitopes were inserted near the N-terminus of the endogenous C1QL3 protein. This model enables purification, detection, and subcellular localization of native C1QL3 protein (C1QL3-2HA) with high specificity, eliminating the need for overexpression or custom antibodies. We validated that C1ql32HA mice maintain normal mRNA expression, biochemical properties, and behavior. Using native PAGE, we determined the endogenous oligomeric state of C1QL3-2HA. Brain-wide light-sheet microscopy uncovered an expanded neuroanatomical map of C1QL3-2HA expression, including newly identified populations in cortical and subcortical regions as well as the retina. Dual immunohistochemistry confirmed cell type-specific expression patterns, and super-resolution STED microscopy localized C1QL3-2HA to hippocampal mossy fiber synapses, positioned between pre- and post-synaptic markers, supporting its hypothesized role in trans-synaptic complexes. This knock-in mouse line is a powerful tool for studying the anatomical, molecular, and synaptic biology of C1QL3 in all cellular/tissue contexts, enabling future studies into its potential roles in the nervous system and beyond.