Supplementary Material for: Developmental changes in the expression of lysophosphatidic acid receptor 4 in the mouse brain

Introduction: Lysophosphatidic acid (LPA) is a bioactive phospholipid that mediates a variety of biological actions through binding to G protein-coupled receptors (GPCRs) known as LPA receptors (LPARs). In mammals, six LPAR subtypes (LPAR1–6) have been identified. This study aims to determine the expression of LPAR4 in the developing mouse brain. Methods: Brains samples were prepared from mice in various stages of development and biochemical and immunohistochemical analyses were conducted using anti-LPAR4. Results: Western blot analysis detected two LPAR4-immunoreactive species at ~50 kDa and ~42 kDa from embryonic day 16.5 (E16.5). The ~50 kDa molecule increased during development, reaching a peak at postnatal day 3 (P3), and then gradually decreased through P22. In contrast, the ~42 kDa molecule continued to increase up to P22. Immunohistochemical analyses demonstrated strong LPAR4 expression in neural cells in the intermediate zone and cortical plate of the E15.5 cerebral cortex, whereas neural progenitors in the ventricular and subventricular zones exhibited weaker expression. At P15, fiber-like staining resembling the apical dendrites of cortical neurons and hippocampal pyramidal cells was also observed. Conclusion: This study demonstrated dynamic, spatiotemporal changes of LPAR4 expression in the brain from embryonic to postnatal stages. These findings support a potential role for LPAR4 in neural development.