Generation and characterization of a Cre-inducible MAP3K1 gain-of-function model.

MAP3K1 is a multifunctional signaling kinase implicated in diverse biological processes. Although its gain-of-function variants contribute to multiple human diseases, including 46,XY disorders of sex development, mechanistic studies have been limited owing to the lack of physiologically relevant in vivo models. To address this gap, we generated a Cre-inducible Map3k1TG transgenic mouse carrying V5- and TurboID-tagged Map3k1 cDNA. Upon tamoxifen-induced Cre activation, Map3k1TG;Rosa26-CreERT2 fetuses displayed tamoxifen dose-dependent lethality and developmental abnormalities, including reduced body size, digit fusion, tail shortening and epidermal thickening, demonstrating broad developmental impact of MAP3K1 overexpression. Male fetuses additionally exhibited impaired sexual differentiation, with reduced anogenital distance and decreased Sertoli and germ cell populations. Mechanistically, MAP3K1 induction activated both MAPK and WNT/β-catenin pathways, leading to β-catenin displacement from plasma membrane in keratinocytes in vitro, and in epidermal and germ cells in vivo. TurboID-based proximity labeling further revealed cytoskeletal-associated proteins as MAP3K1 interactors, consistent with biochemical and imaging evidence showing MAP3K1 colocalization with actin filaments and centrosomes. These findings establish Map3k1TG as a versatile in vivo platform for dissecting MAP3K1 gain-of-function mechanisms in development and diseases.