Gata4 regulates fibroblast migration in a fibroblast-source-dependent manner under physiological matrix stiffness

Abstract

Fibroblast migration contributes to the spatial organization of tissue repair and extracellular matrix remodeling, but the transcriptional mechanisms regulating this process under physiologically relevant mechanical conditions remain incompletely understood. We investigated whether GATA4 regulates fibroblast migration in a tissue-of-origin-dependent manner. Cardiac fibroblasts (CFs), lung fibroblasts (LFs), and tail-tip fibroblasts (TTFs) were isolated from neonatal male and female Gata4fl/fl mice and cultured on 8-kPa polydimethylsiloxane substrates. CF behavior on 8-kPa substrates was first compared with that on conventional tissue-culture plastic. Gata4 was subsequently deleted by adenoviral Cre transduction, and multiple features of steady-state migration were quantified by live-cell imaging. CFs cultured on 8-kPa substrates exhibited a smaller cell area and greater migration speed, displacement, and directional persistence than cells cultured on plastic, establishing a physiologically relevant mechanical baseline. Baseline Gata4 expression was highest in CFs, intermediate in LFs, and lowest in TTFs, whereas adenoviral Cre reduced Gata4 expression by more than 90% in all three populations. Gata4 deletion produced distinct migratory responses according to fibroblast source. In CFs, Gata4 loss reduced instantaneous speed, line speed, segment length, and directional persistence while increasing cell area. LFs showed modest increases in instantaneous speed, path speed, normalized 1-h path distance, and segment length without a clear change in directional persistence, whereas TTF migration was comparatively insensitive to Gata4 loss. Integrated effect-size analysis confirmed that the coordinated alteration of migration rate and directionality was most pronounced in CFs. RNA-seq analysis of adult CFs further showed enrichment of general motility and actin-remodeling programs following Gata4 deletion, without coordinated enrichment of lamellipodium and focal-adhesion programs. Together, these findings indicate that GATA4 supports efficient and persistent fibroblast migration in a fibroblast-source-dependent manner under physiologically relevant mechanical conditions.