Supplementary Material for: Androgen Regulates Dimorphic F-Actin Assemblies in the Genital Organogenesis

Impaired androgen activity induces defective sexual differentiation
of the male reproductive tract, including hypospadias, an abnormal
formation of the penile urethra. Androgen signaling in the urethral
mesenchyme cells (UMCs) plays essential roles in driving dimorphic
urethral development. However, cellular events for sexual
differentiation remain virtually unknown. In this study, histological
analyses, fluorescent staining, and transmission electron microscopy
(TEM) were performed to reveal the cellular dimorphisms of UMCs. F-actin
dynamics and migratory behaviors of UMCs were further analyzed by
time-lapse imaging. We observed a prominent accumulation of F-actin with
poorly assembled extracellular matrix (ECM) in female UMCs. In
contrast, thin fibrils of F-actin co-aligning with the ECM through
membrane receptors were identified in male UMCs. Processes for dimorphic
F-actin assemblies were temporally identified during an
androgen-regulated masculinization programming window and spatially
distributed in several embryonic reproductive tissues. Stage-dependent
modulation of the F-actin sexual patterns by androgen in UMCs was also
demonstrated by time-lapse analysis. Moreover, androgen regulates
coordinated migration of UMCs. These results suggest that androgen
signaling regulates the assembly of F-actin from cytoplasmic
accumulation to membranous fibrils. Such alteration appears to promote
the ECM assembly and the mobility of UMCs, contributing to male type
genital organogenesis.