Hedgehog pathway disruption alters urothelial differentiation and continence mechanisms in the female mouse lower urinary tract

Urinary incontinence (UI) imposes a significant healthcare burden and reduces quality of life. Contributing factors such as aging, pregnancy/childbirth, stress, and injury are recognized, but incomplete understanding of underlying mechanisms limits new therapies. Hedgehog (Hh) signaling has been implicated in lower urinary tract development, but its specific role in female continence mechanisms has not been fully characterized. Here we investigate the functional and molecular consequences of reduced Hh signaling using <i>Gli2</i>^+/-;<i>Gli3</i>^Δ699/+ (<i>Gli</i> mutant) female mice. We assessed spontaneous voiding through void spot assays and uroflowmetry, then assessed contractility in bladder and urethral tissues ex vivo. Female <i>Gli</i> mutant mice display more small volume voids than wild-type mice. <i>Gli</i> mutant female bladder had reduced strength of contraction to electrical field and cholinergic stimuli, whereas the urethra had reduced sensitivity to serotonin-mediated contraction, but not to phenylephrine. Thus, unique changes to bladder and urethral contractility dynamics are present in <i>Gli</i> mutant mice and are dependent upon types of stimuli. Furthermore, expression of serotonin transporter (<i>Sert</i>) mRNA was increased in <i>Gli</i> mutant urethra compared with wild type. Uroplakin IIIa, typically localized to bladder urothelium, was ectopically expressed in distal urethral urothelium of adult but not embryonic (E) <i>day 16 Gli</i> mutant mice. These findings highlight a previously uncharacterized role of Hh signaling in maintaining female lower urinary tract function and urothelial patterning, and support further investigation of its contribution to continence.<b>NEW & NOTEWORTHY</b> This study identifies disrupted Hh signaling as a key determinant of female bladder and urethral contractility, providing new insights into molecular mechanisms maintaining continence. We observe impaired contractile responses to multiple stimuli, including urethral response to serotonin. In addition, we identify ectopic expression of uroplakin IIIa in urethra of <i>Gli</i> mutant mice, arising after prenatal development. By reducing-but not completely ablating-Hh signaling, we elucidate essential roles of this pathway in determining continence.