Adult Gli2+/–;Gli3Δ699/+ Male and Female Mice Display a Spectrum of Genital Malformation

Abstract

Disorders of sexual development (DSD) encompass a broad spectrum of urogenital malformations and are amongst the most common congenital birth defects. Although key genetic factors such as the hedgehog (Hh) family have been identified, a unifying postnatally viable model displaying the spectrum of male and female urogenital malformations has not yet been reported. Since human cases are diagnosed and treated at various stages postnatally, equivalent mouse models enabling analysis at similar stages are of significant interest. Additionally, all non-Hh based genetic models investigating DSD display normal females, leaving female urogenital development largely unknown. Here, we generated compound mutant mice, Gli2+/–;Gli3Δ699/+, which exhibit a spectrum of urogenital malformations in both males and females upon birth, and also carried them well into adulthood. Analysis of embryonic day (E)18.5 and adult mice revealed shortened anogenital distance (AGD), open ventral urethral groove, incomplete fusion of scrotal sac, abnormal penile size and structure, and incomplete testicular descent with hypoplasia in male mice, whereas female mutant mice displayed reduced AGD, urinary incontinence, and a number of uterine anomalies such as vaginal duplication. Male and female fertility was also investigated via breeding cages, and it was identified that male mice were infertile while females were unable to deliver despite becoming impregnated. We propose that Gli2+/–;Gli3Δ699/+ mice can serve as a genetic mouse model for common DSD such as cryptorchidism, hypospadias, and incomplete fusion of the scrotal sac in males, and a spectrum of uterine and vaginal abnormalities along with urinary incontinence in females, which could prove essential in revealing new insights into their equivalent diseases in humans.

GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation

“…Sex steroid receptors are known to stimulate both Sonic and Indian hedgehog in numerous cell types, including those in the developing genital tubercle. Further, evidence that the hedgehog pathway is downstream of the androgen receptor is supported by findings that Gli2 and Gli3 mutant mouse models, in addition to temporally controlled deletion of Shh between E13.5 and E15.5, cause hypospadias and a urethral phenotype that is strikingly similar to the hypospadias observed in Gli3 XtJ mutants [10,11,52]. It is currently not known whether exogenous androgens could alleviate or bypass these hedgehog-mediated defects.…”

“…Each factor has been shown to contribute to male sex differentiation in distinct ways. For example, while mutations in Gli2 alone caused external genitalia defects, compound mutation with Gli1 showed no additional defects, but its combination with Gli3 exhibited more severe defects [5,[9][10][11]. Further, some studies showed that Gli3 is not essential for the development of external genitalia but others found that Gli3 disruption causes significant genital malformations [5,10,11].…”

“…For example, while mutations in Gli2 alone caused external genitalia defects, compound mutation with Gli1 showed no additional defects, but its combination with Gli3 exhibited more severe defects [5,[9][10][11]. Further, some studies showed that Gli3 is not essential for the development of external genitalia but others found that Gli3 disruption causes significant genital malformations [5,10,11]. Meanwhile, within the fetal testis, elimination of Gli1 or Gli2 individually is not sufficient to reproduce the failure in fetal Leydig cell differentiation seen in Dhh knockout mice; Gli3 has not been tested [12].…”

Hedgehog Signaling for Urogenital Organogenesis and Prostate Cancer: An Implication for the Epithelial–Mesenchyme Interaction (EMI)

“…The urogenital system includes the urinary tract: composed of the kidney, the ureter, the bladder, and the urethra; and the reproductive tract: composed of the testes, the accessory glands, and the external genitalia (EXG). Recently, an increasing number of regulatory genes for EXG development has been reported [13][14][15][16][17]. Thus, possible insights gleaned from comparisons between urogenital organogenesis with other medical topics, such as prostate cancer, is valuable.…”

A Human Accelerated Region is a Leydig cellGLI2Enhancer that Affects Male-Typical Behavior

“…We further characterized one HAR, 2xHAR.238, which showed increased activity in the human ortholog relative to the chimpanzee ortholog. 2xHAR.238 is adjacent to Gli2 , a transcriptional effector of Hedgehog signaling ( 12, 22–25 ). We found that 2xHAR.238 regulates Leydig cell expression of Gli2 and physically interacts with the Gli2 promoter, indicating that it is a Gli2 enhancer.…”