Increasing Sufu gene dosage reveals its unorthodox role in promoting polydactyly and medulloblastoma tumorigenesis.

Suppressor of fused (SUFU) is widely regarded as a key negative regulator of the sonic hedgehog (SHH) morphogenic pathway and a known tumor suppressor of medulloblastoma (MB). However, we report here that SUFU expression was markedly increased in 75% of specimens compiled in a tissue array comprising 49 unstratified MBs. The SUFU and GLI1 expression levels in this MB array showed strong positive correlation, which was also identified in a large public data set containing 736 MBs. We further report that increasing Sufu gene dosage in mice caused preaxial polydactyly, which was associated with the expansion of the Gli3 domain in the anterior limb bud and heightened Shh signaling responses during embryonic development. Increasing Sufu gene dosage also led to accelerated cerebellar development and, when combined with ablation of the Shh receptor encoded by Patched1 (Ptch1), promoted MB tumorigenesis. These data reveal multifaceted roles of SUFU in promoting MB tumorigenesis by enhancing SHH signaling. This revelation clarifies potentially counterintuitive clinical observation of high SUFU expression in MBs and may pave way for novel strategies to reduce or reverse MB progression.

Transcriptomics of Hirschsprung disease patient-derived enteric neural crest cells reveals a role for oxidative phosphorylation.

Hirschsprung disease is characterized by the absence of enteric neurons caused by the defects of enteric neural crest cells, leading to intestinal obstruction. Here, using induced pluripotent stem cell-based models of Hirschsprung and single-cell transcriptomic analysis, we identify a gene set of 118 genes commonly dysregulated in all patient enteric neural crest cells, and suggest HDAC1 may be a key regulator of these genes. Furthermore, upregulation of RNA splicing mediators and enhanced alternative splicing events are associated with severe form of Hirschsprung. In particular, the higher inclusion rate of exon 9 in PTBP1 and the perturbed expression of a PTBP1-target, PKM, are significantly enriched in these patient cells, and associated with the defective oxidative phosphorylation and impaired neurogenesis. Hedgehog-induced oxidative phosphorylation significantly enhances the survival and differentiation capacity of patient cells. In sum, we define various factors associated with Hirschsprung pathogenesis and demonstrate the implications of oxidative phosphorylation in enteric neural crest development and HSCR pathogenesis.

IRX5 promotes DNA damage repair and activation of hair follicle stem cells.

The molecular mechanisms allowing hair follicles to periodically activate their stem cells (HFSCs) are incompletely characterized. Here, we identify the transcription factor IRX5 as a promoter of HFSC activation. Irx5-/- mice have delayed anagen onset, with increased DNA damage and diminished HFSC proliferation. Open chromatin regions form near cell cycle progression and DNA damage repair genes in Irx5-/- HFSCs. DNA damage repair factor BRCA1 is an IRX5 downstream target. Inhibition of FGF kinase signaling partially rescues the anagen delay in Irx5-/- mice, suggesting that the Irx5-/- HFSC quiescent phenotype is partly due to failure to suppress Fgf18 expression. Interfollicular epidermal stem cells also show decreased proliferation and increased DNA damage in Irx5-/-mice. Consistent with a role for IRX5 as a promoter of DNA damage repair, we find that IRX genes are upregulated in many cancer types and that there is a correlation between IRX5 and BRCA1 expression in breast cancer.

Mechanosensitive brain tumor cells construct blood-tumor barrier to mask chemosensitivity.

Major obstacles in brain cancer treatment include the blood-tumor barrier (BTB), which limits the access of most therapeutic agents, and quiescent tumor cells, which resist conventional chemotherapy. Here, we show that Sox2+ tumor cells project cellular processes to ensheathe capillaries in mouse medulloblastoma (MB), a process that depends on the mechanosensitive ion channel Piezo2. MB develops a tissue stiffness gradient as a function of distance to capillaries. Sox2+ tumor cells perceive substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion to promote cellular process growth and cell surface sequestration of ß-catenin. Piezo2 knockout reverses WNT/ß-catenin signaling states between Sox2+ tumor cells and endothelial cells, compromises the BTB, reduces the quiescence of Sox2+ tumor cells, and markedly enhances the MB response to chemotherapy. Our study reveals that mechanosensitive tumor cells construct the BTB to mask tumor chemosensitivity. Targeting Piezo2 addresses the BTB and tumor quiescence properties that underlie treatment failures in brain cancer.

A study on etiology of incontinence in double knockout mouse model.

INTRODUCTION AND OBJECTIVE: Stress urinary incontinence is of concern in both pediatric and adult population. Double mutant GLI family zinc finger Gli2+/-; Gli3Δ699/+ murine model of stress incontinence has been recently developed as a reliable model which does not require surgical manipulation to create incontinence and is shown to survive to adulthood. The aim of this study was to establish the etiology of incontinence in the double mutant Gli2+/-; Gli3Δ699/+ mice. STUDY DESIGN: We used 13 cluster of differentiation 1 (CD-1) mice (7-9 weeks) for demonstration of histology of the bladder and urethra. There were 3 Wild Gli2+/- females, 2 Wild Gli2+/- males, 4 Gli2+/-;Gli3Δ699/+ females and 4 Gli2+/-;Gli3Δ699/+ males. The Wild Gli2+/- mice served as the control group and Gli2+/-;Gli3Δ699/+ mice served as the test group. Additionally, eight 16.5 days mice (2 each of Wild Gli2+/- females, Wild Gli2+/- males, double knockout (DKO) Gli2+/-;Gli3Δ699/+ females and Gli2+/-;Gli3Δ699/+ males) were used to assess the histology of the spinal cord. The gross appearance of bladder and urethra was studied using ink injection assays. Immunohistochemistry was done for smooth muscle actin and cytokeratin. RESULTS: Gross and histologic appearance confirmed the previously reported widening of bladder outlet and hypoplasia of smooth muscles in female urethra and also established them in the male urethra of Gli2+/-;Gli3Δ699/+ mice compared to Gli2+/- mice. The double knockout mice were smaller than the Gli2 mice (5.2 vs 6.1 cm, p = 0.002). Immunohistochemistry demonstrated epithelial hyperplasia and smooth muscle hypoplasia. Additionally, there was prostatic hypoplasia in the Gli2+/-;Gli3Δ699/+ male mice. The spinal cord length for body size appeared comparable between the Gli2+/- and Gli2+/-;Gli3Δ699/+ mice but histological evaluation revealed abnormal development of the caudal end of the vertebral body with premature termination of the spinal cord (Figure). DISCUSSION: The histological changes in the bladder neck and urethra were consistent to those previously reported. While previous report described the findings in female mice only, we confirmed that these findings are also present in males as well as prostatic hypoplasia, a possible additional factor leading to stress incontinence. The most important finding in the present study however, was the detection of premature termination of spinal cord in the DKO Gli2+/-; Gli3Δ699/+ mice which has not been reported previously and is likely a major contributor to incontinence in this model. CONCLUSION: The incontinence in male as well as female Gli2+/-; Gli3Δ699/+ mice is due to both myogenic and neurogenic involvement. These double knockout mice are a valuable model of stress incontinence related to neurogenic bladder due to low outlet resistance.

Comparative dietary effectiveness of a modified government-recommended diet with avoidance of ultra-processed foods on weight and metabolic management in children and adolescents: An open-label, randomized study.

BACKGROUND AND OBJECTIVES: Childhood obesity is rapidly rising in China and effective diet interventions are needed. Here, we determine whether the Chinese government-recommended diet (GRD) or a modified diet of further restriction of sugar and ultra-processed food but without energy restriction, minimally processed diet (MPD) is effective on weight loss in children and adolescents with obesity/overweight. METHODS AND STUDY DESIGN: This open-label, randomized study included 60 children and adolescents between 5-18 years old with overweight/obesity. Participants were randomized 1:1 to the GRD or MPD and self-managed at home for 12 weeks. Both groups received general recommendations in physical activities. The changes were evaluated in body weight, fasting glucose and insulin, lipid metabolism and serum uric acid between baseline and week 12. RESULTS: The results indicated great reductions by time for BMI, BMI z-score, fat mass percentage and fat mass index in both groups. An obvious decrease by time for weight was found in the MPD group (p<0.001) as well as fasting glucose (p=0.005), fasting insulin (p=0.001), total cholesterol (p=0.007) and serum uric acid (p=0.006). As for the amount of visceral fat, greater reduction by time was observed in MPD group compared with GRD group. CONCLUSIONS: A 12-week self-intervention combining the Chinese government-recommended diet with physical activities was effective on weight loss in children and adolescents with overweight/obesity. The minimally processed diet was more effective on decreasing visceral fat mass and may be beneficial to improving insulin resistance. Further studies are required to assess long-term outcomes of the general public.

Distinct roles of UVRAG and EGFR signaling in skeletal muscle homeostasis.

OBJECTIVE: Autophagy is a physiological self-eating process that can promote cell survival or activate cell death in eukaryotic cells. In skeletal muscle, it is important for maintaining muscle mass and function that is critical to sustain mobility and regulate metabolism. The UV radiation resistance-associated gene (UVRAG) regulates the early stages of autophagy and autophagosome maturation and plays a key role in endosomal trafficking. This study investigated the essential in vivo role of UVRAG in skeletal muscle biology. METHODS: To determine the role of UVRAG in skeletal muscle in vivo, we generated muscle-specific UVRAG knockout mice using the Cre-loxP system driven by Myf6 promoter that is exclusively expressed in skeletal muscle. Myf6-Cre+ UVRAGfl/fl (M-UVRAG-/-) mice were compared to littermate Myf6-Cre+ UVRAG+/+ (M-UVRAG+/+) controls under basal conditions on a normal chow diet. Body composition, muscle function, and mitochondria morphology were assessed in muscles of the WT and KO mice at 24 weeks of age. RESULTS: M-UVRAG-/- mice developed accelerated sarcopenia and impaired muscle function compared to M-UVRAG+/+ littermates at 24 weeks of age. Interestingly, these mice displayed improved glucose tolerance and increased energy expenditure likely related to upregulated Fgf21, a marker of muscle dysfunction. Skeletal muscle of the M-UVRAG-/- mice showed altered mitochondrial morphology with increased mitochondrial fission and EGFR accumulation reflecting defects in endosomal trafficking. To determine whether increased EGFR signaling had a causal role in muscle dysfunction, the mice were treated with an EGFR inhibitor, gefitinib, which partially restored markers of muscle and mitochondrial deregulation. Conversely, constitutively active EGFR transgenic expression in UVRAG-deficient muscle led to further detrimental effects with non-overlapping distinct defects in muscle function, with EGFR activation affecting the muscle fiber type whereas UVRAG deficiency impaired mitochondrial homeostasis. CONCLUSIONS: Our results show that both UVRAG and EGFR signaling are critical for maintaining muscle mass and function with distinct mechanisms in the differentiation pathway.

Identification of ALK in Thinness.

There is considerable inter-individual variability in susceptibility to weight gain despite an equally obesogenic environment in large parts of the world. Whereas many studies have focused on identifying the genetic susceptibility to obesity, we performed a GWAS on metabolically healthy thin individuals (lowest 6th percentile of the population-wide BMI spectrum) in a uniquely phenotyped Estonian cohort. We discovered anaplastic lymphoma kinase (ALK) as a candidate thinness gene. In Drosophila, RNAi mediated knockdown of Alk led to decreased triglyceride levels. In mice, genetic deletion of Alk resulted in thin animals with marked resistance to diet- and leptin-mutation-induced obesity. Mechanistically, we found that ALK expression in hypothalamic neurons controls energy expenditure via sympathetic control of adipose tissue lipolysis. Our genetic and mechanistic experiments identify ALK as a thinness gene, which is involved in the resistance to weight gain.

Hedgehog-Activated Fat4 and PCP Pathways Mediate Mesenchymal Cell Clustering and Villus Formation in Gut Development.

During development, intestinal epithelia undergo dramatic morphogenesis mediated by mesenchymal signaling to form villi, which are required for efficient nutrient absorption and host defense. Although both smooth-muscle-induced physical forces and mesenchymal cell clustering beneath emerging villi are implicated in epithelial folding, the underlying cellular mechanisms are unclear. Hedgehog (Hh) signaling can mediate both processes. We therefore analyzed its direct targetome and revealed GLI2 transcriptional activation of atypical cadherin and planar cell polarity (PCP) genes. By examining Fat4 and Dchs1 knockout mice, we demonstrate their critical roles in villus formation. Analyses of PCP-mutant mice and genetic interaction studies show that the Fat4-Dchs1 axis acts in parallel to the core-Vangl2 PCP axis to control mesenchymal cell clustering. Moreover, live light-sheet fluorescence microscopy and cultured PDGFRα+ cells reveal a requirement for PCP in their oriented cell migration guided by WNT5A. Therefore, mesenchymal PCP induced by Hh signaling drives cell clustering and subsequent epithelial remodeling.

Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches.

Stomach and intestinal stem cells are located in discrete niches called the isthmus and crypt, respectively. Recent studies have demonstrated a surprisingly conserved role for Wnt signaling in gastrointestinal development. Although intestinal stromal cells secrete Wnt ligands to promote stem cell renewal, the source of stomach Wnt ligands is still unclear. Here, by performing single cell analysis, we identify gastrointestinal stromal cell populations with transcriptome signatures that are conserved between the stomach and intestine. In close proximity to epithelial cells, these perictye-like cells highly express telocyte and pericyte markers as well as Wnt ligands, and they are enriched for Hh signaling. By analyzing mice activated for Hh signaling, we show a conserved mechanism of GLI2 activation of Wnt ligands. Moreover, genetic inhibition of Wnt secretion in perictye-like stromal cells or stromal cells more broadly demonstrates their essential roles in gastrointestinal regeneration and development, respectively, highlighting a redundancy in gastrointestinal stem cell niches.

The impact of sex hormones on genital wound healing in mice: a comparative study.

INTRODUCTION AND OBJECTIVE: The effects estrogen and testosterone have on penile wound healing are still uncertain. This study evaluated the effects of these hormones on the wound healing process of penile and non-penile skin in wild-type (Mus musculus species) 4-5-week-old mice. METHODOLOGY: Seventy wild-type Mus musculus species were randomly assigned to four groups control (n = 17), 1-week post-operative topical estrogen (n = 18), 1-week pre-operative testosterone (n = 17), and immediate post-operative testosterone (n = 18). Incisions were made on the ventrum of the penis and dorsal neck skin. On post-operative day 3, 7, and 14, incision sites were harvested. Evaluation was performed grossly for postsurgical penile edema and histologically for inflammatory cell concentration, presence of fibrinopurulent materials and distribution of collagen-fibroblastic cells. Each treatment group was compared at the three post-operative time points using the Fisher-Freeman--Halton exact test. CD34 and androgen receptor immunohistostaining was performed for between-group differences to assess microvascular density or vasodilatation and androgen receptor upregulation. RESULTS: In this study, the experiment noted significant penile edema on post-operative day 7 in the testosterone groups, whereas less edema in the estrogen group (P = 0.010; Figure). On histologic evaluation of the penile wounds, a significantly increased inflammatory cell concentration was noted for both pre-operative and post-operative testosterone groups on post-operative day 14 (P = 0.023). The estrogen group revealed significantly increased fibrinopurulent material on the 3rd and 7th post-operative days (P = 0.045 and P = 0.005, respectively). No significant between-group differences in the collagen-fibroblastic distribution were noted over the three-time phases. On histologic evaluation of the skin wounds, no significant differences were noted between the groups for inflammatory cell concentration and presence of fibrinopurulent materials. However, compared with the testosterone treatment groups, a significant higher collagen-fibroblast distribution was noted in the estrogen groups on post-operative day 3 and 14 (P = 0.001 and P = 0.044, respectively). CONCLUSION: Sex hormones, when given peri-operatively, may affect the wound healing process in mice. Testosterone appears to stimulate a prolonged inflammatory effect on penile wounds. Conversely, estrogen induces a fibrinopurulent congregation early in the penile wound healing process. For general skin healing, estrogen induces earlier collagen and fibroblast distribution, whereas testosterone has a delayed effect. The findings of this study should be further investigated in larger animal model with longer follow-up period.

GLI2 Modulated by SUFU and SPOP Induces Intestinal Stem Cell Niche Signals in Development and Tumorigenesis.

Gut mesenchyme provides key stem cell niche signals such as Wnt ligands, but how these signals are regulated is unclear. Because Hedgehog (Hh) signaling is critical for gut mesenchymal development and tumorigenesis, we investigated Hh-mediated mechanisms by analyzing mice deleted for key negative regulators of Hh signaling, Sufu and/or Spop, in the gut mesenchyme, and demonstrated their dosage-dependent roles. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction. Consistent with increased epithelial proliferation caused by abnormal Hh activation in development, Sufu reduction promotes intestinal tumorigenesis, whereas Gli2 heterozygosity suppresses it. Our analyses of chromatin and GLI2 binding genomic regions reveal its transcriptional regulation of stem cell niche signals through enhancers, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis.

The Iroquois homeobox proteins IRX3 and IRX5 have distinct roles in Wilms tumour development and human nephrogenesis.

Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock-out Irx3- /Irx5- mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3-/- cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/ß-catenin-signalling. In contrast, IRX5-/- cells displayed activation of Hippo and non-canonical WNT-signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Single Nucleotide Polymorphisms of HAAO and IRX6 Genes as Risk Factors for Hypospadias.

PURPOSE: We evaluated the association of hypospadias and 17 susceptibility loci previously identified by a European genome-wide association study in a cohort of Japanese patients. We also examined the expression of candidate genes in male mouse embryos to determine the possible underlying mechanisms of this disease. MATERIALS AND METHODS: We enrolled 169 Japanese patients (mean age at surgery 3.7 years) who underwent repair of hypospadias. Genotyping of 17 single nucleotide polymorphisms was performed using a multiplex polymerase chain reaction invader assay. We also performed in situ hybridization to determine whether candidate genes were expressed in the male genital tubercle during embryonic development of the external genitalia in mice. RESULTS: Single nucleotide polymorphism rs3816183 of HAAO was significantly associated with susceptibility to hypospadias in general (p = 0.0019) and to anterior/middle hypospadias (p = 0.0283) and posterior hypospadias (p = 0.0226), while single nucleotide polymorphism rs6499755 of IRX6 showed an association with susceptibility to anterior/middle hypospadias (p = 0.0472). In mouse embryos there was no significant upregulation of Haao expression in the developing male external genitalia. Irx3 and Irx5, which are linked to Irx6 within the IrxB cluster, were expressed in the mesenchyme remote from the urethral plate epithelium during the critical embryonic period for masculinization. Irx6 was expressed in the ectodermal epithelium, demonstrating prominent dorsal ectodermal expression without expression in the ventral ectoderm adjacent to the urethral plate during the same period. CONCLUSIONS: Genetic variations of HAAO and IRX6 influence susceptibility to hypospadias in the Japanese population. Further research is needed to clarify the mechanism by which variations in these genes contribute to the pathogenesis of hypospadias.

Dual Regulatory Functions of SUFU and Targetome of GLI2 in SHH Subgroup Medulloblastoma.

SUFU alterations are common in human Sonic Hedgehog (SHH) subgroup medulloblastoma (MB). However, its tumorigenic mechanisms have remained elusive. Here, we report that loss of Sufu alone is unable to induce MB formation in mice, due to insufficient Gli2 activation. Simultaneous loss of Spop, an E3 ubiquitin ligase targeting Gli2, restores robust Gli2 activation and induces rapid MB formation in Sufu knockout background. We also demonstrated a tumor-promoting role of Sufu in Smo-activated MB (∼60% of human SHH MB) by maintaining robust Gli activity. Having established Gli2 activation as a key driver of SHH MB, we report a comprehensive analysis of its targetome. Furthermore, we identified Atoh1 as a target and molecular accomplice of Gli2 that activates core SHH MB signature genes in a synergistic manner. Overall, our work establishes the dual role of SUFU in SHH MB and provides mechanistic insights into transcriptional regulation underlying Gli2-mediated SHH MB tumorigenesis.

A Feedforward Mechanism Mediated by Mechanosensitive Ion Channel PIEZO1 and Tissue Mechanics Promotes Glioma Aggression.

Alteration of tissue mechanical properties is a physical hallmark of solid tumors including gliomas. How tumor cells sense and regulate tissue mechanics is largely unknown. Here, we show that mechanosensitive ion channel Piezo regulates mitosis and tissue stiffness of Drosophila gliomas, but not non-transformed brains. PIEZO1 is overexpressed in aggressive human gliomas and its expression inversely correlates with patient survival. Deleting PIEZO1 suppresses the growth of glioblastoma stem cells, inhibits tumor development, and prolongs mouse survival. Focal mechanical force activates prominent PIEZO1-dependent currents from glioma cell processes, but not soma. PIEZO1 localizes at focal adhesions to activate integrin-FAK signaling, regulate extracellular matrix, and reinforce tissue stiffening. In turn, a stiffer mechanical microenvironment elevates PIEZO1 expression to promote glioma aggression. Therefore, glioma cells are mechanosensory in a PIEZO1-dependent manner, and targeting PIEZO1 represents a strategy to break the reciprocal, disease-aggravating feedforward circuit between tumor cell mechanotransduction and the aberrant tissue mechanics. VIDEO ABSTRACT.

Genetic interaction between Gli3 and Ezh2 during limb pattern formation.

Anteroposterior polarity of the early limb bud is essential for proper skeletal pattern formation. In order to establish anterior identity, hedgehog signalling needs to be repressed by GLI3 repressor activity, although the mechanism of repression is not well defined. Here we describe genetic interaction between Gli3 and Enhancer of Zeste 2 (Ezh2) that encodes the histone methyltransferase subunit of Polycomb Repressive Complex 2. Loss of anterior limb identity was evident in both Gli3 and conditional Ezh2 single mutant embryos. This phenotype was enhanced in Ezh2;Gli3 double mutant embryos, but more closely resembled that of Ezh2 single mutants. Absent anterior skeletal elements in the Ezh2 mutant background were not rescued by either reduction of Gli activator or forced expression of Gli repressor. The data imply that Ezh2 is epistatic to Gli3 and suggest the possibility that hedghehog activation is repressed by the recruitment of polycomb repressive complex 2.

Intermittent fasting promotes adipose thermogenesis and metabolic homeostasis via VEGF-mediated alternative activation of macrophage.

Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction. However, our understanding of the underlying mechanisms of IF-mediated metabolic benefits is limited. Here we show that isocaloric IF improves metabolic homeostasis against diet-induced obesity and metabolic dysfunction primarily through adipose thermogenesis in mice. IF-induced metabolic benefits require fasting-mediated increases of vascular endothelial growth factor (VEGF) expression in white adipose tissue (WAT). Furthermore, periodic adipose-VEGF overexpression could recapitulate the metabolic improvement of IF in non-fasted animals. Importantly, fasting and adipose-VEGF induce alternative activation of adipose macrophage, which is critical for thermogenesis. Human adipose gene analysis further revealed a positive correlation of adipose VEGF-M2 macrophage-WAT browning axis. The present study uncovers the molecular mechanism of IF-mediated metabolic benefit and suggests that isocaloric IF can be a preventive and therapeutic approach against obesity and metabolic disorders.

A genetic female mouse model with congenital genitourinary anomalies and adult stages of urinary incontinence.

AIMS: To characterize the urinary incontinence observed in adult Gli2+/- ; Gli3Δ699/+ female mice and identify the defects underlying the condition. METHODS: Gli2+/- and Gli3Δ699/+ mice were crossed to generate: wild-type, mutant Gli2 (Gli2+/- ), mutant Gli3 (Gli3Δ699/+ ), and double mutant (Gli2+/- ; Gli3Δ699/+ ) female mice, verified via Polymerase Chain Reactions. Bladder functional studies including cystometrogram (CMG), leak point pressure (LPP), and voiding testing were performed on adult female mice. Female bladders and urethras were also analyzed via ink injection and histological assays. RESULTS: CMG tracing showed no signal corresponding to the filling of the Gli2+/- ; Gli3Δ699/+ bladders. LPP were significantly reduced in Gli2+/- ; Gli3Δ699/+ mice compared to wild-type mice. CMG studies revealed a decrease in peak micturition pressure values in Gli2+/- ; Gli3Δ699/+ mice compared with all other groups. No significant differences between mutant and wild-type mice were detected in urinary output. Histological analyses revealed Gli2+/- ; Gli3Δ699/+ mice exhibited a widened urethra and a decrease in smooth muscle layer thickness in the bladder outlet and urethra, with increased mucosal folding. CONCLUSIONS: Gli2+/- ; Gli3Δ699/+ adult female mice display persistent urinary incontinence due to the malformation of the bladder outlet and urethra. This presents a consistent and reliable genetic mouse model for female urinary incontinence and alludes to the key role of genetic factors involved in the condition.