Left ventricular non-compaction cardiomyopathy (LVNC) is a rare heart condition characterized by excessive trabeculae and intertrabecular recesses in the left ventricle of the heart. It is believed to be caused by abnormal trabecular compaction process, which involves trabecular remodelling to form a thick myocardial wall. Embryonic trabeculae consist of bipotent progenitors that differentiate into the ventricular conduction system (VCS) and contractile cardiomyocytes, yet it remains to be further understood how the pathways involved in trabecular cell specifications play a role in the compaction process. Here we present two transcription factors, Iroquois homeobox 3 ( Irx3 ) and Irx4 , playing antagonistic and cooperative roles during trabecular specification and maturation, thereby facilitating proper ventricular compaction. Our single-cell RNA sequencing analysis revealed that the differentiation of trabecular cells into VCS cells was accompanied by increased Irx3 but decreased Irx4 expression. In addition, we found that the loss of Irx4 led to both an increased expression level of Irx3 and an expansion in the number of Irx3 -expressing cells. Conversely, the loss of Irx3 resulted in an upregulation of Irx4 expression within VCS cells. Next, to investigate the functional importance of the antagonistic relationship between Irx3 and Irx4 in trabecular differentiation, we generated Irx3 and Irx4 compound knockout (KO) mice and examined the structure and function of their hearts. Notably, unlike Irx3 KO hearts showing normal ventricular myocardium, Irx4 KO hearts exhibited mild LVNC, and Irx3 and Irx4 double knockouts ( Irx3 /4dKO) hearts displayed severe LVNC. Multiome sequencing of E14.5 ventricles revealed that Irx3 /4dKO ventricles contained an increased number of trabecular cells, expressing higher levels of cell proliferation markers and altered trabecular identity genes, compared to littermate controls. Collectively, our results demonstrate that balanced antagonism between Irx3 and Irx4 is crucial for establishing trabecular identity. This highlights the importance of proper differentiation of trabeculae into the VCS and non-VCS contractile cells during ventricular compaction, thereby preventing noncompaction cardiomyopathy.

Introduction and objectiveStress urinary incontinence is of concern in both pediatric and adult population. Double mutant GLI family zinc finger Gli2 +/-/-; Gli3A699/+/- 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 +/-/-; Gli3A699/+/- mice.Study designWe 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 +/-/- fe-males, 2 Wild Gli2 +/-/-males, 4 Gli2 +/-/-;Gli3A699/+/- females and 4 Gli2 +/-/-;Gli3A699/+/- males. The Wild Gli2 +/-/-mice served as the control group and Gli2 +/-/-;Gli3A699/+/- 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 +/-/-;Gli3A699/+/- females and Gli2 +/-/-;Gli3A699/+/- 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.ResultsGross and histologic appearance confirmed the pre-viously reported widening of bladder outlet and hy-poplasia of smooth muscles in female urethra and also established them in the male urethra ofGli2 +/-/-;Gli3A699/+/- mice compared to Gli2 +/-/-mice. The double knockout mice were smaller than the Gli2 mice (5.2 vs 6.1 cm, p Z 0.002). Immunohis-tochemistry demonstrated epithelial hyperplasia and smooth muscle hypoplasia. Additionally, there was prostatic hypoplasia in the Gli2 +/-/-;Gli3A699/+/- male mice. The spinal cord length for body size appeared comparable between the Gli2 +/-/-and Gli2 +/-/-;Gli3A699/+/- mice but histological evaluation revealed abnormal development of the caudal end of the vertebral body with premature termination of the spinal cord (Figure).DiscussionThe histological changes in the bladder neck and urethra were consistent to those previously re-ported. While previous report described the findings in female mice only, we confirmed that these find-ings 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 +/-/-; Gli3A699/+/- mice which has not been re-ported previously and is likely a major contributor to incontinence in this model.ConclusionThe incontinence in male as well as female Gli2 +/-/-; Gli3A699/+/- 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.

Irx1 and Irx2 ( Irx1/2 ) are two closely linked and widely expressed members of the conserved Iroquois homeobox family of transcription factors. Despite mounting evidence suggesting the importance of homologs of these genes in many aspects of vertebrate development and function, the role of Irx1/2 in mammals has remained largely unknown. Here, we used mice carrying our newly generated Irx1 flox and Irx1 flox Irx2 del mutant alleles to perform a stepwise genetic ablation of Irx1 and Irx2 levels. Our analysis revealed reduced postnatal growth and viability of Irx1 KO mice with gross histological defects in the lung and gut and demonstrated that ablation of one copy of Irx2 in these mice results in neonatal lethality with exacerbated phenotypic defects. Conversely, while Irx2 KO mice appear normal, ablation of one copy of Irx1 in these mutants leads to lethality at weaning. Furthermore, we found that homozygous deletion of both Irx1 and Irx2 results in embryonic lethality by mid-gestation with defective extraembryonic vasculature. Our results illustrate that Irx1 and Irx2 play distinct dose-dependent cooperative functions during both the early and late stages of mouse development.

Obesity is mainly due to excessive food intake. IRX3 and IRX5 have been suggested as determinants of obesity in connection with the intronic variants of FTO, but how these genes contribute to obesity via changes in food intake remains unclear. Here, we show that mice doubly heterozygous for Irx3 and Irx5 mutations exhibit lower food intake with enhanced hypothalamic leptin response. By lineage tracing and single-cell RNA sequencing using the Ins2-Cre system, we identify a previously unreported radial glia-like neural stem cell population with high Irx3 and Irx5 expression in early postnatal hypothalamus and demonstrate that reduced dosage of Irx3 and Irx5 promotes neurogenesis in postnatal hypothalamus leading to elevated numbers of leptin-sensing arcuate neurons. Furthermore, we find that mice with deletion of Irx3 in these cells also exhibit a similar food intake and hypothalamic phenotype. Our results illustrate that Irx3 and Irx5 play a regulatory role in hypothalamic postnatal neurogenesis and leptin response.

Left ventricular noncompaction (LVNC) is a primary congenital cardiomyopathy characterized by prominent trabeculation and intra-trabecular recesses in the ventricular wall. Its genetic basis and pathologic mechanism remain largely unknown. Here it is shown that Irx3;Irx4 double knockout (DKO) mouse mutants represent a novel model of LVNC. Irx3 and Irx4 belong to the family of Iroquois homeobox (Irx) genes, encoding transcription factors important for cardiac function. Mice lacking either Irx3 or Irx4 do not have gross cardiac malformations. In contrast, Irx3;Irx4DKO mice exhibit postnatal lethality and thinner ventricular walls as early as E14.5. By P14, DKO mice have prominent noncompacted trabeculation and abnormal muscular growth that resemble human LVNC. Furthermore, Irx3;Irx4DKO hearts exhibit increased expression of heart failure marker genes, elevated Bmp10 pathway activity, and altered pattern of cardiomyocyte proliferation, each of which are implicated in the pathogenesis of LVNC. Taken together, these studies suggest that Irx3 and Irx4 possess overlapping functions, which are essential to myocardial compaction.%%%%M.Sc.%%%%2019-11-07 00:00:00

The paraventricular nucleus of the hypothalamus (PVH) contains a heterogeneous cluster of Sim1-expressing neurons critical for feeding regulation. Sim1 haploinsufficiency results in hyperphagic obesity with disruption of PVH neurons, yet the molecular profiles of PVH neurons and the mechanism underlying the defects of Sim1 haploinsufficiency are not well understood. By single-cell RNA sequencing, we identified two major populations of Sim1+ PVH neurons, which are differentially affected by Sim1 haploinsufficiency. The Iroquois homeobox genes Irx3 and Irx5 have been implicated in the hypothalamic control of energy homeostasis. We found that Irx3 and Irx5 are ectopically expressed in the Sim1+ PVH cells of Sim1+/− mice. By reducing their dosage and PVH-specific deletion of Irx3, we demonstrate that misexpression of Irx3 and Irx5 contributes to the defects of Sim1+/− mice. Our results illustrate abnormal hypothalamic activities of Irx3 and Irx5 as a central mechanism disrupting PVH development and feeding regulation in Sim1 haploinsufficiency.

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 postoperative 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 histo-logically 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.

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.

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.

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology II1 Apr 2017MP82-01 A GENETIC FEMALE MOUSE MODEL WITH CONGENITAL GENITOURINARY ANOMALIES AND URINARY INCONTINENCE Akbari Pedram, Ali Fathollahi, Rong Mo, Michael Chua, Michael Kavran, Nicole Episalla, Chi-Chung Hui, Walid Farhat, and Adonis Hijaz Akbari PedramAkbari Pedram More articles by this author , Ali FathollahiAli Fathollahi More articles by this author , Rong MoRong Mo More articles by this author , Michael ChuaMichael Chua More articles by this author , Michael KavranMichael Kavran More articles by this author , Nicole EpisallaNicole Episalla More articles by this author , Chi-Chung HuiChi-Chung Hui More articles by this author , Walid FarhatWalid Farhat More articles by this author , and Adonis HijazAdonis Hijaz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2548AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hedgehog signaling pathway is known to have important role in the urogenital development. Transcription mediators of the pathway, Gli2 and Gli3, have been shown to be heavily involved in proper urogenital sinus formation. Both Gli2 and Gli3 null mice are non-viable, and display severe urogenital ad hindgut malformations. Here, we have generated a compound genetic mutant, Gli2+/-;Gli3Δ699/+, that is viable well into adulthood, and displaying variable urogenital malformations including urinary incontinence in its females. We aim to characterize the urinary incontinence observed in Gli2+/-; Gli3Δ699/+ female mice and assess its functional, anatomical, and histological characteristics. METHODS Gli2+/- and Gli3Δ699/+ mice were crossed to generate the double mutant (Gli2+/-; Gli3Δ699/+) female mice and wild type female mice were used as comparison controls; which all were verified via Polymerase Chain Reactions. Void measurements, Cystometrogram (CMG) and leak point pressure (LPP) were performed in all genotypes to assess bladder functions. The mice were then sacrificed to harvest the bladders and urethras for gross characterization via ink injection and histological assays. Differences were reported as mean and standard errors of mean (SEM) and analyzed using univariate analysis. Statistical significance set at 0.05. RESULTS No significant differences between the mutant and wild type mice were detected for 24 hour urinary output [(n= 13) mean 26.5cc±5 vs ( n=7) mean 22.15cc±6, p=0.13]. CMG studies revealed a decrease in peak micturition pressure values and significantly reduced LPP in Gli2+/-; Gli3Δ699/+ mice compared to wild type mice [(n=5) 4.28 cmH2O±2.4 vs (n=4) 20.24 cmH2O±6.45, p<0.0001; (n=5) 6.66 cmH2O±1.6 vs (n=5) 26.5cmH2O±5, p<0.05; respectively]. Gross characterization revealed that the ano-genital distance was severely reduced in double mutant mice; however, the urethra, vagina, and anus all remain separate and distinctly identifiable in these mice. 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/+ female mice display persistent urinary incontinence with evident malformation of the bladder outlet and urethra. This presents a genetic mouse model for female urinary incontinence and alludes to potential genetic factors involved in the human condition. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1097 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Akbari Pedram More articles by this author Ali Fathollahi More articles by this author Rong Mo More articles by this author Michael Chua More articles by this author Michael Kavran More articles by this author Nicole Episalla More articles by this author Chi-Chung Hui More articles by this author Walid Farhat More articles by this author Adonis Hijaz More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

AIMSTo characterize the urinary incontinence observed in adult Gli2(+/-); Gli3(699/+) female mice and identify the defects underlying the condition. METHODSGli2(+/-) and Gli3(699/+) mice were crossed to generate: wild-type, mutant Gli2 (Gli2(+/-)), mutant Gli3 (Gli3(699/+)), and double mutant (Gli2(+/-); Gli3(699/+)) 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. RESULTSCMG tracing showed no signal corresponding to the filling of the Gli2(+/-); Gli3(699/+) bladders. LPP were significantly reduced in Gli2(+/-); Gli3(699/+) mice compared to wild-type mice. CMG studies revealed a decrease in peak micturition pressure values in Gli2(+/-); Gli3(699/+) mice compared with all other groups. No significant differences between mutant and wild-type mice were detected in urinary output. Histological analyses revealed Gli2(+/-); Gli3(699/+) mice exhibited a widened urethra and a decrease in smooth muscle layer thickness in the bladder outlet and urethra, with increased mucosal folding. CONCLUSIONSGli2(+/-); Gli3(699/+) 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.

Left ventricular noncompaction (LVNC) is a primary congenital cardiomyopathy characterized by prominent trabeculations and intra-trabecular recesses in the ventricular wall. This disease is suggest...

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.

Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.

The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh), a morphogen that regulates the activity of Gli transcription factors. However, Shh expression is not observed during the first 12 hr of limb development. During this phase, the limb bud is prepatterned into anterior and posterior regions through the antagonistic actions of transcription factors Gli3 and Hand2. We demonstrate that precocious activation of Shh signaling during this early phase interferes with the Gli3-dependent specification of anterior progenitors, disturbing establishment of signaling centers and normal outgrowth of the limb. Our findings illustrate that limb development requires a sweet spot in the level and timing of pathway activation that allows for the Shh-dependent expansion of posterior progenitors without interfering with early prepatterning functions of Gli3/Gli3R or specification of anterior progenitors.

Limb skeletal pattern relies heavily on graded Sonic hedgehog (Shh) signaling. As a morphogen and growth cue, Shh regulates identities of posterior limb elements, including the ulna/fibula and digits 2 through 5. In contrast, proximal and anterior structures, including the humerus/femur, radius/tibia, and digit 1, are regarded as Shh independent, and mechanisms governing their specification are unclear. Here, we show that patterning of the proximal and anterior limb skeleton involves two phases. Irx3 and Irx5 (Irx3/5) are essential in the initiating limb bud to specify progenitors of the femur, tibia, and digit 1. However, these skeletal elements can be restored in Irx3/5 null mice when Shh signaling is diminished, indicating that Shh negatively regulates their formation after initiation. Our data provide genetic evidence supporting the concept of early specification and progressive determination of anterior limb pattern.