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1.
Cell ; 181(6): 1246-1262.e22, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32442405

RESUMO

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.


Assuntos
Quinase do Linfoma Anaplásico/genética , Magreza/genética , Tecido Adiposo/metabolismo , Adulto , Animais , Linhagem Celular , Estudos de Coortes , Drosophila/genética , Estônia , Feminino , Humanos , Leptina/genética , Lipólise/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Interferência de RNA/fisiologia , Adulto Jovem
2.
Cell ; 140(1): 148-60, 2010 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-20074523

RESUMO

Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. We performed a systems-level genetic dissection of adiposity regulation using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo and revealed hedgehog signaling as the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, we generated fat-specific hedgehog-activation mutant mice. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals.


Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Obesidade/genética , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Adipogenia , Animais , AMP Cíclico/metabolismo , Glucocorticoides/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células Musculares/metabolismo , Proteínas Repressoras/genética
3.
Annu Rev Cell Dev Biol ; 27: 513-37, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21801010

RESUMO

Gli zinc-finger proteins are transcription factors involved in the intracellular signal transduction controlled by the Hedgehog family of secreted molecules. They are frequently mutated in human congenital malformations, and their abnormal regulation leads to tumorigenesis. Genetic studies in several model systems indicate that their activity is tightly regulated by Hedgehog signaling through various posttranslational modifications, including phosphorylation, ubiquitin-mediated degradation, and proteolytic processing, as well as through nucleocytoplasmic shuttling. In vertebrate cells, primary cilia are required for the sensing of Hedgehog pathway activity and involved in the processing and activation of Gli proteins. Two evolutionarily conserved Hedgehog pathway components, Suppressor of fused and Kif7, are core intracellular regulators of mammalian Gli proteins. Recent studies revealed that Gli proteins are also regulated transcriptionally and posttranslationally through noncanonical mechanisms independent of Hedgehog signaling. In this review, we describe the regulation of Gli proteins during development and discuss possible mechanisms for their abnormal activation during tumorigenesis.


Assuntos
Proteínas Hedgehog/metabolismo , Proteínas Oncogênicas/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transformação Celular Neoplásica , Cromatina/metabolismo , Cílios/metabolismo , Anormalidades Congênitas , Extremidades/anatomia & histologia , Extremidades/crescimento & desenvolvimento , Proteínas Hedgehog/genética , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/metabolismo , Tubo Neural/metabolismo , Proteínas Oncogênicas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Transativadores/genética , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco , Dedos de Zinco
4.
Proc Natl Acad Sci U S A ; 119(43): e2206571119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36252002

RESUMO

Development of mammalian auditory epithelium, the organ of Corti, requires precise control of both cell cycle withdrawal and differentiation. Sensory progenitors (prosensory cells) in the cochlear apex exit the cell cycle first but differentiate last. Sonic hedgehog (Shh) signaling is required for the spatiotemporal regulation of prosensory cell differentiation, but the underlying mechanisms remain unclear. Here, we show that suppressor of fused (Sufu), a negative regulator of Shh signaling, is essential for controlling the timing and progression of hair cell (HC) differentiation. Removal of Sufu leads to abnormal Atoh1 expression and a severe delay of HC differentiation due to elevated Gli2 mRNA expression. Later in development, HC differentiation defects are restored in the Sufu mutant by the action of speckle-type PDZ protein (Spop), which promotes Gli2 protein degradation. Deletion of both Sufu and Spop results in robust Gli2 activation, exacerbating HC differentiation defects. We further demonstrate that Gli2 inhibits HC differentiation through maintaining the progenitor state of Sox2+ prosensory cells. Along the basal-apical axis of the developing cochlea, the Sox2 expression level is higher in the progenitor cells than in differentiating cells and is down-regulated from base to apex as differentiation proceeds. The dynamic spatiotemporal change of Sox2 expression levels is controlled by Shh signaling through Gli2. Together, our results reveal key functions of Gli2 in sustaining the progenitor state, thereby preventing HC differentiation and in turn governing the basal-apical progression of HC differentiation in the cochlea.


Assuntos
Células Ciliadas Auditivas , Proteínas Hedgehog , Animais , Diferenciação Celular/genética , Cóclea/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , Proteínas Hedgehog/metabolismo , Mamíferos/genética , RNA Mensageiro/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo
5.
Development ; 147(19)2020 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-32907847

RESUMO

Pattern formation is influenced by transcriptional regulation as well as by morphogenetic mechanisms that shape organ primordia, although factors that link these processes remain under-appreciated. Here we show that, apart from their established transcriptional roles in pattern formation, IRX3/5 help to shape the limb bud primordium by promoting the separation and intercalation of dividing mesodermal cells. Surprisingly, IRX3/5 are required for appropriate cell cycle progression and chromatid segregation during mitosis, possibly in a nontranscriptional manner. IRX3/5 associate with, promote the abundance of, and share overlapping functions with co-regulators of cell division such as the cohesin subunits SMC1, SMC3, NIPBL and CUX1. The findings imply that IRX3/5 coordinate early limb bud morphogenesis with skeletal pattern formation.


Assuntos
Cromátides/metabolismo , Proteínas de Homeodomínio/metabolismo , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Segregação de Cromossomos/genética , Segregação de Cromossomos/fisiologia , Feminino , Imunofluorescência , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Imunoprecipitação , Espectrometria de Massas , Camundongos , Mitose/genética , Mitose/fisiologia , Gravidez , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genética
6.
Am J Physiol Heart Circ Physiol ; 322(5): H725-H741, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35245131

RESUMO

Previous studies have established that transmural gradients of the fast transient outward K+ current (Ito,f) correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high Ito,f expression in the epimyocardium (EPI) being associated with short APs and low contractility and vice versa. Herein, we investigated the effects of altering the Ito,f gradients on transmural contractile properties using mice lacking Irx5 (Irx5-KO) or lacking Kcnd2 (KV4.2-KO) or both. Irx5-KO mice exhibited decreased global LV contractility in association with elevated Ito,f, as well as reduced cell shortening and Ca2+ transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of Irx5 ablation on ECC-related genes was to increase Ito,f gene expression (i.e., Kcnd2 and Kcnip2) in the ENDO, but not the EPI. By contrast, KV4.2-KO mice showed selective increases in cell shortening and Ca2+ transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both Irx5 and Kcnd2 displayed elevated ventricular contractility, comparable to KV4.2-KO mice, demonstrating a dominant role of Irx5-dependent modulation of Ito,f in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the Irx5-dependent Ito,f gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart.NEW & NOTEWORTHY Irx5 is a vital transcription factor that establishes the transmural heterogeneity of ventricular myocyte contractility, thereby ensuring proper contractile function in the healthy heart. Regional differences in excitation-contraction coupling in the ventricular myocardium are primarily mediated through the inverse relationship between Irx5 and the fast transient outward K+ current (Ito,f) across the ventricular wall.


Assuntos
Ventrículos do Coração , Miocárdio , Potenciais de Ação/fisiologia , Animais , Ventrículos do Coração/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Int J Obes (Lond) ; 46(11): 2029-2039, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36115924

RESUMO

OBJECTIVE: Obesity, a leading cause of several metabolic abnormalities, is mainly caused by imbalanced energy homeostasis. IRX3 and IRX5 have been suggested as genetic determinants of obesity in connection with the intronic variants of the FTO gene, the strongest genetic risk factor of polygenic obesity in humans. Although the causal effects of Irx3 and its cooperation with Irx5 in obesity and associated metabolic abnormalities have been demonstrated in vivo, the function of Irx5 in energy homeostasis remains unclear. Here we aim to decipher the actions of Irx5 in the regulation of obesity and metabolic abnormalities. METHODS: We employed a mouse model homozygous for an Irx5-knockout (Irx5KO) allele and determined its metabolic phenotype in the presence or absence of a high-fat diet challenge. To investigate the function of Irx5 in the regulation of energy homeostasis, adipose thermogenesis and hypothalamic leptin response were assessed, and single-cell RNA sequencing (scRNA-seq) in the hypothalamic arcuate-median eminence (ARC-ME) was conducted. RESULTS: Irx5KO mice were leaner and resistant to diet-induced obesity as well as associated metabolic abnormalities, primarily through loss of adiposity. Assessments of energy expenditure and long-term dietary intake revealed that an increase in basal metabolic rate with adipose thermogenesis and a reduction of food intake with improved hypothalamic leptin response in Irx5KO mice may contribute to the anti-obesity effects. Utilizing scRNA-seq and marker gene analyses, we demonstrated the number of ARC-ME neurons was elevated in Irx5KO mice, suggesting a direct role for Irx5 in hypothalamic feeding control. CONCLUSIONS: Our study demonstrates that Irx5 is a genetic factor determining body mass/composition and obesity and regulates both energy expenditure and intake.


Assuntos
Leptina , Obesidade , Humanos , Animais , Camundongos , Leptina/metabolismo , Obesidade/genética , Obesidade/metabolismo , Dieta Hiperlipídica , Hipotálamo/metabolismo , Metabolismo Energético/genética , Camundongos Knockout , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo
8.
Asia Pac J Clin Nutr ; 31(2): 282-293, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35766564

RESUMO

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.


Assuntos
Sobrepeso , Obesidade Infantil , Adolescente , Índice de Massa Corporal , Criança , Pré-Escolar , Dieta , Glucose , Governo , Humanos , Insulina , Sobrepeso/terapia , Obesidade Infantil/prevenção & controle , Ácido Úrico , Redução de Peso
9.
PLoS Genet ; 14(8): e1007488, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30071018

RESUMO

Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan.


Assuntos
Células da Granulosa/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Comunicação Celular , Conexinas/genética , Conexinas/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Nus , Oócitos/fisiologia , Fatores de Transcrição/genética
10.
Gastroenterology ; 157(6): 1556-1571.e5, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31442438

RESUMO

BACKGROUND & AIMS: It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs. METHODS: We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation. Intestine tissues were collected from Tg(GBS-GFP), Sufuf/f; Wnt1-cre, Ptch1+/-, and Gli3Δ699/Δ699 mice and analyzed by flow cytometry and immunofluorescence for levels of messenger RNAs encoding factors in the hedgehog signaling pathway during differentiation of enteric NCs. Human NC cells (HNK-1+p75NTR+) were derived from IMR90 and UE02302 hPSC lines. hPSCs were incubated with a hedgehog agonist (smoothened agonist [SAG]) and antagonists (cyclopamine) and analyzed for differentiation. hPSC-based innervated colonic organoids were derived from these hPSC lines and analyzed by immunofluorescence and neuromuscular coupling assay for expression of neuronal subtype markers and assessment of the functional maturity of the hPSC-derived neurons, respectively. RESULTS: Single-cell RNA sequencing analysis showed that neural fate acquisition by human and mouse enteric NC cells requires reduced expression of NC- and cell cycle-specific genes and up-regulation of neuronal or glial lineage-specific genes. Activation of the hedgehog pathway was associated with progression of mouse enteric NCs to the more mature state along the neuronal and glial lineage differentiation trajectories. Activation of the hedgehog pathway promoted development of cultured hPSCs into NCs of greater neurogenic potential by activating expression of genes in the neurogenic lineage. The hedgehog agonist increased differentiation of hPSCs into cells of the neuronal lineage by up-regulating expression of GLI2 target genes, including INSM1, NHLH1, and various bHLH family members. The hedgehog agonist increased expression of late neuronal markers and neuronal activities in hPSC-derived neurons. CONCLUSIONS: In enteric NCs from humans and mice, activation of hedgehog signaling promotes differentiation into neurons by promoting cell-state transition, expression of genes in the neurogenic lineage, and functional maturity of enteric neurons.


Assuntos
Diferenciação Celular , Proteínas Hedgehog/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Sistema Nervoso Entérico/citologia , Perfilação da Expressão Gênica/métodos , Proteínas Hedgehog/genética , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/inervação , Masculino , Camundongos , Camundongos Transgênicos , Crista Neural/citologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos
11.
Biol Reprod ; 103(3): 620-629, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32507881

RESUMO

Healthy development of ovarian follicles depends on appropriate interactions and function between oocytes and their surrounding granulosa cells. Previously, we showed that double knockout of Irx3 and Irx5 (Irx3/5 DKO) in mice resulted in abnormal follicle morphology and follicle death. Further, female mouse models of individual Irx3 or Irx5 knockouts were both subfertile but with distinct defects. Notably, the expression profile of each gene suggests independent roles for each; first, they are colocalized in pre-granulosa cells during development that then progresses to include oocyte expression during germline nest breakdown and primordial follicle formation. Thereafter, their expression patterns diverge between oocytes and granulosa cells coinciding with the formulation and maturation of intimate oocyte-granulosa cell interactions. The objective of this study was to investigate the contributions of Irx5 and somatic cell-specific expression of Irx3 during ovarian development. Our results show that Irx3 and Irx5 contribute to female fertility through different mechanisms and that Irx3 expression in somatic cells is important for oocyte quality and survival. Based on evaluation of a series of genetically modified mouse models, we conclude that IRX3 and IRX5 collaborate in the same cells and then in neighboring cells to foster a healthy and responsive follicle. Long after these two factors have extinguished, their legacy enables these intercellular connections to mature and respond to extracellular signals to promote follicle maturation and ovulation.


Assuntos
Células da Granulosa/fisiologia , Proteínas de Homeodomínio/genética , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , Fatores de Transcrição/genética , Animais , Feminino , Fertilidade/genética , Infertilidade/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Folículo Ovariano/citologia , Ovário/citologia , Gravidez , Diferenciação Sexual
12.
J Pathol ; 247(1): 86-98, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30246301

RESUMO

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.


Assuntos
Proteínas de Homeodomínio/metabolismo , Neoplasias Renais/metabolismo , Néfrons/metabolismo , Fatores de Transcrição/metabolismo , Tumor de Wilms/metabolismo , Animais , Carcinogênese , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Camundongos Knockout , Morfogênese , Néfrons/crescimento & desenvolvimento , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Tumor de Wilms/genética , Tumor de Wilms/patologia , Via de Sinalização Wnt , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo
13.
Nature ; 507(7492): 371-5, 2014 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-24646999

RESUMO

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.


Assuntos
Proteínas de Homeodomínio/genética , Íntrons/genética , Oxigenases de Função Mista/genética , Obesidade/genética , Oxo-Ácido-Liases/genética , Proteínas/genética , Fatores de Transcrição/genética , Tecido Adiposo/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Animais , Metabolismo Basal/genética , Índice de Massa Corporal , Peso Corporal/genética , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta , Genes Dominantes/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Magreza/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética
14.
J Urol ; 201(2): 386-392, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30063927

RESUMO

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.


Assuntos
3-Hidroxiantranilato 3,4-Dioxigenase/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hipospadia/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenase/metabolismo , Adolescente , Animais , Povo Asiático/genética , Criança , Pré-Escolar , Ectoderma/metabolismo , Embrião de Mamíferos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Lactente , Recém-Nascido , Japão , Masculino , Camundongos , Camundongos Endogâmicos ICR , Organogênese/genética , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Uretra/crescimento & desenvolvimento
15.
N Engl J Med ; 373(10): 895-907, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-26287746

RESUMO

BACKGROUND: Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. METHODS: We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR-Cas9 genome editing in samples from patients. RESULTS: Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR-Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. CONCLUSIONS: Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.).


Assuntos
Adipócitos/metabolismo , Obesidade/genética , Proteínas/genética , Termogênese/genética , Alelos , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Animais , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epigenômica , Expressão Gênica , Engenharia Genética , Humanos , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Obesidade/metabolismo , Fenótipo , Edição de RNA , Risco , Termogênese/fisiologia
16.
Neurourol Urodyn ; 36(8): 1981-1987, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28244147

RESUMO

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.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Incontinência Urinária/genética , Anormalidades Urogenitais/genética , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/genética , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Transdução de Sinais/fisiologia
17.
Genes Dev ; 23(16): 1910-28, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19684112

RESUMO

A central question in Hedgehog (Hh) signaling is how evolutionarily conserved components of the pathway might use the primary cilium in mammals but not fly. We focus on Suppressor of fused (Sufu), a major Hh regulator in mammals, and reveal that Sufu controls protein levels of full-length Gli transcription factors, thus affecting the production of Gli activators and repressors essential for graded Hh responses. Surprisingly, despite ciliary localization of most Hh pathway components, regulation of Gli protein levels by Sufu is cilium-independent. We propose that Sufu-dependent processes in Hh signaling are evolutionarily conserved. Consistent with this, Sufu regulates Gli protein levels by antagonizing the activity of Spop, a conserved Gli-degrading factor. Furthermore, addition of zebrafish or fly Sufu restores Gli protein function in Sufu-deficient mammalian cells. In contrast, fly Smo is unable to translocate to the primary cilium and activate the mammalian Hh pathway. We also uncover a novel positive role of Sufu in regulating Hh signaling, resulting from its control of both Gli activator and repressor function. Taken together, these studies delineate important aspects of cilium-dependent and cilium-independent Hh signal transduction and provide significant mechanistic insight into Hh signaling in diverse species.


Assuntos
Cílios/metabolismo , Evolução Molecular , Proteínas Hedgehog/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Animais , Proteína Axina , Linhagem Celular Transformada , Drosophila , Proteínas de Drosophila/metabolismo , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Receptores Patched , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Repressoras/genética , Receptor Smoothened , Complexos Ubiquitina-Proteína Ligase , Regulação para Cima , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de Zinco
18.
Dev Biol ; 397(2): 191-202, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25448692

RESUMO

Ptch1 and Ptch2 are highly conserved vertebrate homologs of Drosophila ptc, the receptor of the Hedgehog (Hh) signaling pathway. The vertebrate Ptch1 gene encodes a potent tumor suppressor and is well established for its role in embryonic development. In contrast, Ptch2 is poorly characterized and dispensable for embryogenesis. In flies and mice, ptc/Ptch1 controls Hh signaling through the regulation of Smoothened (Smo). In addition, Hh pathway activation also up-regulates ptc/Ptch1 expression to restrict the diffusion of the ligand. Recent studies have implicated Ptch2 in this ligand dependent antagonism, however whether Ptch2 encodes a functional Shh receptor remains unclear. In this report, we demonstrate that Ptch2 is a functional Shh receptor, which regulates Smo localization and activity in vitro. We also show that Ptch1 and Ptch2 are co-expressed in the developing mouse limb bud and loss of Ptch2 exacerbates the outgrowth defect in the limb-specific Ptch1 knockout mutants, demonstrating that Ptch1 and Ptch2 co-operate in regulating cellular responses to Shh in vivo.


Assuntos
Extremidades/embriologia , Morfogênese/fisiologia , Receptores de Superfície Celular/metabolismo , Animais , Western Blotting , Linhagem Celular , Proteínas Hedgehog/metabolismo , Hibridização In Situ , Camundongos , Camundongos Knockout , Morfogênese/genética , Receptores Patched , Receptor Patched-1 , Receptor Patched-2 , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened
19.
Gastroenterology ; 149(7): 1837-1848.e5, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26261006

RESUMO

BACKGROUND & AIMS: Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. METHODS: We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. RESULTS: We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. CONCLUSIONS: We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.


Assuntos
Sistema Nervoso Entérico/anormalidades , Doença de Hirschsprung/genética , Doença de Hirschsprung/patologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/patologia , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Estudos de Casos e Controles , Linhagem da Célula , Movimento Celular , Análise Mutacional de DNA/métodos , Modelos Animais de Doenças , Sistema Nervoso Entérico/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Doença de Hirschsprung/diagnóstico , Doença de Hirschsprung/metabolismo , Humanos , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Crista Neural/metabolismo , Neurogênese , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de Zinco
20.
J Hum Genet ; 61(5): 443-6, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26791356

RESUMO

Tibial hemimelia is a rare, debilitating and often sporadic congenital deficiency. In syndromic cases, mutations of a Sonic hedgehog (SHH) enhancer have been identified. Here we describe an ~5 kb deletion within the SHH repressor GLI3 in two patients with bilateral tibial hemimelia. This deletion results in a truncated GLI3 protein that lacks a DNA-binding domain and cannot repress hedgehog signaling. These findings strengthen the concept that tibial hemimelia arises because of failure to restrict SHH activity to the posterior aspect of the limb bud.


Assuntos
Ectromelia/diagnóstico , Ectromelia/genética , Fatores de Transcrição Kruppel-Like , Mutação , Proteínas do Tecido Nervoso , Fenótipo , Tíbia/anormalidades , Animais , Linhagem Celular , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Éxons , Estudos de Associação Genética , Humanos , Mutação INDEL , Camundongos , Polimorfismo de Nucleotídeo Único , Esqueleto/diagnóstico por imagem , Esqueleto/patologia , Proteína Gli3 com Dedos de Zinco
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