RESUMEN
The aim of this study was to evaluate whether high levels of exogenous testosterone (T) interfere in prostate morphogenesis. Pregnant females were exposed to subcutaneous injections of T cypionate (500 µg/animal) at gestational days 20 and 22. Male and female pups were euthanized at postnatal days 1 and 15. 15-day-old males had only fibroblast growth factor 10 (FGF10) immunostaining and nuclear form factor altered by the treatment, whereas treated females (T1 and T15) had almost all analyzed parameters changed. T1 females showed an increased anogenital distance (AGD), whereas T15 females had both AGD and ovary weight increased. T1 females had a higher number of epithelial buds emerging from the urethral and vaginal epithelium. We observed ectopic prostatic tissue surrounding the vagina in both T1 and T15 females. Moreover, the ectopic acini of T15 females showed delayed luminal formation, and there was a thickening of the periacinar smooth muscle layer (SML). Finally, FGF10 immunostaining intensity decreased in both T15 male and female prostates. Indeed, Sonic hedgehog (Shh) was upregulated in T15 female prostates, whereas no difference was observed between the male groups. These data showed that exogenous T changed the nuclear morphology of prostate epithelial cells in both males and females. Surprisingly, smooth muscle hyperplasia was also observed in the ectopic female prostate. Moreover, T downregulated FGF10 in both male and female prostates. Interestingly, the results suggest that FGF10 downregulation is mediated by the upregulation of Shh in females. In conclusion, exogenous T disrupts prostate development, particularly, affecting, the female.
Asunto(s)
Epitelio/metabolismo , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Proteínas Hedgehog/biosíntesis , Músculo Liso/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Próstata/metabolismo , Testosterona/toxicidad , Animales , Animales Recién Nacidos , Epitelio/efectos de los fármacos , Epitelio/patología , Femenino , Factor 10 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Gerbillinae , Proteínas Hedgehog/genética , Masculino , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Próstata/efectos de los fármacos , Próstata/patologíaRESUMEN
BACKGROUND: ALI/ARDS is the major cause of acute respiratory failure in critically ill patients. As human chorionic villi-derived MSCs (hCMSCs) could attenuate ALI in the airway injury model, and liraglutide, glucagon-like peptide 1 (GLP-1) agonist, possesses anti-inflammatory and proliferation promotion functions, we proposed to probe the potential combinatory effect of hCMSCs and liraglutide on ALI. METHODS: We examined the time- and dose-dependent manner of GLP-1R, SPC, Ang-1, and FGF-10 with LPS via western blot and qRT-PCR. Western blot and chromatin immunoprecipitation assay detected the effects of liraglutide on GLP-1R, SPC, Ang-1, and FGF-10 through PKAc/ß-catenin pathway and cAMP pathway. In the ALI animal model, we detected the effects of MSC and liraglutide combination on ALI symptoms by H&E staining, western blot, ELISA assays, calculating wet-to-dry ratio of the lung tissue, and counting neutrophils, leukocytes, and macrophages in mouse bronchoalveolar lavage fluid (BALF). RESULTS: The data demonstrated that LPS reduced hCMSC proliferation and GLP-1R, SPC, Ang-1, and FGF-10 levels in a dose- and time-dependent manner. Liraglutide significantly dampened the reduction of GLP-1R, SPC, Ang-1, and FGF-10 and reversed the effect of LPS on hCMSCs, which could be regulated by GLP-1R and its downstream cAMP/PKAc/ß-catenin-TCF4 signaling. Combination of hCMSCs with liraglutide showed more therapeutic efficacy than liraglutide alone in reducing LPS-induced ALI in the animal model. CONCLUSIONS: These results reveal that the combination of hCMSCs and liraglutide might be an effective strategy for ALI treatment.
Asunto(s)
Lesión Pulmonar Aguda/terapia , Hipoglucemiantes/uso terapéutico , Liraglutida/farmacología , Trasplante de Células Madre Mesenquimatosas/métodos , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Animales , Vellosidades Coriónicas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/biosíntesis , Humanos , Hipoglucemiantes/farmacología , Lipopolisacáridos/farmacología , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Transfección , beta Catenina/metabolismoRESUMEN
Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.
Asunto(s)
Haploinsuficiencia , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Factores de Transcripción Otx/metabolismo , Enfermedades de la Hipófisis/metabolismo , Hipófisis/metabolismo , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/genética , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/patología , Factores de Transcripción Otx/genética , Enfermedades de la Hipófisis/congénito , Enfermedades de la Hipófisis/patología , Hipófisis/patologíaRESUMEN
INTRODUCTION: Fibroblast growth factors (FGFs) play a crucial role in early embryogenesis of the genital tubercle and are involved in the development of hypospadias, affecting both endo- and ectodermally derived tissues. It was hypothesized that expression of FGFs could be qualitatively or quantitatively altered in skin of children with hypospadias. OBJECTIVE: The objective of the study was to investigate expression patterns and transcription levels of FGF8, FGF10, and FGF Receptor 2 (FGFR2) in patients with hypospadias compared to normal controls. PATIENTS AND METHODS: Skin samples from the ventro-lateral aspect of the foreskin of 32 patients with hypospadias (17 distal and 15 proximal, mean age 25 months) and 10 normal foreskin samples (mean age 77 months) were analyzed by immunohistochemistry. Staining, localization, and distribution of positive cells in epidermis and dermis were categorized independently by two researchers. Complementary DNA (cDNA) samples prepared from messenger RNA (mRNA) isolates of the same samples were analyzed by quantitative polymerase chain reaction (qPCR), comparing expressions of FGF8, FGF10, and FGFR2 with loading controls. RESULTS: Patients with hypospadias consistently showed aberrant immunohistochemical staining patterns for FGF8/FGF10/FGFR2 in epidermis and dermis compared to patients without penile malformation (p < 0.01 for all markers). qPCR displayed no difference in expression levels on mRNA level (FGFR2 p = 0.44, FGF8 p = 0.77, and FGF10 p = 0.17) comparing normal foreskin with foreskin from patients with hypospadias. Figure. DISCUSSION: The results point at an impact of FGF signaling during embryological development of hypospadias on skin, as an ectodermally derived tissue. Similar to the urethral development, this might be a result of mesothelial-epithelial interactions. The differing expression patterns in immunohistochemistry are not matched by a quantitative difference in marker expression on the mRNA level, putatively caused by post-translational modifications or alterations of the downstream pathway. FGFs, particularly FGF10 and FGFR2, are critically involved in wound healing. CONCLUSIONS: There are significant differences in localization and distribution of FGF8, FGF10, and FGFR2 in comparisons of normal foreskin to foreskin of patients with hypospadias, whereas there is no difference in the quantitative expression of these markers on the mRNA level. This confirms the notion that penile skin is affected as well by the embryological aberrations during the embryogenesis of hypospadias.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 8 de Crecimiento de Fibroblastos/biosíntesis , Factor 8 de Crecimiento de Fibroblastos/genética , Prepucio/metabolismo , Hipospadias/genética , Hipospadias/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/biosíntesis , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transcripción Genética , Niño , Preescolar , Factor 10 de Crecimiento de Fibroblastos/análisis , Factor 8 de Crecimiento de Fibroblastos/análisis , Prepucio/química , Expresión Génica , Humanos , Hipospadias/patología , Inmunohistoquímica , Lactante , Masculino , Estudios Prospectivos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/análisisRESUMEN
TGF beta is a multifunctional cytokine that is important in the pathogenesis of pulmonary fibrosis. The ability of TGF beta to stimulate smooth muscle actin and extracellular matrix gene expression in fibroblasts is well established. In this report, we evaluated the effect of TGF beta on the expression of HGF, FGF7 (KGF), and FGF10, important growth and survival factors for the alveolar epithelium. These growth factors are important for maintaining type II cells and for restoration of the epithelium after lung injury. Under conditions of normal serum supplementation or serum withdrawal TGF beta inhibited fibroblast expression of HGF, FGF7, and FGF10. We confirmed these observations with genome wide RNA sequencing of the response of control and IPF fibroblasts to TGF beta. In general, gene expression in IPF fibroblasts was similar to control fibroblasts. Reduced expression of HGF, FGF7, and FGF10 is another means whereby TGF beta impairs epithelial healing and promotes fibrosis after lung injury.
Asunto(s)
Fibroblastos/efectos de los fármacos , Fibrosis Pulmonar Idiopática/patología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Factor de Crecimiento Transformador beta/farmacología , Anciano , Anciano de 80 o más Años , Células Cultivadas , Medio de Cultivo Libre de Suero , Femenino , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 7 de Crecimiento de Fibroblastos/biosíntesis , Factor 7 de Crecimiento de Fibroblastos/genética , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Crecimiento de Hepatocito/biosíntesis , Factor de Crecimiento de Hepatocito/genética , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Genital malformations are among the most common human birth defects, and both genetic and environmental factors can contribute to these malformations. Development of the external genitalia in mammals relies on complex signaling networks, and disruption of these signaling pathways can lead to genital defects. Islet-1 (ISL1), a member of the LIM/Homeobox family of transcription factors, has been identified as a major susceptibility gene for classic bladder exstrophy in humans, a common form of the bladder exstrophy-epispadias complex (BEEC), and is implicated in a role in urinary tract development. We report that deletion of Isl1 from the genital mesenchyme in mice led to hypoplasia of the genital tubercle and prepuce, with an ectopic urethral opening and epispadias-like phenotype. These mice also developed hydroureter and hydronephrosis. Identification of ISL1 transcriptional targets via ChIP-Seq and expression analyses revealed that Isl1 regulates several important signaling pathways during embryonic genital development, including the BMP, WNT, and FGF cascades. An essential function of Isl1 during development of the external genitalia is to induce Bmp4-mediated apoptosis in the genital mesenchyme. Together, these studies demonstrate that Isl1 plays a critical role during development of the external genitalia and forms the basis for a greater understanding of the molecular mechanisms underlying the pathogenesis of BEEC and urinary tract defects in humans.
Asunto(s)
Proteína Morfogenética Ósea 4/genética , Factor 10 de Crecimiento de Fibroblastos/genética , Genitales/anomalías , Genitales/embriología , Proteínas con Homeodominio LIM/genética , Factores de Transcripción/genética , Proteína Wnt-5a/genética , Animales , Extrofia de la Vejiga/genética , Extrofia de la Vejiga/metabolismo , Proteína Morfogenética Ósea 4/biosíntesis , Proteína Morfogenética Ósea 4/metabolismo , Desarrollo Embrionario , Femenino , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genitales/metabolismo , Proteínas con Homeodominio LIM/biosíntesis , Proteínas con Homeodominio LIM/metabolismo , Masculino , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Organogénesis/genética , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/metabolismo , Anomalías Urogenitales/genética , Anomalías Urogenitales/metabolismo , Proteína Wnt-5a/biosíntesis , Proteína Wnt-5a/metabolismoRESUMEN
Fibroblast growth factor 10 (FGF10) is a member of the FGF superfamily. It exhibits diverse biological functions, and is extensively used for fundamental research and clinical applications involving hair growth, tissue repair, and burn wounds. Oil bodies, obtained from oil seeds, have been exploited for a variety of biotechnology applications. The use of oil bodies reduces purification steps and costs associated with the production of heterogonous proteins. Here, recombinant human FGF10 (rhFGF10) was expressed in safflower (Carthamus tinctorius L.) seeds using oilbody-oleosin technology. A plant expression vector, pOTBar-oleosin-rhFGF10, was constructed and introduced into safflower using Agrobacterium tumefaciens transformation, and mature safflower plants were obtained by grafting. Oleosin-rhFGF10 was successfully transformed and expressed in safflower seeds and inherited to the T3 generation. Moreover, MTT assays demonstrated that oil bodies expressed oleosin-FGF10 had a dose-dependent effect on cellular proliferation. In conclusion, this may provide a method of producing oleosin-rhFGF10, and help us meet the increasing pharmacological demands for the protein.
Asunto(s)
Carthamus tinctorius/genética , Factor 10 de Crecimiento de Fibroblastos/genética , Vectores Genéticos/química , Proteínas de Plantas/genética , Semillas/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Carthamus tinctorius/química , Carthamus tinctorius/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/aislamiento & purificación , Factor 10 de Crecimiento de Fibroblastos/farmacología , Expresión Génica , Vectores Genéticos/metabolismo , Humanos , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Ratones , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Semillas/química , Semillas/metabolismoRESUMEN
PURPOSE: In an attempt to improve in vitro embryo production, we investigated the effect of fibroblast growth factor 10 (FGF10) during in vitro maturation on the developmental capacity of bovine oocytes. MATERIAL AND METHODS: Cumulus-oocyte complexes (COCs) were aspirated from follicles of 3-8 mm diameter. After selection, the COCs were matured in medium with or without 0.5 ng/mL of FGF10. The effect of FGF10 during in vitro maturation (IVM) on nuclear maturation kinetics and expansion of the cumulus cells was investigated. Oocyte competence was assessed by the production and development speed of embryos and the relative expression of genes associated with embryo quality. RESULTS: FGF10 delayed the resumption of meiosis from 8 h onwards, but did not affect the percentage of oocytes reaching metaphase II, nor did it increase cumulus expansion at 22 h of maturation. We found no difference between treatments regarding embryo production, developmental speed, and gene expression. CONCLUSION: In conclusion, the presence of FGF10 during IVM had no effect on embryo production, developmental speed, and gene expression.
Asunto(s)
Desarrollo Embrionario/genética , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Técnicas de Maduración In Vitro de los Oocitos , Oocitos/crecimiento & desarrollo , Animales , Blastocisto/metabolismo , Bovinos , Células del Cúmulo/metabolismo , Técnicas de Cultivo de Embriones , Femenino , Regulación del Desarrollo de la Expresión Génica , Meiosis/genética , Oocitos/metabolismoRESUMEN
The continuous growth of mouse incisors depends on epithelial stem cells (SCs) residing in the SC niche, called labial cervical loop (LaCL). The homeostasis of the SCs is subtly regulated by complex signaling networks. In this study, we focus on retinoic acid (RA), a derivative of Vitamin A and a known pivotal signaling molecule in controlling the functions of stem cells (SCs). We analyzed the expression profiles of several key molecules of the RA signaling pathway in cultured incisor explants upon exogenous RA treatment. The expression patterns of these molecules suggested a negative feedback regulation of RA signaling in the developing incisor. We demonstrated that exogenous RA had negative effects on incisor SCs and that this was accompanied by downregulation of Fgf10, a mesenchymally expressed SC survival factor in the mouse incisor. Supplement of Fgf10 in incisor cultures completely blocked RA effects by antagonizing apoptosis and increasing proliferation in LaCL epithelial SCs. In addition, Fgf10 obviously antagonized RA-induced downregulation of the SC marker Sox2 in incisor epithelial SCs. Our findings suggest that the negative effects of RA on incisor SCs result from inhibition of mesenchymal Fgf10.
Asunto(s)
Incisivo/citología , Incisivo/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacos , Tretinoina/toxicidad , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Incisivo/metabolismo , Ratones , Transducción de SeñalRESUMEN
Lhx9 is a member of the LIM-homeodomain gene family necessary for the correct development of many organs including gonads, limbs, heart and the nervous system. In the context of limb development, Lhx9 has been implicated as an integrator for Fibroblast growth factor (FGF) and Sonic hedgehog (Shh) signalling required for proximal-distal (PD) and anterior-posterior (AP) development of the limb. Three splice variants of the Lhx9 transcript are expressed during development, two of which are predicted to act in a dominant negative fashion, competing with the DNA binding version of Lhx9 for binding to cofactors via the LIM-domain. We examined the expression pattern for the three alternative splice forms of Lhx9; Lhx9α, Lhx9ß and Lhx9c during early limb development. We have found that of the three Lhx9 isoforms, only Lhx9α and Lhx9c (intact homeodomain) are expressed during early limb development, each with their own distinct expression pattern. Additionally we determined that Lhx9 expression overlaps with FGF10 expression in the developing limb bud mesenchyme. Limb bud explant cultures, in the presence of signalling pathway inhibitors, also indicated that Lhx9 mRNA expression in the limb bud was dependent on FGF signalling.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/metabolismo , Proteínas con Homeodominio LIM/biosíntesis , Proteínas con Homeodominio LIM/genética , Esbozos de los Miembros/fisiología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Animales , Extremidades/embriología , Femenino , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/genética , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Esbozos de los Miembros/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Embarazo , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Transactivadores/metabolismoRESUMEN
Fibroblast growth factors (FGFs) expressed in the apical ectodermal ridge (AER) and FGF10 expressed in the underlying mesoderm are essential for limb bud outgrowth. Their expression is maintained through a positive feedback loop. We identified the cis-regulatory element and trans-acting factors involved in the AER-FGF-dependent transactivation of Fgf10. Etv1 and Ewsr1 stimulated transcription from the Fgf10 promoter in the sub-AER mesenchyme of mouse and chick limb buds in a conserved AGAAAR cluster-dependent manner. We found that both Etv1 and Ewsr1 were necessary for Fgf10 expression and elongation of the limb bud. In addition, Etv1 and AER-FGF synergistically stimulated Fgf10 promoter activity in an Ewsr1-dependent manner. We also found that Etv1 and Ewsr1 bound to the segment of DNA containing the AGAAAR cluster in vivo and in vitro. Moreover, Etv1 directly bound to the AGAAAR sequence in vitro. Our results suggest that Etv1 and Ewsr1 transactivate Fgf10 directly and cooperatively in response to AER-FGFs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factores de Crecimiento de Fibroblastos/metabolismo , Esbozos de los Miembros/crecimiento & desarrollo , Proteína EWS de Unión a ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Células Cultivadas , Embrión de Pollo , Ectodermo/metabolismo , Factor 10 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteínas Quinasas/metabolismo , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-ß1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-ß1 on salivary gland cell differentiation. TGF-ß1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-ßR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-ß signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-ß1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.
Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular , Glándulas Salivales/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Células Epiteliales/citología , Células Epiteliales/metabolismo , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 7 de Crecimiento de Fibroblastos/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Células Madre Mesenquimatosas , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Glándulas Salivales/citología , Transducción de Señal , Factor de Crecimiento Transformador beta1/antagonistas & inhibidoresRESUMEN
Cleft palate represents one of the most common congenital birth defects in humans. TGFß signaling, which is mediated by Smad-dependent and Smad-independent pathways, plays a crucial role in regulating craniofacial development and patterning, particularly in palate development. However, it remains largely unknown whether the Smad-independent pathway contributes to TGFß signaling function during palatogenesis. In this study, we investigated the function of TGFß activated kinase 1 (Tak1), a key regulator of Smad-independent TGFß signaling in palate development. We show that Tak1 protein is expressed in both the epithelium and mesenchyme of the developing palatal shelves. Whereas deletion of Tak1 in the palatal epithelium or mesenchyme did not give rise to a cleft palate defect, inactivation of Tak1 in the neural crest lineage using the Wnt1-Cre transgenic allele resulted in failed palate elevation and subsequently the cleft palate formation. The failure in palate elevation in Wnt1-Cre;Tak1(F/F) mice results from a malformed tongue and micrognathia, resembling human Pierre Robin sequence cleft of the secondary palate. We found that the abnormal tongue development is associated with Fgf10 overexpression in the neural crest-derived tongue tissue. The failed palate elevation and cleft palate were recapitulated in an Fgf10-overexpressing mouse model. The repressive effect of the Tak1-mediated noncanonical TGFß signaling on Fgf10 expression was further confirmed by inhibition of p38, a downstream kinase of Tak1, in the primary cell culture of developing tongue. Tak1 thus functions to regulate tongue development by controlling Fgf10 expression and could represent a candidate gene for mutation in human PRS clefting.
Asunto(s)
Linaje de la Célula , Fisura del Paladar/embriología , Embrión de Mamíferos/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Cresta Neural/embriología , Lengua/anomalías , Lengua/embriología , Animales , Fisura del Paladar/genética , Fisura del Paladar/patología , Modelos Animales de Enfermedad , Embrión de Mamíferos/patología , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Quinasas Quinasa Quinasa PAM/genética , Ratones , Ratones Mutantes , Cresta Neural/patología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The analysis of Fgf10 mouse mutants has demonstrated a critical role for this ligand in neurosensory development of the vertebrate inner ear, and we have been looking to define the direct upstream regulators of Fgf10 in this sensory organ, as part of constructing the programme of early inner ear development. Through the analysis of reporter constructs in transgenic mouse embryos and neonatal mice, in this report we define a minimal 1400 bp enhancer from the 5' flanking region of Fgf10. This enhancer drives reporter transgene expression in a manner that recapitulates endogenous expression of Fgf10, from its initial onset in the invaginating otic placode and onwards throughout gestation, controlling Fgf10 expression in all developing sensory patches and in the developing VIIIth ganglion. This regulatory region includes three putative Gata3 binding sites that we demonstrate directly interacts with Gata3 protein through the DNA binding domain with differing affinities. Site directed mutagenesis of all three sites and functional testing in transgenic embryos using reporter transgenes reveals an absolute requirement for Gata3 in controlling Fgf10 expression. Transgenic analysis of individual Gata3 binding site mutations illustrates that only one of these binding sites is necessary for reporter expression. Together these data demonstrate that Gata3 directly activates Fgf10 in the early inner ear, and does so through a single binding site.
Asunto(s)
Oído Interno/embriología , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor de Transcripción GATA3/fisiología , Regulación del Desarrollo de la Expresión Génica , Animales , Sitios de Unión , Elementos de Facilitación Genéticos , Factor 10 de Crecimiento de Fibroblastos/genética , Factor de Transcripción GATA3/metabolismo , Ganglios/metabolismo , Genes Reporteros , Ratones , Ratones Transgénicos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Transgenes , Tretinoina/metabolismoRESUMEN
Blood vessels have been shown to play perfusion-independent roles in organogenesis. Here, we examined whether blood vessels determine branching stereotypy of the mouse lung airways in which coordinated branching of epithelial and vascular tubes culminates in their co-alignment. Using different ablative strategies to eliminate the lung vasculature, both in vivo and in lung explants, we show that proximity to the vasculature is indeed essential for patterning airway branching. Remarkably, although epithelial branching per se proceeded at a nearly normal rate, branching stereotypy was dramatically perturbed following vascular ablation. Specifically, branching events requiring a rotation to change the branching plane were selectively affected. This was evidenced by either the complete absence or the shallow angle of their projections, with both events contributing to an overall flat lung morphology. Vascular ablation also led to a high frequency of ectopic branching. Regain of vascularization fully rescued arrested airway branching and restored normal lung size and its three-dimensional architecture. This role of the vasculature is independent of perfusion, flow or blood-borne substances. Inhibition of normal branching resulting from vascular loss could be explained in part by perturbing the unique spatial expression pattern of the key branching mediator FGF10 and by misregulated expression of the branching regulators Shh and sprouty2. Together, these findings uncovered a novel role of the vasculature in organogenesis, namely, determining stereotypy of epithelial branching morphogenesis.
Asunto(s)
Pulmón/irrigación sanguínea , Pulmón/embriología , Organogénesis , Proteínas Adaptadoras Transductoras de Señales , Animales , Comunicación Celular , Células Endoteliales/fisiología , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/biosíntesis , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Morfogénesis , Neovascularización Fisiológica , Técnicas de Cultivo de Órganos , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Embryonic stem (ES) cell technology may serve as a platform for the discovery of drugs to treat diseases such as diabetes. However, because of difficulties in establishing reliable ES cell differentiation methods and in creating cost-effective plating conditions for the high-throughput format, screening for molecules that regulate pancreatic beta cells and their immediate progenitors has been limited. A relatively simple and inexpensive differentiation protocol that allows efficient generation of insulin-expressing cells from murine ES cells was previously established in our laboratories. In this report, this system is characterized in greater detail to map developmental cell stages for future screening experiments. Our results show that sequential activation of multiple gene markers for undifferentiated ES cells, epiblast, definitive endoderm, foregut, and pancreatic lineages was found to follow the sequence of events that mimics pancreatic ontogeny. Cells that expressed enhanced green fluorescent protein, driven by pancreatic and duodenal homeobox 1 or insulin 1 promoter, correctly expressed known beta cell lineage markers. Overexpression of Sox17, an endoderm fate-determining transcription factor, at a very early stage of differentiation (days 2-3) enhanced pancreatic gene expression. Overexpression of neurogenin3, an endocrine progenitor cell marker, induced glucagon expression at stages when pancreatic and duodenal homeobox 1 message was present (days 10-16). Forced expression (between days 16 and 25) of MafA, a pancreatic maturation factor, resulted in enhanced expression of insulin genes, glucose transporter 2 and glucokinase, and glucose-responsive insulin secretion. Day 20 cells implanted in vivo resulted in pancreatic-like cells. Together, our differentiation assay recapitulates the proceedings and behaviors of pancreatic development and will be valuable for future screening of beta cell effectors.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/fisiología , Células Secretoras de Insulina/metabolismo , Páncreas/crecimiento & desarrollo , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/genética , Células Cultivadas , Células Madre Embrionarias/citología , Endodermo/citología , Endodermo/metabolismo , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/biosíntesis , Insulina/genética , Insulina/metabolismo , Masculino , Ratones , Ratones SCID , Páncreas/citología , Páncreas/metabolismo , Factores de Transcripción SOXF/análisis , Factores de Transcripción SOXF/biosíntesis , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Células Madre/citología , Células Madre/metabolismo , Transactivadores/análisis , Transactivadores/biosíntesis , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/análisis , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The pituitary gland is an endocrine organ that is developmentally derived from a fold in the oral ectoderm and a juxtaposed fold in the neural ectoderm. Here, we show that the absence of Vax1, a homeodomain transcription factor known for its role in eye and optic chiasm development, causes the rostral oral ectoderm to form an ectopic fold that eventually develops into a separate second pituitary with all the pituitary cell types and neuronal fibers characteristic of the normal pituitary. The induction of the second pituitary is associated with a localized ectopic expression of Fgf10, a gene encoding a growth factor known to recruit oral ectodermal cells into the pituitary. Interestingly, there are rare cases of pituitary duplications in humans that are also associated with optic nerve dysplasia, suggesting that VAX1 might be involved in the pathogenesis of this disorder.
Asunto(s)
Ectodermo/citología , Neuropéptidos/deficiencia , Hipófisis/crecimiento & desarrollo , Factores de Transcripción/deficiencia , Animales , Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Fibras Nerviosas , Enfermedades del Nervio Óptico/etiología , Hipófisis/inervación , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Bronchopulmonary dysplasia (BPD) is a frequent complication of preterm birth. This chronic lung disease results from arrested saccular airway development and is most common in infants exposed to inflammatory stimuli. In experimental models, inflammation inhibits expression of fibroblast growth factor-10 (FGF-10) and impairs epithelial-mesenchymal interactions during lung development; however, the mechanisms connecting inflammatory signaling with reduced growth factor expression are not yet understood. In this study we found that soluble inflammatory mediators present in tracheal fluid from preterm infants can prevent saccular airway branching. In addition, LPS treatment led to local production of mediators that inhibited airway branching and FGF-10 expression in LPS-resistant C.C3-Tlr4(Lpsd)/J fetal mouse lung explants. Both direct NF-κB activation and inflammatory cytokines (IL-1ß and TNF-α) that activate NF-κB reduced FGF-10 expression, whereas chemokines that signal via other inflammatory pathways had no effect. Mutational analysis of the FGF-10 promoter failed to identify genetic elements required for direct NF-κB-mediated FGF-10 inhibition. Instead, NF-κB activation appeared to interfere with the normal stimulation of FGF-10 expression by Sp1. Chromatin immunoprecipitation and nuclear coimmunoprecipitation studies demonstrated that the RelA subunit of NF-κB and Sp1 physically interact at the FGF-10 promoter. These findings indicate that inflammatory signaling through NF-κB disrupts the normal expression of FGF-10 in fetal lung mesenchyme by interfering with the transcriptional machinery critical for lung morphogenesis.
Asunto(s)
Factor 10 de Crecimiento de Fibroblastos/biosíntesis , Pulmón/embriología , FN-kappa B/metabolismo , Proteínas Quinasas/metabolismo , Animales , Corioamnionitis/metabolismo , Inmunoprecipitación de Cromatina , Femenino , Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunoprecipitación , Recién Nacido , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Embarazo , Nacimiento Prematuro , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
RATIONALE: The genes encoding fibroblast growth factor (FGF) 8 and 10 are expressed in the anterior part of the second heart field that constitutes a population of cardiac progenitor cells contributing to the arterial pole of the heart. Previous studies of hypomorphic and conditional Fgf8 mutants show disrupted outflow tract (OFT) and right ventricle (RV) development, whereas Fgf10 mutants do not have detectable OFT defects. OBJECTIVES: Our aim was to investigate functional overlap between Fgf8 and Fgf10 during formation of the arterial pole. METHODS AND RESULTS: We generated mesodermal Fgf8; Fgf10 compound mutants with MesP1Cre. The OFT/RV morphology in these mutants was affected with variable penetrance; however, the incidence of embryos with severely affected OFT/RV morphology was significantly increased in response to decreasing Fgf8 and Fgf10 gene dosage. Fgf8 expression in the pharyngeal arch ectoderm is important for development of the pharyngeal arch arteries and their derivatives. We now show that Fgf8 deletion in the mesoderm alone leads to pharyngeal arch artery phenotypes and that these vascular phenotypes are exacerbated by loss of Fgf10 function in the mesodermal core of the arches. CONCLUSIONS: These results show functional overlap of FGF8 and FGF10 signaling from second heart field mesoderm during development of the OFT/RV, and from pharyngeal arch mesoderm during pharyngeal arch artery formation, highlighting the sensitivity of these key aspects of cardiovascular development to FGF dosage.