RESUMO
BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme's activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation.
Assuntos
Ameloblastos , Amelogênese , Amelogênese/genética , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular , Epitélio , Camundongos , Transdução de SinaisRESUMO
Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning.
Assuntos
Família 26 do Citocromo P450/genética , Desenvolvimento Embrionário/genética , Proteínas Hedgehog/genética , Ácido Retinoico 4 Hidroxilase/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Embrião de Mamíferos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Organogênese/genética , Transdução de Sinais/genética , Dente/crescimento & desenvolvimento , Dente/metabolismo , Tretinoína/metabolismoRESUMO
The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Tretinoína/farmacologia , Alelos , Animais , Linhagem Celular , Família 26 do Citocromo P450/genética , Família 26 do Citocromo P450/metabolismo , Células Epiteliais/metabolismo , Epitélio/crescimento & desenvolvimento , Feminino , Proteínas Hedgehog/genética , Masculino , Células de Merkel/efeitos dos fármacos , Células de Merkel/metabolismo , Camundongos , Ácido Retinoico 4 Hidroxilase/genética , Ácido Retinoico 4 Hidroxilase/metabolismo , Transdução de Sinais , Papilas Gustativas/metabolismo , Língua/crescimento & desenvolvimento , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.
Assuntos
Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Odontogênese/genética , Dente/metabolismo , Animais , Animais Recém-Nascidos , Imuno-Histoquímica , Hibridização In Situ , Isoenzimas , Lisossomos/metabolismo , Camundongos , Especificidade de Órgãos/genética , Transporte Proteico , Dente/embriologia , Dente/crescimento & desenvolvimentoRESUMO
We show that removing the Shh signal tranducer Smoothened from skin epithelium secondarily results in excess Shh levels in the mesenchyme. Moreover, the phenotypes we observe reflect decreased epithelial Shh signaling, yet increased mesenchymal Shh signaling. For example, the latter contributes to exuberant hair follicle (HF) induction, while the former depletes the resulting follicular stem cell niches. This disruption of the niche apparently also allows the remaining stem cells to initiate hair formation at inappropriate times. Thus, the temporal structure of the hair cycle may depend on the physical structure of the niche. Finally, we find that the ablation of epithelial Shh signaling results in unexpected transformations: the follicular outer root sheath takes on an epidermal character, and certain HFs disappear altogether, having adopted a strikingly mammary gland-like fate. Overall, our study uncovers a multifaceted function for Shh in sculpting and maintaining the integrity and identity of the developing HF.
Assuntos
Folículo Piloso/anormalidades , Folículo Piloso/embriologia , Proteínas Hedgehog/metabolismo , Glândulas Mamárias Animais/patologia , Transdução de Sinais , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Transformada , Ectoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/patologia , Proteínas Hedgehog/genética , Hiperplasia , Integrases/metabolismo , Queratinócitos/citologia , Glândulas Mamárias Animais/citologia , Mesoderma/citologia , Metaplasia , Camundongos , Morfogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Células-Tronco/citologia , beta Catenina/metabolismoRESUMO
Compared with the embryonic development of other organs, development of the secondary palate is seemingly simple. However, each step of palatogenesis, from initiation until completion, is subject to a tight molecular control that is governed by epithelial-mesenchymal interactions. The importance of a rigorous molecular regulation of palatogenesis is reflected when loss of function of a single protein generates cleft palate, a frequent malformation with a complex etiology. Genetic studies in humans and targeted mutations in mice have identified numerous factors that play key roles during palatogenesis. This review highlights the current understanding of the molecular and cellular mechanisms involved in normal and abnormal palate development with special respect to recent advances derived from studies of mouse models.
Assuntos
Fissura Palatina/embriologia , Fissura Palatina/metabolismo , Animais , Células Epiteliais/metabolismo , Humanos , Mutação/genética , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
Different sodium-dependent inorganic phosphate (P(i)) uptake mechanisms play a major role in cellular P(i) homeostasis. The function and detailed distribution patterns of the type III Na(+)-phosphate cotransporter, PiT-2, in different organs during development are still largely unknown. We therefore examined the temporospatial expression patterns of Pit2 during murine odontogenesis. Odontoblasts were always devoid of Pit2 expression, whereas a transient, but strong, expression was detected in young secretory ameloblasts. However, the stratum intermedium and, later on, the papillary layer and cells of the subodontoblastic layer, exhibited high levels of Pit2 mRNA, which increased gradually as the tooth matured. Hormonal treatment or P(i) starvation of tooth germs in vitro did not alter Pit2 levels or patterns of expression, indicating mechanisms of regulation different from those of PiT-1 or other cell types. PiT-2 also functions as a retroviral receptor, and functional membrane-localized protein was confirmed throughout the dental papilla/pulp by demonstrating cellular permissiveness to infection by a gammaretrovirus that uses PiT-2 as a receptor. The distinct pattern of Pit2 expression during odontogenesis suggests that its P(i)-transporter function may be important for homeostasis of dental cells and not specifically for mineralization of the dental extracellular matrices. The expression of viral receptors in enamel-forming cells and the dental pulp may be of pathological significance.
Assuntos
Ameloblastos/metabolismo , Papila Dentária/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Odontogênese/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/biossíntese , Animais , Química Encefálica , Gammaretrovirus/metabolismo , Hibridização In Situ , Camundongos , Receptores Virais/biossíntese , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/fisiologiaRESUMO
During palatogenesis, fusion of the palatine shelves is a crucial event, the failure of which results in the birth defect, cleft palate. The fate of the midline epithelial seam (MES), which develops transiently upon contact of the two palatine shelves, is still strongly debated. Three major mechanisms underlying the regression of the MES upon palatal fusion have been proposed: (1) apoptosis has been evidenced by morphological and molecular criteria; (2) epithelial-mesenchymal transformation has been suggested based on ultrastructural and lipophilic dye cell labeling observations; and (3) migration of MES cells toward the oral and nasal areas has been proposed following lipophilic dye cell labeling. To verify whether epithelial-mesenchymal transformation of MES cells takes place during murine palatal fusion, we used the Cre/lox system to genetically mark Sonic hedgehog- and Keratin-14-expressing palatal epithelial cells and to identify their fate in vivo. Our analyses provide conclusive evidence that rules out the occurrence of epithelial-mesenchymal transformation of MES cells.
Assuntos
Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Mesoderma/citologia , Morfogênese/fisiologia , Palato/embriologia , Animais , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Proteínas de Fluorescência Verde , Proteínas Hedgehog , Imuno-Histoquímica , Queratina-14 , Queratinas/metabolismo , Camundongos , Camundongos Transgênicos , Transativadores/metabolismoRESUMO
We have previously demonstrated that the transcription factor nuclear factor (NF)1-C2 plays an important role in the mammary gland for the activation of the tumor suppressor gene p53. It also activates the milk genes carboxyl ester lipase and whey acidic protein, implying that NF1-C2 participates both in the establishment of a functional gland and in protection of the gland against tumorigenesis during proliferation. In this study, we have developed a new sensitive NF1-C2-specific antiserum for immunohistochemical analyses of the NF1-C2 distribution during mammary gland development. We show that the NF1-C2 protein is present in the epithelial compartment at the virgin stage and throughout mammary gland development. However, in the lactation stage the NF1-C2 protein levels strongly decreased, and many epithelial nuclei stained negative. In situ hybridization shows that NF1-C2 transcripts are expressed in the whole epithelium at pregnancy as well as the lactation stage, indicating that the reduction in protein levels is posttranscriptionally regulated. At involution, the NF1-C2 proteins are back to high levels. Based on studies using NMuMG cells and mammary tissue from heterozygous prolactin receptor knockout mice, we also demonstrate that prolactin has a direct effect in the maintenance of the NF1-C2 protein levels in the mammary epithelial nuclei at the virgin stage and during pregnancy. Hence, we have identified another transcription factor in the mammary gland, besides signal transducer and activator of transcription 5, through which prolactin may control mammary gland development. Furthermore, our data suggest a link between prolactin and p53 in the mammary gland, through NF1-C2.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glândulas Mamárias Animais/crescimento & desenvolvimento , Prolactina/farmacologia , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/análise , Proteínas Estimuladoras de Ligação a CCAAT/genética , Núcleo Celular/química , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Imunoquímica , Lactação/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fatores de Transcrição NFI , Gravidez , Prolactina/genética , Prolactina/metabolismo , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Sharpe and colleagues unveil a crucial role for NF-kappaB activity in tooth development, and show that IKKalpha functions both within and independently from the NF-kappaB pathway during molar and incisor morphogenesis, respectively (in the February issue of Developmental Cell).
Assuntos
Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Anormalidades Dentárias/genética , Dente/embriologia , Fatores de Transcrição , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Quinase I-kappa B , Organogênese/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor Notch1 , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/genética , Dente/citologia , Dente/metabolismo , Anormalidades Dentárias/metabolismoRESUMO
Sonic hedgehog (Shh), a member of the mammalian Hedgehog (Hh) family, plays a key role during embryogenesis and organogenesis. Tooth development, odontogenesis, is governed by sequential and reciprocal epithelial-mesenchymal interactions. Genetic removal of Shh activity from the dental epithelium, the sole source of Shh during tooth development, alters tooth growth and cytological organization within both the dental epithelium and mesenchyme of the tooth. In this model it is not clear which aspects of the phenotype are the result of the direct action of Shh on a target tissue and which are indirect effects due to deficiencies in reciprocal signalings between the epithelial and mesenchymal components. To distinguish between these two alternatives and extend our understanding of Shh's actions in odontogenesis, we have used the Cre-loxP system to remove Smoothened (Smo) activity in the dental epithelium. Smo, a seven-pass membrane protein is essential for the transduction of all Hh signals. Hence, removal of Smo activity from the dental epithelium should block Shh signaling within dental epithelial derivatives while preserving normal mesenchymal signaling. Here we show that Shh-dependent interactions occur within the dental epithelium itself. The dental mesenchyme develops normally up until birth. In contrast, dental epithelial derivatives show altered proliferation, growth, differentiation and polarization. Our approach uncovers roles for Shh in controlling epithelial cell size, organelle development and polarization. Furthermore, we provide evidence that Shh signaling between ameloblasts and the overlying stratum intermedium may involve subcellular localization of Patched 2 and Gli1 mRNAs, both of which are targets of Shh signaling in these cells.