RESUMEN
Ciliogenesis precedes lineage-determining signaling in skin development. To understand why, we performed shRNA-mediated knockdown of seven intraflagellar transport proteins (IFTs) and conditional ablation of Ift-88 and Kif3a during embryogenesis. In both cultured keratinocytes and embryonic epidermis, all of these eliminated cilia, and many (not Kif3a) caused hyperproliferation. Surprisingly and independent of proliferation, ciliary mutants displayed defects in Notch signaling and commitment of progenitors to differentiate. Notch receptors and Notch-processing enzymes colocalized with cilia in wild-type epidermal cells. Moreover, differentiation defects in ciliary mutants were cell autonomous and rescued by activated Notch (NICD). By contrast, Shh signaling was neither operative nor required for epidermal ciliogenesis, Notch signaling, or differentiation. Rather, Shh signaling defects in ciliary mutants occurred later, arresting hair follicle morphogenesis in the skin. These findings unveil temporally and spatially distinct functions for primary cilia at the nexus of signaling, proliferation, and differentiation.
Asunto(s)
Diferenciación Celular , Cilios/metabolismo , Epidermis/embriología , Epidermis/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Proteínas Portadoras/genética , Polaridad Celular , Proliferación Celular , Proteínas del Citoesqueleto/metabolismo , Células Epidérmicas , Técnicas de Silenciamiento del Gen , Folículo Piloso/citología , Proteínas Hedgehog/metabolismo , Cinesis , Ratones , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Although in vitro studies of embryonic stem cells have identified polycomb repressor complexes (PRCs) as key regulators of differentiation, it remains unclear as to how PRC-mediated mechanisms control fates of multipotent progenitors in developing tissues. Here, we show that an essential PRC component, Ezh2, is expressed in epidermal progenitors but diminishes concomitant with embryonic differentiation and with postnatal decline in proliferative activity. We show that Ezh2 controls proliferative potential of basal progenitors by repressing the Ink4A-Ink4B locus and tempers the developmental rate of differentiation by preventing premature recruitment of AP1 transcriptional activator to the structural genes that are required for epidermal differentiation. Together, our studies reveal that PRCs control epigenetic modifications temporally and spatially in tissue-restricted stem cells. They maintain their proliferative potential and globally repressing undesirable differentiation programs while selectively establishing a specific terminal differentiation program in a stepwise fashion.
Asunto(s)
Diferenciación Celular , Células Epidérmicas , Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre/metabolismo , Animales , Núcleo Celular/metabolismo , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Histonas/metabolismo , Humanos , Metilación , Ratones , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Represoras/metabolismoRESUMEN
Hair follicles (HFs) undergo cyclical periods of growth, which are fueled by stem cells (SCs) at the base of the resting follicle. HF-SC formation occurs during HF development and requires transcription factor SOX9. Whether and how SOX9 functions in HF-SC maintenance remain unknown. By conditionally targeting Sox9 in adult HF-SCs, we show that SOX9 is essential for maintaining them. SOX9-deficient HF-SCs still transition from quiescence to proliferation and launch the subsequent hair cycle. However, once activated, bulge HF-SCs begin to differentiate into epidermal cells, which naturally lack SOX9. In addition, as HF-SC numbers dwindle, outer root sheath production is not sustained, and HF downgrowth arrests prematurely. Probing the mechanism, we used RNA sequencing (RNA-seq) to identify SOX9-dependent transcriptional changes and chromatin immunoprecipitation (ChIP) and deep sequencing (ChIP-seq) to identify SOX9-bound genes in HF-SCs. Intriguingly, a large cohort of SOX9-sensitive targets encode extracellular factors, most notably enhancers of Activin/pSMAD2 signaling. Moreover, compromising Activin signaling recapitulates SOX9-dependent defects, and Activin partially rescues them. Overall, our findings reveal roles for SOX9 in regulating adult HF-SC maintenance and suppressing epidermal differentiation in the niche. In addition, our studies expose a role for SCs in coordinating their own behavior in part through non-cell-autonomous signaling within the niche.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Folículo Piloso/citología , Folículo Piloso/metabolismo , Factor de Transcripción SOX9/metabolismo , Transducción de Señal , Activinas/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Epidérmicas , Perfilación de la Expresión Génica , Ratones , Receptores Notch/metabolismo , Factor de Transcripción SOX9/genética , Proteína Smad2/metabolismo , Células Madre/citología , Proteínas Wnt/metabolismoRESUMEN
Polycomb protein group (PcG)-dependent trimethylation on H3K27 (H3K27me3) regulates identity of embryonic stem cells (ESCs). How H3K27me3 governs adult SCs and tissue development is unclear. Here, we conditionally target H3K27 methyltransferases Ezh2 and Ezh1 to address their roles in mouse skin homeostasis. Postnatal phenotypes appear only in doubly targeted skin, where H3K27me3 is abolished, revealing functional redundancy in EZH1/2 proteins. Surprisingly, while Ezh1/2-null hair follicles (HFs) arrest morphogenesis and degenerate due to defective proliferation and increased apoptosis, epidermis hyperproliferates and survives engraftment. mRNA microarray studies reveal that, despite these striking phenotypic differences, similar genes are up-regulated in HF and epidermal Ezh1/2-null progenitors. Featured prominently are (1) PcG-controlled nonskin lineage genes, whose expression is still significantly lower than in native tissues, and (2) the PcG-regulated Ink4a/Inkb/Arf locus. Interestingly, when EZH1/2 are absent, even though Ink4a/Arf/Ink4b genes are fully activated in HF cells, they are only partially so in epidermal progenitors. Importantly, transduction of Ink4b/Ink4a/Arf shRNAs restores proliferation/survival of Ezh1/2-null HF progenitors in vitro, pointing toward the relevance of this locus to the observed HF phenotypes. Our findings reveal new insights into Polycomb-dependent tissue control, and provide a new twist to how different progenitors within one tissue respond to loss of H3K27me3.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Folículo Piloso/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Homeostasis/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Factores de Transcripción/metabolismo , Cicatrización de Heridas/genética , Factor 1 de Ribosilacion-ADP/genética , Apoptosis/genética , Proliferación Celular , Supervivencia Celular/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Células Epidérmicas , Epidermis/trasplante , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Folículo Piloso/citología , N-Metiltransferasa de Histona-Lisina/genética , Metilación , Complejo Represivo Polycomb 2 , Trasplante de Piel , Células Madre/metabolismo , Factores de Transcripción/genéticaRESUMEN
Adult stem cells sustain tissue maintenance and regeneration throughout the lifetime of an animal. These cells often reside in specific signalling niches that orchestrate the stem cell's balancing act between quiescence and cell-cycle re-entry based on the demand for tissue regeneration. How stem cells maintain their capacity to replenish themselves after tissue regeneration is poorly understood. Here we use RNA-interference-based loss-of-function screening as a powerful approach to uncover transcriptional regulators that govern the self-renewal capacity and regenerative potential of stem cells. Hair follicle stem cells provide an ideal model. These cells have been purified and characterized from their native niche in vivo and, in contrast to their rapidly dividing progeny, they can be maintained and passaged long-term in vitro. Focusing on the nuclear proteins and/or transcription factors that are enriched in stem cells compared with their progeny, we screened â¼2,000 short hairpin RNAs for their effect on long-term, but not short-term, stem cell self-renewal in vitro. To address the physiological relevance of our findings, we selected one candidate that was uncovered in the screen: TBX1. This transcription factor is expressed in many tissues but has not been studied in the context of stem cell biology. By conditionally ablating Tbx1 in vivo, we showed that during homeostasis, tissue regeneration occurs normally but is markedly delayed. We then devised an in vivo assay for stem cell replenishment and found that when challenged with repetitive rounds of regeneration, the Tbx1-deficient stem cell niche becomes progressively depleted. Addressing the mechanism of TBX1 action, we discovered that TBX1 acts as an intrinsic rheostat of BMP signalling: it is a gatekeeper that governs the transition between stem cell quiescence and proliferation in hair follicles. Our results validate the RNA interference screen and underscore its power in unearthing new molecules that govern stem cell self-renewal and tissue-regenerative potential.
Asunto(s)
Interferencia de ARN , Regeneración/fisiología , Células Madre/citología , Proteínas de Dominio T Box/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proliferación Celular , Células Epidérmicas , Femenino , Folículo Piloso/citología , Masculino , Ratones , Regeneración/genética , Transducción de Señal , Células Madre/metabolismo , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genéticaRESUMEN
MicroRNAs (miRNAs) play important roles in differentiation of stem cells. However, the precise dynamics of miRNA induction during stem cell differentiation have not been visualized and molecular mechanisms through which miRNAs execute their function remain unclear. Using high-resolution in situ hybridization together with cell lineage and proliferation markers in mouse skin, we show that miR-203 is transcriptionally activated in the differentiating daughter cells upon the asymmetric cell division of interfollicular progenitor cells. Once induced, miR-203 rapidly promotes the cell cycle exit within 6 hours and abolishes self-renewal of the progenitor cells. With an inducible mouse model, we identify numerous miR-203 in vivo targets that are highly enriched in regulation of cell cycle and cell division, as well as in response to DNA damage. Importantly, co-suppression of individual targets, including p63, Skp2 and Msi2 by miR-203 is required for its function of promoting the cell cycle exit and inhibiting the long-term proliferation. Together, our findings reveal the rapid and widespread impact of miR-203 on the self-renewal program and provide mechanistic insights into the potent role of miR-203 during the epidermal differentiation. These results should also contribute to understanding the role of miR-203 in the development of skin cancer.
Asunto(s)
Diferenciación Celular , Células Epidérmicas , MicroARNs/metabolismo , Animales , División Celular Asimétrica , Biomarcadores/metabolismo , Linaje de la Célula , Proliferación Celular , Desarrollo Embrionario , Epidermis/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Células Madre/citología , Células Madre/metabolismo , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
Here, we show that the tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) sensitizes cells to ferroptosis, an iron-dependent form of cell death, by restraining the expression and activity of the cystine/glutamate antiporter system Xc- (xCT). Loss of PTEN activates AKT kinase to inhibit GSK3ß, increasing NF-E2 p45-related factor 2 (NRF2) along with transcription of one of its known target genes encoding xCT. Elevated xCT in Pten-null mouse embryonic fibroblasts increases the flux of cystine transport and synthesis of glutathione, which enhances the steady-state levels of these metabolites. A pan-cancer analysis finds that loss of PTEN shows evidence of increased xCT, and PTEN-mutant cells are resistant to ferroptosis as a consequence of elevated xCT. These findings suggest that selection of PTEN mutation during tumor development may be due to its ability to confer resistance to ferroptosis in the setting of metabolic and oxidative stress that occurs during tumor initiation and progression.
Asunto(s)
Cistina , Ferroptosis , Animales , Ratones , Cistina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Fibroblastos/metabolismoRESUMEN
Co-expression and gene linkage have hampered elucidating the physiological relevance of cadherins in mammalian tissues. Here, we combine conditional gene ablation and transgenic RNA interference to uncover new roles for E- and P-cadherins in epidermal sheet formation in vitro and maintenance of epidermal integrity in vivo. By devising skin-specific RNAi technology, we demonstrate that cadherin inhibition in vivo impairs junction formation and intercellular adhesion and increases apoptosis. These defects compromise epidermal barrier function and tissue integrity. In vitro, with only E-cadherin missing, epidermal sheet formation is delayed, but when both cadherins are suppressed, defects extend to adherens junctions, desmosomes, tight junctions and cortical actin dynamics. Using different rescue strategies, we show that cadherin level rather than subtype is critical. Finally, by comparing conditional loss-of-function studies of epidermal catenins and cadherins, we dissect cadherin-dependent and independent roles of adherens junction components in tissue physiology.
Asunto(s)
Cadherinas/metabolismo , Epidermis/metabolismo , Uniones Adherentes/metabolismo , Animales , Cadherinas/genética , Células Epidérmicas , Epidermis/ultraestructura , Técnica del Anticuerpo Fluorescente , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Interferencia de ARNRESUMEN
The Saccharomyces cerevisiae chromatin silencing factor Sir2 suppresses genomic instability and extends replicative life span. In contrast, we find that mouse embryonic fibroblasts (MEFs) deficient for SIRT1, a mammalian Sir2 homolog, have dramatically increased resistance to replicative senescence. Extended replicative life span of SIRT1-deficient MEFs correlates with enhanced proliferative capacity under conditions of chronic, sublethal oxidative stress. In this context, SIRT1-deficient cells fail to normally upregulate either the p19(ARF) senescence regulator or its downstream target p53. However, upon acute DNA damage or oncogene expression, SIRT1-deficient cells show normal p19(ARF) induction and cell cycle arrest. Together, our findings demonstrate an unexpected SIRT1 function in promoting replicative senescence in response to chronic cellular stress and implicate p19(ARF) as a downstream effector in this pathway.
Asunto(s)
Senescencia Celular , Daño del ADN , Sirtuinas/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Daño del ADN/efectos de los fármacos , Doxorrubicina/farmacología , Fibroblastos , Genes ras/genética , Peróxido de Hidrógeno/farmacología , Ratones , Ratones Noqueados , Células 3T3 NIH , Estrés Oxidativo/efectos de los fármacos , Fase S/efectos de los fármacos , Sirtuina 1 , Sirtuinas/deficiencia , Sirtuinas/genética , Proteína p14ARF Supresora de Tumor/metabolismoRESUMEN
During mouse development, core planar cell polarity (PCP) proteins become polarized in the epidermal plane to guide angling/morphogenesis of hair follicles. How PCP is established is poorly understood. Here, we identify a key role for Wdr1 (also known as Aip1), an F-actin-binding protein that enhances cofilin/destrin-mediated F-actin disassembly. We show that cofilin and destrin function redundantly in developing epidermis, but their combined depletion perturbs cell adhesion, cytokinesis, apicobasal polarity and PCP. Although Wdr1 depletion accentuates single-loss-of-cofilin/destrin phenotypes, alone it resembles core PCP mutations. Seeking a mechanism, we find that Wdr1 and cofilin/destrin-mediated actomyosin remodelling are essential for generating or maintaining cortical tension within the developing epidermal sheet and driving the cell shape and planar orientation changes that accompany establishment of PCP in mammalian epidermis. Our findings suggest intriguing evolutionary parallels but mechanistic modifications to the distal wing hinge-mediated mechanical forces that drive cell shape change and orient PCP in the Drosophila wing disc.
Asunto(s)
Polaridad Celular , Forma de la Célula , Epidermis/metabolismo , Queratinocitos/metabolismo , Mecanotransducción Celular , Proteínas de Microfilamentos/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Actomiosina/metabolismo , Animales , Adhesión Celular , Línea Celular , Citocinesis , Citoesqueleto/metabolismo , Destrina/deficiencia , Destrina/genética , Células Epidérmicas , Evolución Molecular , Genotipo , Terapia por Láser , Ratones Noqueados , Proteínas de Microfilamentos/genética , Microscopía por Video , Fenotipo , Transporte de Proteínas , Interferencia de ARN , Estrés Mecánico , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development. METHODOLOGY/PRINCIPAL FINDINGS: The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. µCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ. CONCLUSIONS/SIGNIFICANCE: The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.
Asunto(s)
Ameloblastos/metabolismo , Amelogénesis/genética , Cadherinas/genética , Esmalte Dental/metabolismo , Órgano del Esmalte/metabolismo , Ameloblastos/citología , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Cadherinas/deficiencia , Cadherinas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Movimiento Celular , Esmalte Dental/citología , Esmalte Dental/crecimiento & desarrollo , Órgano del Esmalte/citología , Órgano del Esmalte/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Dureza , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Cultivo Primario de Células , Transducción de Señal , Propiedades de SuperficieRESUMEN
In adult skin, self-renewing, undifferentiated hair follicle stem cells (HF-SCs) reside within a specialized niche, where they spend prolonged times as a single layer of polarized, quiescent epithelial cells. When sufficient activating signals accumulate, HF-SCs become mobilized to fuel tissue regeneration and hair growth. Here, we show that architectural organization of the HF-SC niche by transcription factor LHX2 plays a critical role in HF-SC behavior. Using genome-wide chromatin and transcriptional profiling of HF-SCs in vivo, we show that LHX2 directly transactivates genes that orchestrate cytoskeletal dynamics and adhesion. Conditional ablation of LHX2 results in gross cellular disorganization and HF-SC polarization within the niche. LHX2 loss leads to a failure to maintain HF-SC quiescence and hair anchoring, as well as progressive transformation of the niche into a sebaceous gland. These findings suggest that niche organization underlies the requirement for LHX2 in hair follicle structure and function.
Asunto(s)
Células Madre Adultas/fisiología , Folículo Piloso/fisiología , Cabello/crecimiento & desarrollo , Proteínas con Homeodominio LIM/metabolismo , Glándulas Sebáceas/fisiología , Factores de Transcripción/metabolismo , Animales , Adhesión Celular/genética , Transdiferenciación Celular , Células Cultivadas , Microambiente Celular , Citoesqueleto/metabolismo , Perfilación de la Expresión Génica , Proteínas con Homeodominio LIM/genética , Ratones , Ratones Mutantes , Ratones Transgénicos , Mutación/genética , Regeneración , Nicho de Células Madre/genética , Factores de Transcripción/genéticaRESUMEN
Increasing evidence suggests that microRNAs may play important roles in regulating self-renewal and differentiation in mammalian stem cells (SCs). Here, we explore this issue in skin. We first characterize microRNA expression profiles of skin SCs versus their committed proliferative progenies and identify a microRNA subset associating with "stemness." Of these, miR-125b is dramatically downregulated in early SC progeny. We engineer an inducible mice system and show that when miR-125b is sustained in SC progenies, tissue balance is reversibly skewed toward stemness at the expense of epidermal, oil-gland, and HF differentiation. Using gain- and loss-of-function in vitro, we further implicate miR-125b as a repressor of SC differentiation. In vivo, transcripts repressed upon miR-125b induction are enriched >700% for predicted miR-125b targets normally downregulated upon SC-lineage commitment. We verify some of these miR-125b targets, and show that Blimp1 and VDR in particular can account for many tissue imbalances we see when miR-125b is deregulated.
Asunto(s)
Linaje de la Célula/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Piel/citología , Células Madre/citología , Células Madre/metabolismo , Regiones no Traducidas 3'/genética , Animales , Sitios de Unión , Diferenciación Celular/genética , Proliferación Celular , Secuencia Conservada/genética , Citometría de Flujo , Cabello/citología , Cabello/metabolismo , Ratones , Ratones Transgénicos , MicroARNs/metabolismo , Organogénesis/genética , Fenotipo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Receptores de Calcitriol/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.
Asunto(s)
Cateninas/genética , Cateninas/metabolismo , Esmalte Dental/crecimiento & desarrollo , Esmalte Dental/metabolismo , Marcación de Gen , Ameloblastos/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Diente/crecimiento & desarrollo , Diente/metabolismo , Catenina deltaRESUMEN
Hair follicles (HFs) undergo cyclic bouts of degeneration, rest, and regeneration. During rest (telogen), the hair germ (HG) appears as a small cell cluster between the slow-cycling bulge and dermal papilla (DP). Here we show that HG cells are derived from bulge stem cells (SCs) but become responsive quicker to DP-promoting signals. In vitro, HG cells also proliferate sooner but display shorter-lived potential than bulge cells. Molecularly, they more closely resemble activated bulge rather than transit-amplifying (matrix) cells. Transcriptional profiling reveals precocious activity of both HG and DP in late telogen, accompanied by Wnt signaling in HG and elevated FGFs and BMP inhibitors in DP. FGFs and BMP inhibitors participate with Wnts in exerting selective and potent stimuli to the HG both in vivo and in vitro. Our findings suggest a model where HG cells fuel initial steps in hair regeneration, while the bulge is the engine maintaining the process.
Asunto(s)
Folículo Piloso , Regeneración/fisiología , Células Madre/fisiología , Animales , Linaje de la Célula , Células Cultivadas , Femenino , Factor 7 de Crecimiento de Fibroblastos/genética , Factor 7 de Crecimiento de Fibroblastos/metabolismo , Folículo Piloso/citología , Folículo Piloso/fisiología , Ratones , Ratones Transgénicos , Morfogénesis/fisiología , Transducción de Señal/fisiología , Células Madre/citología , Transcripción Genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
During the hair cycle, follicle stem cells (SCs) residing in a specialized niche called the "bulge" undergo bouts of quiescence and activation to cyclically regenerate new hairs. Developmental studies have long implicated the canonical bone morphogenetic protein (BMP) pathway in hair follicle (HF) determination and differentiation, but how BMP signaling functions in the hair follicle SC niche remains unknown. Here, we use loss and gain of function studies to manipulate BMP signaling in the SC niche. We show that when the Bmpr1a gene is conditionally ablated, otherwise quiescent SCs are activated to proliferate, causing an expansion of the niche and loss of slow-cycling cells. Surprisingly, follicle SCs are not lost, however, but rather, they generate long-lived, tumor-like branches that express Sox4, Lhx2, and Sonic Hedgehog but fail to terminally differentiate to make hair. A key component of BMPR1A-deficient SCs is their elevated levels of both Lef1 and beta-catenin, which form a bipartite transcription complex required for initiation of the hair cycle. Although beta-catenin can be stabilized by Wnt signaling, we show that BMPR1A deficiency enhances beta-catenin stabilization in the niche through a pathway involving PTEN inhibition and PI3K/AKT activation. Conversely, sustained BMP signaling in the SC niche blocks activation and promotes premature hair follicle differentiation. Together, these studies reveal the importance of balancing BMP signaling in the SC niche.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/deficiencia , Proteínas Morfogenéticas Óseas/metabolismo , Transducción de Señal , Células Madre/citología , Células Madre/fisiología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Proteínas Morfogenéticas Óseas/genética , Proliferación Celular , Regulación de la Expresión Génica , Folículo Piloso/citología , Folículo Piloso/fisiología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM , Factor de Unión 1 al Potenciador Linfoide/análisis , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción SOXC , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta Catenina/análisis , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Immunoglobulin H class-switch recombination (CSR) occurs between switch regions and requires transcription and activation-induced cytidine deaminase (AID). Transcription through mammalian switch regions, because of their GC-rich composition, generates stable R-loops, which provide single-stranded DNA substrates for AID. However, we show here that the Xenopus laevis switch region S(mu), which is rich in AT and not prone to form R-loops, can functionally replace a mouse switch region to mediate CSR in vivo. X. laevis S(mu)-mediated CSR occurred mostly in a region of AGCT repeats targeted by the AID-replication protein A complex when transcribed in vitro. We propose that AGCT is a primordial CSR motif that targets AID through a non-R-loop mechanism involving an AID-replication protein A complex.