RESUMO
Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs results in severe hearing impairment. Damaged HCs can be regenerated by neighboring supporting cells (SCs), thus the functional regeneration of HCs is the main goal for the restoration of auditory function in vivo. Here, cochlear SC trans-differentiation into outer and inner HC by the induced expression of the key transcription factors Atoh1 and its co-regulators Gfi1, Pou4f3, and Six1 (GPAS), which are necessary for SCs that are destined for HC development and maturation via the AAV-ie targeting the inner ear stem cells are successfully achieved. Single-cell nuclear sequencing and lineaging tracing results showed that the majority of new Atoh1-derived HCs are in a state of initiating differentiation, while GP (Gfi1, Pou4f3) and GPS (Gfi1, Pou4f3, and Six1) enhanced the Atoh1-induced new HCs into inner and outer HCs. Moreover, the patch-clamp analysis indicated that newborn inner HCs induced by GPAS forced expression have similar electrophysiological characteristics to those of native inner HCs. Also, GPAS can induce HC regeneration in the HC-damaged mice model. In summary, the study demonstrates that AAV-mediated co-regulation of multiple genes, such as GPAS, is an effective means to achieve functional HC regeneration in the mouse cochlea.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Reprogramação Celular , Dependovirus , Células Ciliadas Auditivas , Regeneração , Animais , Camundongos , Dependovirus/genética , Reprogramação Celular/genética , Regeneração/genética , Regeneração/fisiologia , Células Ciliadas Auditivas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator de Transcrição Brn-3C/genética , Fator de Transcrição Brn-3C/metabolismo , Diferenciação Celular/genética , Vetores Genéticos/genética , Proteínas de Ligação a DNA , Fatores de Transcrição , Proteínas de HomeodomínioRESUMO
Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Terapia Genética , Hepatócitos , Hepatopatias , Hepatócitos/metabolismo , Hepatócitos/transplante , Sistemas CRISPR-Cas/genética , Humanos , Animais , Hepatopatias/terapia , Hepatopatias/genética , Hepatopatias/patologia , Camundongos , Terapia Genética/métodos , Tirosinemias/terapia , Tirosinemias/genética , Proliferação de Células , HidrolasesRESUMO
Hair cell (HC) damage is a leading cause of sensorineural hearing loss, and in mammals supporting cells (SCs) are unable to divide and regenerate HCs after birth spontaneously. Procollagen C-endopeptidase enhancer 2 (Pcolce2), which encodes a glycoprotein that acts as a functional procollagen C protease enhancer, was screened as a candidate regulator of SC plasticity in our previous study. In the current study, we used adeno-associated virus (AAV)-ie (a newly developed adeno-associated virus that targets SCs) to overexpress Pcolce2 in SCs. AAV-Pcolce2 facilitated SC re-entry into the cell cycle both in cultured cochlear organoids and in the postnatal cochlea. In the neomycin-damaged model, regenerated HCs were detected after overexpression of Pcolce2, and these were derived from SCs that had re-entered the cell cycle. These findings reveal that Pcolce2 may serve as a therapeutic target for the regeneration of HCs to treat hearing loss.
Assuntos
Animais Recém-Nascidos , Reprogramação Celular , Cóclea , Animais , Camundongos , Cóclea/metabolismo , Cóclea/citologia , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/citologia , Dependovirus/genética , Ciclo Celular , Camundongos Endogâmicos C57BL , Regeneração , Células Labirínticas de Suporte/metabolismo , Neomicina/farmacologiaRESUMO
Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four-transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV-ie-mediated Gpm6b overexpression promoted SC-derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re-entry cell cycle and facilitating the SC-to-HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans-differentiation of Lgr5+ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.
Assuntos
Dependovirus , Células Ciliadas Auditivas , Animais , Dependovirus/genética , Camundongos , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/citologia , Reprogramação Celular , Camundongos Endogâmicos C57BL , Cóclea/metabolismo , Cóclea/citologia , Transdiferenciação Celular , Organoides/metabolismo , Organoides/citologiaRESUMO
Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.
Assuntos
Surdez , Perda Auditiva Neurossensorial , Humanos , Animais , Camundongos , Dependovirus/genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/terapia , Audição , Surdez/genética , Surdez/terapia , Terapia GenéticaRESUMO
Inner ear hair cells detect sound vibration through the deflection of mechanosensory stereocilia. Cytoplasmic protein TPRN has been shown to localize at the taper region of the stereocilia, and mutations in TPRN cause hereditary hearing loss through an unknown mechanism. Here, using biochemistry and dual stimulated emission depletion microscopy imaging, we show that the TPRN, together with its binding proteins CLIC5 and PTPRQ, forms concentric rings in the taper region of stereocilia. The disruption of TPRN rings, triggered by the competitive inhibition of the interaction of TPRN and CLIC5 or exogenous TPRN overexpression, leads to stereocilia degeneration and severe hearing loss. Most importantly, restoration of the TPRN rings can rescue the damaged auditory function of Tprn knockout mice by exogenously expressing TPRN at an appropriate level in HCs via promoter recombinant adeno-associated virus (AAV). In summary, our results reveal highly structured TPRN rings near the taper region of stereocilia that are crucial for stereocilia function and hearing. Also, TPRN ring restoration in stereocilia by AAV-Tprn effectively repairs damaged hearing, which lays the foundation for the clinical application of AAV-mediated gene therapy in patients with TPRN mutation.
Assuntos
Surdez , Perda Auditiva , Animais , Humanos , Camundongos , Surdez/genética , Audição/genética , Perda Auditiva/genética , Perda Auditiva/terapia , Camundongos Knockout , Proteínas/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Estereocílios/metabolismoRESUMO
OTOF mutations are the principal causes of auditory neuropathy. There are reports on Otof-related gene therapy in mice, but there is no preclinical research on the drug evaluations. Here, Anc80L65 and the mouse hair cell-specific Myo15 promoter (mMyo15) are used to selectively and effectively deliver human OTOF to hair cells in mice and nonhuman primates to evaluate the efficacy and safety of OTOF gene therapy drugs. A new dual-AAV-OTOF-hybrid strategy to transfer full-length OTOF is generated, which can stably restore hearing in adult OTOFp.Q939*/Q939* mice with profound deafness, with the longest duration being at least 150 days, and the best therapeutic effect without difference in hearing from wild-type mice. An AAV microinjection method into the cochlea of cynomolgus monkeys without hearing impairment is further established and found the OTOF can be safely and effectively driven by the mMyo15 promoter in hair cells. In addition, the therapeutic dose of AAV drugs has no impact on normal hearing and does not cause significant systemic toxicity both in mouse and nonhuman primates. In summary, this study develops a potential gene therapy strategy for DFNB9 patients in the clinic and provides complete, standardized, and systematic research data for clinical research and application.
Assuntos
Perda Auditiva Neurossensorial , Perda Auditiva , Adulto , Humanos , Camundongos , Animais , Proteínas de Membrana/genética , Perda Auditiva Neurossensorial/genética , PrimatasRESUMO
Hair cells (HCs) in mammals cannot spontaneously regenerate after damage. Atoh1 overexpression can promote HC regeneration in the postnatal cochlea, but the regenerated HCs do not possess the structural and functional characteristics of HCs in situ. The stereocilia on the apical surface of HCs are the first-level structure for sound conduction, and regeneration of functional stereocilia is the key basis for the reproduction of functional HCs. Espin, as an actin bundling protein, plays an important role in the development and structural maintenance of the stereocilia. Here, we found that the upregulation of Espin by AAV-ie was able to induced the aggregation of actin fibres in Atoh1-induced HCs in both cochlear organoids and explants. In addition, we found that persistent Atoh1 overexpression resulted in impaired stereocilia in both endogenous and newly formed HCs. In contrast, the forced expression of Espin in endogenous and regenerative HCs was able to eliminate the stereocilia damage caused by persistent Atoh1 overexpression. Our study shows that the enhanced expression of Espin can optimize the developmental process of stereocilia in Atoh1-induced HCs and can attenuate the damage to native HCs induced by Atoh1 overexpression. These results suggest an effective method to induce the maturation of stereocilia in regenerative HCs and pave the way for functional HC regeneration via supporting cell transdifferentiation.
Assuntos
Actinas , Estereocílios , Animais , Estereocílios/metabolismo , Actinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Cóclea , Epitélio/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mamíferos/metabolismoRESUMO
Mechanosensitive hair cells (HCs) in the cochlear sensory epithelium are critical for sound detection and transduction. Mammalian HCs in the cochlea undergo cytogenesis during embryonic development, and irreversible damage to hair cells postnatally is a major cause of deafness. During the development of the organ of Corti, HCs and supporting cells (SCs) originate from the same precursors. In the neonatal cochlea, damage to HCs activates adjacent SCs to act as HC precursors and to differentiate into new HCs. However, the plasticity of SCs to produce new HCs is gradually lost with cochlear development. Here, we delineate an essential role for the guanine nucleotide exchange factor Net1 in SC trans-differentiation into HCs. Net1 overexpression mediated by AAV-ie in SCs promoted cochlear organoid formation and HC differentiation under two and three-dimensional culture conditions. Also, AAV-Net1 enhanced SC proliferation in Lgr5-EGFPCreERT2 mice and HC generation as indicated by lineage tracing of HCs in the cochleae of Lgr5-EGFPCreERT2/Rosa26-tdTomatoloxp/loxp mice. We further found that the up-regulation of Wnt/ß-catenin and Notch signaling in AAV-Net1-transduced cochleae might be responsible for the SC proliferation and HC differentiation. Also, Net1 overexpression in SCs enhanced SC proliferation and HC regeneration and survival after HC damage by neomycin. Taken together, our study suggests that Net1 might serve as a potential target for HC regeneration and that AAV-mediated gene regulation may be a promising approach in stem cell-based therapy in hearing restoration.
Assuntos
Transdiferenciação Celular , Células Ciliadas Auditivas , Animais , Camundongos , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Cóclea , Camundongos TransgênicosRESUMO
The cochlea consists of multiple types of cells, including hair cells, supporting cells and spiral ganglion neurons, and is responsible for converting mechanical forces into electric signals that enable hearing. Genetic and environmental factors can result in dysfunctions of cochlear and auditory systems. In recent years, gene therapy has emerged as a promising treatment in animal deafness models. One major challenge of the gene therapy for deafness is to effectively deliver genes to specific cells of cochleae. Here, we screened and identified an AAV-ie mutant, AAV-ie-K558R, that transduces hair cells and supporting cells in the cochleae of neonatal mice with high efficiency. AAV-ie-K558R is a safe vector with no obvious deficits in the hearing system. We found that AAV-ie-K558R can partially restore the hearing loss in Prestin KO mice and, importantly, deliver Atoh1 into cochlear supporting cells to generate hair cell-like cells. Our results demonstrate the clinical potential of AAV-ie-K558R for treating the hearing loss caused by hair cell death.
Assuntos
Surdez , Perda Auditiva , Animais , Cóclea/metabolismo , Surdez/metabolismo , Surdez/terapia , Terapia Genética , Células Ciliadas Auditivas/metabolismo , Perda Auditiva/genética , Perda Auditiva/metabolismo , Perda Auditiva/terapia , CamundongosRESUMO
G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB1), we discovered periodically repeating clusters of CB1 hotspots within the axons of neurons. We observed these CB1 hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB1 signaling. Furthermore, we found that CB1 hotspot periodicity increased upon CB1 agonist application, and these activated CB1 displayed less dynamic movement compared to non-activated CB1. Our results suggest that CB1 forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.
Assuntos
Encéfalo/citologia , Imagem Molecular/métodos , Neurônios/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Citoesqueleto/metabolismo , Feminino , Recuperação de Fluorescência Após Fotodegradação , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência/métodos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/análise , Receptor CB1 de Canabinoide/genéticaRESUMO
Transcription growth factor ß (TGF-ß) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-ß-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-ß-induced EMT process. Enhancers gained upon TGF-ß treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-ß stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-ß signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-ß signaling-associated cancer metastasis.
Assuntos
Reprogramação Celular/efeitos dos fármacos , Elementos Facilitadores Genéticos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Células A549 , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Fator 3-beta Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Histona Desacetilase 1/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Proteínas Musculares/metabolismo , Metástase Neoplásica , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
Auditory hair cells are the mechanical sensors of sound waves in the inner ear, and the stereocilia, which are actin-rich protrusions of different heights on the apical surfaces of hair cells, are responsible for the transduction of sound waves into electrical signals. As a crucial actin-binding and bundling protein, espin is able to cross-link actin filaments and is therefore necessary for stereocilia morphogenesis. Using advanced super-resolution stimulated emission depletion microscopy, we imaged espin expression at the sub-diffraction limit along the whole length of the stereocilia in outer hair cells and inner hair cells in order to better understand espin's function in the development of stereocilia.
RESUMO
Hearing loss is the most common sensory disorder. While gene therapy has emerged as a promising treatment of inherited diseases like hearing loss, it is dependent on the identification of gene delivery vectors. Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the US for treating a rare inherited eye disease but no safe and efficient vectors have been identified that can target the diverse types of inner ear cells. Here, we identify an AAV variant, AAV-inner ear (AAV-ie), for gene delivery in mouse inner ear. Our results show that AAV-ie transduces the cochlear supporting cells (SCs) with high efficiency, representing a vast improvement over conventional AAV serotypes. Furthermore, after AAV-ie-mediated transfer of the Atoh1 gene, we find that many SCs trans-differentiated into new HCs. Our results suggest that AAV-ie is a useful tool for the cochlear gene therapy and for investigating the mechanism of HC regeneration.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Dependovirus/genética , Terapia Genética/métodos , Células Ciliadas Auditivas Internas/citologia , Perda Auditiva/genética , Perda Auditiva/terapia , Animais , Células Cultivadas , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Clinical therapies of pluripotent stem cells (PSCs)-based transplantation have been hindered by frequent development of teratomas or tumors in animal models and clinical patients. Therefore, clarifying the mechanism of carcinogenesis in stem cell therapy is of great importance for reducing the risk of tumorigenicity. Here we differentiate Oct4-GFP mouse embryonic stem cells (mESCs) into neural progenitor cells (NPCs) and find that a minority of Oct4+ cells are continuously sustained at Oct4+ state. These cells can be enriched and proliferated in a standard ESC medium. Interestingly, the differentiation potential of these enriched cells is tightly restricted with much higher tumorigenic activity, which are thus defined as differentiation-resistant ESCs (DR-ESCs). Transcriptomic and epigenomic analyses show that DR-ESCs are characterized by primordial germ cell-like gene signatures (Dazl, Rec8, Stra8, Blimp1, etc.) and specific epigenetic patterns distinct from mESCs. Moreover, the DR-ESCs possess germ cell potential to generate Sycp3+ haploid cells and are able to reside in sperm-free spermaduct induced by busulfan. Finally, we find that TGFß signaling is overactivated in DR-ESCs, and inhibition of TGFß signaling eliminates the tumorigenicity of mESC-derived NPCs by inducing the full differentiation of DR-ESCs. These data demonstrate that these TGFß-hyperactivated germ cell-like DR-ESCs are the main contributor for the tumorigenicity of ESCs-derived target cell therapy and that inhibition of TGFß signaling in ESC-derived NPC transplantation could drastically reduce the risk of tumor development.
Assuntos
Carcinogênese/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neurais/citologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinogênese/genética , Diferenciação Celular , Linhagem Celular , Epigênese Genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Neurais/metabolismo , TranscriptomaRESUMO
Mouse pluripotent stem cells (PSCs), such as ES cells and induced PSCs (iPSCs), are an excellent system to investigate the molecular and cellular mechanisms involved in early embryonic development. The signaling pathways orchestrated by leukemia inhibitor factor/STAT3, Wnt/ß-catenin, and FGF/MEK/ERK play key roles in the generation of pluripotency. However, the function of TGF-ß signaling in this process remains elusive. Here we show that inhibiting TGF-ß signaling with its inhibitor SB431542 can substitute for Oct4 during reprogramming. Moreover, inhibiting TGF-ß signaling can sustain the pluripotency of iPSCs and ES cells through modulating FGF/MEK/ERK signaling. Therefore, this study reveals a novel function of TGF-ß signaling inhibition in the generation and maintenance of PSCs.
Assuntos
Benzamidas/farmacologia , Reprogramação Celular/efeitos dos fármacos , Dioxóis/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Imunofluorescência , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 3 de Transcrição de Octâmero/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
RESUMO
Proliferation of the neural/neuronal progenitor cells (NPCs) at the ventricular zone of the dorsal spinal cord requires the stimuli of Wnt and bone morphogenic protein (BMP). However, how these two signaling pathways are regulated to initiate differentiation in the NPCs as they enter the intermediate zone is not known. Here, we show that Smad6, a negative regulator of BMP signaling, is expressed in the intermediate zone of the chick dorsal spinal cord. Knockdown experiments show that Smad6 is required for promoting NPCs to exit the cell cycle and differentiate into neurons. Although we find that Smad6 inhibits BMP signaling, as expected, we also find that Smad6 unexpectedly inhibits the Wnt/ß-catenin pathway. The inhibition of the Wnt/ß-catenin pathway by Smad6 is independent of its effect on the BMP pathway. Rather, Smad6 through its N-terminal domain and link region enhances the interaction of C-terminal binding protein with the ß-catenin/T cell factor (TCF) complex and the TCF-binding element to inhibit ß-catenin-mediated transcriptional activation. Our study provides evidence that transition of NPCs from a proliferative state to a differentiating state is controlled by the dual inhibitory role of Smad6 to both BMP and Wnt signaling at the level of transcription.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Tubo Neural/embriologia , Neurônios/citologia , Transdução de Sinais/fisiologia , Proteína Smad6/metabolismo , Análise de Variância , Animais , Western Blotting , Bromodesoxiuridina , Embrião de Galinha , Imunoprecipitação da Cromatina , Primers do DNA/genética , Eletroporação , Imunofluorescência , Regulação da Expressão Gênica/genética , Imunoprecipitação , Hibridização In Situ , Luciferases , Tubo Neural/metabolismo , Reação em Cadeia da Polimerase , Interferência de RNA , Proteína Smad6/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Bone morphogenetic protein (BMP) signaling plays a crucial role in maintaining the pluripotency of mouse embryonic stem cells (ESCs) and has negative effects on ESC neural differentiation. However, it remains unclear when and how BMP signaling executes those different functions during neural commitment. Here, we show that a BMP4-sensitive window exists during ESC neural differentiation. Cells at this specific period correspond to the egg cylinder stage epiblast and can be maintained as ESC-derived epiblast stem cells (ESD-EpiSCs), which have the same characteristics as EpiSCs derived from mouse embryos. We propose that ESC neural differentiation occurs in two stages: first from ESCs to ESD-EpiSCs and then from ESD-EpiSCs to neural precursor cells (NPCs). We further show that BMP4 inhibits the conversion of ESCs into ESD-EpiSCs during the first stage, and suppresses ESD-EpiSC neural commitment and promotes non-neural lineage differentiation during the second stage. Mechanistic studies show that BMP4 inhibits FGF/ERK activity at the first stage but not at the second stage; and IDs, as important downstream genes of BMP signaling, partially substitute for BMP4 functions at both stages. We conclude that BMP signaling has distinct functions during different stages of ESC neural commitment.