RESUMEN
Mutations in, and the altered expression of, epigenetic modifiers are pervasive in human tumours, making epigenetic factors attractive antitumour targets. The open-versus-closed chromatin state within the cells-of-origin of cancer correlates with the uneven distribution of mutations. However, the long-term effect of targeting epigenetic modifiers on mutability in patients with cancer is unclear. Here, we increased chromatin accessibility by deleting the histone H3 lysine 9 (H3K9) methyltransferase G9a in murine epidermis and show that this does not alter the single nucleotide variant burden or global genomic distribution in chemical mutagen-induced squamous tumours. G9a-depleted tumours develop after a prolonged latency compared with their wild-type counterparts, but are more aggressive and have an expanded cancer progenitor pool, pronounced genomic instability and frequent loss-of-function p53 mutations. Thus, we call for caution when assessing long-term therapeutic benefits of chromatin modifier inhibitors, which may promote more aggressive disease.
Asunto(s)
Cromatina/genética , Inestabilidad Genómica , N-Metiltransferasa de Histona-Lisina/genética , Mutación , Neoplasias Cutáneas/genética , Animales , Línea Celular , Cromatina/metabolismo , Epidermis/metabolismo , Epidermis/patología , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Invasividad Neoplásica , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The pancreatic ß-cell transcriptome is highly sensitive to external signals such as glucose oscillations and stress cues. MicroRNAs (miRNAs) have emerged as key factors in gene expression regulation. Here, we aimed to identify miRNAs that are modulated by glucose in mouse pancreatic islets. We identified miR-708 as the most upregulated miRNA in islets cultured at low glucose concentrations, a setting that triggers a strong stress response. miR-708 was also potently upregulated by triggering endoplasmic reticulum (ER) stress with thapsigargin and in islets of ob/ob mice. Low-glucose induction of miR-708 was blocked by treatment with the chemical chaperone 4-phenylbutyrate, uncovering the involvement of ER stress in this response. An integrative analysis identified neuronatin (Nnat) as a potential glucose-regulated target of miR-708. Indeed, Nnat expression was inversely correlated with miR-708 in islets cultured at different glucose concentrations and in ob/ob mouse islets and was reduced after miR-708 overexpression. Consistent with the role of Nnat in the secretory function of ß-cells, miR-708 overexpression impaired glucose-stimulated insulin secretion (GSIS), which was recovered by NNAT overexpression. Moreover, miR-708 inhibition recovered GSIS in islets cultured at low glucose. Finally, miR-708 overexpression suppressed ß-cell proliferation and induced ß-cell apoptosis. Collectively, our results provide a novel mechanism of glucose regulation of ß-cell function and growth by repressing stress-induced miR-708.
Asunto(s)
Estrés del Retículo Endoplásmico , Células Secretoras de Insulina/fisiología , MicroARNs/fisiología , Animales , Apoptosis , Células Cultivadas , Células Secretoras de Insulina/metabolismo , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , MicroARNs/análisis , Proteínas del Tejido Nervioso/análisis , Factor de Transcripción CHOP/genéticaRESUMEN
The DNA methyltransferase Dnmt3a suppresses tumorigenesis in models of leukemia and lung cancer. Conversely, deregulation of Dnmt3b is thought to generally promote tumorigenesis. However, the role of Dnmt3a and Dnmt3b in many types of cancer remains undefined. Here, we show that Dnmt3a and Dnmt3b are dispensable for homeostasis of the murine epidermis. However, loss of Dnmt3a-but not Dnmt3b-increases the number of carcinogen-induced squamous tumors, without affecting tumor progression. Only upon combined deletion of Dnmt3a and Dnmt3b, squamous carcinomas become more aggressive and metastatic. Mechanistically, Dnmt3a promotes the expression of epidermal differentiation genes by interacting with their enhancers and inhibits the expression of lipid metabolism genes, including PPAR-γ, by directly methylating their promoters. Importantly, inhibition of PPAR-γ partially prevents the increase in tumorigenesis upon deletion of Dnmt3a. Altogether, we demonstrate that Dnmt3a and Dnmt3b protect the epidermis from tumorigenesis and that squamous carcinomas are sensitive to inhibition of PPAR-γ.
Asunto(s)
Carcinoma de Células Escamosas/fisiopatología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Epidermis/fisiología , Homeostasis , PPAR gamma/metabolismo , Animales , ADN Metiltransferasa 3A , Ratones , ADN Metiltransferasa 3BRESUMEN
The fact that the identity of the cells that initiate metastasis in most human cancers is unknown hampers the development of antimetastatic therapies. Here we describe a subpopulation of CD44bright cells in human oral carcinomas that do not overexpress mesenchymal genes, are slow-cycling, express high levels of the fatty acid receptor CD36 and lipid metabolism genes, and are unique in their ability to initiate metastasis. Palmitic acid or a high-fat diet specifically boosts the metastatic potential of CD36+ metastasis-initiating cells in a CD36-dependent manner. The use of neutralizing antibodies to block CD36 causes almost complete inhibition of metastasis in immunodeficient or immunocompetent orthotopic mouse models of human oral cancer, with no side effects. Clinically, the presence of CD36+ metastasis-initiating cells correlates with a poor prognosis for numerous types of carcinomas, and inhibition of CD36 also impairs metastasis, at least in human melanoma- and breast cancer-derived tumours. Together, our results indicate that metastasis-initiating cells particularly rely on dietary lipids to promote metastasis.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antígenos CD36/antagonistas & inhibidores , Neoplasias de la Boca/patología , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Antígenos CD36/genética , Antígenos CD36/inmunología , Antígenos CD36/metabolismo , Proliferación Celular , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores de Hialuranos/metabolismo , Metabolismo de los Lípidos/genética , Metástasis Linfática/genética , Metástasis Linfática/patología , Masculino , Ratones , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/metabolismo , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/genética , Células Madre Neoplásicas/metabolismo , Ácido Palmítico/administración & dosificación , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacología , Penetrancia , Pronóstico , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The most common thyroid abnormality among Down syndrome (DS) children corresponds to a mildly elevated TSH, with T4 decreased or in the normal range and thyroid hypoplasia, from the neonatal period onward, which aggravate their mental impairment. Transgenic Dyrk1A mice, obtained by bacterial artificial chromosome engineering (mBACTgDyrk1A), have 3 copies of the Dyrk1A gene. The objective is to determine whether this transgenic Dyrk1A (Dyrk1A(+/++)) mouse is an adequate murine model for the study of thyroid dysgenesis in DS. Embryonic thyroid development from embryonic day 13.5 (E13.5) to E17.5 was analyzed in wild-type (WT) and Dyrk1A(+/++) mice by immunofluorescence with anti-Nkx2-1, anti-thyroglobulin, and anti-T4 antibodies, markers of early thyroid development, hormonogenesis, and final differentiation, respectively. The expression of transcription factors Nkx2-1, Pax8, and Foxe1 involved in thyroidogenesis were studied by quantitative RT-PCR at the same embryonic stages. We then compared the adult phenotype at 8 to 12 weeks in Dyrk1A(+/++) and WT mice for T4 and TSH levels, thyroidal weight, and histological analysis. Regarding thyroidal development, at E15.5, Dyrk1A(+/++) thyroid lobes are double the size of WT thyroids (P = .01), but the thyroglobulin stained surface in Dyrk1A(+/++) thyroids is less than a third as large at E17.5 (P = .04) and their differentiated follicular surface half the size (P = .004). We also observed a significant increase in Nkx2-1, Foxe1, and Pax8 RNA levels in E13.5 and E17.5 Dyrk1A(+/++) embryonic thyroids. Dyrk1A(+/++) young adult mice have significantly lower plasma T4 (2.4 ng/mL versus WT, 3.7 ng/mL; P = 0.019) and nonsignificantly higher plasma TSH (114 mUI/L versus WT, 73mUI/L; P = .09). In addition, their thyroids are significantly heavier (P = .04) and exhibit large disorganized regions. Dyrk1A overexpression directly leads to thyroidal embryogenetic, functional and morphological impairment. The young adult thyroid phenotype is probably a result of embryogenetic impairment. The Dyrk1A(+/++) mouse can be considered a suitable study model for thyroid dysgenesis in DS.
Asunto(s)
Modelos Animales de Enfermedad , Síndrome de Down/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Disgenesias Tiroideas/metabolismo , Glándula Tiroides/embriología , Animales , Cromosomas Artificiales Bacterianos , Síndrome de Down/complicaciones , Síndrome de Down/patología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Disgenesias Tiroideas/complicaciones , Disgenesias Tiroideas/genética , Quinasas DyrKRESUMEN
Diabetes is caused by loss or dysfunction of pancreatic beta cells. Generation of beta cells in vitro is a promising strategy to develop a full-scale cell therapy against diabetes, and the development of methods without gene transfer may provide safer protocols for human therapy. Here we show that thyroid hormone receptors are expressed in embryonic murine pancreas. Addition of the thyroid hormone T3 in an ex vivo culture model of embryonic (E12.5) dorsal pancreas, mimicking embryonic pancreatic development, promoted an increase of ductal cell number at expenses of the acinar compartment. Double labeled cells expressing specific markers for ductal and acinar cells were observed, suggesting cell reprogramming. Increased mRNA levels of the pro-endocrine gene Ngn3 and an increased number of beta cells were detected in cultures treated previously with T3 suggesting that ductal cells promoted by T3 can subsequently differentiate into endocrine cells. So, indirectly, T3 induced endocrine differentiation. Moreover, T3 induced the expression of the pro-endocrine gene Ngn3 in the acinar 266-6 cell line. The pro-endocrine effect of T3 in the pancreatic explants and in the acinar cell line, was abrogated by the Akt inhibitor Ly294002 indicating the involvement of Akt signaling in this process. Altogether we show numerous evidences that define T3 as a promising candidate to generate endocrine cells from exocrine tissue, using ectopically gene expression free protocols, for cell therapy against diabetes.
Asunto(s)
Células Acinares/citología , Células Secretoras de Insulina/citología , Páncreas/embriología , Conductos Pancreáticos/citología , Triyodotironina/farmacología , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Ratones , Morfolinas/farmacología , Técnicas de Cultivo de Órganos , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Conductos Pancreáticos/efectos de los fármacos , Conductos Pancreáticos/metabolismo , Embarazo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Hormona Tiroidea/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3(+) progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3(CreERT/+)) and Neurog3-deficient (Neurog3(CreERT/CreERT)) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities.
Asunto(s)
Células Acinares/citología , Tipificación del Cuerpo , Sistema Endocrino/citología , Sistema Endocrino/embriología , Conductos Pancreáticos/citología , Conductos Pancreáticos/embriología , Células Madre/citología , Células Acinares/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Linaje de la Célula , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Epitelio/embriología , Epitelio/metabolismo , Hormonas/metabolismo , Ratones , Modelos Biológicos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/metabolismo , Conductos Pancreáticos/metabolismo , Células Madre/metabolismo , Factores de Tiempo , Torso/embriologíaRESUMEN
A longstanding unsettled question is whether pancreatic beta cells originate from exocrine duct cells. We have now used genetic labeling to fate map embryonic and adult pancreatic duct cells. We show that Hnf1beta+ cells of the trunk compartment of the early branching pancreas are precursors of acinar, duct, and endocrine lineages. Hnf1beta+ cells subsequent form the embryonic duct epithelium, which gives rise to both ductal and endocrine lineages, but not to acinar cells. By the end of gestation, the fate of Hnf1beta+ duct cells is further restrained. We provide compelling evidence that the ductal epithelium does not make a significant contribution to acinar or endocrine cells during neonatal growth, during a 6 month observation period, or during beta cell growth triggered by ligation of the pancreatic duct or by cell-specific ablation with alloxan followed by EGF/gastrin treatment. Thus, once the ductal epithelium differentiates it has a restricted plasticity, even under regenerative settings.
Asunto(s)
Células Secretoras de Insulina/citología , Páncreas/embriología , Animales , Femenino , Factor Nuclear 1-beta del Hepatocito/genética , Factor Nuclear 1-beta del Hepatocito/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Páncreas/citología , Páncreas Exocrino/embriologíaRESUMEN
Through the analysis of genetically modified mice a hierarchy of transcription factors regulating pancreas specification, endocrine destiny as well as endocrine subtype specification and differentiation has been established. In addition to conventional approaches such as transgenic technologies and gene targeting, recombinase fate mapping in mice has been key in establishing the lineage relationship between progenitor cells and their progeny in understanding pancreas formation. Moreover, the design of specific mouse models to conditionally express transcription factors in different populations of progenitor cells has revealed to what extent transcription factors required for islet cell development are also sufficient to induce endocrine differentiation and the importance of the competence of progenitor cells to respond to the genetic program implemented by these factors. Taking advantage of this basic science knowledge acquired in rodents, immature insulin-producing cells have recently been differentiated in vitro from human embryonic stem cells. Taken together these major advances emphasize the need to gain further in-depth knowledge of the molecular and cellular mechanisms controlling beta-cell differentiation in mice to generate functional beta-cells in the future that could be used for cell therapy in diabetes.
Asunto(s)
Desarrollo Embrionario/fisiología , Páncreas/embriología , Factores de Transcripción/genética , Animales , Diferenciación Celular , Diabetes Mellitus Tipo 1/terapia , Endodermo/fisiología , Femenino , Ratones , Modelos Animales , Páncreas/crecimiento & desarrollo , EmbarazoRESUMEN
Diabetes results from the absolute or relative deficiency of insulin-producing beta cells. The prospect that non-beta pancreatic cells could be harnessed to become beta cells has led to interest in understanding the plasticity of pancreatic cells. Recent studies, however, have shown that adult beta cells are largely derived from preexisting beta cells. In this issue of the JCI, Desai et al. show that acinar cells, the major cell type in the pancreas, do not contribute to new beta cells formed during pancreatic regeneration (see the related article beginning on page 971). These studies suggest that the fate of adult pancreatic cell lineages is immutable. However, also in this issue of the JCI, Collombat et al. demonstrate that inducing a single transcription factor named Arx in adult beta cells causes these cells to undergo massive transdifferentiation into alpha and pancreatic polypeptide endocrine cells (see the related article beginning on page 961). This finding points to an unexpected plasticity of postnatal pancreatic endocrine cells.
Asunto(s)
Diabetes Mellitus/etiología , Células Secretoras de Insulina/fisiología , Páncreas/citología , Páncreas/fisiología , Diabetes Mellitus/fisiopatología , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/fisiopatología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , RegeneraciónRESUMEN
The deleted in colorectal cancer (DCC) gene encodes a 170- to 190-kDa protein of the Immunoglobulin superfamily. Firstly identified as a tumor suppressor gene in human colorectal carcinomas, the main function for DCC has been described in the nervous system as part of a receptor complex for netrin-1. Moreover, roles in mucosecretory cell differentiation and as inducer of apoptosis have also been reported. DCC knockout mice supported a crucial role for this gene in axonal migration, yet questioned its implication in tumor suppression and mucosecretory differentiation. The work presented here demonstrates that a DCC-transfected HT-29 colonic human cell line (HT-29/DCC) displays an increase in cell-cell adhesion to the detriment of cell-matrix interactions: HT-29/DCC cells exhibit more and better-structured desmosomes while focal adhesions and hemidesmosomes are disrupted. HT-29/DCC cells show no changes in adherent junctions but upon treatment with TPA, HT-29/DCC cells show resistance to scattering, and maintain E-cadherin in the membrane. In addition, the actin cytoskeleton is affected in HT-29/DCC cells: stress fibers are disrupted while cortical actin remains intact. We identified a putative ERM-M (ezrin/radixin/moesin and merlin) binding domain in the juxtamembrane region of the DCC protein. In vitro pull-down assays demonstrate the interaction of the DCC cytoplasmic domain with the N-terminal region of ezrin and merlin, and co-immunoprecipitation assays in transiently DCC-transfected COS-1 cells showed that the interaction between DCC and ezrin also takes place in vivo. Altogether, our results suggest that DCC could regulate cell adhesion and migration through its association with ERM-M proteins.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Western Blotting , Adhesión Celular/fisiología , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Receptor DCC , Desmosomas/metabolismo , Desmosomas/ultraestructura , Matriz Extracelular/metabolismo , Células HT29 , Humanos , Inmunohistoquímica , Inmunoprecipitación , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica , Modelos Genéticos , Datos de Secuencia Molecular , Neurofibromina 2/metabolismo , Unión Proteica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología , Transfección/métodos , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.
Asunto(s)
Aldehído Oxidorreductasas/deficiencia , Aldehído Oxidorreductasas/metabolismo , Páncreas/embriología , Tretinoina/metabolismo , Aldehído Oxidorreductasas/genética , Animales , Diferenciación Celular/efectos de los fármacos , Endodermo/citología , Endodermo/metabolismo , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Heterocigoto , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Metaloproteínas/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes , Páncreas/citología , ARN Mensajero/metabolismo , Transactivadores/deficiencia , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transgenes , Tretinoina/administración & dosificación , Tretinoina/farmacología , beta-Galactosidasa/metabolismoRESUMEN
The basic helix-loop-helix transcription factor Neurogenin 3 (NGN3) controls endocrine cell fate specification in uncommitted pancreatic progenitor cells. Ngn3-deficient mice do not develop any islet cells and are diabetic. All the major islet cell types, including insulin-producing beta-cells, derive from Ngn3-positive endocrine progenitor cells. Therefore, the characterization of this population of immature cells is of particular interest for the development of novel strategies for cell replacement therapies in type 1 diabetes. To explore further the biology of islet progenitor cells we have generated a mouse in which Ngn3-expressing cells are labeled with the enhanced yellow fluorescent protein (EYFP) using a knock-add-on strategy. In this approach, the EYFP cDNA is introduced into the 3'-untranslated region of the proendocrine transcription factor, Neurogenin 3, without deleting any endogenous coding or regulatory sequences. In Ngn3(EYFP/+) and Ngn3(EYFP/EYFP) mice, the EYFP protein is targeted to Ngn3-expressing progenitors in the developing pancreas, and islets develop normally. Islet progenitors can be purified from whole embryonic pancreas by fluorescence-activated cell sorting from Ngn3(EYFP/+) mice and their development can be monitored in real time in pancreas explant cultures. These experiments showed that endocrine progenitors can form de novo and expand, in vitro, in the absence of signals from the surrounding mesenchyme, suggesting that endocrine commitment is a default pathway. The Ngn3(EYFP) mice represent a valuable tool to study islet cell development and neogenesis in normal and diabetic animals as well as for the determination of the conditions to generate beta-cells in vitro.