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1.
Artigo em Inglês | MEDLINE | ID: mdl-31310834

RESUMO

BACKGROUND & AIMS: Activating mutation of the KRAS gene is common in some cancers, such as pancreatic cancer, but rare in other cancers. Chronic pancreatitis is a predisposing condition for pancreatic ductal adenocarcinoma (PDAC), but how it synergizes with KRAS mutation is not known. METHODS: We used a mouse model to express an activating mutation of Kras in conjunction with obstruction of the main pancreatic duct to recapitulate a common etiology of human chronic pancreatitis. Because the cell of origin of PDAC is not clear, Kras mutation was introduced into either duct cells or acinar cells. RESULTS: Although KrasG12D expression in both cell types was protective against damage-associated cell death, chronic pancreatitis induced p53, p21, and growth arrest only in acinar-derived cells. Mutant duct cells did not elevate p53 or p21 expression and exhibited increased proliferation driving the appearance of PDAC over time. CONCLUSIONS: One mechanism by which tissues may be susceptible or resistant to KRASG12D-initiated tumorigenesis is whether they undergo a p53-mediated damage response. In summary, we have uncovered a mechanism by which inflammation and intrinsic cellular programming synergize for the development of PDAC.

2.
Cell Rep ; 22(10): 2667-2676, 2018 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-29514095

RESUMO

Many patients with type 1 diabetes (T1D) have residual ß cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual ß cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant ß cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and ß cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-ß cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.

3.
Development ; 142(21): 3637-48, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26534984

RESUMO

Homozygous Mnx1 mutation causes permanent neonatal diabetes in humans, but via unknown mechanisms. Our systematic and longitudinal analysis of Mnx1 function during murine pancreas organogenesis and into the adult uncovered novel stage-specific roles for Mnx1 in endocrine lineage allocation and ß-cell fate maintenance. Inactivation in the endocrine-progenitor stage shows that Mnx1 promotes ß-cell while suppressing δ-cell differentiation programs, and is crucial for postnatal ß-cell fate maintenance. Inactivating Mnx1 in embryonic ß-cells (Mnx1(Δbeta)) caused ß-to-δ-like cell transdifferentiation, which was delayed until postnatal stages. In the latter context, ß-cells escaping Mnx1 inactivation unexpectedly upregulated Mnx1 expression and underwent an age-independent persistent proliferation. Escaper ß-cells restored, but then eventually surpassed, the normal pancreatic ß-cell mass, leading to islet hyperplasia in aged mice. In vitro analysis of islets isolated from Mnx1(Δbeta) mice showed higher insulin secretory activity and greater insulin mRNA content than in wild-type islets. Mnx1(Δbeta) mice also showed a much faster return to euglycemia after ß-cell ablation, suggesting that the new ß-cells derived from the escaper population are functional. Our findings identify Mnx1 as an important factor in ß-cell differentiation and proliferation, with the potential for targeting to increase the number of endogenous ß-cells for diabetes therapy.


Assuntos
Diabetes Mellitus/patologia , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Transdiferenciação Celular , Senescência Celular , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Hiperplasia/metabolismo , Células Secretoras de Insulina/citologia , Camundongos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Células Secretoras de Somatostatina/metabolismo , Fatores de Transcrição/genética
4.
Artigo em Inglês | MEDLINE | ID: mdl-26257608

RESUMO

Commissural neurons project across the midline at all levels of the central nervous system (CNS), providing bilateral communication critical for the coordination of motor activity and sensory perception. Midline crossing at the spinal ventral midline has been extensively studied and has revealed that multiple developmental lineages contribute to this commissural neuron population. Ventral midline crossing occurs in a manner dependent on Robo3 regulation of Robo/Slit signaling and the ventral commissure is absent in the spinal cord and hindbrain of Robo3 mutants. Midline crossing in the spinal cord is not limited to the ventral midline, however. While prior anatomical studies provide evidence that commissural axons also cross the midline dorsally, little is known of the genetic and molecular properties of dorsally-crossing neurons or of the mechanisms that regulate dorsal midline crossing. In this study, we describe a commissural neuron population that crosses the spinal dorsal midline during the last quarter of embryogenesis in discrete fiber bundles present throughout the rostrocaudal extent of the spinal cord. Using immunohistochemistry, neurotracing, and mouse genetics, we show that this commissural neuron population includes spinal inhibitory neurons and sensory nociceptors. While the floor plate and roof plate are dispensable for dorsal midline crossing, we show that this population depends on Robo/Slit signaling yet crosses the dorsal midline in a Robo3-independent manner. The dorsally-crossing commissural neuron population we describe suggests a substrate circuitry for pain processing in the dorsal spinal cord.


Assuntos
Padronização Corporal/fisiologia , Proteínas de Membrana/metabolismo , Atividade Motora/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural/fisiologia , Nociceptores/fisiologia , Medula Espinal , Fatores Etários , Aminoácidos/metabolismo , Animais , Axônios/fisiologia , Padronização Corporal/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Inibição Neural/genética , Transdução de Sinais/fisiologia , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Curr Opin Endocrinol Diabetes Obes ; 21(2): 77-82, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24569548

RESUMO

PURPOSE OF REVIEW: We highlight some of the major recent advances in characterizing human pancreas development and endocrine cell differentiation. RECENT FINDINGS: Extensive research efforts have helped to define crucial events in the mouse pancreas organogenesis. Information gained from these studies was used to develop human embryonic stem cell (hESC) differentiation protocols with the goal of generating functional glucose-responsive, insulin-producing human ß-cells. In spite of remarkable progress in hESC differentiation, current protocols based on mouse developmental biology can produce human ß-cells only in vivo. New differentiation markers and recently generated reagents may provide an unprecedented opportunity to develop a high-density expression map of human fetal pancreas and pancreatic islets that could serve as a reference point for in vitro hESC differentiation. SUMMARY: Integrating an increased knowledge of human pancreas development into hESC differentiation protocols has the potential to greatly advance our ability to generate functional insulin-producing cells for ß-cell replacement therapy.


Assuntos
Células-Tronco Embrionárias/citologia , Sistema Endócrino/citologia , Células Secretoras de Insulina/citologia , Pâncreas/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Sistema Endócrino/embriologia , Glucose/metabolismo , Humanos , Camundongos , Organogênese , Pâncreas/embriologia , Pâncreas/crescimento & desenvolvimento , Transdução de Sinais
6.
Gastroenterology ; 146(1): 233-44.e5, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23999170

RESUMO

BACKGROUND & AIMS: Metaplasias often have characteristics of developmentally related tissues. Pancreatic metaplastic ducts are usually associated with pancreatitis and pancreatic ductal adenocarcinoma. The tuft cell is a chemosensory cell that responds to signals in the extracellular environment via effector molecules. Commonly found in the biliary tract, tuft cells are absent from normal murine pancreas. Using the aberrant appearance of tuft cells as an indicator, we tested if pancreatic metaplasia represents transdifferentiation to a biliary phenotype and what effect this has on pancreatic tumorigenesis. METHODS: We analyzed pancreatic tissue and tumors that developed in mice that express an activated form of Kras (Kras(LSL-G12D/+);Ptf1a(Cre/+) mice). Normal bile duct, pancreatic duct, and tumor-associated metaplasias from the mice were analyzed for tuft cell and biliary progenitor markers, including SOX17, a transcription factor that regulates biliary development. We also analyzed pancreatic tissues from mice expressing transgenic SOX17 alone (ROSA(tTa/+);Ptf1(CreERTM/+);tetO-SOX17) or along with activated Kras (ROSAtT(a/+);Ptf1a(CreERTM/+);tetO-SOX17;Kras(LSL-G12D;+)). RESULTS: Tuft cells were frequently found in areas of pancreatic metaplasia, decreased throughout tumor progression, and absent from invasive tumors. Analysis of the pancreatobiliary ductal systems of mice revealed tuft cells in the biliary tract but not the normal pancreatic duct. Analysis for biliary markers revealed expression of SOX17 in pancreatic metaplasia and tumors. Pancreas-specific overexpression of SOX17 led to ductal metaplasia along with inflammation and collagen deposition. Mice that overexpressed SOX17 along with Kras(G12D) had a greater degree of transformed tissue compared with mice expressing only Kras(G12D). Immunofluorescence analysis of human pancreatic tissue arrays revealed the presence of tuft cells in metaplasia and early-stage tumors, along with SOX17 expression, consistent with a biliary phenotype. CONCLUSIONS: Expression of Kras(G12D) and SOX17 in mice induces development of metaplasias with a biliary phenotype containing tuft cells. Tuft cells express a number of tumorigenic factors that can alter the microenvironment. Expression of SOX17 induces pancreatitis and promotes Kras(G12D)-induced tumorigenesis in mice.


Assuntos
Ductos Biliares/citologia , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/patologia , Proteínas HMGB/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/patologia , Fatores de Transcrição SOXF/metabolismo , Animais , Ductos Biliares/metabolismo , Carcinoma Ductal Pancreático/complicações , Carcinoma Ductal Pancreático/metabolismo , Transformação Celular Neoplásica/metabolismo , Humanos , Metaplasia/complicações , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Camundongos Transgênicos , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/metabolismo , Pancreatite/metabolismo , Lesões Pré-Cancerosas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais
7.
Diabetes ; 62(12): 4154-64, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23884891

RESUMO

Pancreatic islets are highly vascularized mini-organs, and vascular endothelial growth factor (VEGF)-A is a critical factor in the development of islet vascularization. To investigate the role of VEGF-A and endothelial cells (ECs) in adult islets, we used complementary genetic approaches to temporally inactivate VEGF-A in developing mouse pancreatic and islet progenitor cells or in adult ß-cells. Inactivation of VEGF-A early in development dramatically reduced pancreatic and islet vascularization, leading to reduced ß-cell proliferation in both developing and adult islets and, ultimately, reduced ß-cell mass and impaired glucose clearance. When VEGF-A was inactivated in adult ß-cells, islet vascularization was reduced twofold. Surprisingly, even after 3 months of reduced islet vascularization, islet architecture and ß-cell gene expression, mass, and function were preserved with only a minimal abnormality in glucose clearance. These data show that normal pancreatic VEGF-A expression is critical for the recruitment of ECs and the subsequent stimulation of endocrine cell proliferation during islet development. In contrast, although VEGF-A is required for maintaining the specialized vasculature observed in normal adult islets, adult ß-cells can adapt and survive long-term reductions in islet vascularity. These results indicate that VEGF-A and islet vascularization have a lesser role in adult islet function and ß-cell mass.


Assuntos
Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/irrigação sanguínea , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Camundongos
8.
Development ; 140(4): 751-64, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23325761

RESUMO

Pancreatic multipotent progenitor cells (MPCs) produce acinar, endocrine and duct cells during organogenesis, but their existence and location in the mature organ remain contentious. We used inducible lineage-tracing from the MPC-instructive gene Ptf1a to define systematically in mice the switch of Ptf1a(+) MPCs to unipotent proacinar competence during the secondary transition, their rapid decline during organogenesis, and absence from the mature organ. Between E11.5 and E15.5, we describe tip epithelium heterogeneity, suggesting that putative Ptf1a(+)Sox9(+)Hnf1ß(+) MPCs are intermingled with Ptf1a(HI)Sox9(LO) proacinar progenitors. In the adult, pancreatic duct ligation (PDL) caused facultative reactivation of multipotency factors (Sox9 and Hnf1ß) in Ptf1a(+) acini, which undergo rapid reprogramming to duct cells and longer-term reprogramming to endocrine cells, including insulin(+) ß-cells that are mature by the criteria of producing Pdx1(HI), Nkx6.1(+) and MafA(+). These Ptf1a lineage-derived endocrine/ß-cells are likely formed via Ck19(+)/Hnf1ß(+)/Sox9(+) ductal and Ngn3(+) endocrine progenitor intermediates. Acinar to endocrine/ß-cell transdifferentiation was enhanced by combining PDL with pharmacological elimination of pre-existing ß-cells. Thus, we show that acinar cells, without exogenously introduced factors, can regain aspects of embryonic multipotentiality under injury, and convert into mature ß-cells.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Multipotentes/fisiologia , Organogênese/fisiologia , Pâncreas/embriologia , Recuperação de Função Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Células Acinares/citologia , Animais , Pesos e Medidas Corporais , Técnicas de Introdução de Genes , Camundongos , Microscopia Confocal , Células-Tronco Multipotentes/metabolismo , Pâncreas/fisiologia , Tamoxifeno , Fatores de Tempo
9.
Cancer Cell ; 22(6): 737-50, 2012 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-23201164

RESUMO

Tumors are largely classified by histologic appearance, yet morphologic features do not necessarily predict cellular origin. To determine the origin of pancreatic ductal adenocarcinoma (PDA), we labeled and traced pancreatic cell populations after induction of a PDA-initiating Kras mutation. Our studies reveal that ductal and stem-like centroacinar cells are surprisingly refractory to oncogenic transformation, whereas acinar cells readily form PDA precursor lesions with ductal features. We show that formation of acinar-derived premalignant lesions depends on ectopic induction of the ductal gene Sox9. Moreover, when concomitantly expressed with oncogenic Kras, Sox9 accelerates formation of premalignant lesions. These results provide insight into the cellular origin of PDA and suggest that its precursors arise via induction of a duct-like state in acinar cells.


Assuntos
Células Acinares/patologia , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/genética , Fatores de Transcrição SOX9/genética , Células Acinares/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Genes ras , Humanos , Metaplasia , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Fatores de Transcrição SOX9/metabolismo
10.
Dev Biol ; 367(1): 40-54, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22546694

RESUMO

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the ß cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and ß cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and ß cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in ß cell proliferation and ß cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in ß cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in ß cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs ß cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.


Assuntos
Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Animais , Contagem de Células , Células Endoteliais/metabolismo , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/metabolismo , Camundongos
11.
Dev Biol ; 362(1): 57-64, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22146645

RESUMO

The Notch signaling pathway regulates embryonic development of the pancreas, inhibiting progenitor differentiation into exocrine acinar and endocrine islet cells. The adult pancreas appears to lack progenitor cells, and its mature cell types are maintained by the proliferation of pre-existing differentiated cells. Nonetheless, Notch remains active in adult duct and terminal duct/centroacinar cells (CACs), in which its function is unknown. We previously developed mice in which cells expressing the Notch target gene Hes1 can be labeled and manipulated, by expression of Cre recombinase, and demonstrated that Hes1(+) CACs do not behave as acinar or islet progenitors in the uninjured pancreas, or as islet progenitors after pancreatic duct ligation. In the current study, we assessed the function of Notch signaling in the adult pancreas by deleting the transcription factor partner of Notch, Rbpj, specifically in Hes1(+) cells. We find that loss of Rbpj depletes the pancreas of Hes1-expressing CACs, abrogating their ongoing contribution to growth and homeostasis of more proximal duct structures. Upon Rbpj deletion, CACs undergo a rapid transformation into acinar cells, suggesting that constitutive Notch activity suppresses the acinar differentiation potential of CACs. Together, our data provide direct evidence of an endogenous genetic program to control interconversion of cell fates in the adult pancreas.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Pâncreas Exócrino/fisiologia , Fenótipo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Células Acinares/metabolismo , Animais , Diferenciação Celular/fisiologia , Técnicas Histológicas , Camundongos , Oligonucleotídeos/genética , Fatores de Transcrição HES-1
12.
Mol Cell Endocrinol ; 339(1-2): 144-50, 2011 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-21539888

RESUMO

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile.


Assuntos
Antígenos de Superfície/metabolismo , Separação Celular/métodos , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Ductos Pancreáticos/metabolismo , Animais , Anticorpos Monoclonais Murinos , Antígenos de Superfície/imunologia , Diacilglicerol Quinase/metabolismo , Dipeptidil Peptidase 4/metabolismo , Citometria de Fluxo/métodos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/citologia , Pâncreas/embriologia , Ductos Pancreáticos/citologia , Ductos Pancreáticos/embriologia , Pré-Albumina/metabolismo , Ratos , Ratos Endogâmicos F344 , Coloração e Rotulagem
13.
Dev Dyn ; 240(3): 530-65, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21337462

RESUMO

Pancreas oganogenesis comprises a coordinated and highly complex interplay of signaling events and transcriptional networks that guide a step-wise process of organ development from early bud specification all the way to the final mature organ state. Extensive research on pancreas development over the last few years, largely driven by a translational potential for pancreatic diseases (diabetes, pancreatic cancer, and so on), is markedly advancing our knowledge of these processes. It is a tenable goal that we will one day have a clear, complete picture of the transcriptional and signaling codes that control the entire organogenetic process, allowing us to apply this knowledge in a therapeutic context, by generating replacement cells in vitro, or perhaps one day to the whole organ in vivo. This review summarizes findings in the past 5 years that we feel are amongst the most significant in contributing to the deeper understanding of pancreas development. Rather than try to cover all aspects comprehensively, we have chosen to highlight interesting new concepts, and to discuss provocatively some of the more controversial findings or proposals. At the end of the review, we include a perspective section on how the whole pancreas differentiation process might be able to be unwound in a regulated fashion, or redirected, and suggest linkages to the possible reprogramming of other pancreatic cell-types in vivo, and to the optimization of the forward-directed-differentiation of human embryonic stem cells (hESC), or induced pluripotential cells (iPSC), towards mature ß-cells.


Assuntos
Organogênese/fisiologia , Pâncreas/citologia , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Humanos , Organogênese/genética
14.
Methods Mol Biol ; 561: 65-72, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19504064

RESUMO

The possibility of generating transgenic animals is of obvious advantage for the analysis of gene function in development and disease. One of the established vertebrate model systems in developmental biology is the amphibian Xenopus laevis. Different techniques have been successfully applied to create Xenopus transgenics; in this chapter, the so-called meganuclease method is described. This technique is not only technically simple, but also comparably efficient and applicable to both Xenopus laevis and Xenopus tropicalis. The commercially available endonuclease I-SceI (meganuclease) mediates the integration of foreign DNA into the frog genome after coinjection into fertilized eggs. Tissue-specific gene expression, as well as germline transmission, has been observed.


Assuntos
Animais Geneticamente Modificados , Desoxirribonucleases de Sítio Específico do Tipo II/administração & dosagem , Fertilização In Vitro/métodos , Técnicas de Transferência de Genes , Proteínas de Saccharomyces cerevisiae/administração & dosagem , Transgenes/fisiologia , Xenopus laevis/genética , Animais , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Embrião não Mamífero/metabolismo , Feminino , Mutação em Linhagem Germinativa , Microinjeções/métodos , Oócitos/citologia , Oócitos/fisiologia , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Distribuição Tecidual , Xenopus laevis/embriologia
15.
Mech Dev ; 124(7-8): 518-31, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17643968

RESUMO

Early patterning of the endoderm as a prerequisite for pancreas specification involves retinoic acid (RA) as a critical signalling molecule in gastrula stage Xenopus embryos. In extension of our previous studies, we made systematic use of early embryonic endodermal and mesodermal explants. We find RA to be sufficient to induce pancreas-specific gene expression in dorsal but not ventral endoderm. The differential expression of retinoic acid receptors (RARs) in gastrula stage endoderm is important for the distinct responsiveness of dorsal versus ventral explants. Furthermore, BMP signalling, that is repressed dorsally, prevents the formation of pancreatic precursor cells in the ventral endoderm of gastrula stage Xenopus embryos. An additional requirement for mesoderm suggests the production of one or more further pancreas inducing signals by this tissue. Finally, recombination of manipulated early embryonic explants, and also inhibition of RA activity in whole embryos, reveal that RA signalling, as it is relevant for pancreas development, operates simultaneously on both mesodermal and endodermal germ layers.


Assuntos
Endoderma/fisiologia , Pâncreas/embriologia , Tretinoína/farmacologia , Xenopus laevis/embriologia , Animais , Padronização Corporal/fisiologia , Pâncreas/fisiologia , Transdução de Sinais , Xenopus laevis/fisiologia
16.
Dev Dyn ; 235(1): 247-52, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16258935

RESUMO

Several experimental approaches have been described to generate transgenic frogs. Here, we report on the application of a novel method in Xenopus, making use of I-SceI meganuclease. The characteristic feature of this endonuclease is that it has an extended recognition site of 18 bp, which is expected to exist only once in 7 x 10(10) bp of random DNA sequences. Various reporter constructs flanked by two I-SceI recognition sites were injected together with the I-SceI meganuclease into one-cell stage Xenopus embryos. We observed an overall transgenesis frequency of 10% or more under optimized condition. The injected genes were integrated into the genome and transmitted to F1 offspring. Southern blot analysis showed that between one and eight copies of the transgene were integrated. Meganuclease-aided transgenesis, thus, provides a simple and highly efficient tool for transgenesis in Xenopus.


Assuntos
Animais Geneticamente Modificados , Desoxirribonucleases de Sítio Específico do Tipo II , Técnicas de Transferência de Genes , Animais , Southern Blotting , DNA , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Transgenes , Xenopus laevis/embriologia
17.
Dev Biol ; 271(1): 144-60, 2004 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15196957

RESUMO

How and when the vertebrate endoderm is first subdivided into discrete progenitor cell populations that will give rise to the different major organs, including pancreas and liver, are only poorly understood. We have used Xenopus laevis as a model system to characterize these events, since it is particularly suited to study the early embryonic patterning in vertebrates. Our experimental results support the notion that retinoic acid (RA) functions as an essential endodermal patterning signal in Xenopus and that it acts as early as during gastrulation. As a result of RA treatment, the expression of Sonic Hedgehog (Shh), a known inhibitor of pancreas development in other vertebrate systems, is negatively regulated in the dorsal prepancreatic endoderm. Furthermore, RA is found to promote endocrine at the expense of exocrine differentiation in the dorsal pancreas, correlating with a specific inhibition of Notch signaling activities in this territory. Conversely, RA enhances exocrine marker gene expression in the ventral pancreas.


Assuntos
Diferenciação Celular/fisiologia , Epigênese Genética , Pâncreas/embriologia , Transdução de Sinais/fisiologia , Tretinoína/fisiologia , Proteínas de Xenopus , Animais , Contagem de Células , Primers do DNA , Endoderma/fisiologia , Células Enteroendócrinas/fisiologia , Glândulas Exócrinas/embriologia , Proteínas Hedgehog , Hibridização In Situ , Insulina/metabolismo , Microinjeções , Morfogênese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/metabolismo , Transativadores/metabolismo , Tretinoína/metabolismo , Xenopus laevis
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