RESUMO
Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.
Assuntos
Genes Neoplásicos/genética , Genoma Humano/genética , Genômica , Mutação/genética , Neoplasias Pancreáticas/classificação , Neoplasias Pancreáticas/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Ductal Pancreático/classificação , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Metilação de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/genética , Histona Desmetilases/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Receptores Citoplasmáticos e Nucleares/genética , Análise de Sobrevida , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Transcriptoma , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Peixe-ZebraRESUMO
Pancreatitis is caused by inflammatory injury to the exocrine pancreas, from which both humans and animal models appear to recover via regeneration of digestive enzyme-producing acinar cells. This regenerative process involves transient phases of inflammation, metaplasia, and redifferentiation, driven by cell-cell interactions between acinar cells, leukocytes, and resident fibroblasts. The NFκB signaling pathway is a critical determinant of pancreatic inflammation and metaplasia, whereas a number of developmental signals and transcription factors are devoted to promoting acinar redifferentiation after injury. Imbalances between these proinflammatory and prodifferentiation pathways contribute to chronic pancreatitis, characterized by persistent inflammation, fibrosis, and acinar dedifferentiation. Loss of acinar cell differentiation also drives pancreatic cancer initiation, providing a mechanistic link between pancreatitis and cancer risk. Unraveling the molecular bases of exocrine regeneration may identify new therapeutic targets for treatment and prevention of both of these deadly diseases.
Assuntos
Células Acinares/citologia , Células Acinares/fisiologia , Pâncreas Exócrino/fisiologia , Regeneração/fisiologia , Animais , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Humanos , Pâncreas Exócrino/citologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Pancreatite/patologia , Pancreatite/fisiopatologia , Transdução de Sinais/fisiologiaRESUMO
The formation of epithelial tubes underlies the development of diverse organs. In the skin, hair follicles resemble tube-like structures with lumens that are generated through poorly understood cellular rearrangements. Here, we show that creation of the hair follicle lumen is mediated by early outward movement of keratinocytes from within the cores of developing hair buds. These migratory keratinocytes express keratin 79 (K79) and stream out of the hair germ and into the epidermis prior to lumen formation in the embryo. Remarkably, this process is recapitulated during hair regeneration in the adult mouse, when K79(+) cells migrate out of the reactivated secondary hair germ prior to formation of a new hair canal. During homeostasis, K79(+) cells line the hair follicle infundibulum, a domain we show to be multilayered, biochemically distinct and maintained by Lrig1(+) stem cell-derived progeny. Upward movement of these cells sustains the infundibulum, while perturbation of this domain during acne progression is often accompanied by loss of K79. Our findings uncover previously unappreciated long-distance cell movements throughout the life cycle of the hair follicle, and suggest a novel mechanism by which the follicle generates its hollow core through outward cell migration.
Assuntos
Acne Vulgar/metabolismo , Folículo Piloso/embriologia , Queratinócitos/metabolismo , Queratinas/metabolismo , Regeneração , Animais , Linhagem Celular , Movimento Celular , Células HEK293 , Cabelo/embriologia , Folículo Piloso/metabolismo , Humanos , Queratinas/genética , Queratinas Específicas do Cabelo , Queratinas Tipo II , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Morfogênese , Proteínas do Tecido Nervoso/metabolismoRESUMO
Wnt glycoproteins control key processes during development and disease by activating various downstream pathways. Wnt secretion requires post-translational modification mediated by the O-acyltransferase encoded by the Drosophila porcupine homolog gene (PORCN). In humans, PORCN mutations cause focal dermal hypoplasia (FDH, or Goltz syndrome), an X-linked dominant multisystem birth defect that is frequently accompanied by ocular abnormalities such as coloboma, microphthalmia, or even anophthalmia. Although genetic ablation of Porcn in mouse has provided insight into the etiology of defects caused by ectomesodermal dysplasia in FDH, the requirement for Porcn and the actual Wnt ligands during eye development have been unknown. In this study, Porcn hemizygosity occasionally caused ocular defects reminiscent of FDH. Conditional inactivation of Porcn in periocular mesenchyme led to defects in mid- and hindbrain and in craniofacial development, but was insufficient to cause ocular abnormalities. However, a combination of conditional Porcn depletion in optic vesicle neuroectoderm, lens, and neural crest-derived periocular mesenchyme induced severe eye abnormalities with high penetrance. In particular, we observed coloboma, transdifferentiation of the dorsal and ventral retinal pigment epithelium, defective optic cup periphery, and closure defects of the eyelid, as well as defective corneal morphogenesis. Thus, Porcn is required in both extraocular and neuroectodermal tissues to regulate distinct Wnt-dependent processes during morphogenesis of the posterior and anterior segments of the eye.
Assuntos
Olho/embriologia , Hipoplasia Dérmica Focal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Aciltransferases , Alelos , Animais , Modelos Animais de Doenças , Olho/metabolismo , Feminino , Genótipo , Glicoproteínas/metabolismo , Hemizigoto , Hibridização In Situ , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Recombinação Genética , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/metabolismo , Proteínas Wnt/metabolismoRESUMO
Pancreatic exocrine and endocrine lineages arise from multipotent pancreatic progenitor cells (MPCs). Exploiting the mechanisms that govern expansion and differentiation of these cells could enhance efforts to generate ß-cells from stem cells. Although our prior work indicates that the canonical Wnt signaling component ß-catenin is required qualitatively for exocrine acinar but not endocrine development, precisely how this requirement plays out at the level of MPCs and their lineage-restricted progeny is unknown. In addition, the contribution of ß-catenin function to ß-cell development remains controversial. To resolve the potential roles of ß-catenin in development of MPCs and ß-cells, we generated pancreas- and pre-endocrine-specific ß-catenin knockout mice. Pancreas-specific loss of ß-catenin produced not only a dramatic reduction in acinar cell numbers, but also a significant reduction in ß-cell mass. The loss of ß-cells is due not to a defect in the differentiation of endocrine precursors, but instead correlates with an early and specific loss of MPCs. In turn, this reflects a novel role for ß-catenin in maintaining proximal-distal patterning of the early epithelium, such that distal MPCs resort to a proximal, endocrine-competent "trunk" fate when ß-catenin is deleted. Moreover, ß-catenin maintains proximal-distal patterning, in part, by inhibiting Notch signaling. Subsequently, ß-catenin is required for proliferation of both distal and proximal cells, driving overall organ growth. In distinguishing two distinct roles for ß-catenin along the route of ß-cell development, we suggest that temporally appropriate positive and negative manipulation of this molecule could enhance expansion and differentiation of stem cell-derived MPCs.
Assuntos
Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Pâncreas/embriologia , Pâncreas/metabolismo , beta Catenina/genética , beta Catenina/fisiologia , Animais , Padronização Corporal , Diferenciação Celular , Proliferação de Células , Epitélio/metabolismo , Genótipo , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Camundongos , Camundongos Knockout , Tamanho do Órgão , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/citologiaRESUMO
In this issue of Cancer Cell, Guerra and colleagues provide important new insights regarding the ability of specific pancreatic cell types to generate invasive pancreatic cancer. First, they demonstrate that classical pancreatic "ductal" neoplasia can be induced by activation of oncogenic Kras in nonductal exocrine cells. Second, they show that, while Kras activation in immature acinar and centroacinar cells is readily able to induce ductal neoplasia, Kras-mediated tumorigenesis in mature exocrine pancreas requires the induction of chronic epithelial injury. The results shed new light on the "cell of origin" of pancreatic ductal cancer and demonstrate that chronic pancreatitis provides a permissive environment for Kras-induced pancreatic neoplasia.
Assuntos
Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/patologia , Genes ras , Neoplasias Pancreáticas/patologia , Pancreatite Crônica/patologia , Animais , Carcinoma in Situ/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Linhagem da Célula , Transformação Celular Neoplásica , Ceruletídeo , Humanos , Camundongos , Mutação , Invasividade Neoplásica , Neoplasias Pancreáticas/metabolismo , Pancreatite Crônica/induzido quimicamenteRESUMO
The LIM-Homeodomain transcription factor Lhx2 is an essential organizer of early eye development and is subsequently expressed in retinal progenitor cells (RPCs). To determine its requirement in RPCs, we performed a temporal series of conditional inactivations in mice with the early RPC driver Pax6 α-Cre and the tamoxifen-inducible Hes1(CreERT2) driver. Deletion of Lhx2 caused a significant reduction of the progenitor population and a corresponding increase in neurogenesis. Precursor fate choice correlated with the time of inactivation; early and late inactivation led to the overproduction of retinal ganglion cells (RGCs) and rod photoreceptors, respectively. In each case, however, the overproduction was selective, occurring at the expense of other cell types and indicating a role for Lhx2 in generating cell type diversity. RPCs that persisted in the absence of Lhx2 continued to generate RGC precursors beyond their normal production window, suggesting that Lhx2 facilitates a transition in competence state. These results identify Lhx2 as a key regulator of RPC properties that contribute to the ordered production of multiple cell types during retinal tissue formation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Retina/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular/fisiologia , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Mutantes , Células-Tronco Neurais/citologia , Gravidez , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologiaRESUMO
Notch signaling regulates numerous developmental processes, often acting either to promote one cell fate over another or else to inhibit differentiation altogether. In the embryonic pancreas, Notch and its target gene Hes1 are thought to inhibit endocrine and exocrine specification. Although differentiated cells appear to downregulate Hes1, it is unknown whether Hes1 expression marks multipotent progenitors, or else lineage-restricted precursors. Moreover, although rare cells of the adult pancreas express Hes1, it is unknown whether these represent a specialized progenitor-like population. To address these issues, we developed a mouse Hes1(CreERT2) knock-in allele to inducibly mark Hes1(+) cells and their descendants. We find that Hes1 expression in the early embryonic pancreas identifies multipotent, Notch-responsive progenitors, differentiation of which is blocked by activated Notch. In later embryogenesis, Hes1 marks exocrine-restricted progenitors, in which activated Notch promotes ductal differentiation. In the adult pancreas, Hes1 expression persists in rare differentiated cells, particularly terminal duct or centroacinar cells. Although we find that Hes1(+) cells in the resting or injured pancreas do not behave as adult stem cells for insulin-producing beta (ß)-cells, Hes1 expression does identify stem cells throughout the small and large intestine. Together, these studies clarify the roles of Notch and Hes1 in the developing and adult pancreas, and open new avenues to study Notch signaling in this and other tissues.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Pâncreas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/citologia , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Mutantes , Microscopia de Fluorescência , Pâncreas/embriologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição HES-1RESUMO
The past several decades have seen great effort devoted to mimicking the key features of pancreatic ductal adenocarcinoma (PDAC) in animals and have produced 2 robust models of this deadly cancer. Carcinogen-treated Syrian hamsters develop PDAC with genetic lesions, which reproduce those of human, including activation of the Kras oncogene, and early studies in this species validated nongenetic risk factors for PDAC including pancreatitis, obesity, and diabetes. More recently, PDAC research has been invigorated by the development of genetically engineered mouse models based on tissue-specific Kras activation and deletion of tumor suppressor genes. Surprisingly, mouse PDAC appears to arise from exocrine acinar rather than ductal cells, via a process of phenotypic reprogramming that is accelerated by inflammation. Studies in both models have uncovered molecular mechanisms by which inflammation promotes and sustains PDAC and identified targets for chemoprevention to suppress PDAC in high-risk individuals. The mouse model, in particular, has also been instrumental in developing new approaches to early detection as well as treatment of advanced disease. Together, animal models enable diverse approaches to basic and preclinical research on pancreatic cancer, the results of which will accelerate progress against this currently intractable cancer.
Assuntos
Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Neoplasias Pancreáticas/patologia , Animais , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Cricetinae , Humanos , Camundongos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fatores de RiscoRESUMO
The Drosophila porcupine gene is required for secretion of wingless and other Wnt proteins, and sporadic mutations in its unique human ortholog, PORCN, cause a pleiotropic X-linked dominant disorder, focal dermal hypoplasia (FDH, also known as Goltz syndrome). We generated a conditional allele of the X-linked mouse Porcn gene and analyzed its requirement in Wnt signaling and embryonic development. We find that Porcn-deficient cells exhibit a cell-autonomous defect in Wnt ligand secretion but remain responsive to exogenous Wnts. Consistent with the female-specific inheritance pattern of FDH, Porcn hemizygous male embryos arrest during early embryogenesis and fail to generate mesoderm, a phenotype previously associated with loss of Wnt activity. Heterozygous Porcn mutant females exhibit a spectrum of limb, skin, and body patterning abnormalities resembling those observed in human patients with FDH. Many of these defects are recapitulated by ectoderm-specific deletion of Porcn, substantiating a long-standing hypothesis regarding the etiology of human FDH and extending previous studies that have focused on downstream elements of Wnt signaling, such as ß-catenin. Conditional deletion of Porcn thus provides an experimental model of FDH, as well as a valuable tool to probe Wnt ligand function in vivo.
Assuntos
Ectoderma/metabolismo , Hipoplasia Dérmica Focal/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Wnt/metabolismo , Aciltransferases , Sequência de Aminoácidos , Animais , Western Blotting , Padronização Corporal/genética , Células Cultivadas , Modelos Animais de Doenças , Ectoderma/embriologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Hipoplasia Dérmica Focal/genética , Deleção de Genes , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Proteínas Wnt/genética , Proteína Wnt-5a , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Proteína Wnt3 , beta Catenina/genética , beta Catenina/metabolismoRESUMO
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-1RESUMO
Proper maintenance of mature cellular phenotypes is essential for stable physiology, suppression of disease states, and resistance to oncogenic transformation. We describe the transcriptional regulatory roles of four key DNA-binding transcription factors (Ptf1a, Nr5a2, Foxa2 and Gata4) that sit at the top of a regulatory hierarchy controlling all aspects of a highly differentiated cell-type-the mature pancreatic acinar cell (PAC). Selective inactivation of Ptf1a, Nr5a2, Foxa2 and Gata4 individually in mouse adult PACs rapidly altered the transcriptome and differentiation status of PACs. The changes most emphatically included transcription of the genes for the secretory digestive enzymes (which conscript more than 90% of acinar cell protein synthesis), a potent anabolic metabolism that provides the energy and materials for protein synthesis, suppressed and properly balanced cellular replication, and susceptibility to transformation by oncogenic KrasG12D. The simultaneous inactivation of Foxa2 and Gata4 caused a greater-than-additive disruption of gene expression and uncovered their collaboration to maintain Ptf1a expression and control PAC replication. A measure of PAC dedifferentiation ranked the effects of the conditional knockouts as Foxa2+Gata4 > Ptf1a > Nr5a2 > Foxa2 > Gata4. Whereas the loss of Ptf1a or Nr5a2 greatly accelerated Kras-mediated transformation of mature acinar cells in vivo, the absence of Foxa2, Gata4, or Foxa2+Gata4 together blocked transformation completely, despite extensive dedifferentiation. A lack of correlation between PAC dedifferentiation and sensitivity to oncogenic KrasG12D negates the simple proposition that the level of differentiation determines acinar cell resistance to transformation.
Assuntos
Pâncreas Exócrino , Neoplasias Pancreáticas , Camundongos , Animais , Células Acinares/metabolismo , Epitélio/metabolismo , Fatores de Transcrição/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Fenótipo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismoRESUMO
Understanding the origin of pancreatic ß cells has profound implications for regenerative therapies in diabetes. For over a century, it was widely held that adult pancreatic duct cells act as endocrine progenitors, but lineage-tracing experiments challenged this dogma. Gribben et al. recently used two existing lineage-tracing models and single-cell RNA sequencing to conclude that adult pancreatic ducts contain endocrine progenitors that differentiate to insulin-expressing ß cells at a physiologically important rate. We now offer an alternative interpretation of these experiments. Our data indicate that the two Cre lines that were used directly label adult islet somatostatin-producing ∂ cells, which precludes their use to assess whether ß cells originate from duct cells. Furthermore, many labeled ∂ cells, which have an elongated neuron-like shape, were likely misclassified as ß cells because insulin-somatostatin coimmunolocalizations were not used. We conclude that most evidence so far indicates that endocrine and exocrine lineage borders are rarely crossed in the adult pancreas.
Assuntos
Células Secretoras de Insulina , Lacunas de Evidências , Diferenciação Celular , Pâncreas/fisiologia , Ductos Pancreáticos , Insulina , SomatostatinaRESUMO
A crucial step in eye organogenesis is the transition of the optic vesicle into the optic cup. Several transcription factors and extracellular signals mediate this transition, but whether a single factor links them into a common genetic network is unclear. Here, we provide evidence that the LIM homeobox gene Lhx2, which is expressed in the optic neuroepithelium, fulfils such a role. In Lhx2(-/-) mouse embryos, eye field specification and optic vesicle morphogenesis occur, but development arrests prior to optic cup formation in both the optic neuroepithelium and lens ectoderm. This is accompanied by failure to maintain or initiate the expression patterns of optic-vesicle-patterning and lens-inducing determinants. Of the signaling pathways examined, only BMP signaling is noticeably altered and Bmp4 and Bmp7 mRNAs are undetectable. Lhx2(-/-) optic vesicles and lens ectoderm upregulate Pax2, Fgf15 and Sox2 in response to BMP treatments, and Lhx2 genetic mosaics reveal that transcription factors, including Vsx2 and Mitf, require Lhx2 cell-autonomously for their expression. Our data indicate that Lhx2 is required for optic vesicle patterning and lens formation in part by regulating BMP signaling in an autocrine manner in the optic neuroepithelium and in a paracrine manner in the lens ectoderm. We propose a model in which Lhx2 is a central link in a genetic network that coordinates the multiple pathways leading to optic cup formation.
Assuntos
Olho/embriologia , Proteínas de Homeodomínio/fisiologia , Organogênese/fisiologia , Fatores de Transcrição/fisiologia , Animais , Padronização Corporal/genética , Embrião de Mamíferos , Técnica Direta de Fluorescência para Anticorpo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Técnicas de Cultura de Órgãos , Organogênese/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Microbiome dysbiosis is a feature of diabetes, but how microbial products influence insulin production is poorly understood. We report the mechanism of BefA, a microbiome-derived protein that increases proliferation of insulin-producing ß cells during development in gnotobiotic zebrafish and mice. BefA disseminates systemically by multiple anatomic routes to act directly on pancreatic islets. We detail BefA's atomic structure, containing a lipid-binding SYLF domain, and demonstrate that it permeabilizes synthetic liposomes and bacterial membranes. A BefA mutant impaired in membrane disruption fails to expand ß cells, whereas the pore-forming host defense protein, Reg3, stimulates ß cell proliferation. Our work demonstrates that membrane permeabilization by microbiome-derived and host defense proteins is necessary and sufficient for ß cell expansion during pancreas development, potentially connecting microbiome composition with diabetes risk.
Assuntos
Diabetes Mellitus , Microbiota , Camundongos , Animais , Peixe-Zebra , Pâncreas/metabolismo , Insulina/metabolismo , Diabetes Mellitus/metabolismo , Proteínas/metabolismoRESUMO
Efforts to model pancreatic cancer in mice have focused on mimicking genetic changes found in the human disease, particularly the activating KRAS mutations that occur in pancreatic tumors and their putative precursors, pancreatic intraepithelial neoplasia (PanIN). Although activated mouse Kras mutations induce PanIN lesions similar to those of human, only a small minority of cells that express mutant Kras go on to form PanINs. The basis for this selective response is unknown, and it is similarly unknown what cell types in the mature pancreas actually contribute to PanINs. One clue comes from the fact that PanINs, unlike most cells in the adult pancreas, exhibit active Notch signaling. We hypothesize that Notch, which inhibits differentiation in the embryonic pancreas, contributes to PanIN formation by abrogating the normal differentiation program of tumor-initiating cells. Through conditional expression in the mouse pancreas, we find dramatic synergy between activated Notch and Kras in inducing PanIN formation. Furthermore, we find that Kras activation in mature acinar cells induces PanIN lesions identical to those seen upon ubiquitous Kras activation, and that Notch promotes both initiation and dysplastic progression of these acinar-derived PanINs, albeit short of invasive adenocarcinoma. At the cellular level, Notch/Kras coactivation promotes rapid reprogramming of acinar cells to a duct-like phenotype, providing an explanation for how a characteristically ductal tumor can arise from nonductal acinar cells.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/metabolismo , Proteínas ras/metabolismo , Animais , Carcinoma Ductal Pancreático/patologia , Antagonistas de Estrogênios/metabolismo , Feminino , Humanos , Camundongos , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Gravidez , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Receptores Notch/genética , Transdução de Sinais/fisiologia , Tamoxifeno/metabolismo , Transgenes , Proteínas ras/genéticaRESUMO
OBJECTIVE: To identify novel disease associated loci for amyotrophic lateral sclerosis (ALS), we utilized sequencing data and performed in vitro and in vivo experiments to demonstrate pathogenicity of mutations identified in TP73. METHODS: We analyzed exome sequences of 87 sporadic ALS patients and 324 controls, with confirmatory sequencing in independent ALS cohorts of >2,800 patients. For the top hit, TP73, a regulator of apoptosis, differentiation, and a binding partner as well as homolog of the tumor suppressor gene TP53, we assayed mutation effects using in vitro and in vivo experiments. C2C12 myoblast differentiation assays, characterization of myotube appearance, and immunoprecipitation of p53-p73 complexes were perform in vitro. In vivo, we used CRISPR/Cas9 targeting of zebrafish tp73 to assay motor neuron number and axon morphology. RESULTS: Five heterozygous rare, nonsynonymous mutations in TP73 were identified in our sporadic ALS cohort. In independent ALS cohorts, we identified an additional 19 rare, deleterious variants in TP73. Patient TP73 mutations caused abnormal differentiation and increased apoptosis in the myoblast differentiation assay, with abnormal myotube appearance. Immunoprecipitation of mutant ΔN-p73 demonstrated that patient mutations hinder ΔN-p73's ability to bind p53. CRISPR/Cas9 knockout of tp73 in zebrafish led to impaired motor neuron development and abnormal axonal morphology, concordant with ALS pathology. CONCLUSION: Together, these results strongly suggest that variants in TP73 correlate with risk for ALS and indicate a novel role for apoptosis in ALS disease pathology.
RESUMO
BACKGROUND: Histological evidence suggests that insulin-producing beta (beta)-cells arise in utero from duct-like structures of the fetal exocrine pancreas, and genetic lineage tracing studies indicate that they are maintained in the adult by self-renewal. These studies have not addressed the origin of the new beta-cells that arise in large numbers shortly after birth, and contradictory lineage tracing results have been published regarding the differentiation potential of duct cells in this period. We established an independent approach to address this question directly. RESULTS: We generated mice in which duct and acinar cells, comprising the exocrine pancreas, can be genetically marked by virtue of their expressing the mucin gene Muc1. Using these mice, we performed time-specific lineage tracing to determine if these cells undergo endocrine transdifferentiation in vivo. We find that Muc1+ cells do give rise to beta-cells and other islet cells in utero, providing formal proof that mature islets arise from embryonic duct structures. From birth onwards, Muc1 lineage-labeled cells are confined to the exocrine compartment, with no detectable contribution to islet cells. CONCLUSIONS: These results argue against a significant contribution by exocrine transdifferentiation to the normal postnatal expansion and maintenance of beta-cell mass. Exocrine transdifferentiation has been proposed to occur during injury and regeneration, and our experimental model is suited to test this hypothesis in vivo.