Wt1 is involved in pancreas development and adult pancreatic homeostasis

The embryonic mesothelium lining the visceral organs gives rise to mesenchymal cells through a localized epithelial-mesenchymal transition (EMT). This has been extensively studied in some cases, such as the heart, where the epicardium gives rise to epicardial-derived cells that contribute to the cardiac vascular and connective tissues. In other organs, such as the lungs, liver and gut, the developmental fate of the mesothelial-derived mesenchyme and their importance for visceral morphogenesis has also been demonstrated (reviewed in Ariza et al., Dev Dyn, 2016, 245:307-22). Hepatic stellate cells (HSC) are located in the perisinusoidal space of the liver. It has been described that cells derived from the liver mesothelium through an EMT contributes to the HSC population and also to the sinusoidal endothelium during development (IJpenberg et al. Dev Biol, 2007, 312: 157–170; Asahina et al., Hepatology, 2011, 53:983-95). Thus, we checked is a similar developmental origin accounts for pancreatic stellate cells (PSC), a population of pancreatic stromal cells that share many features with HSC. In normal adult pancreas, PSC are quiescent, star-shaped cells with a periacinar distribution. When activated by profibrogenic stimuli such as inflammatory cytokines or oxidative stress, PSC transform into myofibroblast-like cells. Thus, PSC are the major source of extracellular matrix in the adult pancreas, but their embryonic origin remains unknown. The Wilms’ tumor suppressor gene (Wt1) is highly expressed in the embryonic mesothelium. For this reason, we have used two lines of transgenic mice for lineage tracing of mesothelial-derived cells, systemic (Wt1Cre; R26REYFP), tamoxifen-inducible (Wt1ERT2; R26REYFP) and we have also used the inducible driver for conditional deletion of Wt1 (Wt1ERT2; Wt1 flox) in adult mice. Our results confirm that WT1 protein is only expressed in the mesothelium of the developing pancreas, allowing for reliable tracing of the mesothelial-derived cells. During the early stages of pancreas morphogenesis, its mesothelium shows the typical features of EMT. Mesothelial-derived cells, identified by constitutive YFP expression, differentiate into a major part of the PSCs and also contribute to other connective and vascular cell type, including endothelium. Thus, mesothelial-derived cells originated by EMT seem to constitute an important subpopulation of mesodermal cells during pancreas development, contributing to its morphogenesis. On the other hand, systemic deletion of Wt1 in adult mice causes a severe atrophy of the pancreas, although this factor is only expressed in the pancreatic mesothelium. In addition, we have observed that adult PSC express Wt1 in the caerulein-induced pancreatitis model. Our results suggest that: 1) normal pancreatic function is maintained by a Wt1-dependent signaling mechanism acting from the mesothelium and 2) Wt1 plays a role in PSC activation in adult mice. These observations point to a relevant function of the Wt1 gene in pancreatic development and function.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Expression of the Wilms tumor suppressor gene (Wt1) in a subpopulation of embryonic cardiomyocytes is required for cardiac development

The Wilms’ tumour gene, WT1, encodes a zinc-finger transcription factor involved in the development of several organs. WT1 is expressed during mammalian embryonic development in many tissues, including the urogenital system, spleen, spinal cord, diaphragm, coelomic epithelium and epicardium (Armstrong et al., 1993; Moore et al., 1999; Cano et al., 2016; Ariza et al., 2016; Carmona et al., 2016). Post-transcriptional modifications of the Wt1 pre-mRNA lead to the production of up to 24 different isoforms, which seem to serve distinct but overlapping cellular and developmental functions. We have checked if Wt1 is expressed by the embryonic myocardium. Using transgenic mice lines for lineage tracing (mWt1/IRES/GFPCre;ROSA26REYFP), we have detected a small population of cardiomyocytes from a Wt1-expressing cell lineage in early developmental stages (E8.5-E9.5). These cardiomyocytes were mainly located in the inflow tract, but some of them were observed in the ventricles. We confirmed Wt1 expression by RT-PCR in hearts before the attachment of proepicardial cells, when the cardiac tube is only constituted of myocardium and endocardium. We have also studied the mRNA levels of the four main isoforms of Wt1 in a reverse transcriptase-polymerase chain reaction (RT- PCR) assay. We have found differential expression of these Wt1 isoforms in the embryonic heart. Conditional deletion of Wt1 in cardiac Troponin T expressing cells caused severe damage in the developing heart, particularly muscular defects in the interventricular septum and free ventricular walls, as well as defective sinus venous formation. These embryos did not survive after birth. Likewise, conditional deletion of GATA4 in Wt1-expressing cells causes a similar phenotype in the myocardium, but also defects in the proepicardium, epicardium and subepicardial space, causing embryonic death around E11.5. Thus, we conclude that Wt1 is expressed in a subpopulation of early embryonic cardiomyocytes, and this expression seems to be essential for heart development.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Three embryonic cell lineages related with the epicardium show distinct developmental fates

The embryonic epicardium generates a population of mesenchymal cells that contribute to the coronary vessels and the connective tissue of the adult heart. We have used murine cell-tracing models to compare the developmental fate of three different lineages related with the epicardium. Mice bearing R26R-EYFP reporters were crossed with mice expressing Cre-recombinase under control of the promoters of the cardiac troponin gene (cTnT), the Wilms tumor suppressor gene (Wt1), and the G2 enhancer of the GATA4 gene. Thus, we could trace, using confocal microscopy and flow cytometry, the lineage of the cardiac cells expressing Wt1, cTnT and GATA4 under control of the G2 enhancer, from midgestation to adults. Additionally we have studied a knockin Wt1-GFP reporter model to detect cells with Wt1 expression in the developing and adult heart. During development, Wt1 is expressed in a major part of the proepicardium and epicardium, and also in some epicardial-derived mesenchymal cells, in part of the mesenchymal and coronary endothelial cells and also in a fraction of the cardiomyocytes. GATA4 expression is activated by the enhancer G2 in lateral mesoderm and pro/epicardium, but not in epicardial-derived cells, although most of these cells as well as some cardiomyocytes originate from a G2-GATA4 expressing lineage. cTnT is expressed in all the cardiomyocytes, but a part of the epicardial cells also derives from a cTnTCre-EYFP positive cell lineage. The developmental fate of these linages reveals interesting differences, according to our preliminary results. For example, the G2-GATA4 cell linage contributes more than the Wt1 cell lineage to the coronary endothelium during development. However, both lineages are highly represented in the adult cardiac endothelium, suggesting postnatal expression of Wt1 in the coronary endothelium and incorporation of endothelial progenitor cells from bone marrow (where the G2-GATA4 reporter is active in 20% of hematopoietic stem cells).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Aplicação do Controlo Estatístico do Processo numa Indústria Automóvel

A Qualidade auxilia na compreensão da variabilidade dos processos, permitindo a tomada de decisão ponderada com base em dados factuais. Em diversos processos industriais, os dados não seguem os pressupostos de aplicabilidade das cartas de controlo, nomeadamente a independência e a Normalidade, e caso sejam aplicadas as cartas de controlo tradicionais, o número de falsos alarmes irá sofrer um aumento drástico conduzindo a conclusões erróneas sobre o processo. A presente dissertação tem como principal objetivo a melhoria do desempenho do processo da produção, fazendo uso do Controlo Estatístico do Processo. Para alcançar o objetivo proposto foi necessário, em primeiro lugar, verificar os pressupostos de aplicabilidade das cartas de controlo. Caso o pressuposto de independência não se verifique, isto é, os dados apresentem auto-correlação significativa, a metodologia proposta sugere ajustar os processos através do modelo ARIMA mais adequado e extrair os resíduos independentes para aplicação das cartas de controlo baseadas nos resíduos e erros de previsão. Caso o pressuposto da Normalidade não se verifique, isto é, os dados não sigam uma distribuição Normal, a metodologia proposta sugere a sua transformação através da Transformação Box-Cox e aplicação das cartas de controlo baseadas nos dados transformados. Posteriormente, caso se verifique a necessidade de estudar simultaneamente diversas características da qualidade de um determinado produto, sugere-se a aplicação do estudo multivariado. A metodologia proposta foi validada através de um caso de estudo desenvolvido na Volkswagen Autoeuropa. Os resultados da aplicação da metodologia proposta sugerem que de facto, a modelação dos dados auto-correlacionados através do modelo ARIMA e a transformação dos dados não-normais através da Transformação Box-Cox funcionam efetivamente para garantir os pressupostos da independência e Normalidade respetivamente, e permitem a correta aplicação das cartas de controlo e consequente identificação das causas especiais responsáveis pelo aumento da variabilidade do processo

Role of the Wilms' tumor suppressor gene Wt1 in pancreatic development

The Wilms tumor suppressor gene (Wt1) encodes a transcription factor involved in the development of a number of organs, but the role played by Wt1 in pancreatic development is unknown. The pancreas contains a population of pancreatic stellate cells (PSC) very important for pancreatic physiology. We described elsewhere that hepatic stellate cells originate from the WT1‐expressing liver mesothelium. Thus, we checked if the origin of PSCs was similar. WT1 expression is restricted to the pancreatic mesothelium. Between embryonic day (E) 10.5 and E15.5, this mesothelium gives rise to mesenchymal cells that contribute to a major part of the PSC and other cell types including endothelial cells. Most WT1 systemic mutants show abnormal localization of the dorsal pancreas within the mesentery and intestinal malrotation by E14.0. Embryos with conditional deletion of WT1 between E9.5 and E12.5 showed normal dorsal pancreatic bud and intestine, but the number of acini in the ventral bud was reduced approximately 30% by E16.5. Proliferation of acinar cells was reduced in WT1 systemic mutants, but pancreatic differentiation was not impaired. Thus, mesothelial‐derived cells constitute an important subpopulation of pancreatic mesodermal cells. WT1 expression is not essential for pancreas development, although it influences intestinal rotation and correct localization of the dorsal pancreas within the mesogastrium

Procura da boa norma para a localização industrial

Mestrado em Planeamento do Território - Ordenamento da CidadeO processo de localização industrial foi influenciado ao longo da História, por diversos factores, que ditaram a sua implantação e organização no território. Se, numa etapa inicial, estes factores dependiam, sobretudo, das necessidades físicas da produção, com a crescente complexidade e evolução tecnológica, registadas no último século, impõem-se, cada vez mais, os de natureza intangível, relacionados com as formas de acesso à informação, o conhecimento, as externalidades, entre outros. No processo de tomada de decisão de localização são identificados, por diversos autores, factores de localização industrial emergentes, que decorrem do conjunto de características inerentes ao local e meio envolvente. Conjuntamente com as características biofísicas, funcionais e fundiárias do local de implantação, são ponderados outros factores, com abrangências territoriais distintas. O desafio, que actualmente, se coloca ao ordenamento do território, de articulação das diferentes escalas de intervenção, aponta para a necessidade de utilizar metodologias mais integradoras, que identifiquem, por um lado, as existências, contextualizadas num referencial histórico e institucional, e por outro lado, as dinâmicas de localização industrial, sobretudo, através dos seus principais agentes. Esta investigação visa, precisamente, contribuir em termos metodológicos, para a aferição de tipologias de localização industrial, tendo por base, a realidade territorial do concelho da Batalha, como caso de estudo. Do cruzamento entre as abordagens teórica e prática, resulta uma proposta de modelo de intervenção, que se espera, que possa servir de guia na procura da boa norma para a localização industrial. ABSTRACT: Along History, the process of industrial location was influenced by several factors, which dictated its settlement and organization in the territory. If, in an initial stage, these factors were depended, especially, on the physical necessities of the production, with the growing complexity and technological evolution registered in the last century, there are imposed, more and more, those of intangible nature, related to means of information access, the knowledge, the externalities, and others. In the process of making decisions on location, several authors identify emergent factors of industrial location, which result from the set of inherent characteristics of the place and surrounding setting. Together with the biophysical environment, functional and land policy characteristics of the industrial settlement, other factors are considered, with different territorial ranges. The challenge, which at present is put to territorial management strategies of articulation of the different scales of intervention, points to the necessity of applying more integrated methodologies, which identify, on one hand, the existences, contextualized in an historical and institutional reference system, and on the other hand, the dynamics of industrial location, especially, through its main agents. Hence, this investigation aims to be a methodological tool for the determination of industrial location typologies, having for support the territorial reality of the municipality of Batalha, as case study. From the crosses between theoretical and practical approaches, results a proposal of an intervention model that is expected to serve as guide in the search of a good industrial location criterion

A population of hematopoietic stem cells derives from GATA4-expressing progenitors located in the placenta and lateral mesoderm of mice

GATA transcription factors are expressed in the mesoderm and endoderm during development. GATA1-3, but not GATA4, are critically involved in hematopoiesis. An enhancer (G2) of the mouse Gata4 gene directs its expression throughout the lateral mesoderm and the allantois, beginning at embryonic day 7.5, becoming restricted to the septum transversum by embryonic day 10.5, and disappearing by midgestation. We have studied the developmental fate of the G2-Gata4 cell lineage using a G2-Gata4Cre;R26REYFP mouse line. We found a substantial number of YFP+ hematopoietic cells of lymphoid, myeloid and erythroid lineages in embryos. Fetal CD41+ /cKit+ /CD34+ and Lin– /cKit+ /CD31+ YFP+ hematopoietic progenitors were much more abundant in the placenta than in the aorta-gonad-mesonephros area. They were clonogenic in the MethoCult assay and fully reconstituted hematopoiesis in myeloablated mice. YFP+ cells represented about 20% of the hematopoietic system of adult mice. Adult YFP+ hematopoietic stem cells constituted a long-term repopulating, transplantable population. Thus, a lineage of adult hematopoietic stem cells is characterized by the expression of GATA4 in their embryonic progenitors and probably by its extraembryonic (placental) origin, although GATA4 appeared not to be required for hematopoietic stem cell differentiation. Both lineages basically showed similar physiological behavior in normal mice, but clinically relevant properties of this particular hematopoietic stem cell population should be checked in physiopathological conditions.España Ministerio de Ecomomía BFU2014-52299-PEspaña Instituto de Salud Carlos III RD12/0019-002

Role of vitamin A/retinoic acid in regulation of embryonic and adult hematopoiesis

Vitamin A is an essential micronutrient throughout life. Its physiologically active metabolite retinoic acid (RA), acting through nuclear retinoic acid receptors (RARs), is a potent regulator of patterning during embryonic development, as well as being necessary for adult tissue homeostasis. Vitamin A deficiency during pregnancy increases risk of maternal night blindness and anemia and may be a cause of congenital malformations. Childhood Vitamin A deficiency can cause xerophthalmia, lower resistance to infection and increased risk of mortality. RA signaling appears to be essential for expression of genes involved in developmental hematopoiesis, regulating the endothelial/blood cells balance in the yolk sac, promoting the hemogenic program in the aorta-gonad-mesonephros area and stimulating eryrthropoiesis in fetal liver by activating the expression of erythropoietin. In adults, RA signaling regulates differentiation of granulocytes and enhances erythropoiesis. Vitamin A may facilitate iron absorption and metabolism to prevent anemia and plays a key role in mucosal immune responses, modulating the function of regulatory T cells. Furthermore, defective RA/RARα signaling is involved in the pathogenesis of acute promyelocytic leukemia due to a failure in differentiation of promyelocytes. This review focuses on the different roles played by vitamin A/RA signaling in physiological and pathological mouse hematopoiesis duddurring both, embryonic and adult life, and the consequences of vitamin A deficiency for the blood system