Genetic identification of a novel NeuroD1 function in the early differentiation of islet α, PP and ε cells

AbstractNkx2.2 and NeuroD1 are vital for proper differentiation of pancreatic islet cell types. Nkx2.2-null mice fail to form β cells, have reduced numbers of α and PP cells and display an increase in ghrelin-producing ε cells. NeuroD1-null mice display a reduction of α and β cells after embryonic day (e) 17.5. To begin to determine the relative contributions of Nkx2.2 and NeuroD1 in islet development, we generated Nkx2.2−/−;NeuroD1−/− double knockout (DKO) mice. As expected, the DKO mice fail to form β cells, similar to the Nkx2.2-null mice, suggesting that the Nkx2.2 phenotype may be dominant over the NeuroD1 phenotype in the β cells. Surprisingly, however, the α, PP and ε phenotypes of the Nkx2.2-null mice are partially rescued by the simultaneous elimination of NeuroD1, even at early developmental time points when NeuroD1 null mice alone do not display a phenotype. Our results indicate that Nkx2.2 and NeuroD1 interact to regulate pancreatic islet cell fates, and this epistatic relationship is cell-type dependent. Furthermore, this study reveals a previously unappreciated early function of NeuroD1 in regulating the specification of α, PP and ε cells

Novel computational analysis of protein binding array data identifies direct targets of Nkx2.2 in the pancreas

<p>Abstract</p> <p>Background</p> <p>The creation of a complete genome-wide map of transcription factor binding sites is essential for understanding gene regulatory networks <it>in vivo</it>. However, current prediction methods generally rely on statistical models that imperfectly model transcription factor binding. Generation of new prediction methods that are based on protein binding data, but do not rely on these models may improve prediction sensitivity and specificity.</p> <p>Results</p> <p>We propose a method for predicting transcription factor binding sites in the genome by directly mapping data generated from protein binding microarrays (PBM) to the genome and calculating a moving average of several overlapping octamers. Using this unique algorithm, we predicted binding sites for the essential pancreatic islet transcription factor <it>Nkx2.2 </it>in the mouse genome and confirmed >90% of the tested sites by EMSA and ChIP. Scores generated from this method more accurately predicted relative binding affinity than PWM based methods. We have also identified an alternative core sequence recognized by the <it>Nkx2.2 </it>homeodomain. Furthermore, we have shown that this method correctly identified binding sites in the promoters of two critical pancreatic islet β-cell genes, <it>NeuroD1 </it>and <it>insulin2</it>, that were not predicted by traditional methods. Finally, we show evidence that the algorithm can also be applied to predict binding sites for the nuclear receptor <it>Hnf4α</it>.</p> <p>Conclusions</p> <p>PBM-mapping is an accurate method for predicting Nkx2.2 binding sites and may be widely applicable for the creation of genome-wide maps of transcription factor binding sites.</p

Long non-coding RNAs as local regulators of pancreatic islet transcription factor genes

The transcriptional programs of differentiated cells are tightly regulated by interactions between cell type-specific transcription factors and cis-regulatory elements. Long non-coding RNAs (lncRNAs) have emerged as additional regulators of gene transcription. Current evidence indicates that lncRNAs are a very heterogeneous group of molecules. For example, selected lncRNAs have been shown to regulate gene expression in cis or trans, although in most cases the precise underlying molecular mechanisms is unknown. Recent studies have uncovered a large number of lncRNAs that are selectively expressed in pancreatic islet cells, some of which were shown to regulate β cell transcriptional programs. A subset of such islet lncRNAs appears to control the expression of β cell-specific transcription factor (TF) genes by local cis-regulation. In this review, we discuss current knowledge of molecular mechanisms underlying cis-regulatory lncRNAs and discuss challenges involved in using genetic perturbations to define their function. We then discuss known examples of pancreatic islet lncRNAs that appear to exert cis-regulation of TF genes. We propose that cis-regulatory lncRNAs could represent a molecular target for modulation of diabetes-relevant genes

Generation and Characterization of Functional Human Hypothalamic Neurons.

Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Defects in the function of hypothalamic neurons cause a spectrum of human diseases, including obesity, infertility, growth defects, sleep disorders, social disorders, and stress disorders. These diseases have been studied in animal models such as mice, but the rarity and relative inaccessibility of mouse hypothalamic neurons and species-specific differences between mice and humans highlight the need for human cellular models of hypothalamic diseases. We and others have developed methods to differentiate human pluripotent stem cells (hPSCs) into hypothalamic neurons and related cell types, such as astrocytes. This protocol builds on published studies by providing detailed step-by-step instructions for neuronal differentiation, quality control, long-term neuronal maintenance, and the functional interrogation of hypothalamic cells by calcium imaging. Together, these protocols should enable any group with appropriate facilities to generate and study human hypothalamic cells. © 2017 by John Wiley & Sons, Inc.The work described in this publication was supported by funds from the Wellcome Trust, the Medical Research Council (MR/P501967/1), and the Academy of Medical Sciences (SBF001\1016)

Nkx2.2 and Arx genetically interact to regulate pancreatic endocrine cell development and endocrine hormone expression

AbstractNkx2.2 and Arx are essential pancreatic transcription factors. Nkx2.2 is necessary for the appropriate specification of the islet alpha, beta, PP and epsilon cell lineages, whereas Arx is required to form the correct ratio of alpha, beta, delta and PP cells. To begin to understand the cooperative functions of Nkx2.2 and Arx in the development of endocrine cell lineages, we generated progenitor cell-specific deletions of Arx on the Nkx2.2 null background. The analysis of these mutants demonstrates that expansion of the ghrelin cell population in the Nkx2.2 null pancreas is not dependent on Arx; however, Arx is necessary for the upregulation of ghrelin mRNA levels in Nkx2.2 mutant epsilon cells. Alternatively, in the absence of Arx, delta cell numbers are increased and Nkx2.2 becomes essential for the repression of somatostatin gene expression. Interestingly, the dysregulation of ghrelin and somatostatin expression in the Nkx2.2/Arx compound mutant (Nkx2.2null;ArxΔpanc) results in the appearance of ghrelin+/somatostatin+ co-expressing cells. These compound mutants also revealed a genetic interaction between Nkx2.2 and Arx in the regulation of the PP cell lineage; the PP cell population is reduced when Nkx2.2 is deleted but is restored back to wildtype numbers in the Nkx2.2null;ArxΔpanc mutant. Moreover, conditional deletion of Arx in specific pancreatic cell populations established that the functions of Arx are necessary in the Neurog3+ endocrine progenitors. Together, these experiments identify novel genetic interactions between Nkx2.2 and Arx within the endocrine progenitor cells that ensure the correct specification and regulation of endocrine hormone-producing cells

Efeito da época de aplicação de fungicida na redução da intensidade da brusone do trigo.

A brusone (Pyricularia grisea) é responsável por grandes perdas de qualidade de grão e de produtividade na cultura do trigo. Como estratégia de manejo é indicado o uso de cultivares com resistência e pulverização de fungicidas. Neste trabalho objetivou-se avaliar o efeito da aplicação de fungicida a base de triciclazol em diferentes estádios da planta sobre a intensidade (incidência e severidade) da doença na espiga. A cultivar Brilhante de trigo foi semeada no campo em fevereiro de 2012 no Distrito Federal. O fungicida utilizado foi o triciclazol na dose de 188g de ingrediente ativo/ha. Os tratamentos utilizados foram: (1) testemunha sem aplicação de fungicida; (2) aplicação no estádio 55 (metade das inflorescências emergidas) repetida duas vezes a cada 10 dias, (3) aplicação no estádio 60 (início da antese) repetida duas vezes a cada 10 dias; (4) aplicação no estádio 69 (antese completa) repetida duas vezes a cada 10 dias. O delineamento experimental foi em blocos casualizados com seis repetições. Aos 88 dias após a semeadura, no estágio 85 (grão macio), foram coletadas 100 espigas por parcela, determinado a incidência e severidade com uma escala de cinco níveis, 0, 25, 50, 75 e 100%. A aplicação de fungicida realizada no período de emergência da inflorescência colaborou com a redução significativa de 32,8% na incidência e 42,8% na severidade da brusone em relação à testemunha e demais tratamentos.Título em inglês: Effect of fungicide application timing on wheat blast intensity reduction. Resumo 539