347 research outputs found
DNA methyltransferase 3B regulates duration of neural crest production via repression of Sox10
No full textNeural crest stem cells arise within the central nervous system but then undergo an epithelial-to-mesenchymal transition to migrate away and contribute to the peripheral nervous system and craniofacial skeleton. Here we show that DNA methyltransferase 3B (DNMT3B) is responsible for the loss of competence of dorsal neural tube cells to generate emigrating neural crest cells. DNMT3B knockdown results in up-regulation of neural crest markers, prolonged neural crest emigration, and subsequent precocious neuronal differentiation of the trigeminal ganglion. We find that DNMT3B binds to the promoter of Sox10, known to be important for neural crest emigration and lineage acquisition. Bisulfite sequencing further reveals methylation of the Sox10 promoter region upon cessation of emigration in normal embryos, whereas this mark is reduced after DNMT3B loss. Taken together, these results reveal the importance of DNA methylation in regulating the ability of neural tube cells to produce neural crest cells and the timing of peripheral neuron differentiation.Fil: Hu, Na. California Institute of Technology; Estados UnidosFil: Strobl Mazulla, Pablo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Simoes Costa, Marcos. California Institute of Technology; Estados UnidosFil: Sanchez Vasquez, Estefania. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Bronner, Marianne E.. California Institute of Technology; Estados Unido
In situ hybridization as a method to examine gene regulatory activity in vivo
No full textTranscription factor-enhancer binding events are among the most well-studied protein-DNA interactions, allowing researchers to determine mechanisms of transcriptional activation or repression during development. While large-scale ChIP-sequence datasets, together with computational predictions and chromatin accessibility data, yield information on potential transcription factor binding activities, reporter gene assays provide measurable information on whether these binding activities are functional in particular cell types during development. Here, we present a detailed protocol to examine enhancer activity in Drosophila embryos using cloning, transgenesis, and in situ hybridization
Parrhesía e loucura no exemplo de Estamira
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Filosofia e Ciências Humanas, Programa de Pós-Graduação em Filosofia, Florianópolis, 2015.Um autor como Michel Foucault (1926-1984), ao apresentar um estudo extenso sobre saber, poder e ética, deixa o leitor com dúvidas e indagações: O sujeitado ao poder não teria como se contrapor à submissão? O poder psiquiátrico, com seus saberes sobre a loucura, seria tão poderoso assim? Na tentativa de responder estas questões, estabeleceu-se como objetivo geral investigar a parrhesía e a loucura no exemplo de Estamira. Como pressuposto metodológico, optou-se pelos escritos de Michel Foucault sobre a temática da loucura. Na análise do documentário a respeito de Estamira, produzido por Marcos Prado em 2004, foram analisadas algumas cenas, fundamentando-se, sobretudo, em seus últimos escritos, de 1980 a 1984, do Collège de France. O pensamento último de Foucault evidencia uma preocupação histórica do que seria o cuidado de si e a parrhesía no período da Grécia Clássica, por volta do séc. IV a.C., o período helênico romano séc. I e II d.C. e os primeiros cristãos (IV a V d.C.). O autor consegue perceber, na antiguidade grega, a existência de uma subjetividade que não segue normas, a qual se torna uma prática de liberdade (práticas ascéticas), uma busca de como dizer a verdade (parrhesía) que leva a uma constituição ética atrelada à estética da existência da vida do sujeito. Conclui-se, com base nas análises históricas foucaultianas, que o exemplo de Estamira nos remete a uma ?ontologia do presente?, ?ontologia dos discursos verdadeiros?, uma concepção de subjetividade, de verdade e de filosofia de vida para o indivíduo, com sua loucura, transformar sua vida e ser diferente do que é, governando a si mesmo pela parrhesía, ser franco, falar a verdade.Abstract : An author such as Michel Foucault (1926-1984) when presenting an extensive study of knowledge, power and ethics, leaves the reader with doubts and questions: The subjected to power could not possibly oppose itself from submission? The psychiatric power, with its knowledge about insanity, would be that powerful? In the attempt to answer these questions, it was established as a general objective to investigate the parrhesia and insanity in the case of Estamira. For the methodological presupposition, it was chosen the writings of Michel Foucault on the theme of madness. In the documentary film analysis about Estamira, produced by Marcos Prado in 2004, some scenes were analyzed mostly based in his later writings, from 1980 to 1984, from the Collège de France. The final thought of Foucault reveals a historical concern of what would be the self care and the Parrhesia in the Classical Greece period from around the IV century B.C., the Roman Hellenistic period during the I and II century A.D. and the early Christians (IV to V A.D.). The author is able to perceive that in Greek antiquity, the existence of a subjectivity that does not follow rules, which turns to a practice of freedom (ascetic practices), a search for how to tell the truth (parrhesia) leading to an ethical constitution tied to the aesthetics of existence of the subject´s life. The conclusion is, based on Foucault's historical analysis, that the example of Estamiranos refers to an "ontology of the present", "ontology of real discourses," a conception of subjectivity, truth and life philosophy for the individual, with his insanity, to transform his life and be different than it is, ruling himself through parrhesia, be honest and to tell the truth
Developmental regulatory networks controlling neural crest multipotency and differentiation
No full text154 pagesNeural crest cells are a migratory and multipotent stem cell population that are specified at the dorsal aspect of the neural tube in developing vertebrate embryos. The neural crest has been studied for centuries and was first identified for their ability to migrate broadly throughout the embryo. Classical lineage tracing studies, now affirmed by genetic fate mapping, have confirmed that multipotent neural crest progenitors can contribute to a diverse array of fates including sensory neurons and glia in the peripheral nervous system, the dermis and craniofacial skeleton, melanocytes, Schwann cells, the enteric nervous system, cardiac septum, the outflow tract of the heart, and more. To better understand how neural crest cells achieve such varied developmental potential, extensive work has been performed to create hierarchical and temporal gene regulatory networks (GRNs) that both identify distinct cell states and their functional output. One limitation of the current state of the neural crest GRN is that it is generated from a compilation of experimental evidence in mice, chick, frog, and zebrafish, leading to inconsistencies due to functional divergence or gene duplication events. Furthermore, despite advances in genomic profiling, network-level reconstructions of the neural crest GRN are unable to resolve the precise timing and complexity of multiple circuits using a single assay. My thesis research focuses on the cranial subpopulation of neural crest, which is unique in its ability to give rise to ectomesenchyme – or mesenchymal cells derived from the ectoderm. In this work, I characterize the role of the canonical pluripotent transcription factors OCT4 and SOX2 and their co-opted role to promote neural crest multipotency. Additionally, I examined developing neural crest cells using single cell ATAC-Seq to identify the transcription factors and developmental gene regulatory circuits driving differentiation in neural crest. This work unveiled the regulatory circuits behind seven different lineages within the earliest stages of cranial neural crest development. Understanding these regulatory networks provide valuable insight into the control of stem cell multipotency and may be used to guide directed differentiation of neural crest derivatives in future studies
NEW CONNECTIONS IN THE GENE REGULATORY NETWORK UNDERLYING CELL FATE SPECIFICATION IN C. ELEGANS POSTEMBRYONIC MESODERM DEVELOPMENT
No full text194 pagesCell fate specification during development requires the combinatorial actions of transcription factors and cell-cell signaling. The C. elegans postembryonic mesoderm, or M lineage, is derived from a single multipotent precursor cell, which during hermaphrodite postembryonic development produces 32 cells of six different types. The M lineage therefore provides an excellent system to dissect the regulatory mechanisms underlying fate specification. My thesis work centers around a key transcription factor, SEM-2, which belongs to the SoxC group, and its roles in M lineage development. By analyzing animals carrying a partial loss-of-function mutation in SEM-2, I uncovered previously unappreciated functions and interactions of SEM-2 in M lineage development. First, in addition to its role in specifying a ventral M lineage fate, the sex myoblast (SM) fate, SEM-2 also functions in the dorsal M lineage, where it antagonizes the expression and function of the forkhead transcription factor LET-381/FoxF/C. Second, SEM-2 is not only required for specifying the SM fate, but it is also essential for the proliferation and diversification of the SM lineage, particularly the differentiation of type II vulval muscles required for egg-laying. Third, SEM-2 appears to directly regulate the expression of hlh-8, which encodes a basic helix-loop-helix Twist transcription factor that plays critical roles in the proper patterning of the M lineage. My findings suggest that the SoxC-Twist axis, including the downstream targets of Twist, represents an evolutionarily conserved regulatory cassette important in metazoan development. There are three SoxC proteins in mammals, Sox4, Sox11 and Sox12. Mutations in Sox4 and Sox11 are associated with a neurodevelopmental disorder called Coffin-Siris syndrome (CSS). Many CSS-associated mutations affect conserved residues in SoxC proteins. I introduced a SoxC CSS-associated mutation into sem-2 in worms and confirmed that it is a partial loss-of-function mutation that likely causes defects in humans due to haploinsufficiency. Further phenotypic analysis of the mutant worms revealed that SEM-2 not only functions in the M lineage, but also in other mesodermal tissues, specifically in the defecation muscles, possibly by acting through hlh-8/CeTwist. Altogether, my work identified new interactions in the gene regulatory network underlying C. elegans postembryonic development and adds to the general understanding of the structure-function relationship of SoxC proteins.2027-06-1
GENOME-WIDE PROTEIN MOLECULAR ARCHITECTURE OF HUMAN PROMOTERS
No full text140 pagesTranscription factors (TFs) bind DNA cis-regulatory elements (CREs) in the context of chromatin to regulate the transcription machinery and gene expression. The precise positioning of the pre-initiation complex (PIC), TFs, cofactors, and nucleosomes within the human genome—and their interactions on a genomic scale—has yet to be thoroughly investigated. In this study, we developed an advanced version of the ChIP-exo assay (v6), utilizing a combination of nucleases as high-precision structural probes to reconstruct these interactions with single-base genomic resolution. We show that for many TFs, their unbound CREs are typically translationally and/or rotationally buried on a nucleosome. For CTCF/Cohesin binding, not only do nucleosomes become translationally phased at its flanks, but CTCF/Cohesin retains interactions with each of them. For pioneer factor FoxA binding, the sites generally lack a rotational and translational setting on nucleosome. For NFIA binding, the CRE becomes rotationally exposed and translationally constrained at the nucleosome edge. Nuclease probes also reveal precise and general architectural relationships among TFs (SP1, GABPA), cofactors (P300), the transcription machinery (TBP, TFIIB, Pol II), and nucleosomes at promoters. We propose a refined model of human promoters characterized by bidirectional transcription, where two distinct PICs initiate on opposing strands while sharing common upstream regulatory regions. Our findings revealed that different general transcription factors (GTFs) exhibit specific ChIP exonuclease stop patterns at TATA boxes, which are precisely located 30 bp upstream of genome-wide transcription start sites (TSSs), irrespective of strand orientation. Furthermore, when RNA polymerase II (Pol II) transitions into a transcriptionally paused state, TBP and TFIIA show significantly higher occupancy at TATA-like core promoters compared to TATA-less core promoters. Additionally, we discovered that genome-wide +1 nucleosomes exhibit a stable in vivo architecture, with paused Pol II embedded within these rotationally constrained nucleosomes. The rotational setting of the +1 nucleosome is influenced primarily by the periodic occurrence of specific dinucleotides but is also regulated by cofactors. Notably, we identified a novel motif that is both translationally and rotationally phased on the +1 nucleosome. When this motif is bound by WDR5/RbBP5, the DNA is rotated by 5 bp (or 10n + 5 bp), leading to an alteration in its rotational phasing
DEVELOPMENTAL FUNCTION OF ACTIVE TRANSPOSABLE ELEMENTS IN ZEBRAFISH
No full textTransposable elements (TEs) are highly active during vertebrate embryogenesis. The deleterious effects of active TEs have been studied extensively, but despite their selfish origins, once TEs are no longer mobile, their sequences can be coopted by their hosts for a variety of cellular functions. However, whether active TEs can have a developmental function remains an open and provocative question. Zebrafish (Danio rerio) is a powerful system to study embryonic development due to the accessibility of its transparent embryos. Furthermore, the zebrafish genome harbors an abundance and wide diversity of TEs, including many recently active families. Using single-cell RNA-seq data, I identified a small set of TE families with somatic expression during zebrafish embryogenesis, primarily comprising endogenous retroviruses. Among those, I further characterized BHIKHARI (Bik-1), an endogenous lokiretrovirus expressed specifically in mesendoderm cell lineages. I found that Bik-1 is mostly composed of nonautonomous copies which have been transposing very recently but only encode a predicted Gag protein. To knock down Bik-1 expression, I made use of locked nucleic acid (LNAs). Upon LNA injection in zebrafish embryos, Bik-1 mRNA levels were reduced by ~70%, whereas the expression of host genes was not significantly affected. Strikingly, we observed developmental defects in embryos depleted of Bik-1, including smaller heads, lack of eyes and shortened anterior- posterior axis. Rescue experiments indicate that these phenotypes are caused by the depletion of the Gag protein encoded by Bik, likely due to cellular activities mediated by viral-like particles. Another Gag protein from a closely related TE family, BHIKHARI-2 (Bik-2), is also important for the migration of its hosts, the neural crest cells. Taken together, we propose an “addiction” model to explain how organismal development can be become progressively dependent on products encoded by active TEs without fundamentally altering conserved developmental processes. By proving that the mobile and replicative nature of TEs is not incompatible with having a beneficial impact on host fitness, our findings challenge the long-held view that active TEs are merely in conflict with their host. In doing so, it forces a reevaluation of the ‘selfish DNA’ theory proposed four decades ago
Decoding the cis-regulatory principles of signaling-responsive neural crest enhancers
No full text126 pagesThe establishment and maintenance of discrete domains of gene expression are critical for early embryonic development. In vertebrates, embryonic patterning is mediated by the combinatorial action of signaling systems and transcription factors. Cooperation between inductive signals is essential for the establishment of the progenitor cell populations that will give rise to distinct tissues and organs. For example, Wnts and BMPs are both required for the formation of the neural crest, a migratory, multipotent cell population that gives rise to important structures like the craniofacial skeleton and the peripheral nervous system. These signals converge at the neural plate border, the embryonic region where neural crest cells will form, to activate the genes that endow these cells with their unique properties. Neural crest formation is controlled by a gene regulatory network (GRN) composed of dozens of genes expressed in a specific spatial domain within the neural plate border. However, the cis-regulatory principles that allow for the emergence of these discrete patterns of gene expression remain poorly understood. To address this, I examined the regulation of GRN components in avian embryos to identify and dissect genomic elements that are regulated by signaling systems. I focused on signaling-responsive enhancers that control the expression of MSX1 and ZFHX4, which are genes important for the formation of the neural plate border and the neural crest, respectively. I employed these genes and their respective regulatory elements as a model to elucidate the cis-regulatory principles that allow enhancers to interpret multiple signaling inputs and produce specific patterns of gene expression
A MATEMÁTICA DO ENSINO DE FRAÇÕES NA COLEÇÃO 'MATEMÁTICA, METODOLOGIA E COMPLEMENTOS' DE RUY MADSEN BARBOSA (1966).
No full textThis dissertation aims to identify elements of the mathematics of fractions teaching in the collection of textbooks "Mathematics, Methodology and Complements for Primary Teachers" by Ruy Madsen Barbosa published in 1966. We delimited a period for this study, namely 1960 to 1970. This demarcation is due to the creation of the Law of Directives and Bases of Education of 1961 and a year before the unification of primary education in 1st grade by Law Nº. 5.692 of 1971, both considered important milestones in the history of Brazilian education. The studies were guided by the following questions: What guidelines were conveyed, in the collection "Mathematics, Methodology and Complements for primary teachers" by Ruy Madsen Barbosa (1966), concerning the teaching of fractions? The theoretical and methodological framework is based on the socio-historical perspective, considering elements such as: History of Mathematics Education (COSTA, 2017); knowledge to teach and knowledge to teach (HOFSTETTER and SCHNEUWLY, 2017); mathematics to teach and mathematics to teach (VALENTE, 2017); and teaching mathematics (MORAIS; BERTINI; VALENTE, 2020). The collection of sources was carried out in the Institutional Repository of the Federal University of Santa Catarina on the page of the Research Group in History of Mathematics Education (Ghemat/Brazil). After asepsis of the sources found, we selected the collection "Matemática, Metodologia e Complementos para professores primários", published in 1966, by Ruy Madsen Barbosa, containing three manuals for teacher's training. We considered as categories of analysis: knowing how to teach, knowing how to teach, sequence, grading, meaning, didactic devices, and exercises and problems. From the studies carried out, it is possible to infer that, in the analyzed textbooks, the teaching of fractions starts from the concrete to the abstract, using manipulative materials; the fractional numbers were presented before the content of decimal numbers; the textbooks present a well-defined mathematical language, in the sense of a language using symbols, definitions, etc., using set theory; emphasis on equivalent fractions for the teaching of operations; the exercises and problems with repetition characteristics, among other points that we discussed during the preparation of this dissertation.Esta dissertação tem como objetivo identificar elementos da matemática do ensino de frações na coleção de manuais “Matemática, Metodologia e Complementos para professores primários” de Ruy Madsen Barbosa publicadas no ano de 1966. Delimitamos um período para este estudo, a saber: 1960 a 1970. Esta demarcação se deve pela criação da Leis de Diretrizes e Bases da Educação de 1961 e um ano anterior à unificação do ensino primário em ensino de 1º grau pela Lei Nº. 5.692 de 1971, ambos considerados marcos importantes da história da educação brasileira. Os estudos foram conduzidos pelos seguintes questionamentos: Que orientações foram veiculadas, na coleção “Matemática, Metodologia e Complementos para professores primários” de Ruy Madsen Barbosa (1966), relativos ao ensino de frações? O referencial teórico-metodológico está embasado na perspectiva sócio-histórica, considerando elementos como: História da Educação Matemática (COSTA, 2017); saber a ensinar e saber para ensinar (HOFSTETTER e SCHNEUWLY, 2017); matemática a ensinar e matemática para ensinar (VALENTE, 2017); e matemática do ensino (MORAIS; BERTINI; VALENTE, 2020). A coleta de fontes foi realizada no Repositório Institucional da Universidade Federal de Santa Catarina na página do Grupo de Pesquisa em História da Educação Matemática (Ghemat/Brasil). Após uma assepsia das fontes encontradas, selecionamos a coleção “Matemática, Metodologia e Complementos para professores primários”, publicada no ano de 1966, por Ruy Madsen Barbosa, contendo três manuais destinados à formação de professores. Foram consideradas como categorias de análise: saber a ensinar, saber para ensinar, sequência, graduação, significado, dispositivos didáticos e exercícios e problemas. A partir dos estudos realizados, é possível inferir que, nos livros analisados, o ensino de frações parte do concreto para o abstrato, utilizando materiais manipuláveis; os números fracionários eram apresentados anterior ao conteúdo de números decimais; os manuais apresentam uma linguagem matemática bem definida, no sentido de uma linguagem utilizando símbolos, definições, etc., utilizando a teoria dos conjuntos; ênfase nas frações equivalentes para o ensino das operações; os exercícios e problemas com características de repetição, entre outros pontos que discutimos durante a elaboração desta dissertação
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