Fatiga en Drosophila Melanogaster. Un sensor molecular de oxígeno que regula el crecimiento celular y la ramificación del sistema respiratorio

Los niveles tisulares de oxígeno regulan la expresión de genes que intervienen en procesos celulares y fisiológicos básicos como la angiogénesis, la eritropoiesis, y el ciclo celular. Dicha expresión depende de HIF-1 (Hipoxia Inducible Factor-1), un factor de transcripción heterodimérico α/β que es degradado en normoxia y estabilizado en hipoxia. La destrucción de HIF-1α depende de la hidroxilación de dos residuos de prolina, catalizada por prolil-4-hidroxilasas (PHDs) específicas que por emplear oxígeno molecular como co-sustrato son consideradas Sensores Moleculares de Oxígeno de la célula. Las PHDs fueron recientemente identificadas y se ignoraba qué tipos de procesos biológicos controlan en metazoos superiores. En la presente Tesis, se identificó el sensor de oxígeno de Drosophila, al que denominamos Fatiga y se caracterizó su función en la regulación del crecimiento celular y en la ramificación del sistema traqueal, un proceso que presenta rasgos en común con la angiogénesis de los mamíferos. También se observó, mediante experimentos genéticos, que los principales fenotipos de pérdida de función de Fatiga se revierten en ausencia de la proteína Sima, homólogo de HIF en Drosophila. Sin embargo, los doble mutantes fatiga sima presentan anomalías en el desarrollo del ala y del ovario, evidenciando posibles funciones de Fatiga independientes de Sima.Oxygen levels control the expression of a plethora of genes involved in cellular and physiological processes such as angiogenesis, eritropoiesis and cell cycle. In mammals, the transcriptional response to hypoxia depends on HIF-1 (Hypoxia Inducible Factor-1), an α/β heterodimeric transcription factor that is rapidly degraded in normoxia and stabilized in hypoxia. Oxygen Dependent destruction of the HIF-1α subunit is mediated by the hydroxylation of two key prolyl residues, a reaction catalyzed by specific prolyl-4-hydroxylases that utilize molecular oxygen as a co-substrate thus, functioning as oxygen sensors. Since PHDs have been recently identified, the developmental consequences of their inactivation in higher metazoans had not been reported so far. In this Thesis, we have identified the Drosophila oxygen sensor, which we have named Fatiga. We have characterized its effects on cell growth and tracheal sprouting, a process related to mammalian angiogenesis. Finally, we have observed that the defects occurring in Fatiga mutants are reverted by concomitant mutation of the sima gene, the Drosophila HIF-α homologue, suggesting that the main functions of PHDs during Drosophila development involve downregulation of HIF. However, fga sima adult flies show defects in wing and ovary development, which imply that Fga is involved in patterning these organs in a Sima-independent manner.Fil: Centanin, Lázaro. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Teaching with Learner Corpus Data

One type of resource that can facilitate teachers’ access to texts is the language corpus (plural corpora), which is defined as an electronically searchable collection of texts organized by text types and context representation, such as news articles, blog posts, dialogues, and others. While access to these texts provides teachers with an array of possible topics to inform both grammar and writing classes, it may be difficult to select texts that are appropriate to course content and students’ proficiency levels. In other words, texts from native-speaker corpora may lack examples of text types relevant to learners' proficiency levels as they are full of vocabulary and grammar that is unfamiliar to students, especially at the novice and intermediate levels. For this reason, we turn to learner corpora as an alternative. In this article, we discuss how to create corpus-informed activities using learner corpus data to teach language in context, facilitating analysis of grammar and writing in an integrated manner. First, we briefly discuss the basic elements of corpus search and their pedagogical applications. Then, we share a sample hands-off lesson created using the corpus Multilingual Academic Corpus of Assignments Writing and Speech (MACAWS), which contains written and oral assignments produced by students in the University of Arizona's Portuguese and Russian foreign language programs (Staples et al., 2019-, available at macaws.corporaproject.org). We hope that teachers use these examples as a springboard to expand this approach to other languages. To do so, we conclude by giving directions on where to find similar corpora resources and how to adapt these principles to their own contexts

Sox2, Tlx, Gli3, and Her9 converge on Rx2 to define retinal stem cells in vivo

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 License.-- et al.Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina.DI received fellowships from the Human Frontier Science Program (HFSP) and the Japan Society for the Promotion of Science (JSPS); TT received a fellowship of the Hartmut Hoffmann Berling International Graduate School (HBIGS) in Heidelberg. The project was supported by the Collaborative Research Center SFB 873 (J.W.) of the German Research Foundation (DFG).Peer Reviewe

Identification, visualization and clonal analysis of intestinal stem cells in fish.

Recently, a stochastic model of symmetrical stem cell division followed by neutral drift has been proposed for intestinal stem cells (ISCs), which has been suggested to represent the predominant mode of stem cell progression in mammals. In contrast, stem cells in the retina of teleost fish show an asymmetric division mode. To address whether the mode of stem cell division follows phylogenetic or ontogenetic routes, we analysed the entire gastrointestinal tract of the teleost medaka (Oryzias latipes). X-ray microcomputed tomography shows a correlation of 3D topography with the functional domains. Analysis of ISCs in proliferation assays and via genetically encoded lineage tracing highlights a stem cell niche in the furrow between the long intestinal folds that is functionally equivalent to mammalian intestinal crypts. Stem cells in this compartment are characterized by the expression of homologs of mammalian ISC markers - sox9, axin2 and lgr5 - emphasizing the evolutionary conservation of the Wnt pathway components in the stem cell niche of the intestine. The stochastic, sparse initial labelling of ISCs ultimately resulted in extended labelled or unlabelled domains originating from single stem cells in the furrow niche, contributing to both homeostasis and growth. Thus, different modes of stem cell division co-evolved within one organism, and in the absence of physical isolation in crypts, ISCs contribute to homeostatic growth

Oxygen Sensing in Drosophila: Multiple Isoforms of the Prolyl Hydroxylase Fatiga Have Different Capacity to Regulate HIFα/Sima

Background: The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIFprolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. Methodology and Principal Findings: We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. Conclusions and Significance: Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.Fil:Acevedo, J.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Centanin, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Dekanty, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Wappner, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Hypoxia activates IKK-NF-κB and the immune response in <em>Drosophila melanogaster</em>

Hypoxia, or low oxygen availability, is an important physiological and pathological stimulus for multicellular organisms. Molecularly, hypoxia activates a transcriptional programme directed at restoration of oxygen homoeostasis and cellular survival. In mammalian cells, hypoxia not only activates the HIF (hypoxia-inducible factor) family, but also additional transcription factors such as NF-κB (nuclear factor κB). Here we show that hypoxia activates the IKK–NF-κB [IκB (inhibitor of nuclear factor κB)–NF-κB] pathway and the immune response in Drosophila melanogaster. We show that NF-κB activation is required for organism survival in hypoxia. Finally, we identify a role for the tumour suppressor Cyld, as a negative regulator of NF-κB in response to hypoxia in Drosophila. The results indicate that hypoxia activation of the IKK–NF-κB pathway and the immune response is an important and evolutionary conserved response

Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina.

The potency of post-embryonic stem cells can only be addressed in the living organism, by labeling single cells after embryonic development and following their descendants. Recently, transplantation experiments involving permanently labeled cells revealed multipotent neural stem cells (NSCs) of embryonic origin in the medaka retina. To analyze whether NSC potency is affected by developmental progression, as reported for the mammalian brain, we developed an inducible toolkit for clonal labeling and non-invasive fate tracking. We used this toolkit to address post-embryonic stem cells in different tissues and to functionally differentiate transient progenitor cells from permanent, bona fide stem cells in the retina. Using temporally controlled clonal induction, we showed that post-embryonic retinal NSCs are exclusively multipotent and give rise to the complete spectrum of cell types in the neural retina. Intriguingly, and in contrast to any other vertebrate stem cell system described so far, long-term analysis of clones indicates a preferential mode of asymmetric cell division. Moreover, following the behavior of clones before and after external stimuli, such as injuries, shows that NSCs in the retina maintained the preference for asymmetric cell division during regenerative responses. We present a comprehensive analysis of individual post-embryonic NSCs in their physiological environment and establish the teleost retina as an ideal model for studying adult stem cell biology at single cell resolution.This is the final version of the article. It has been published by The Company of Biologists Ltd in Development here: http://dev.biologists.org/content/early/2014/08/19/dev.109892.long

Drosophila Genome-Wide RNAi Screen Identifies Multiple Regulators of HIF–Dependent Transcription in Hypoxia

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF–dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF–related pathologies, including heart attack, cancer, and stroke

A Systematic Screen for Tube Morphogenesis and Branching Genes in the Drosophila Tracheal System

Many signaling proteins and transcription factors that induce and pattern organs have been identified, but relatively few of the downstream effectors that execute morphogenesis programs. Because such morphogenesis genes may function in many organs and developmental processes, mutations in them are expected to be pleiotropic and hence ignored or discarded in most standard genetic screens. Here we describe a systematic screen designed to identify all Drosophila third chromosome genes (∼40% of the genome) that function in development of the tracheal system, a tubular respiratory organ that provides a paradigm for branching morphogenesis. To identify potentially pleiotropic morphogenesis genes, the screen included analysis of marked clones of homozygous mutant tracheal cells in heterozygous animals, plus a secondary screen to exclude mutations in general “house-keeping” genes. From a collection including more than 5,000 lethal mutations, we identified 133 mutations representing ∼70 or more genes that subdivide the tracheal terminal branching program into six genetically separable steps, a previously established cell specification step plus five major morphogenesis and maturation steps: branching, growth, tubulogenesis, gas-filling, and maintenance. Molecular identification of 14 of the 70 genes demonstrates that they include six previously known tracheal genes, each with a novel function revealed by clonal analysis, and two well-known growth suppressors that establish an integral role for cell growth control in branching morphogenesis. The rest are new tracheal genes that function in morphogenesis and maturation, many through cytoskeletal and secretory pathways. The results suggest systematic genetic screens that include clonal analysis can elucidate the full organogenesis program and that over 200 patterning and morphogenesis genes are required to build even a relatively simple organ such as the Drosophila tracheal system