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Ruolo del gene Xotx2 nel mesendoderma anteriore di Xenopus laevis
No full textRIASSUNTO
Lo sviluppo dell'occhio dei vertebrati richiede una complessa sequenza di induzioni planari e verticali. La prima evidenza morfologica di tale processo è visibile alla fine della neurulazione. Dalle pareti laterali del tubo neurale appena chiuso evaginano le vescicole ottiche, che con il proseguimento dello sviluppo daranno origine a retina, epitelio pigmentato e nervo ottico. Le vescicole ottiche inducono inoltre l'ectoderma soprastante a diventare placode lentogeno, ovvero cristallino presuntivo. Quest’ultimo induce la vescicola ottica ad invaginarsi, formando una struttura a due strati a forma di coppa: lo strato esterno diverrà epitelio pigmentato, mentre quello interno darà origine alla retina neurale, che attraverso fenomeni di delaminazione assumerà la morfologia della retina adulta. La coppa ottica è connessa al diencefalo tramite il peduncolo ottico, che darà origine nell’adulto al nervo ottico.
Gli esperimenti di embriologia classica hanno chiarito come lo sviluppo dell’occhio sia determinato primariamente alla fine della gastrulazione dall’effetto di induzione verticale del mesendoderma anteriore, costituito dalla placca precordale, sull’ectoderma soprastante. Tale specificazione avviene tramite l’espressione di proteine di secrezione che hanno per lo più la funzione di antagonizzare i morfogeni, che definiscono, attraverso gradienti di concentrazione, gli assi antero-posteriore e dorso-ventrale dell’embrione. Questi fattori così in competizione con i recettori membranali degli stessi morfogeni, portando all’inibizione delle cascate di trasduzione del segnale da essi stimolate. In questo modo l’ectoderma assume la competenza a diventare placca neurale prima, vescicola ottica poi. Entrambe le vescicole ottiche, comunque, derivano da una regione impari mediana della placca neurale, che prende il nome di campo dell’occhio
( “eyefield” ).
Recentemente è stata scoperta la correlazione fra origine di tale campo morfogenetico ed inibizione di una delle più importanti cascate di trasduzione del segnale dello sviluppo dell'organismo: la "Wnt pathway". Gli Wnt sono proteine secrete isolate per la prima volta in topo (int-1) e in Drosophila (wingless) che hanno un'azione posteriorizzante a livello della piastra neurale. Essi agiscono su dei recettori di membrana, detti Frizzled, e tramite una complessa rete di interazioni citoplasmatiche aumentano la concentrazione cellulare di beta-catenina. Questa entra nel nucleo e lega un repressore trascrizionale, la proteina LEF/TCF, convertendolo in attivatore: in questo modo viene promossa l'espressione di un gran numero di geni nelle diverse fasi dello sviluppo.
I geni codificanti gli antagonisti secreti dalla placca precordale cominciano ad essere espressi nella gastrula precoce nella zona dell’organizzatore di Spemann. I geni Cerberus, Dkk-1, Frzb-1 e Crescent codificano per proteine che hanno propriamente la funzione di inibire la via degli Wnt, creando probabilmente un territorio di competenza per la formazione del campo dell’occhio. Tuttavia, i fattori dell’organizzatore in grado di regolare la trascrizione dei geni suddetti restano al momento sconosciuti. In tale ottica, in questo lavoro di tesi è stato preso in considerazione Xotx2, un gene materno successivamente espresso nell’organizzatore e codificante per un fattore di trascrizione, come possibile regolatore dei geni per proteine secrete dall’organizzatore.
A tale scopo, sono state effettuate microiniezioni di mRNA sintetizzato in vitro per il gene Xotx2 nel blastomero ventrale vegetativo di embrioni a 4 blastomeri. Gli embrioni sono stati fatti crescere fino allo stadio di gastrula precoce (st. 10+ / 10 ¼ ), fissati, emisezionati con bisturi da microdissezione a livello del labbro dorsale del blastoporo e processati per ibridazione in situ “whole mount”. In questo modo si può verificare se il trascritto iniettato sia sufficiente per far esprimere in posizione ectopica i geni presi in esame, ovvero codificanti per inibitori secreti di Wnt. Come controllo dell’attività del trascritto è stata saggiata la capacità da parte di Otx2 di inibire i movimenti di estensione convergente durante la gastrulazione e di indurre la formazione di organi adesivi supplementari. In questo modo è stato possibile osservare un’espressione ectopica del gene Dkk-1.
L’esperimento di perdita di funzione del gene Otx2 nel topo genera gravi mutazioni anteriori, quali l’assenza della testa. Con lo stesso scopo è stata operata in Xenopus l’iniezione di RNA codificanti per una proteina Xotx2 nella quale il dominio di transattivazione viene sostituito con il dominio di repressione della trascrizione contenuto nella proteina Engrailed. I fenotipi generati mostrano riduzioni parziali o totali del prosencefalo e, in generale, di tutte le strutture cefaliche più anteriori, occhi compresi. Nel nostro caso il costrutto “Engrailed-repressor” è stato microiniettato nel blastomero vegetativo dorsale, in modo da indirizzare il trascritto nel futuro territorio dell’organizzatore. Ne è risultata una abolizione praticamente totale dell'espressione normale del gene Dkk-1. Il gene Xotx2 è risultato quindi necessario e sufficiente a dirigere l’espressione del gene Dkk-1, il quale, da dati presenti in letteratura, è capace peraltro di dirigere lo sviluppo della placca precordale.
Sono tuttora in corso esperimenti per mettere in luce la precisa funzione mesendodermica del gene Xotx2 attraverso l'osservazione fenotipica di embrioni iniettati.
Questi dati gettano nuova luce su un possibile meccanismo di induzione verticale attraverso il quale Xotx2 può portare alla specificazione dell’ ”eyefield” nella placca neurale anteriore
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Plasticity and postnatal development of the mammalian retina: pathological effects of mutations in retinal-specific genes
No full textMicroRNAs (miRNAs) are small double-stranded RNAs that, in vertebrates, function as negative regulators of gene expression by base pairing with the 3’-UTR of target mRNAs while part of RNA-induced silencing complexes (RISCs). More than 400 miRNAs have been identified in both mice and humans, each of them having the potential to regulate hundreds of target genes. It has been proposed that more than one third of all human genes may be regulated by miRNAs. Most of experimentally detectable miRNAs are expressed in the brain and, among those upregulated in a tissue specific expression pattern in the embryo, half are brain-specific/enriched.
miRNAs are processed by Dicer, a dsRNA-specific endonuclease, from an inactive ~70-nt precursor RNA to a functional ~25-nt molecule.
The development of therapies for inherited retinal degenerations relies upon a better understanding of the cell biology of this heterogeneous family of diseases. In particular, it is of relevance to study alterations occurring in the retina of various lines of transgenic, knock-out and spontaneous-mutant mice, which mimic various types of retinal degeneration.
For this reason, the first aim of this project is to characterize the retinal phenotype in a mouse in which the production of retinal microRNAs has been greatly impaired by knocking out Dicer, the gene encoding for the key enzyme for the synthesis of miRNAs. Our final goal is to test whether miRNAs could represent a novel class of candidate disease genes.
Dicer null mice have been reported to die at embryonic day 7.5 rendering it impossible to study the role Dicer may play in later stages of development or in adult tissues. To bypass that problem, a floxed Dicer allele has been recently created. Using this allele in conjunction with a retinal-specific cre (Chx10-Cre) allele, it has been possible to inactivate Dicer in the retina selectively, thus creating a conditional knock-out (CKO) mouse. Since Dicer is required for the processing of miRNAs, removal of this enzyme results in a significant decrease in mature miRNAs.
Retinal sections from CKO and wt mice aged postnatal (P) 16, 30, 45 days and 3, 4, 5, and 7 months were processed for immunocytochemistry to cell type specific antibodies. Eyes used for morphological analysis were from animals previously used for ERG recordings. Moreover, retinas from CKO and wt animals of different ages were processed by Northern blot analysis to confirm the decrease of mature microRNA levels.
As previously reported, the Chx10Cre transgenic allele does not drive Cre expression in all retinal cells; thus, not all cells in CKO retinas are expected to lack a functional Dicer protein. For this, we performed in situ hybridization on retinal sections with a probe specific for the floxed exon in the Dicer conditional allele. We found that native Dicer is expressed in the vast majority of retinal cells of control mice, while in the CKO retinas Dicer expression is patchy, with groups of labeled cells interdigitated with unlabeled cells.
Northern blot and in situ hybridization analysis confirmed that Dicer CKO retinas are indeed deficient in mature miRNAs (miR-183, miR-124a and miR-96).
Development of Dicer CKO retinas is grossly normal. All cell types analyzed are present at postnatal day 16 (P16). However, all retinas examined show the presence of rosettes, typical circular structures in which photoreceptors are oriented toward an internal lumen. Rosettes have previously been described in retinoblastoma, diabetic retinopathy and retinitis pigmentosa in association with retinal degeneration and/or abnormal proliferation. Nevertheless, BrdU and phosphohistone H3 staining fails to detect dividing cells in Dicer CKO retinas. Thus, rosette formation is not caused by increased proliferation of retinal cells.
At P16, rosettes are scattered along an otherwise normal retinal surface and are primarily composed of photoreceptors and their synaptic terminals.
The time period from P16 to P45 is characterized by progressive alteration and remodelling of the laminar retinal structure: the number of rosettes increases and their structure becomes more complex, with migration of second order neurons into the outer nuclear layer.
At 3 months, rosettes decrease in size and eventually disappear. Photoreceptors composing the rosettes degenerate and the outer nuclear layer becomes progressively thinner. DNA-binding dyes reveal pycnotic nuclei in the outer and inner retina. Simultaneously, Müller cells become hypertrophic: radial processes increase in size and GFAP reactivity raises, indicating a generalized glial activation. At the same time, Cre positive cells become scarce, possibly because a large fraction of these Dicer null cells degenerate. Surviving rod bipolars are often in clusters and have altered morphologies with hypertrophic axonal arborizations. Most of the residual rod bipolar cells are Cre-negative and therefore most likely originated from precursors in which Dicer was not inactivated.
The retinal-specific Dicer-KO mouse we studied is the first example of miRNAs inactivation in an area of the mammalian Central Nervous System and demonstrates that such a genetic manipulation leads to an aberrant retinal layering eventually culminating with extensive retinal degeneration.
Summarizing, inactivation of Dicer results in a decrease of retinal-expressed miRNAs. Dicer CKO animals show a unique pattern of progressive retinal disorganization and degeneration. These results represent the first step towards identifying the role miRNAs contribute to retinal cell function and survival. The reported data also provide insight into the molecular mechanisms of retinal degeneration and the maintenance of normal retinal function and architecture and point out to miRNAs as likely candidate disease genes.
Retinal degeneration in Dicer CKO mice does not parallel precisely other known retinal pathologies. However, some similarities could be noticed. For instance, as occurs in retinitis pigmentosa (RP), photoreceptors are the first neurons to be hit by miRNA withdrawal. In addition, in both RP and Dicer CKO mice inner retinal neurons like ganglion cells appear to be preserved.
Retinitis Pigmentosa is a family of inherited diseases leading to photoreceptor death and represents one of the major causes of blindness in the world.
Among therapeutic strategies under study, the devise and implant of electronic prostheses has reached the stage of clinical trials. Retinal ganglion cells (RGCs) form the biological substrate that epiretinal prostheses stimulate to restore vision in RP; hence, the efficacy of such electronic implants depends upon RGCs viability. For all these reasons, we focused our attention on
the morphology and survival of RGCs in the rd1 mouse, an established model of autosomic recessive RP, at various stages from the onset of photoreceptor death.
In order to study single RGCs, we generated a transgenic rd1/Thy1-GFP-M mouse line, crossing GFP-M mice, expressing GFP in a small number of heterogeneous RGCs of various types, with rd1 mice, in which rod death starts in the 2nd week of life. As the two founder strains are on C57BL/6J and C3H backgrounds, respectively, repeated backcrossing was performed to isolate double mutants free of background effects. We are presently studying the F9 generation. Retinal whole mounts from animals aged 1 to 12 months were stained with anti GFP antibodies and examined by confocal microscopy. After classification, each cell was studied quantitatively after Neurolucida tracings and Metamorph analysis. To estimate survival in the ganglion cell layer (GCL), retinal whole mounts were stained with ethidium and cell nuclei counted in serial optical sections.
The GCL in rd1 mutant mice aged 1 year displayed at a first sight clear signs of reorganization. Cell bodies were irregularly spaced, with empty areas suggestive of neuronal degeneration. Often cells appeared arranged in circular domains resembling the initial stage of rosette formation. In addition, chromatin condensation was observed in a considerable number of cells.
Extensive cell counting in rd1 mice aged 1 year reveals excellent survival of cells in the ganglion cell layer (94.6%). Nevertheless, a dramatic increase in the number of condensed nuclei (+ 85%) was observed in these retinas with respect to a previous time point at 4.5 months.
In the novel rd1/Thy-1 GFP strain, single RGCs exhibiting GFP labeling occurred in lower number as compared to the wild type founder. This characteristic is not caused by degeneration of the ganglion cell layer, but simply for the mice analyzed were heterozigous unlike the founder. We empirically discovered that the number of green ganglion cells is sensitive to the number of alleles of the Thy-1 GFP transgene.
Our studies suggest that these neurons have also undergone cell body shrinkage and dendritic tree retraction. This finding could be misleading, as it’s very similar to regressive remodeling previously described for bipolar cells as an effect of deafferentation. By measuring ganglion cells from 1 month-old mice, we discovered how the problem described is attributable more to an unrealized postnatal growth rather than a remodeling for retinal degeneration.
This study is telling us how the postnatal development of their dendritic trees is affected in the rd1/Thy-1 GFP mouse, a new strain of mouse dedicated to the study of retinal ganglion cells in retinal degeneration.
The results of these two projects together give light to different mechanism of plasticity and remodelling of retinal cells in postnatal development and adult life of normal and pathological mammalian models
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression
Get PDFMilk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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