Thèses sur le sujet « Xenopus laevi »

DNA polymerase-theta (Polq) is a class A family DNA polymerase comprising of a helicase-like domain on the N-terminus and a DNA polymerase domain on the C-terminus. Pol-theta overexpression in breast and ovarian cancer patients correlate with high tumor grade and poor response to chemotherapeutic drugs. Consistently, Pol-theta inhibition is synthetically lethal with inactivation of BRCA1/2 genes, which are often found mutated in these tumors. The ability of Pol-theta to sustain viability of BRCA1/2 defective cancer cells has been attributed to its role in alternative end-joining repair of double strand breaks (DSBs) resulting from collapsed replication forks. In addition to DSBs a major role in BRCA1/2 activity is the suppression of defects associated with faulty DNA replication. Indeed, the occurrence of extensive DNA replication defects ranging from single stranded DNA gap accumulation to nascent DNA degradation in the absence of BRCA1/2 and RAD51 has been previously demonstrated. However, the role of Pol-theta in counteracting defective DNA replication in the absence of functional BRCA1/2 and RAD51 is poorly understood. To address this question, we cloned and purified the full length and different domains of Xenopus laevis Pol-theta and generated antibody to study Pol-theta function in replicating Xenopus egg extracts. Our preliminary findings indicate that Pol-theta has replication dependent and independent functions. Significantly, although dispensable for normal chromosomal DNA replication, Pol-theta is strongly enriched at stalled forks upon replication stress conditions induced by aphidicolin. Using DNA electron microscopy, we discovered that Pol-theta overexpression suppresses ssDNA gaps at the replication fork and replication fork reversal triggered by aphidicolin-induced fork stalling. Therefore, our results suggest that Pol-theta not only repairs DSBs but also prevents the occurrence of potentially harmful DNA replication intermediates. We are currently investigating Pol-theta function in relation to replicative defects arising in the absence of BRCA1/2 and RAD51. Better understanding of Pol-theta function at stalled forks will help to target breast and ovarian cancers more effectively.

Early development of O2 chemoreception and hypoxic responses under normoxic (150 mmHg) and chronically hypoxic (110 mmHg) conditions were investigated in Xenopus laevis from hatching to 3 weeks post fertilization. Development, growth, O2 consumption, ventilatory and cardiac performance, and branchial neuroepithelial cells (NEC) density and size were determined. At 3 days post fertilization (dpf), larvae started gill ventilation at a rate of 28 ± 4 beats/min and showed increased frequency to 60 ± 2 beats/min at a PO2 of 30 mmHg. Also at 3 dpf, NECs were identified in the gill filament buds using immunohistochemical methods. Lung ventilation began at 5 dpf and exhibited a 3-fold increase in frequency from normoxia to a PO2 of 30 mmHg. Hypoxic tachycardia developed at 5 dpf, causing an increase of 20 beats/min in heart rate, which led to a 2-fold increase in mass-specific cardiac output at a PO2 of 70 mmHg. At 10 dpf, gill ventilatory sensitivity to hypoxia increased, which was associated with the increase in NEC density, from 15 ± 1 to 29 ± 2 cells/mm of filament at 5 and 10 dpf, respectively. Unlike the elevated rate, cardiac and ventilatory volumes were independent of acute hypoxia. Despite increased cardioventilatory frequency, larvae experienced an average of 80% depression in during acute hypoxia. Chronic hypoxia (PO2 of 110 mmHg) decreased mass-specific cardiac performance before 10 dpf. In older larvae (10 to 21 dpf), chronic hypoxia decreased acute branchial and pulmonary hypoxic hyperventilation and increased NEC size. Collectively, these data suggest that larvae exhibit strong O2-driven acute hypoxic responses post-hatching, yet are still O2 conformers. All acute hypoxic responses developed before 5 dpf, and then the effects of chronic hypoxia started to show between 7 and 21 dpf. Thus, the early formation of acute hypoxic responses is susceptible to the environment and can be shaped by the ambient PO2.

The endoderm is the inner germ layer of the vertebrate embryo from which the respiratory and digestive systems are derived. These include organs such as the liver, pancreas, stomach, lungs and intestine. Recent research has helped our understanding of early vertebrate endoderm specification and terminal differentiation of specific endodermal lineages. However, very little is known about the molecular mechanisms that control endoderm patterning and morphogenesis during vertebrate development. As a way to identify genes involved in these elusive steps of development, I performed a differential hybridisation screen in a macroarray tailbud ventral foregut cDNA library coupled with in situ hybridisation analysis. My aim was to identify and characterise new regionally expressed endodermal genes in Xenopus laevis, a classical embryologic model organism. Here, I report the identification and characterisation of a dozen novel regionally expressed endoderm genes. At tailbud stages their expression patterns fall into three re-occurring domains; anterior ventral midgut endoderm, posterior endoderm and dorsal endoderm. In addition, regional expression of some of these genes is observable at gastrula stages, endoderm specification. These are the first early stable endodermal markers for different regions of the gastrula endoderm. This suggests that the earliest steps in endoderm patterning are concurrent with endoderm specification. Furthermore I describe the identification of a mesodermal transcription factor, which appears to be expressed in ‘early embryonic macrophages’ - and a poorly characterised embryonic cell population. I present an overview of endoderm development together with the results from my screen. Overall, these results reveal an unexpected degree of early endodermal patterning and assist our understanding of the link between early and late events of vertebrate endoderm development. In addition, this work provides us with new and very useful markers for endodermal patterning, and potentially some key developmental regulators of endodermal formation.

The ubiquitous human octamer-binding transcription factor, Oct-1, is believed to regulate the expression of a number of ubiquitously expressed genes. These include genes which are expressed throughout the cell-cycle (eg. snRNA genes) and histone H2B genes, whose expression is tightly coupled to nuclear DNA synthesis at S-phase of the cell-cycle. I have isolated and completely sequenced two X. laevis homologues of Oct-1. The high degree of relatedness of the two homologues indicates that these are likely to be copies of the same gene, which arose during the theoretical genome duplication event in X. laevis evolution. X. laevis and human Oct-1 display strong evolutionary conservation (85% Identity over a stretch of 750 amino acids), which presumably means that X. laevis has a similar, if not identical function to human Oct-1. Homology between human and X. laevis does, however, break down shortly before the N terminal end, at a point where alternate splicing is known to occur in hunan Oct-1 (W. Herr, pers. comn.). The full length X. laevis cDNA clone which I have isolated may represent a novel alternately spliced form of Oct-1. Two octamer-binding proteins have been identified (in band shift assays) in X. laevis oocyte, embryo and tissue extract. Oct-1, and a second, previously unidentified octamer-binding protein which has been termed Oct-R, for octamer-related. Oct-1 does not bind to a degenerate octamer motif most often seen in X. laevis H2B promoters. Oct-R binds more strongly to this degenerate motif than the consensus motif, but only in the context of the H2B promoter, and does not bind either motif in another sequence context. This suggests that Oct-R may have a role in regulation of H2B transcription, although no direct evidence has been obtained. Since Oct-1 is believed to stimulate the S-phase specific induction of histone H2B gene transcription the possibility that Oct-1 binding activity is cell-cycle regulated is of interest. X. laevis Oct-1 (and Oct-R) binding activity does not appear to be cell-cycle regulated. Oct-1 and Oct-R are stored in the oocyte (partly in the cytoplasm), in an amount equivalent to at least 80 000 somatic cells. Histone protein and message are stored in the oocyte as part of the mechanism to provide enough histones to keep-up with the high rate of DNA synthesis in early Xenopus development. It is possible that histone gene transcription factors are stored for the same purpose. By mutation of the octamer motif in the promoter of X. laevis histone H2B gene promoter I have tentatively concluded that the octamer motif is required for the expression of a H2B gene (independently of DNA synthesis) in the oocyte. The H2B gene occurs in association with a H2A gene, as part of a divergently expressed gene pair. The octamer motif may be required for the expression of both H2B and H2A genes. The degenerate octamer motif contained in this H2B promoter does not bind efficiently to Oct-1 in vitro, but binds well to Oct-R, indirectly suggesting that Oct-R is required for the expression of the H2B gene. A polyclonal antiserum raised against the N terminal domain of X. laevis Oct-1 reacts to proteins other than Oct-1 on Western blots of oocyte and embryo extract. These proteins, which are antigenically related to the N terminal domain of Oct-1, are entirely located in the cytoplasm of the oocyte, and entirely located in the nucleus of somatic cells. These proteins are synthesised during oogenesis, and stored in the oocyte in an amount equivalent to at least 100 000 somatic cells.

Memoria para optar al Título Profesional de Médico Veterinario
La regulación transcripcional negativa es un mecanismo importante de control de la expresión de genes que contribuyen en el fenotipo neuronal. El elemento represor de la transcripción REST/NRSF ha sido propuesto como un regulador negativo de muchos genes de diferenciación neuronal terminal, expresándose en células no neuronales, precursores neuronales y neuronas en diferenciación. El papel de REST in vivo durante la diferenciación del sistema nervioso aún se desconoce, dada, entre otras, la letalidad de la pérdida de función de REST en ratones. El laboratorio en el que se desarrolló esta memoria de título ha utilizado otro organismo modelo, Xenopus laevis, a partir del cual se han obtenido resultados compatibles con la participación de REST en procesos muy tempranos del desarrollo neural. La interpretación de estos resultados requiere del análisis de los patrones de expresión de la proteína REST, lo que origina el objetivo principal de esta memoria de título: generar anticuerpos policlonales contra REST/NRSF de Xenopus laevis, para luego ser probados en embriones de Xenopus en diferentes estadíos del desarrollo. La electrotransferencia de extractos de embriones, evidenció que el suero antiREST es inmunoreactivo a una proteína de ~200 KDa, la cual está presente en embriones en los estadíos de clivaje, blástula, gástrula, neurula y organogénesis. En embriones inyectados con un morfolino antisentido de REST, el suero antiREST no detectó ninguna proteína; a diferencia de los embriones control no inyectados, en los cuales reconoció una proteína de ~200 KDa. Estos resultados son compatibles con la idea de que el suero antiREST reconoce la proteína REST endógena de Xenopus laevis. Por otra parte, el suero antiREST no resultó ser de utilidad en el reconocimiento de la proteína REST en embriones y en cortes de ellos mediante inmunohistoquímica

I have isolated two D-type cyclins and a putative kinase partner related to Cdk4 from the frog Xenopus laevis. Three D-type cyclins have previously been isolated from mammalian species, where they are thought to be involved in the control of cell proliferation via the regulation the GO-+G 1 and G 1--IS transitions. The RNA and protein levels of D-type cyclins and Cdk4 were followed during Xenopus early embryonic development. The two D-type cyclins and Cdk4 are absent during the early rapid cleavage phase of Xenopus development. Cyclin D1 mRNA and protein can first be detected after the mid-blastula transition (MBT), and while Cdk4 mRNA is present throughout development the protein is only detectable after MBT. I have been unable to detect cyclin D2 mRNA or protein in embryos or adult tissues, although the D2 clone was originally isolated from an oocyte cDNA library. When translated in vitro, Xenopus cyclins D1 and D2 preferentially associate with recombinant Cdk4 protein, and bind less strongly to Cdc2 and Cdk2. Cdk4 in combination with either cyclin Dl or cyclin D2 is also able to form a ternary complex with retinoblastoma protein (pRb), and cyclin D2-Cdk4 was able to phosphorylate the pRb to which it was bound. Whole-mount in situ hybridization revealed that cyclin Dl, Cdk4 and pRb mRNAs are localised to distinct regions of the developing embryo, in contrast to other cyclins which tend to be distributed rather homogeneously, if they are present at all. In particular, cyclin D1 mRNA is strongly localised to the developing eye and other neural regions of the developing embryonic head. Dominant-negative mutants of Cdk4 were constructed by mutating the essential lysine 33 amino acid residue to arginine. The mRNA encoding this kinase-dead Cdk4 was injected into fertilized eggs to look for effects resulting from lack of Cdk4 function after MBT, when Cdk4 protein is first expressed. No discernible abnormal phenotype was seen, however, in agreement with previously reported results obtained in human cultured cells.

Dissertation (PhD)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: Concern has increased that widespread adverse effects are occurring in humans and wildlife populations as a result of exposure to environmental chemicals (mostly man-made) that disrupt the normal functioning of the endocrine system. Many pharmaceutical, agricultural and industrial chemicals, acting as endocrine modulating compounds (EDCs), have been detected in an accumulated form in food, in drinking water and in the environment. Although the levels of these chemicals can be measured analytically, it is important to evaluate biological activity. For this, animal models are used and relevant bioassays developed. These assays are based on biological markers, which are specific xenobiotically-induced physiological responses and are usually deviations in cellular or biochemical components, processes or structures. Vitellogenin is a large protein complex, produced in the liver under estrogen control and circulates in the plasma, destined for incorporation into the developing oocyte in female oviparous vertebrates. Since vitellogenin production is low or nonexistent in males, its presence may therefore be interpreted as evidence of exposure to estrogenic environmental contaminants. In this study the African Clawed Frog, Xenopus laevis was used as model to study the effects of estrogens on biochemical and physiological parameters associated with vitellogenesis. As a starting point the seasonal female reproductive cycle in a natural Xenopus laevis population in terms of ovarian state, plasma vitellogenin and plasma estrogen levels was studied. It was shown that plasma vitellogenin and estrogen levels were seasonal and correlated to a seasonal ovarian cycle, which peaked during spring. However, although seasonality existed, there were reproductively active individuals at any time during the year. Increases in plasma estrogen levels predated increases in plasma vitellogenin levels and ovarian maturation. Lipoprotein profiles, as well as plasma cholesterol, triglyceride and phospholipid concentrations were determined and it was shown that estrogen affected these in such a way that these parameters could be used as additional markers in estrogen contamination studies. In order to develop an in vitro bioassay to screen for estrogenic activity, the use of hepatic tissue cultures was investigated. Optimal culture conditions were established and increased sensitivity in the estrogenic response was obtained by using liver slices from male frogs that were pre-treated with estrogen. Validation studies proved that this bioassay could be employed for routine screening of water and chemical samples. In order to refine the Xenopus laevis vitellogenin ELISA and liver slice bioassay, existing polyclonal anti-vitellogenin antibodies were replaced by culturing monoclonal antibodies. Selected antibodies were characterised and ELISAs developed and validated. This study showed that the newly developed Xenopus laevis vitellogenin ELISA and liver slice bioassay have the potential to be employed in environmental monitoring programmes.
AFRIKAANSE OPSOMMING: Daar is toenemende besorgdheid dat afwykings in mens- en dierbevolkings voorkom as gevolg van blootstelling aan chemikalieë (hoofsaaklik mensgemaak) in die omgewing wat die normale werking van die endokrienstelsel versteur. Verskeie farmaseutiese, landbou- en industriële chemikalieë, wat as endokrienversteurders optree, is in die omgewing gevind. AI kan die vlakke van hierdie stowwe analities bepaal word, is dit belangrik om hulle biologiese aktiwiteit te evalueer. Hiervoor word diermodelle aangewend om toepaslike toetse daarvoor te ontwikkel. Hierdie toetse word baseer op biologiese merkers, spesifieke xenobioties-geïnduseerde fisiologiese reaksies, en is gewoonlik afwykings van sellulêre- of biochemiese komponente, -prosesse of - strukture. Vitellogeen ('n dooiervoorloper) is 'n lipoproteïenkompleks wat, onder estrogeenbeheer, in die lewer vervaardig word en in die plasma sirkuleer vir uiteindelike inkorporasie in ontwikkelende oësiete van vroulike, ovipare werweldiere. Aangesien daar min of geen vitellogeen in manlike diere geproduseer word, is die teenwoordigheid daarvan 'n aanduiding dat die dier aan estrogeniese omgewingsbesoedeling blootgestel is. In hierdie studie is die Platanna, Xenopus laevis, as model gebruik om die gevolge van estrogene op biochemiese en fisiologiese veranderlikes, wat met vitellogenese geassosieer word, te bestudeer. As vertrekpunt is die seisoenale voortplantingsiklus van die wyfie, in terme van vitellogeen en -estrogeen vlakke in die plasma en aktiwiteit van die ovaria bepaal. Daar is aangetoon dat die estrogeen- en vitellogeenvlakke in die plasma met die ovariumsiklus, wat gedurende die lente hoogtepunte bereik, korreleer. Alhoewel daar seisoenaliteit bestaan, was daar dwarsdeur die jaar wyfies wat ovarium dooierneerlegging getoon het. Verhoging in estrogeenvlakke het vitellogeenpieke en rypwording van die ovaria voorafgegaan. Lipoproteïenprofiele, sowel as die cholesterol- , trigliseried- en fosfolipiedkonsentrasies in die plasma is bepaal en daar is aangetoon dat estrogeen hierdie medeveranderlikes in só 'n mate affekteer dat hulle as bykomende biomerkers vir estrogeenblootstelling in besoedelingstudies gebruik kan word. In vitro Xenopus laevis lewersnitte in die weefselkultuur omgewing is ondersoek om 'n biotoets te onwikkel vir die gebruik in vinnige estrogenisiteits-toetsing van watermonsters en chemise stowwe. Die mees gunstige kultuurtoestande is bepaal en die sensitiwiteit van estrogeenreaksies is verhoog deur lewer van mannetjies, wat vooraf met estrogeen behandel is, te gebruik. Hierdie biotoets se geldigheid is gestaaf en kan in roetine eerstevlaktoetsing van watermonsters gebruik word. Die gebruik van poliklonale teenliggaampies in 'n bestaande enzyme-linked immunosorbent assay (ELISA) is vervang deur spesiaal-ontwikkelde monoklonale anti-Xenopus laevis vitellogeen teenliggaampies. Uitgesoekte teenliggaampies, spesifiek teen die estrogeengeïnduseerde proteïene, is gekarakteriseer en ELISAs saamgestel en die geldigheid gestaaf. Hierdie studie het aangetoon dat die nuut-onwikkelde Xenopus laevis vitellogeen-ELISA en lewerkultuurbiotoets die potensiaal het om In omgewingsmoniteringprogramme gebruik te word.

An investigation was made into the spiral waves solutions for the Atri et al model, a partial differential equation model for Ca²⁺ dynamics in the Xenopus laevis oocyte. Spiral wave solutions, both stable and unstable, were found to exist in the oscillatory regime for this model. The spiral wave solutions were found to have a period that decreased as the initial IP₃ bolus increased. Increasing the initial IP₃ bolus also lead to destabilisation of the spiral waves solutions. After the break up of spiral wave solutions complex spatio-temporal patterns occurred. In some cases spirals reformed after breaking up.

Muscarinic acetylcholine receptors (mAChRs) mediate effects of acetylcholine (ACh) in many systems, including those involved in locomotion. In the stage 37/38 Xenopus laevis tadpole, a well-understood model system of vertebrate locomotion, mAChRs have been found to be located on motor neurons with evidence suggesting that mAChRs are involved in swimming behaviour. The current study aimed to further investigate the role of mAChR-mediated cholinergic transmission by employing extracellular and whole-cell patch clamp recordings to examine the effects of mAChR activation on the properties of different types of neurons in the Xenopus laevis tadpole swimming circuit. It was found that mAChR activation can increase the threshold for initiating swimming by skin stimulation and can lead to the generation of spontaneous motor output in the absence of physical stimuli. These effects were found to be a result of direct inhibition of dorsolateral sensory interneurons of the mechanosensory pathway, direct inhibition of glycinergic inhibitory interneurons in the CPG and a decrease in CPG neuron firing reliability during swimming. The data presented here comprise the first whole-cell patch-clamp investigation into mAChR-mediated cholinergic transmission in the Xenopus laevis tadpole swimming circuit and provide novel evidence that mAChRs modulate the properties of mechanosensory pathway and CPG neurons in this model system of vertebrate locomotion.

SOMMARIO Transdifferenziamento lentogeno Le larve di X. laevis possono rigenerare il cristallino per un processo di transdifferenziamento della cornea esterna in fibre della lente. Tale fenomeno è promosso da un induttore retinico. L’ectoderma corneale deriva dallo strato esterno dell’ectoderma presuntivo lentogeno (PLE) per cui, nelle fasi iniziali dello sviluppo dell’occhio, è interessato dalle azioni induttive che portano alla formazione della lente da parte dello strato profondo del PLE (induzioni precoci). L’induzione della cornea avviene nelle fasi finali dello sviluppo dell’occhio, grazie all’azione induttrice esercitata dalla coppa ottica e dalla lente sull’ectoderma antistante (induzioni tardive). Mi sono chiesto in primo luogo quali fossero i segnali responsabili del mantenimento della capacità lentogena nell’area lentogenica larvale. Per tale ragione, sono stati impiantati nella camera vitrea di larve ospiti frammenti di cornea o di epidermide larvale, sviluppati da un ectoderma che era stato raggiunto, durante lo sviluppo embrionale, da un solo tipo di segnale induttivo (precoce o tardivo) o da entrambi i segnali induttivi (precoci e tardivi); altri frammenti impiantati nella camera vitrea derivavano invece da un ectoderma che non aveva ricevuto nessuno di questi segnali. La capacità lentogena degli impianti è stata valutata utilizzando una sonda antisenso per pax6, mentre l’effettiva trasformazione lentogena degli impianti è stata analizzata utilizzando un anticorpo monoclonale anti-lente. Nel complesso, i risultati ottenuti hanno dimostrato che la capacità della cornea esterna e dell’epidermide pericorneale di rigenerare una lente è il risultato sia delle induzioni precoci, che causano lo sviluppo della lente dal PLE, sia delle induzioni tardive, coinvolte nel differenziamento della cornea. Inoltre, le sole induzioni precoci o le sole induzioni tardive sono sufficienti a mantenere la potenzialità lentogena nell’area lentogenica larvale. Nei primi stadi embrionali la capacità lentogena non è confinata unicamente all’area lentogenica (PLE e regioni immediatamente circostanti), ma è presente in tutto l’ectoderma (Henry and Mittleman, 1995). Perciò, successivamente ho analizzato: a) il decremento della capacità lentogena in una regione ectodermica vicina all’area lentogenica (ectoderma della testa) e in una lontana (ectoderma del fianco), b) la capacità dell’epidermide della testa e del fianco di rispondere ai segnali, rilasciati da un occhio trapiantato, promuoventi la competenza lentogena, c) le parti dell’occhio responsabili dell’effetto promuovente la competenza lentogena. I risultati sono stati ottenuti impiantando nella camera vitrea di larve ospiti frammenti di ectoderma o di epidermide e determinando, in seguito, la percentuale di impianti positivi all’anticorpo anti-lente. I risultati hanno dimostrato che, durante lo sviluppo embrionale e larvale, la competenza lentogena decresce molto più rapidamente nell’ectoderma del fianco che in quello della testa. I risultati ottenuti dopo il trapianto di occhi privati di alcune parti dimostrano che i fattori promuoventi la competenza lentogena sono rilasciati sia dalla lente che dalla retina. Infine, l’analisi della distribuzione del recettore FGFR-2, nella cornea, nell’epidermide e nell’epidermide sotto cui è stato trapiantato un occhio, indica che la competenza lentogena potrebbe essere correlata alla presenza di tale recettore nel tessuto rispondente. Transdifferenziamento retinogeno In questa parte della tesi, ho analizzato il transdifferenziamento in senso retinico di frammenti dell’epitelio pigmentato (RPE), impiantati nella camera vitrea. Le diverse fasi del processo di transdifferenziamento (depigmentazione, proliferazione e differenziamento) sono state seguite utilizzando la marcatura con BrdU come marker della fase proliferativa e l’anticorpo monoclonale mAbHp1 come indicatore del completamento della fase differenziativa. Inoltre, ho analizzato durante il processo di transdifferenziamento il pattern spazio-temporale di espressione di pax6. I risultati hanno dimostrato che nella maggior parte degli impianti, localizzati nella camera vitrea dell’occhio di una larva ospite, solo la parte della vescicola rivolta verso la retina ospite transdifferenziava in una retina laminare, mentre la parte rivolta verso la lente rimaneva densamente pigmentata. Il quadro di espressione di pax6 durante il processo di transdifferenziamento retinogeno ricalca quello osservabile durante il normale sviluppo della retina, ciò indica che sviluppo e rigenerazione della retina sono processi strettamente correlati. Espressione di Xdach durante lo sviluppo Lo sviluppo dell’occhio, dai Vertebrati agli Invertebrati, è regolato da uno stesso network di geni interagenti (pax6/eya1/six3/dach1) (Hanson, 2001). In Xenopus laevis, finora sono stati clonati i geni pax6, eya e six3 (Lupo et al., 2000). Nell’ultima parte di questa tesi ho clonato il gene Dachshund in Xenopus (Xdach) e ne ho delineato il quadro di espressione durante lo sviluppo embrionale e larvale. Mediante ibridazione in situ ho dimostrato che tale gene è espresso nel SNC, nella retina, nell’orecchio e nell’arto in sviluppo. In una successiva fase della ricerca, sarà interessante analizzare il ruolo di Xdach nel transdifferenziamento retinogeno.
SUMMARY Lens transdifferentiation After lentectomy through the pupillary hole, the outer cornea of larval X. laevis can undergo transdifferentiation to regenerate a new lens. This process is elicited by inductive factor(s) produced by the neural retina. During embryogenesis, the outer cornea develops from the outer layer of the presumptive lens ectoderm (PLE) under the influence of the eye cup and the lens. In this part of my thesis, we investigated whether the capacity of the outer cornea to regenerate a lens is the result of early inductive signals causing lens-forming bias and lens specification of the PLE, or late inductive signals causing cornea formation or both signals. Fragments of larval epidermis or cornea developed from ectoderm that had undergone only one kind of inductive signals, or both kinds of signals, or none of them, were implanted into the vitreous chamber of host larvae. The regeneration potential and the lens-forming transformations of the implants were tested using an antisense probe for pax6 as an earlier marker of lens formation and a monoclonal antibody anti-lens as a definitive indicator of lens cell differentiation. Results demonstrated that the capacity of the larval outer cornea to regenerate a lens is the result of both early and late inductive signals and that either early inductive signals alone or late inductive signals alone can elicit this capacity. In larval X. laevis the capacity to regenerate a lens is restricted to the outer cornea and pericorneal epidermis (Lentogenic Area, LA). However, in early embryos, the whole ectoderm is capable of responding to inductive factors of the larval eye forming lens cells. In this part, we have analyzed 1) the decrease of the lens-forming capacity in ectodermal regions both near LA (head epidermis) and far from LA (flank epidermis) during development, 2) the capacity of the head epidermis and flank epidermis to respond to lens-competence promoting factors released by an eye transplanted below these epidermal regions, and 3) the eye components responsible for the promoting effect of the transplanted eye. Results were obtained by implanting fragments of ectoderm or epidermis into the vitreous chamber of host tadpoles and by evaluating the percentage of implants positive to a monoclonal antibody anti-lens. We demonstrated that in the flank region the lens-forming competence is lost at the embryonic stage 30/31 and it is weakly restored by eye transplantation; however, in the head region the lens-forming competence is lost at the larval stage 48 and it is strongly restored by eye transplantation. Results obtained after transplantation of eyes deprived of some components indicate that the lens and the retina are the main source of these promoting factors. The immunohistochemical detection of the FGFR-2 protein in the epidermis of stage 53 larvae submitted to eye transplantation at stage 46 showed that the eye transplantation increased the level of FGFR-2 protein in the head epidermis but not in the flank epidermis, indicating that the lens-forming competence in X. laevis epidermis could be related to the presence of an activated FGF receptor system in the responding tissue. Retina transdifferentiation This study examined the retina transdifferetiation (TD) of RPE fragments implanted into the vitreous chamber of not lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles resembling “inverted eyes” in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina.The phases of the TD process were followed using BrdU labeling as marker of the proliferation phase and a monoclonal antibody (mAbHp1) as a definitive indicator of retina formation. Pigmented RPE cells do not express pax6. In the early phase of RPE to retina TD all depigmented and proliferating precursor cells expressed pax6. Changes in the pax6 expression pattern became apparent in the early phase of the outer nuclear layer (ON) differentiation, when pax6 expression decreased in the presumptive ON of the new-forming retina. Finally, during the late differentiation phase, the (ON), which contains photoreceptors, no longer expressed pax6 and this expression was confined to the ganglion cell layer and the inner nuclear layer. Given the substantial identity of pax6 expression during retinal development and RPE to retina TD, these observations suggest that pax6 is necessary for correct retina formation, indicating that retina development and RPE-retina TD are highly related processes. Xdach expression during development Two Xenopus Dachshund genes, XdachA and XdachB, were isolated, whose cDNAs were 2273 and 2117 bp long, respectively. They contained full-length open reading frames of 610 and 558 amino acids and showed an amino acid sequence highly similar to murine Dach1 (70%) and chick Dach1 (60%). The two Xenopus proteins presented a marked similarity (about 95%) at the level of the Dachbox-N of other Dach proteins and a low level of similarity (about 30%) at that of the Dachbox-C. DachA and DachB expression was analyzed by “whole mount” in situ hybridization and RT-PCR on embryos, at stages between 12 and 34, larval brain, eye and limb buds. The two genes are expressed in overlapping patterns during eye, ear, brain and limb development and show a significant expression similarity to mouse and chick Dach1.

In 1871, a unique substance was isolated from the white blood cells of pus. This substance, which later became known as chromatin, was shown to be a nucleoprotein complex which encompasses the majority of genomic DNA in all eukaryotes. Although chromatin was once viewed as primarily a structural component of the nucleus, it is now accepted that it also plays an important role in the modulation of transcription of individual genes. In this study, the 5S rRNA genes in Xenopus laevis were used as a system to investigate potential roles for chromatin structures in transcription regulation. X. laevis produces two major classes of 5S rRNA: the somatic type is present in most cells whereas the oocyte type is produced only during oogenesis and the early stages of embryogenesis. These two gene families share a very similar coding region and employ identical transcription machinery, leading researchers to believe that it is how these genes are packed into chromatin which is responsible for the differential developmental regulation. Initially, this study focused on the binding constraints placed on the RNA polymerase III basal transcription factor, transcription factor IIIA (TFIIIA), by a histone octamer. Five overlapping fragments of the X. laevis oocyte and somatic 5S rRNA genes were reconstituted into nucleosomes and it was shown that each fragment positions a histone octamer at unique translational sites. Using these nucleosomes it was demonstrated that nucleosome translational positioning is the major determinant of the binding of TFIIIA to the 5S rRNA genes. The relationship between core histone acetylation and transcription of the X. laevis 5S rRNA genes was also investigated. By immunopreciptitating chromatin fragments from a X. laevis kidney cell line with an antibody specific for hyperacetylated histone H4, it was shown that the oocyte 5S rRNA genes are packaged with hypoacetylated histone H4 when transcriptionally repressed.This taken together with the results of others, suggests a link between histone acetylation and RNA polymerase III transcription. However this study was unable to shed light on the basis for this relationship as it was found that histone acetylation did not affect the binding of TFIIIA to nucleosomal DNA. In an attempt to understand the mechanism by which transcription factors compete with histone octamers for cognate binding sites in chromatin, the effect of the histone binding protein nucleoplasmin on the binding of TFIIIA to nucleosomal 5S rRNA genes was tested. It was shown that despite the previously reported nucleosome remodeling ability of nucleoplasmin, the binding of TFIIIA to nucleosomal DNA cannot be facilitated by this protein. Furthermore it was demonstrated that nucleoplasmin cannot overcome nucleosome mediated repression of transcription of reconstituted 5S rRNA genes. In contrast to earlier work, this study used a homologous system composed of the 5S rRNA gene, nucleoplasmin and TFIIIA from Xenopus laevis. Finally, it has long been proposed that selective binding of histone H1 is, in part, responsible for the differential developmental regulation of the oocyte and somatic 5S rRNA genes in Xenopus laevis. In this study it was shown that histone H1 bound both oocyte and somatic genes equally after reconstitution into mononucleosomes or oligonucleosome arrays. Furthermore it was shown that the binding of histone H1 selectively repressed only oocyte gene transcription, and that a RNA polymerase III selectively repressed only oocyte gene transcription, and that a RNA polymerase III transcription complex was able to initiate transcription of nucleosomal somatic templates regardless of whether histone H1 was present. These results support a model in which the differential regulation of the 5S rRNA genes is not due simply to the prevention of histone HI binding by transcription complexes on the somatic genes, but rather a difference in the interaction of histone HI with the somatic and oocyte genes.
Graduate

Thesis (M.A.)--Central Connecticut State University, 1999.
Thesis advisor: Kathy Martin. " ... in partial fulfillment of the requirements for the degree of Master of Arts in Biological Sciences." Includes bibliographical references (leaves 42-44).

La voie de biosynthèse des purines est une voie métabolique conservée et essentielle. Chez l’Homme, des mutations dans plusieurs gènes impliqués dans cette voie provoquent de sévères maladies neuro-musculaires à composante développementale. Cependant, le lien entre génotypes et phénotypes n’est pas connu. Afin de mieux comprendre le rôle des gènes de la voie des purines au cours du développement, nous avons utilisé Xenopus laevis comme modèle vertébré. Les principaux gènes de la voie des purines du xénope n’étaient pas connus, ils ont donc tout d’abord été identifiés in sillico, puis les fonctions enzymatiques pour lesquels ils codent ont été validées in vivo en système hétérologue chez S. cerevisiae. Des analyses d’expression spatiotemporelle chez l’embryon de xénope ont montré que ces gènes sont exprimés tout au long du développement et en particulier dans les tissus neuro-musculaires, suggérant un rôle dans le développement de ces tissus. Le knock-down des gènes, ppat, hprt ou adsl, trois gènes clés de la voie des purines, conduit dans chaque cas à de sévères altérations des muscles squelettiques et en particulier des somites et des muscles hypaxiaux des embryons. Ces phénotypes musculaires sont la conséquence d’une altération précoce de l’expression des gènes MRF (Myogenic Regulatory Factors) myoD et myf5. Un défaut de migration des myoblastes précurseurs des muscles hypaxiaux a également été mis en évidence. Pour conclure, X. laevis est un modèle pertinent qui apporte de nouvelles connaissances permettant de mieux comprendre la cause des altérations musculaires développementales associées aux déficiences en purines
The purine biosynthesis pathway is a conserved metabolic pathway essential for many cell functions. In Human, several mutations in genes involved in this pathway lead to severe neuromuscular diseases, which are at least in part caused by unknown developmental impairments. We established a Xenopus laevis model to decipher the role of the purine biosynthesis genes during vertebrate development. As no data was available regarding this pathway, the main Xenopus purine genes were first identified in silico and functionally validated in vivo using the yeast Saccharomyces cerevisiae as a heterologous system. Spatio-temporal analyses revealed that these genes are expressed all along the development, especially in neuromuscular tissues, suggesting an important role during their formation. The knock-down of ppat, adsl or hprt, three key purine genes, leads in each case to severe defects in skeletal muscles embryonic defects, in particular in somites and hypaxial muscles. These muscular phenotypes are the consequence of an early alteration in expression of some crucial Myogenic Regulatory Factors (MRF), such as myoD and myf5. Moreover, an alteration of the hypaxial muscles precursors was observed. In conclusion our results establish X. laevis as an ideal model to get new insights into the neuromuscular developmental alterations associated to purine deficiencies

Observation of some of the phenomena of tolerance to soluble protein antigens and allogeneic tissue transplants in Xenopus laevis has formed the framework of the present study. The method of larval induction of high-zone tolerance used in this laboratory has been confirmed and further analysed. Larvae treated with high doses of Human-γ-globulin (HGG) were unable to produce anti-HGG antibody after challenge. The proliferative response demonstrated in the spleens of tolerant toadlets 21 days after challenge was, however, of similar magnitude to that in normally responding animals. Adoptive transfer of high-zone tolerance specific to HGG was demonstrated by intravenous inoculation of tolerant histocompatible splenocytes simultaneously with an antigenic challenge via the dorsal lymph sac. This is indicative of the active involvement of a suppressor T-cell population. The induction of high-zone tolerance in X. laevis results in changes in spleen cell populations as demonstrated by buoyant density gradient separation. Spleen cell sub-populations taken from the separated layers were not, however, effective in the adoptive transfer of tolerance. A normal lymphocyte transfer reaction was observed in X. laevis to show a number of characteristics seen in the mammalian reaction. The use of mitomycin-C treated donor cells and early thymectomized hosts has demonstrated that the phenomenon is composed of donor and host components which are largely distinct from each other. Implantation of allogeneic larval spleens resulted in the induction of transplantation tolerance or impaired rejection in a significant proportion of skin grafted toadlets in which both the donor and host larvae were up to and including stage 51 at the time of transplantation. The implication of these results is that immunomaturity of the donor and host is important in the induction of transplantation tolerance but that other factors must also be involved.

Mutations of Drosophila bagpipe, an NK3 class homeobox gene, result in failure of visceral mesoderm to differentiate into stomach tissue. Thus bagpipe, in association with other factors, is a good candidate for specification of visceral mesoderm in Drosophila. The cloning and characterisation of a Xenopus bagpipe homologue was therefore of great interest. This study describes the isolation of a full length Xenopus cDNA clone that on the basis of database analysis and sequence comparisons has been assigned as Xenopus bagpipe (XBap). Previous studies had revealed that the majority of homeoproteins recognise DNA sites with a 5'-TAAT-3' core but the NK class of homeoproteins had been shown to bind specifically to sites containing a 5'-CAAG-3' core. Experiments described here, however, show the XBap DNA binding site to be an even more divergent, 5'-TTAAGTGG-- TTAAGTGG-3'. A series of mutant oligonucleotides revealed that the `T' of the 5'- T_{1}A_{2}A_{3}G_{4}-3' core, as well as the presence of two such cores, are indeed essential for optimal XBap DNA binding. The murine NK3 class homeoproteins, Nkx-3.1 and Bapxl, are demonstrated to have the same requirements for optimal DNA binding as XBap. Drosophila Bagpipe, however, was found to have a less stringent requirement for a `T' at position one of the core, binding equally well to a 'C' in this position, but the presence of two such cores is still necessary for optimal DNA binding. Preliminary studies using site directed mutagenesis attempted to define the amino acids responsible for the differences. The effect of XBap on transcription was studied using a Xenopus oocyte assay and two cell transfection assays. XBap was not found to act as a transcriptional activator in any of these assays but evidence was obtained to suggest that a C-terminal truncation of XBap could act as a repressor of transcription.

The eyes are the most important sensory organs for most vertebrates. Their structure and development is conserved between several vertebrate species. The development is regulated by several signalling pathways, including the Wnt/β-catenin signalling pathway. It is required for several aspects of retinal development and it is known to regulate the proliferation of neuro-epithelial stem cells. In Xenopus laevis the intracellular Wnt/β-catenin signalling pathway is activated in the retina by the Wnt receptor Fz5. Fz5 function in the eye was shown to regulate tissue specific gene expression and neuron versus Müller glial cell differentiation. However, no candidate Wnt ligand that could act through the Fz5 receptor in this tissue had been described. Wnt6 was recently found to be expressed in the developing retina, indicating that Wnt6 and Fz5 share temporal and spatial expression. Here, I tested the hypothesis that Wnt6 might function as ligand for Fz5 in the retina. In this thesis I show that a knock down of Wnt6 led to the same eye phenotype seen in Fz5 morphants, including reduced eye size, changed marker gene expression and altered neuron/Müller glia ratio. Rescue experiments show that the observed phenotype is specific and is mediated by altered Wnt/β-catenin signalling pathway function. These findings support a linear model, in which Wnt6 signal interacts with the Fz5 receptor to activate the Wnt/β-catenin pathway to regulate neural and Müller glia cell differentiation in retinal tissue. These results make Wnt6 a candidate for Fz5 ligand.

Há duas populações invasoras da rã Xenopus laevis em Portugal, nas ribeiras da Laje e de Barcarena, em Oeiras. Aparentemente essas duas populações estão isoladas entre si. Estudou-se a documentada diferença de tamanhos das rãs entre as duas ribeiras, usando a esqueletocronologia para avaliar a estrutura etária, longevidade e crescimento delas. A população da ribª da Laje está envelhecida, pois as rãs apresentam maiores valores de tamanho corporal, idade e maturação sexual mais tardia nas fêmeas. Na ribª de Barcarena há uma estrutura etária jovem, com valores menores de tamanho e idade. Isto sugere que a ribª Barcarena pode ter boas condições de reprodução, mas piores condições de sobrevivência de adultos, ao contrário da ribª Laje. Aqui a estrutura etária está envelhecida, porventura em resultado da anterior campanha de erradicação de rãs. Porém, tais resultados também poderão ser justificáveis em face das diferenças nas condições ambientais vigentes nas ribeiras; ### Age structure and growth of invasive populations of the frog Xenopus laevis in Portugal: a skeletochronological approach 2. Abstract: There are two populations of Xenopus laevis in Portugal, living in two streams, Laje and Barcarena, located in Oeiras, Portugal. Previous studies have found differences in the size of individuals of both populations. The present work intends to analyze this size difference through the analysis of the age structure, longevity and the growth of these animals, using skeletochronology. Laje’s is characterized by an aged population with higher values of individual length, age and longevity. Barcarena’s depicted a younger age structure with lower values of individual length, age and longevity. These results suggest that Barcarena may have better conditions for reproduction, but worse conditions for adult survival, while the opposite may happen in Laje. The environmental differences could explain these results, but the aged population of Laje may also be a result of the eradication program. We suggest different approaches for the continuation of the eradication program.

Retinal degeneration is the progressive loss of neurons lining the posterior surface of the eye. Loss of a certain group of neurons called rod photoreceptors can occur as the result of genetic mutation. In humans, and in mammalian models of retinal degeneration, the death of these cells is permanent, and often followed by cone photoreceptor death, which leads to blindness. As a step towards understanding the implications of rod cell death in the retina, we generated transgenic X. laevis that expressed a novel form of caspase-9, with binding domains specific to the compound AP20187. We treated these transgenic animals with AP20187 and caused rod cell death by apoptosis in tadpoles and post metamorphic animals. Peak rod apoptosis occurred two days after drug exposure. We adapted an electroretinography apparatus, and protocols designed for mammals to measure functional changes in X. laevis rod and cone derived responses. We observed delayed secondary cone cell dysfunction after induced rod cell apoptosis, which was subsequently restored. These animals provide a simple and clinically relevant model of diseases like Retinitis pigmentosa, in which we will be able to probe in detail the mechanisms that govern cone cell dysfunction as a consequence of rod apoptosis. The unique ability of this species to recover from this insult will provide clues towards initiating similar recovery in humans.