M.Sc. (Zoology) / An investigation was made into the presence and uptake of seven selected metals by the organs and tissues of the various developmental stages of the African clawed frog Xenopus laevis from industrial, mine and sewerage polluted aquatic environments on the Witwatersrand. These results were compared with conditions in two relatively unpolluted localities where the clawed frog also occurs. Attention was given to the physical and chemical conditions including the concentration of the metals at various localities. Gut content analyses of the frog stages of the plataona was made to identify the particular food items ingested by the animal but also to correlate this with the incidence of the macro invertebrate organisms present. Metal concentrations were determined for both the gut contents as well as the macro invertebrates as a group at two selected localities. The presence and concentration of various metals were determined in specific organs and tissues of the frog and compared with each other. According to the data the spleen, gallbladder, middle & Hind gut as well as the testes were identified as the target organs containing the highest concentrations of the various metals. Shortcomings in the work is pointed out and recommendations made on possible further research.

LBH is a highly conserved protein whose role during vertebrate development is relatively under-studied. In collaboration with the Albertson lab, our lab has previously shown that it is necessary for cranial neural crest cell migration in the zebrafish and in Xenopus laevis. The molecular mechanisms through which it acts are not well understood. In Xenopus, LBH is a maternally deposited protein. As such, studying its role in early development has not been feasible through the morpholino-mediated knockdown techniques that prevent translation of target genes. Recently, a technique for degrading endogenous proteins was developed, called Trim-Away. This was developed in mammalian systems and utilizes the E3 ubiquitin ligase Trim21 in conjunction with an antibody against a protein of interest in order to degrade the protein. In order to observe the effects of a knockdown of LBH during early embryonic development, we sought to modify the technique for use in Xenopus. We injected embryos with mRNA encoding the human form of trim21 along with a monoclonal antibody against LBH that our lab developed (2B8) and tracked degradation of the protein over time, monitoring embryos for any phenotypes arising during early development. Our results demonstrate that Trim-Away can be utilized in Xenopus. LBH depleted embryos display a variety of defects during gastrulation, the process by which the three germ layers are properly organized. These appear to be mainly due to defects in fibronectin fibrillogenesis and mesodermal migration.

No description available.

CK2α is a serine-threonine kinase that is involved in a large number of biological processes, including embryonic development, cancer, and cell proliferation. Recently, it has been found that mutations in CK2α results in a developmental condition known as Okur-Chung neurodevelopmental syndrome (OCNDS). This disorder commonly results in intellectual disability, congenital heart defects (CHDs), gross motor delay, and facial abnormalities. CK2α inhibition has so far primarily been studied in mice, through methods such as knockout, gene floxing, and CRISPR/Cas9 mutations. In this thesis, we provide a proof of principle that chemical inhibition of CK2 in Xenopus laevis embryos can induce a phenocopy similar to the heart phenotype of the CK2α knockout mouse model, and demonstrate the potential of Xenopus laevis as an animal model to study molecular mechanisms that may underlie OCNDS. Here we carefully examined whole embryos, sections stained with multiple antibodies, sections stained with hematoxylin and eosin, and assessment of proliferation and apoptosis rates. The phenotypes observed in the Xenopus laevis model were analyzed and compared to both the CK2α knockout mouse model and OCNDS patients. Results found commonalities among facial features, heart deformities, and muscle patterning between the animal models, which overlapped heavily with patient symptoms. Thus, this work has established Xenopus laevis treated with chemical inhibitors as an appropriate animal model for further characterization of the mechanisms that may underlie OCNDS. / 2023-11-18T00:00:00Z

Chez les eucaryotes supérieurs, la réplication de l'ADN est initiée à partir de plusieurs milliers d'origines. Mais la régulation spatio-temporelle de leur activation reste mal caractérisée. Une voie de contrôle (checkpoint) de la phase S est activée lorsque les fourches de réplication sont bloquées, inhibant ainsi l'activation d'origines tardives. L'objectif de ma thèse consistait à étudier deux facteurs essentiels dans le programme spatio-temporel de la réplication, dans le système du xénope : la protéine « checkpoint » Chk1, qui est un facteur inhibiteur de l'activation des origines, et les désoxyribonucléotides (dNTPs), précurseurs de la synthèse de l'ADN. Chez le xénope, après douze divisions embryonnaires, a lieu la transition mid-blastuléenne (MBT). A cette étape, une augmentation du ratio nucléo-cytosolique va entrainer la titration des facteurs de réplication, ce qui active le point de contrôle et ralentit la phase S. Il est possible de mimer in vitro les phases S rapides des embryons pendant le développement précoce en augmentant la concentration en noyaux dans l'extrait d'œufs.Nous avons pu voir par l'inhibition, la déplétion ou la surexpression de Chk1 que cette protéine régulait l'activation des origines lors d'un stress, mais également dans une phase S non perturbée, grâce à la technique du peignage moléculaire. Ce résultat montre que le niveau de Chk1 doit être finement régulé pour permettre une réplication correcte dans une phase S non perturbée, chez les eucaryotes supérieurs. Nous avons ensuite cherché à savoir si la concentration en dNTPs pouvait être limitante pendant le développement et comment elle modulait le programme de réplication. Nous avons comparé l'effet de l'ajout de dNTPs sur la réplication en mimant plusieurs stades précoces du développement pré-MBT. La variation de la concentration en dNTPs agit sur la réplication en augmentant à la fois l'activation des origines et, en fonction de la concentration en noyaux, aussi la vitesse des fourches. Cet effet est indépendant du checkpoint de la réplication dans ce système et d'autres études sont nécessaires pour comprendre les mécanismes moléculaires. / DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal regulation program which is not well characterized. The S phase checkpoint is activated when replication forks are blocked which inhibits the firing of late origins. The aim of my thesis consisted to study two essentials factors in spatio-temporal replication program in Xenopus system: the checkpoint protein Chk1, inhibitor of origin activation, and the deoxyribonucleotides (dNTPs), DNA synthesis precursors. In Xenopus, the mid-blastula transition (MBT) occurs after twelve embryonic divisions. An increase of the nucleo-cytosolic ratio induces a titration of replication factors, that activates the checkpoint and slows down the S phase. It is possible to mimic in vitro the rapid S phases of early Xenopus development stages by increasing the nuclei concentration. By DNA combing combined with Chk1 inhibition, depletion and overexpression experiments, we show that Chk1 controls origins activation in perturbed but also unperturbed S phase. My results show that Chk1 levels needs to be tightly regulated in order to properly control the replication program during normal S phase in higher eukaryotes. In order to determine whether the concentration of dNTPs could be another limiting replication factor, we compared the effect of dNTPs addition on replication by mimicking in vitro several early stages of pre-MBT development. Addition of dNTPs affects DNA replication, by increasing origin activation and, dependent on nuclei concentration, also the fork speed. This effect is independent of the S phase checkpoint and further studies are needed in order to understand the molecular mechanisms behind.

Thesis advisor: Laura Anne Lowery / Wolf Hirschhorn Syndrome (WHS) is a neurodevelopmental disorder characterized by craniofacial malformations, heart and skeletal defects, intellectual disability as well as seizure disorders. While this disorder is thought to arise from a deletion of a region on the short arm of chromosome 4, which includes the four genes WHSC1, WHSC2, LETM1 and TACC3, the mechanism by which loss of these genes results in WHS is not understood. Given that these genes have been linked to cell migration and that affected tissues include those derived from the neural crest, we propose that WHS results from a defect in neural crest cell migration. Here, we show that WHSC1, WHSC2, TACC3 and LETM1 are all expressed along the neural tube and developing neural folds during Xenopus embryonic development. These genes are additionally enriched in the pharyngeal arches, which are migrating neural crest cells. The knockdown of these WHS-related genes leads to variable defects in craniofacial and cartilage morphology. Moreover, the loss of WHS gene expression causes defects in forebrain and midbrain development. This implicates these four genes in the WHS phenotype. Further analysis of both WHSC1 and TACC3 function show that their individual knockdown causes defective neural crest cell migration both in vivo and in vitro. This supports the notion that the WHS phenotype is a result of erroneous neural crest cell motility. Our analysis shows that the WHS related genes; WHSC1, WHSC2, LETM1 and TACC3, play a role in the WHS phenotype of craniofacial malformation, skeletal abnormality, and microcephaly. Further analysis of these genes will determine the combinatorial effects of their knockdown on neural crest cell migration during embryonic development to further elucidate the mechanism through which WHS develops. / Thesis (BS) — Boston College, 2018. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Cardiac failure is one of the leading cause of deaths worldwide. Potential therapeutic approach, which overcome invasive organ transplantation and delivery of immunosuppressive drugs, is lacking nowadays. However, research of mesenchymal stem cells (MSCs) therapy displays immunomodulation potential, which can further promote variety of organ regeneration without need of drug treatment. Xenopus tropicalis immature Sertoli cells (XtiSCs) culture was established in our laboratory from juvenile Xenopus tropicalis male. XtiSCs possess immunomodulatory capacity and differentiation to cardiomyocytes after the treatment with the inhibitor of glycogen synthase kinase-3 (GSK-3) CHIR99021. To test the survival rate of transplanted XtiSCs we firstly microinjected treated cells directly inside tadpole's heart. XtiSCs proliferated there for the whole tested time period (30 days). However, after direct heart XtiSCs injection and subsequent cardiac injury in adult frog, no cells were localized in wound area. Thus, we focused on remote control of cardiac regeneration using XtiSCs without CHIR99021 treatment. We injected cells inside skeletal muscle bed and confirmed their survival and proliferation. Moreover, if cells were transplanted 3 days before heart injury, it resulted in significant reduction of fibronectin...

Thanks to their ability to differentiate into variable cell types and migrate to the site of an injury mesenchymal stem cells (MSC) are broadly used in regenerative medicine. Their relative easy availability together with the property to control the immune system determines them as a cure of autoimmune diseases or a recovery of wounded tissues. Similar features posses Sertoli cells which take place in the seminiferous tubule of testis. Cell culture of testicular stem cells from juvenile male testes of X. tropicalis (XtTSC) was established in supervisor's laboratory. This cell culture showing both MSC's and SeC's properties was transformed to carry red fluorescent protein RFP. The aim of this diploma thesis was to investigate an behavior of transformed XtTSC in living organism, therefore cells were transplanted into the X. tropicalis tadpoles in stage 41. Subsequently, their migration potential was explored. To study of XtTSC's differentiation potential it was necessary to introduce a reliable sectioning techniques for the subsequent immunohistochemical analysis. Based on our experiments, we found that the XtTSC's cell culture contains precursors of SeC and peri-tubular myoid cells, however in vivo these cells turned into the dedifferentiated MSC-like state allowing a strong migration through the...

Thesis advisor: Laura Anne Lowery / Axon guidance is a critical process in forming the connections between a neuron and its target. Development of a properly functioning nervous system relies heavily on how accurately an axon is guided to the right target. Defects in the guidance machinery may result in neurological disorders. The growth cone that is formed at the tip of a growing axon is responsible for navigating axons to their final targets. The growth cone steers the growing axon towards the appropriate direction by integrating extracellular guidance cues received by membrane-associated receptors at the growth cone periphery. Upon receiving guidance cues, a number of intracellular signal transduction pathways are initiated downstream of the guidance receptors, that can promote or halt growth cone advance. The growth cone generates these responses by remodeling its cytoskeletal components, which are actin network in the periphery and microtubules in the growth cone center. In this thesis, we focus on understanding the role of microtubule dynamics regulation within the growth cone as it makes guidance decisions. Specifically, we examine the role of TACC3 as a microtubule plus-end binding protein during axon outgrowth and guidance. We show that TACC3 localizes at microtubule plus-ends in embryonic Xenopus laevis growth cones and regulates microtubule growth parameters. We also show that TACC3 is important for promoting axon outgrowth in cultured neural tube explants. Furthermore, our data suggests that TACC3 affects axon guidance in vivo and ex vivo. Examination of embryos depleted of TACC3 revealed guidance defects in the spinal cord neurons, while TACC3-overexpressing cultured spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Finally, in an attempt to delineate the mechanism behind TACC3-mediated axon guidance under Slit2, we studied the importance of tyrosine phosphorylation induced by Abelson tyrosine kinase. We find that retaining phosphorylatable tyrosines within the TACC domain is important for its microtubule plus-end tracking behavior and its impact on microtubule dynamics regulation, axon outgrowth and guidance. Together, this thesis contributes new insights to the understanding of the role of TACC3 as a microtubule plus-end binding protein and identifies TACC3 as a potential regulator of axon outgrowth and guidance during Xenopus laevis embryonic development. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Nitric oxide (NO) is an interesting molecule, which is involved in many important biological processes such as vasodilatation, neurotransmission, immune response and cell proliferation. This work presents the crucial role of NO during the Xenopus laevis embryonic epidermis development. The outer layer of the embryonic epidermis is composed of 4 cell types (small secretory cells, multi-ciliated cells, ionocytes and goblet cells). Embryonic epidermis composition reflects specialized epithelia such as a respiratory epithelium of mammals. Therefore, Xenopus embryonic epidermis has become a suitable model for the study of human mucosal and mucociliary epithelium and their defects. I found that NO is mainly produced in ionocytes and multi-ciliated cells by using molecular and immunohistochemical approaches. The study of molecular and cellular phenotype changes in embryos with inhibited NO production revealed the necessity of this molecule for correct formation and function of the mucociliary epithelium. NO inhibition caused reduction of specialized epidermal cell types (small secretory cells, ionocytes and multi-ciliated cells) and structural changes in multi-ciliated cells. I also showed that NO affects the development of the embryonic epidermis through the sGC-cGMP-PKG signaling pathway, probably by...