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Kin-recognition abilities, first demonstrated 25 years ago in toad tadpoles, now appear to be widespread among amphibians. In some vertebrates kin recognition is based, at least in part, on highly polymorphic major histocompatibility complex (MHC) genes. Besides protecting animals from disease resistance, MHC genes regulate social behaviour. They allow relatives to recognise one another so that they can cooperate for mutual benefit. These two seemingly distinct functions of MHC genes may be integrally related, because animals need to outbreed to optimise the immune systems of their offspring. The ability to discriminate MHC-type is therefore likely to facilitate kin discrimination in tadpoles. I tested association preferences of African clawed-frog (Xenopus laevis) tadpoles in a laboratory choice apparatus. As in other anuran species, I found that tadpoles at earlier developmental stages preferentially associate with unfamiliar siblings over unfamiliar non-siblings but that this preference reverses during development. Tadpoles approaching metamorphosis demonstrated a reversal in their preference; they preferentially school with non-kin rather than kin. The ontogenetic switch in larval schooling preferences coincides with the onset of thyroid hormone (TH) controlled development and may be indicative of decreased fitness benefits associated with schooling with kin at later developmental stages. These may result from an increase in intraspecific competition, predation, or disease susceptibilities of prometamorphic individuals. Alternatively, the kin avoidance behaviours observed at later larval stages might reflect disassociative behaviour that facilitates inbreeding avoidance at reproductive maturity. This is the first study to find a shift from an association preference for kin to non-kin during amphibian larval development. Using allele-specific PCR techniques to MHC-type tadpoles, I tested association preferences among siblings based on shared MHC haplotypes. By using only full siblings in experimental tests, I controlled for genetic variation elsewhere in the genome that might influence schooling preferences. I found that X. laevis tadpoles discriminate among familiar full siblings based on differences at MHC genes. Subjects from four families preferentially schooled with MHC-identical siblings over those with which they shared no or one haplotype. Furthermore, the strength of tadpoles’ MHC-assortative schooling preferences significantly correlated with amino acid differences in the peptide-binding region (PBR) of both the MHC class I and II loci. Since MHC-PBR polymorphisms determine the pool of peptides that can serve as ligands for MHC molecules, these findings support the hypothesis that MHC peptide ligands mediate MHC type discrimination. As test subjects were equally familiar with all stimulus groups, tadpole discrimination appears to involve a self-referent genetic recognition mechanism whereby individuals compare their own MHC type with those of conspecifics. I also found that non-MHC-linked genetic differences contribute to tadpole association preferences in tests that contrast MHC and kinship. Tadpoles did not discriminate between MHC-similar non-siblings and MHC-dissimilar siblings and preferentially associated with MHC-dissimilar non-siblings rather than MHC-similar non-siblings. Although the MHC may be not solely responsible for the genetically determined cues that direct tadpole association preferences, it certainly is important in facilitating discrimination among conspecifics in X. laevis tadpoles. MHC-based discrimination may be retained through ontogeny and serve to maintain MHC-polymorphisms by facilitating disassortative mating.

A thesis submitted to the Faculty of Science, University of the Witwatersrand in partial fulfilment of the requirements for the Degree of Master of Science in Biotechnology. / Alkaline phosphatases (AP's) comprise a family of isozymes which are distributed widely in nature. Their function is unknown. There is evidence that in Xenopus tissue non-specific AP (TNAP) plays some role in differentiation and morphogenesis during embryological development. The isolation and elucidation of the primary structure of the TNAP gene is one aspect in the study of the function of TNAP in the embryo and is the primary aim of this work. ( Abbreviation abstract ) / AC2017

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.

Wnt signalling is a key regulator of vertebrate heart development yet the exact requirements of the Wnt signalling components remains unclear. The endogenous Wnt ligand Wnt6 has been identified as a regulator of cardiogenesis required for controlling heart muscle differentiation via canonical Wnt/β-catenin signalling. We show for the first time a requirement for an endogenous Wnt signalling inhibitor for normal heart muscle differentiation. Expression of sfrp1 is strongly induced in differentiating heart muscle. We show that sfrp1 is not only able to promote heart muscle differentiation but is also required for the formation of a normal sized heart muscle in the developing embryo. sfrp1 is functionally able to inhibit Wnt6 signalling and its requirement during heart development relates to relieving the cardiogenesis-restricting function of endogenous wnt6. In turn, we discover that sfrp1 gene expression in the heart is regulated by wnt6 signalling, which for the first time indicates that sfrp genes can function as part of a negative Wnt feedback regulatory loop. Our experiments indicate that sfrp1 controls the size of the differentiating heart muscle primarily by regulating cell fate within the cardiac mesoderm between muscular and non-muscular cell lineages. The cardiac mesoderm is therefore not passively patterned by signals from the surrounding tissue, but regulates its differentiation into muscular and non-muscular tissue using positional information from the surrounding tissue. This regulatory network may ensure that Wnt activation enables expansion and migration of cardiac progenitors, followed by Wnt inhibition permitting cardiomyocyte differentiation.

During early development of Xenopus laevis, gastrulation is a key morphogenetic event which transforms the embryo into three of primary germ layers: ectoderm, mesoderm, and endoderm. In order for the physical separation of these layers to occur, cells have to acquire specific properties that distinguish one layer from another. These properties, which include cell adhesion and migration, should be reflected in the tissue-specific proteome. While genetic analysis has led to the determination of a number of proteins involved in germ layer formation, this method would not have identified those proteins regulated on a translational or post-translational level. In this study, we have developed a two-dimensional gel based comparative proteomic approach employing difference gel electrophoresis (DiGE) to identify proteins involved in germ layer morphogenesis during Xenopus gastrulation. Differences between the physical properties of the ectoderm and mesoderm are likely based on differences in the proteomes of the cell surface and/or cortex. We therefore analyzed plasma membrane enriched fractions, obtained using discontinuous sucrose density gradient centrifugation. The Decyder program was used to quantify expression changes with statistical confidence across multiple DiGE gels, provide independent confirmation of distinct expression patterns from the individual experiments, and demonstrate high reproducibility between replicate samples. The identity of 23 proteins, which were obtained from 33 analyzed spots, was determined using mass spectrometry. Our proteomic analysis of Xenopus ectoderm and mesoderm identified alterations in proteins involved in cytoskeletal organization, signal transduction, protein folding, vesicle trafficking, and in glycolysis. We have also demonstrated the feasibility of proteomics in Xenopus, and have therefore shown that proteomics may be a valuable tool for analysis of early development in this system.

Kin-recognition abilities, first demonstrated 25 years ago in toad tadpoles, now appear to be widespread among amphibians. In some vertebrates kin recognition is based, at least in part, on highly polymorphic major histocompatibility complex (MHC) genes. Besides protecting animals from disease resistance, MHC genes regulate social behaviour. They allow relatives to recognise one another so that they can cooperate for mutual benefit. These two seemingly distinct functions of MHC genes may be integrally related, because animals need to outbreed to optimise the immune systems of their offspring. The ability to discriminate MHC-type is therefore likely to facilitate kin discrimination in tadpoles. I tested association preferences of African clawed-frog (Xenopus laevis) tadpoles in a laboratory choice apparatus. As in other anuran species, I found that tadpoles at earlier developmental stages preferentially associate with unfamiliar siblings over unfamiliar non-siblings but that this preference reverses during development. Tadpoles approaching metamorphosis demonstrated a reversal in their preference; they preferentially school with non-kin rather than kin. The ontogenetic switch in larval schooling preferences coincides with the onset of thyroid hormone (TH) controlled development and may be indicative of decreased fitness benefits associated with schooling with kin at later developmental stages. These may result from an increase in intraspecific competition, predation, or disease susceptibilities of prometamorphic individuals. Alternatively, the kin avoidance behaviours observed at later larval stages might reflect disassociative behaviour that facilitates inbreeding avoidance at reproductive maturity. This is the first study to find a shift from an association preference for kin to non-kin during amphibian larval development. Using allele-specific PCR techniques to MHC-type tadpoles, I tested association preferences among siblings based on shared MHC haplotypes. By using only full siblings in experimental tests, I controlled for genetic variation elsewhere in the genome that might influence schooling preferences. I found that X. laevis tadpoles discriminate among familiar full siblings based on differences at MHC genes. Subjects from four families preferentially schooled with MHC-identical siblings over those with which they shared no or one haplotype. Furthermore, the strength of tadpoles’ MHC-assortative schooling preferences significantly correlated with amino acid differences in the peptide-binding region (PBR) of both the MHC class I and II loci. Since MHC-PBR polymorphisms determine the pool of peptides that can serve as ligands for MHC molecules, these findings support the hypothesis that MHC peptide ligands mediate MHC type discrimination. As test subjects were equally familiar with all stimulus groups, tadpole discrimination appears to involve a self-referent genetic recognition mechanism whereby individuals compare their own MHC type with those of conspecifics. I also found that non-MHC-linked genetic differences contribute to tadpole association preferences in tests that contrast MHC and kinship. Tadpoles did not discriminate between MHC-similar non-siblings and MHC-dissimilar siblings and preferentially associated with MHC-dissimilar non-siblings rather than MHC-similar non-siblings. Although the MHC may be not solely responsible for the genetically determined cues that direct tadpole association preferences, it certainly is important in facilitating discrimination among conspecifics in X. laevis tadpoles. MHC-based discrimination may be retained through ontogeny and serve to maintain MHC-polymorphisms by facilitating disassortative mating.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "December 1996." Typescript. Vita. Includes bibliographical references (leaves 138-152). Also available on the Internet.

The experiments were focussed on three protein-nucleic acid interactions in the Xenopus oocyte: TFIIIA-5S RNA, TFIIIA-5S DNA and ribosomal protein L5-5S RNA. The binding affinities and contact sites of the proteins to the nucleic acids were studied. For studying the TFIIIA-5S RNA interaction, block mutations were constructed in helical stems II, III, IV and V of Xenopus laevis oocyte 5S RNA. The affinities of these mutants for binding to transcription factor IIIA were determined using a nitrocellulose filter binding assay. Mutations in stems III and IV had little or no effect on the binding affinity of TFIIIA for 5S RNA. However, single mutants in stems II and V (positions 16-21, 57-62, 71-72, and 103-104) which disrupt the double helix, reduce the binding of TFIIIA by a factor of two- to three-fold. In contrast, double mutants (16-21/57-62, 71-72/103-104) which restore the helical structure of these stems, but with altered sequences, fully restore the TFIIIA binding affinity. The experiments reported here indicate that the double helical structures of stems II and V, but not the sequences, are required for optimal TFIIIA binding. The effects on TFIIIA binding affinity of a series of substitution mutations in the Xenopus laevis oocyte 5S RNA gene were quantified. These data indicate that TFIIIA binds specifically to 5S DNA by forming sequence-specific contacts with three discrete sites located within the classical A and C boxes and the intermediate element of the internal control region. Substitution of the nucleotide sequence at any of the three sites significantly reduces TFIIIA binding affinity, with a 100-fold reduction observed for substitutions in the box C subregion. These results are consistent with a direct interaction of TFIIIA with specific base pairs within the major groove of the DNA. In contrast, the TFIIIA binding data for the same mutations expressed in 5S RNA indicates that the protein does not make any strong sequence-specific contacts with the RNA. Although the protein footprinting sites on the 5S DNA and 5S RNA are coincident, nucleotide substitutions in 5S RNA which moderately reduce TFIIIA binding affinity do not correspond at all to the three specific TFIIIA interaction sites within the gene. For investigating the L5-5S RNA interaction, a cDNA encoding ribosomal protein L5 of Xenopus laevis was subcloned into a T7 expression vector and expressed in Escherichia coli. The resulting soluble fusion protein with a histidine tag at the N-terminus was purified by affinity chromatography to 95% homogeneity. The equilibrium binding of recombinant L5 to Xenopus 5S ribosomal RNA was characterized, and the affinity of the protein for a set of 5S RNA mutants was quantitatively measured using a nitrocellulose filter binding assay. L5 binds to 5S RNA with properties similar to those of the TFIIIA-5S RNA interaction. However, unlike TFIIIA, L5 was insensitive to changes in either the sequence or the secondary structure of the 5S RNA. The results from these studies indicate that the specific protein-nucleic acid interactions in the biological pathway of 5S RNA use distinct mechanisms. / Graduate

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.