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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 vegetal cortex of the Xenopus oocyte is enriched for several mRNAs critical for early embryonic developmental processes, including germ layer specification and dorsoventral axis formation. A recent microarray screen for other vegetally localized RNAs identified several hundred novel cortex-enriched transcripts, which may have undiscovered roles in early development. In order to better elucidate the functions of localized mRNAs in early development, I characterized the spatiotemporal expression patterns and developmental functions of two novel transcripts, TRIO and F-actin binding protein (triobp) and Cdc42 effector protein 4-like (cep4l). Overexpression and loss-of-function experiments failed to identify a critical role for TrioBP in early Xenopus development. For Cep4l, I found that overexpression of Cep4l induced primary neuron formation throughout the epidermis, preferentially inducing primary sensory neurons. This increase came at the expense of neighboring non-neuronal ciliated and ion-secreting cells, suggesting a role for Cep4l in neural boundary formation. Additionally, I have shown that Cep4l binds specifically to Cdc42 through its known Cdc42/Rac-interactive binding (CRIB) domain, and that this activation was necessary for Cep4l function. Morpholino (MO) oligonucleotide based inhibition of Cep4l protein synthesis resulted in decreased primary sensory neurogenesis. Additionally, I have shown that Cdc42 itself is required for sensory neurogenesis. Furthermore, I find that Fgf8a, an isoform of Fgf8 previously known to regulate neuronal development, but not the Fgf8b isoform, regulates the association of Cep4l and Cdc42. Importantly, I further show that Cep4l and Cdc42 are required for the ability of Fgf8a to induce sensory neurons. Overall, this work suggests a novel role for Cep4l and Cdc42 in the regulation of primary sensory neuronal fate downstream of a unique Fgf8 signaling pathway. I propose that binding of Fgf8a to its receptors activates Cdc42 and recruits Cep4l, which could serve as a scaffold for integrating additional signaling pathways involved in controlling sensory neuron fate.

Polarized cell movements are essential to the cell rearrangements that occur during morphogenesis. In Xenopus, cell polarity is reflected in the directional cell intercalations that drive the morphogenetic movements characterizing gastrulation. While these cell behaviours are well described, the molecular mechanism underlying this cell polarity is unknown. PAR-3 is a multi-domain scaffolding protein and a key regulator of cell polarity. I have isolated a cDNA encoding Xenopus PAR-3 and generated several mutant constructs, each lacking a conserved domain. Initial characterization of GFP-tagged PAR-3 in A6 cells demonstrates localization to points of cell-cell contact in epithelial sheets, as well as at the leading edge of migrating cells. PAR-3 constructs lacking the CR1 or PDZ1 domain fail to compartmentalize properly and are found in the cytoplasm. Eliminating the PDZ3 domain resulted in a loss of contact inhibition. Mutation of the aPKC phosphorylation site created a membrane hyper-accumulation phenotype. Together these data suggest that the CR1 and PDZ1 domains mediate membrane compartmentalization that is modulated through aPKC phosphorylation, while the PDZ3 domain is required for contact inhibition. In embryos, PAR-3 is expressed throughout gastrulation and over-expression of PAR-3 inhibits blastopore closure indicating a requirement during gastrulation. Inhibition is relieved when the construct lacking the CR1 domain is over-expressed. PAR-3 was localized to the cell periphery in axial mesoderm. Localization was abolished with deletion of the CR1 domain indicating that membrane targeting of PAR-3 is required for gastrulation and this targeting is dependent on oligomerization of PAR-3. This investigation also suggests PAR-3 functions independent of the PAR complex in Xenopus embryos indicating involvement of a different PAR-3 signaling pathway.

No description available.

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.

No description available.

No description available.