Morphogenesis, or the development of tissues, structures, and organs, is at the heart of embryonic development. Morphogenesis is a complex, multi-tissue process that requires coordinated cellular communication, migration, and differentiation; due to this complexity, the mechanisms that underlie morphogenesis remain poorly understood. The sea urchin embryo is morphologically and genetically more simple than most other developmental model organisms, and is optically transparent, making it a highly tractable system in which to study morphogenetic processes. The sea urchin larval endoskeleton is a biomineral that is secreted by primary mesenchyme cells (PMCs). The PMCs ingress into the embryo and remain individual, mesenchymal cells that migrate into a stereotypic three-dimensional (3-D) pattern within the blastocoel, prefiguring the form of the ensuing skeleton, which they subsequently secrete. PMC positioning is directed by cues originating in the overlying ectoderm; however, the molecular identity of those cues has remained unknown. The work described in this dissertation combines systems-level approaches with in vivo 3-D spatial analysis to identify novel skeletal patterning genes and to define their functional roles in skeletal patterning. A transcriptomics-based screen identified numerous novel candidate skeletal patterning cues. Of those cues, two were further pursued for detailed functional studies. First, the sulfate transporter SLC26a2/7 (SLC) was found to promote ventral accumulation of sulfated proteoglycans that is both necessary and sufficient to attract PMCs to the ventral territory for ventral skeleton formation. Second, the enzyme 5-lipoxygenase (LOX) was found to be required for ventral and rotational skeletal patterning, and its product, 5(S)-HETE was found to be a chemoattractant for PMCs, thereby identifying a novel role for lipoxygenase enzymes in embryonic patterning and morphogenesis. Recent work from other groups has demonstrated that PMCs diversify their gene expression profiles during skeletal patterning, implying that PMC diversification is involved in skeletal patterning, likely as a response to locally distinct spatial cues. The studies herein identify Tbx2/3 and Pks2 as important PMC subset-specific genes whose spatial expression is modulated by SLC and LOX, respectively. Together, these results provide new mechanistic insights that define our molecular understanding of the regulation of sea urchin ventral skeletal patterning.

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The growing popularity of physical sunscreens will also lead to an increased release of the ingredients from zinc oxide (ZnO) sunscreens into marine environments. Though zinc (Zn) is a necessary micronutrient in the ocean, greater than natural Zn concentrations are being released into marine environments by use of sunscreens. The extent of the consequences of the addition of Zn to the ocean are not fully understood. We investigated effects of materials released by zinc oxide (ZnO) sunscreens on the development of California purple sea urchin, Strongylocentrotus purpuratus. Embryos developed in various concentrations of Zn, the sources of which included zinc-containing compounds: ZnO and ZnSO4; and ZnO sunscreens: All Good, Badger, and Raw Elements. ZnO sunscreens were slightly more toxic than ZnO and ZnSO4, suggesting that the sunscreens may release additional unknown materials that are detrimental to sea urchin embryo development. All concentrations of Zn exposure resulted in significant malformations (skeletal abnormality, stage arrest, axis determination disruption), which were identified using light and fluorescent confocal microscopy. Developing embryos internalize Zn2+in proportion to the concentration of Zn in their environment. Additionally, both ZnO sunscreens and ZnO and ZnSO4at 1ppm Zn, significantly increased calcein-AM (CAM) accumulation, indicating decreased multidrug resistant (MDR) transporter activity. This is the first research that we know of to show that ZnO sunscreens release high concentrations of Zn that are internalized by and have detrimental effects on aquatic organisms.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: South Africa's coastline is in the region of 3650kms and encompasses many different and dynamic marine environments. To enhance our current understanding of the population structure and gene flow patterns of intertidal zone marine species in this region, this study sets out to investigate the phylogeographic population structure of the Cape sea urchin, Parechinus angulosus, using mitochondrial and nuclear DNA sequence data collected in 2007 and 2008. Individuals were sampled from 18 geographic locations between southern Namibia and Durban, covering nearly the full extent of the species range. Sequence data were obtained from a 790bp region of the COI mtDNA gene (n=510) and a 182bp region of the nDNA SpREJ9 gene (n=145), respectively. The mtDNA data revealed 283 polymorphic sites (36%) defining 195 haplotypes, of which 160 were unique and 35 shared among individuals. Haplotype diversity (h) was found to be high both overall (h=0.95) and for individual localities (h=0.75-0.98), with nucleotide diversity (π) being low overall (π=0.013) as well as for individual localities (π=0.0033-0.0254). AMOVA revealed significant population structure among sampling sites in the Namaqua Province biogeographical region, as well as between three of the four respective coastal biogeographic provinces/regions. Gene flow was bi-directional among sampling sites in the south coast Agulhas and East Coast Province biogeographical regions, while gene flow in the Namaqua Province appears to be dominated by northwards movement. BAPS identified a significant break in the Cape Point region, which was also reflected in the gene flow patterns and parsimony networks. This broadly corresponds to previously identified biogeographic regions as well as genetic breaks for other marine species found along this coast. Fu's Fs statistics showed strong signal(s) of population expansion for individual sampling localities as well as for the data set as a whole, while MDIV estimated a time since expansion ranging from 7733-4759 years ago. The nDNA data revealed 54 variable sites (29.7%), defining 72 alleles of which 50 were unique and 22 shared among individuals. Many of the alleles (69.4%) were restricted to single sampling sites, with Betty's Bay on the south coast being the most diverse from a genetic viewpoint. Allelic diversity was high overall (h=0.86) while nucleotide diversity was low (π=0.025). No nuclear sub-group structure was identified by BAPS, although the parsimony network revealed shallow genetic structure between the Namaqua and Agulhas Provinces, with significant pairwise ФST values also recovered between their individual coastal localities. This points to at least one major barrier to gene flow for Parechinus angulosus along the South African coast, namely Cape Point. Several additional, smaller hindrances to gene flow along the coast were also identified, most of which are congruent with findings from studies on both other and sea urchin species. As a standalone study this research elucidated many aspects regarding the phylogeography of the Cape sea urchin, P. angulosus. However, it is when viewed in the broader context of invertebrate phylogeography along the southern African coastline that this research will provide its most critical insight. / AFRIKAANSE OPSOMMING: Geen opsomming

Em ouriços, os esferulócitos vermelhos são responsáveis pela biossíntese do equinocromo, um pigmento naftaquinônico considerado antioxidante e bactericida, no entanto seu papel na resposta imune permanece pouco elucidado. O presente trabalho avaliou a reposta imune inata de ouriços-do-mar Lytechinus variegatus, através da atividade fagocítica frente a diferentes concentrações de equinocromo (50 e 100 µg/ml). Para tanto, o equinocromo foi extraído e purificado por RP-HPLC. Nossos resultados demonstraram que o equinocromo em ambas as concentrações modula positivamente a fagocitose, aumentando a quantidade de células fagocitando. A concentração de 50 µg/ml foi capaz de ativar os amebócitos fagocíticos (AF), e aumentar a quantidade de AF com quatro ou mais leveduras fagocitadas. Já na concentração de 100 µg/ml, além da ativação dos AF, aumentou também, a quantidade de AF com uma, duas, quatro ou mais leveduras fagocitadas, sugerindo uma atuação dose-dependente. Desta forma, os dados apresentados demonstram que o equinocromo exerce um importante papel na resposta imune. / The biosynthesis of echinochrome is mediated by red sphere cell. This naphthoquinonic e pigment presents antioxidant and bactericidal characteristics. However, the echinochrome role in immune response remains unclear. In this study, we evaluated the innate immune response of the sea urchin Lytechinus variegatus. To this purpose, the echinochrome was extracted and purified by RP-HPLC. Finally, phagocytic amoebocytes were exposed to different concentrations of echinochrome (50 and 100 mg/ml), when phagocytic activity was analysed. Here, we showed that echinochrome positively modulate phagocytosis, increasing the number of phagocytizing cells. The concentration of 50 mg/ml activated phagocytic amoebocytes (AF), and increased the number of AF containing four or more phagocytosed yeasts. For the other hand, at 100 mg/ml exposure, the activation of AF also increased the number of AF with one, two, four or more yeast phagocytosed, suggesting a dose-dependent activity. Thus, the data presented demonstrated that echinochrome plays an important role in the immune response.

Although transition metals are essential for life, misregulation of redox-active metal uptake, delivery, storage, and excretion has been linked with a series of neurodegenerative disorders. Alzheimer's disease (AD) is considered an epidemic and is the most widespread of all forms of dementia. Copper ions found in large concentrations localized in amyloid-ß plaques in the brain of AD patients have been linked with the generation of reactive oxygen species which are suspected to be the culprits leading to neuronal cell death. Herein a series of mechanistic and spectroscopic studies elucidate the chemistry about the metal-centered oxidation of biomolecules, including catecholamine neurotransmitters and some analogues by copper-complexes of amyloid-ß peptide. Transition metals can also be useful tools for characterization of metalloproteins due to their unique chemical and spectroscopic features. Herein a series of studies of the native Zn²+ and Cu²+-derivative of recombinant Blastula Protease 10 (BP10) from the sea urchin Paracentrotus lividus are presented in order to elucidate its catalytic mechanism, with the use of enzymology, metal substitution, and electronic absorption spectroscopy.

Marine ecosystems can be exposed to natural and anthropogenic disturbances that can lead to ecological failures. Marine reserves have been lately suggested to protect marine populations and communities that have been affected by habitat destruction and harvest. This research evaluates the potential role of two marine reserves established in Oregon in 1967 (Whale Cove) and 1993 (Gregory Point). The red sea urchin (Strongylocentrotus franciscanus) was selected as indicator of population recovery since it is the only species that is commercially harvested. Changes in density, biomass, average size, size structure, growth and mortality rates were evaluated through time to assess population recovery. These parameters were also compared between reserves and adjacent exploited areas to evaluate the effect of exploitation. Results from Whale Cove (old reserve) indicate that the population in this area is fully recovered. On the contrary, the population in Gregory Point (new reserve) showed signs of recovery after six years of being protected. The importance of red urchins as source populations to provide larvae to adjacent areas was explored by the analysis of drifter's trajectories. Both reserves might be connected in a network where larvae produced in Whale Cove will provide recruits to Gregory Point and adjacent exploited areas, as well as populations in northern California. Gregory Point releases larvae that become recruits for Whale Cove only when spawning takes place in winter, otherwise larvae travel to central California. No clear trends were found in growth and mortality rates between reserves and non-reserves; differences were more related with food availability, competitors, and age specific mortality. We applied qualitative simulations to characterize and differentiate the community network inside reserves and exploited areas. Results suggest that communities from a particular site can be represented by a set of alternative models with consistent species interactions. Differences in predator-prey interactions as well as non-predatory relationships (interference competition, mutualism, amensalism) were found among sites. Each set of models represents a hypothesis of community organization that agreed with natural history information. Alternative models suggest that kelp forest communities are dynamic and can shift from one network configuration to another providing a buffer against a variable environment. / Graduation date: 2002

Epithelial-mesenchymal transitions (EMTs) are fundamental and indispensable to embryonic morphogenesis throughout the animal kingdom. At the onset of gastrulation in the sea urchin embryo, micromere-derived primary mesenchyme cells (PMCs) undergo an EMT process to ingress into the blastocoel, and these cells later become the larval skeleton. Much has been learned about PMC specification in sea urchin embryos. However, much less is known about how states of the sequentially progressing PMC gene regulatory network (GRN) controls the EMT process during PMC ingression. Transcriptional regulators such as Snail and Twist have emerged as important molecules for controlling EMTs in many model systems. Sea urchin snail and twist genes were cloned from Lytechinus variegates, and each has been experimentally connected to the PMC regulatory network; these experiments demonstrate several requirements for PMC ingression, and in doing so, begin to illustrate how a gene regulatory network state controls morphogenesis. Functional knockdown analyses of Snail with morpholino-substituted antisense oligonucleotides (MASO) in whole embryos and chimeras demonstrated that Snail is required in micromeres for PMC ingression. Investigations also show that Snail downregulates cadherin expression as an evolutionarily conserved mechanism, and Snail positively regulates a required endocytic clearance of epithelial membrane molecules during EMT. Perturbation experiments indicate that Twist has accessory roles in regulating PMC ingression, and possibly plays a maintenance role in PMC specification network state. In addition, Twist also functions in the post-EMT network state, particularly in directing PMC differentiation and skeletogenesis. The recently annotated sea urchin genome accelerates the discovery of new genes and holds strong promise of mapping out a complete canvas of the micromere-PMC gene regulatory network. Using the genome resources we successfully cloned several newly identified PMC genes, and found most of them to be expressed in micromeres just prior to ingression of the nascent PMCs. Current experiments focus on the roles of these genes in preparing for, executing, and/or controlling the mesenchymal behavior following PMC ingression. The functions and inter-relationships of these genes will greatly augment our understanding of how a gene regulatory network state controls a crucial morphogenetic event. / Dissertation

<p>Pathogens and host immune systems use strikingly similar methods of diversification. Mechanisms such as point mutations and recombination help pathogens escape the host immune system and similar mechanisms help the host immune system attack rapidly evolving pathogens. Understanding the interplay between pathogen and immune system evolution is crucial to effective drug and vaccine development. In this thesis we employ various computational methods to study diversification in a pathogen, an invertebrate and a vertebrate immune system.</p> <p>First, we develop a technique for phylogenetic inference in the presence of recombination based on the principle of minimum description length, which assigns a cost-the description length-to each network topology given the observed sequence data. We show that the method performs well on simulated data and demonstrate its application on HIV <italic>env</italic> gene sequence data from 8 human subjects.</p> <p>Next, we demonstrate via phylogenetic analysis that the evolution of repeats in an immune-related gene family in <italic>Strongylocentrotus purpuratus</italic> is the result of recombination and duplication and/or deletion. These results support the evidence suggesting that invertebrate immune systems are highly complex and may employ similar mechanisms for diversification as higher vertebrates.</p> <p>Third, we develop a probabilistic model of the immunoglobulin (Ig) rearrangement process and a Bayesian method for estimating posterior probabilities for the comparison of multiple plausible rearrangements. We validate the software using various datasets and in all tests, SoDA2 performed better than other available software.</p> <p>Finally, we characterize the somatic population genetics of the nucleotide sequences of >1000 recombinant Ig pairs derived from the blood of 5 acute HIV-1 infected (AHI) subjects. We found that the Ig genes from the 20 day AHI PC showed extraordinary clonal relatedness among themselves; a single clone comprised of 52 members, with observed and inferred precursor antibodies specific for HIV-1 Env gp41. Antibodies from AHI patients show a decreased CDR3H length and an increased mutation frequency when compared to influenza vaccinated individuals. The high mutation frequency is coupled with a comparatively low synonymous to non-synonymous mutation ratio in the heavy chain. Our results may suggest presence of positive antigenic selection in previously triggered non-HIV-1 memory B cells in AHI.</p> <p>Taken together, the studies presented in this thesis provide methods to study diversification in pathogens, and invertebrate and vertebrate immune systems.</p> / Dissertation

Genes present in multiple copies and genes that contain regions of repetitive sequences can undergo concerted evolution, which results in homogenization of the nucleotide sequence of the genes or repetitive regions. In regions of tandem repeats, this occurs through misalignment of repeat units followed by unequal crossover, which generates two products with differing numbers of repeat units. Gene conversion is thought to lead to one of these products becoming fixed in a species. The homogenous sequence of previously studied genes that have been thought to undergo this process has made it difficult to determine the exact models involved. Here I examine concerted evolution in SM50, a sea urchin gene that encodes a protein involved in biomineralization. The repetitive region in the SM50 gene varies in length between species, and there is variability in each repeat unit as well. I examine the codon usage in SM50 in a variety of species, and discuss how purifying selection, substitutions, concerted evolution, and selection at the level of DNA sequence have played a role in the evolution of this gene. I also examine the structure and sequence of the repeat units, and purpose models that have led to the evolution of the repeat pattern seen in the different species examined. Finally, I have found variation in the number of repeat units within several species. This has allowed us to deduce the specific models of unequal crossover that led to this variation. The unique variation in the repetitive region of SM50 has enabled us to describe a model of how substitutions affect the model of misalignment and unequal crossover.