Made available in DSpace on 2015-05-07T14:49:31Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3590758 bytes, checksum: bf7818f89b1ec249bca8891f256b8f1a (MD5) Previous issue date: 2013-04-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This thesis called Sustainability in Suape Bay: between the Suape Port and Industrial Complex and the Sea Urchin Festival seek to understand the relationship into the realization of the Sea Urchin Festival as a cultural and folk religious manifestation of Suape region. The relationships maintained by the population that inhabits the Suape Bay for generations is a complexity composed by relevant elements as historical, economic, social, cultural or environmental which are analyzed in this study. Through a quantitative and qualitative perspective, we seek to analyze the mode of production and reproduction of theirs lives, which are strongly attached with elements of the local nature, as well the marine territory category and with their cultural manifestations. They are facing the growing social changes resulted by the pressures coming from the enterprises and the development promises of the most emblematic economic policy megaproject of Pernambuco State, the Suape Port and Industrial Complex. We can say that the contemporary scenario showed to the Suape population, who are rooted into a rustic culture and traditional modal of life based on their small scale fishing practice, is a turning point, once some of them realize they are living one social and environmental conflict against the economic development machine, in the same time that others see each other in an uprooting process with their own territory. So there are some of the Sea Urchin Festival expressions that now just belong to the social and folk memory sphere, as well other ones get the drift, getting an uncertain way. / Esta dissertação intitulada Sustentabilidade na Baía de Suape: entre o Complexo Industrial Portuário de Suape e a Festa da Ouriçada busca compreender as relações que transpassam a realização da Festa da Ouriçada, enquanto manifestação da cultura e da religiosidade popular suapense. Estas relações protagonizadas pela população que habita a Baía de Suape há gerações são compostas pela complexidade de sua pertinência seja histórica, econômica, social, cultural ou ambiental, as quais são analisadas na presente pesquisa. Através de uma abordagem quantitativa e qualitativa, buscou-se a análise dos fortes vínculos de seu modo de produção e reprodução de vida com os elementos naturais locais, com a categoria territorialidade marinha e com suas manifestações culturais face às constantes transformações sociais provenientes das pressões exercidas por empreendimentos e pelas promessas de desenvolvimento do megaprojeto mais emblemático da política econômica pernambucana, o Complexo Industrial Portuário Governador Eraldo Gueiros de Suape (Cips). Pode-se considerar que o cenário posto na contemporaneidade para a população suapense enraizada no modelo de cultura rústica e tradicional de sua prática da pesca artesanal é um ponto de mutação, já que alguns se percebem vivendo uma situação de conflito socioambiental diante dos avanços das máquinas do desenvolvimento econômico, enquanto outros se veem em processo de desenlace com sua territorialidade. Na mesma direção, algumas manifestações da Festa da Ouriçada agora só pertencem à memória social de seu povo, enquanto sua prática fica à deriva, tomando um caminho, no mínimo, incerto.

O aquecimento global é uma realidade e seus efeitos são bastante estudados atualmente. No entanto, pouca atenção tem-se dado para as alterações que ocorrem com invertebrados marinhos em decorrência dessa alteração climática. Sabe-se que uma das alterações que ocorrem em consequência do aumento da temperatura da água do mar, é o aumento do aparecimento de doenças no ambiente marinho. Para tanto, é importante que se avalie sua resposta imune inata frente a esse fator estressor. Os ouriços-do-mar foram escolhidos como modelo por serem considerados bons biondicadores ambientais, além de serem filogeneticamente próximos aos cordados, partilhando com estes, amplo repertório de genes e receptores envolvidos no sistema imune inato. O presente trabalho avaliou a resposta imune inata de ouriços-do-mar antárticos (S. neumayeri) e tropicais (L. variegatus e E. lucunter) frente ao estresse térmico por diferentes temperaturas e períodos de exposição. No que se refere aos ouriços-do-mar tropicais, foram estudadas duas espécies, uma que habita regiões menos submetidas a variações de marés (L. variegatus) e outra constantemente exposta a variações de maré (E. lucunter). Constatou-se uma diferença entre a resposta ao estresse térmico nas três espécies estudadas. A espécie antártica demonstrou alterações mais significativas de aumento na porcentagem de esferulócitos vermelhos (EV) e da capacidade fagocítica no período agudo de exposição (24h) quando submetidos a temperaturas intermediárias. Por outro lado, a espécie tropical E. lucunter apresentou alteração somente na porcentagem de EV no período de exposição crônico (7 e 14 dias) a temperaturas altas, enquanto que L. variegatus apresentou alteração neste tipo celular em todas as temperaturas experimentais e períodos avaliados, além de uma importante redução da capacidade fagocítica nos mesmos períodos que foi diretamente proporcional ao aumento da temperatura. Buscando compreender tais diferenças entre animais que habitam uma mesma região, verificou-se que a espécie L. variegatus apresentou alteração na adesão e espraiamento celular, no citoesqueleto de actina dos amebócitos fagocíticos (AF), na migração celular sem adição de substância quimiotática e na resposta quimiotática frente a leveduras S. cerevisiae, além de diminuição do estresse oxidativo em elevadas temperaturas. Constatou-se também que os AF desta espécie apresentaram alterações morfométricas importantes, como diminuição na circunferência celular e alteração na área de espraiamento. Por outro lado, a espécie E. lucunter não apresentou alteração em nenhum parâmetro analisado. Conclui-se, assim, que o estresse térmico atua de maneiras diferentes em espécies polares e tropicais de ouriços-do-mar. No entanto, os mecanismos moleculares pelos quais a espécie E. lucunter apresenta maior termotolerância em comparação à espécie L. variegatus permanecem imcompreendidos e merecem ser estudados. / Global warming is a reality and its effects are widely studied today. However, little attention has been given to the changes that occur to marine invertebrates due to this climate change. It is known that one of the changes that occur as a result of the seawater temperature rise is the increased occurrence of diseases in the marine environment. Therefore, it is important to evaluate the innate immune response against this stressor. The sea urchins were chosen as a model for being considered good environmental bioindicators, and are phylogenetically close to chordates, sharing with them large repertoire of genes and receptors involved in the innate immune system. This study evaluated the innate immune response of Antarctic (S. neumayeri) and tropical (L. variegatus and E. lucunter) sea urchins against thermal stress by different temperatures and exposure periods. With regard to tropical sea urchins, two species, one that inhabits sites less subject to tidal variations (L. variegatus) and the other constantly exposed to tidal oscilations (E. lucunter) were studied. A difference between the response to thermal stress in the three species was observed. The Antarctic species showed more significant changes regarding increase of the percentage of red spherule cells and phagocytic capacity in acute exposure period (24 h) when subjected to mild temperatures. On the other hand, tropical species E. lucunter presented only change in the percentage of red spherule cells during chronic exposure (7 and 14 days) at extreme temperatures, while L. variegatus showed a change in this cell type in all experimental temperatures and exposure periods evaluated, as well as a significant reduction in phagocytic capacity in the same periods that was directly proportional to the temperature increase. Trying to understand the differences between animals that inhabit the same region , it was found that the species L. variegatus showed changes in cell adhesion and spreading, actin cytoskeleton of phagocytic amoebocytes, cell migration without the addition of chemotactic substance and chemotactic response against yeasts S. cerevisiae, besides a reduction of oxidative stress at elevated temperatures. It was also found that the phagocytic amoebocyte of this species showed significant morphological changes, such as reduction in cell circunference and change in the area of spreading. On the other hand, the species E. lucunter presented no change in any parameter analyzed. Thus, it is concluded that the thermal stress acts differently in polar and tropical species of sea urchins. However, the molecular mechanisms by which the species E. lucunter shows higher thermotolerance compared to the species L. variegatus remain obscure and deserve to be studied.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Echinodermata are marine-exclusive animals found in various habitat types and depth.Sea urchins have great relevance in this group because of important control exerted on algal community and reef bioerosion. Population of sea urchins has been experiencing variances on its natural density, thus impacting the marine biodiversity and testing habitats resilience. Anthropogenic or natural aspects are the main drivers of this event once observed only in temperate areas, but now noticed worldwide. As a result of such rapid expansion and new migratory routes, the genetic characteristics of these species might undergo changes in heterozigosity levels as well as in gene flow rates amongst other subpopulations geographically distant. Molecular markers can provide the rates of genetic variability in animals, showing their patterns of diversification, origin and migratory movements. This project analyzed the genetic characteristics of the white sea urchin populations in Fernando de Noronha and Bahia using mtDNA citocrome oxidase 1 from 89 samples, aiming to evaluate the gene flow within Brazilian populations and those spread in the Atlantic ocean. The results showed that sea urchins from F. Noronha form a group genetically diverse and, regardless its recent population expansion haplotype diversity suggests these organisms are not the result of a massive population migration from other areas. The haplotype network well linked with the population from Bahia is a result of continuous gene flow between these groups, taking a continentisland direction and the likely migration rates through the North Equatorial undercurrent. The frequency of unique haplotypes in the island population demonstrates high heterozigosity, and low divergence rates in comparison with Bahia population. / Os Echinodermata sÃo animais exclusivamente marinhos encontrados nos mais diversos tipos de habitat e profundidades. Nesse grupo se destacam os ouriÃos-do-mar por seu papel fundamental no controle da comunidade de algas e bioerosÃo do substrato rÃgido. Suas populaÃÃes vem comumente sofrendo variaÃÃes em sua densidade natural, impactando a biodiversidade marinha e colocando em prova a resiliÃncia dos habitats afetados. Fatores antrÃpicos ou naturais sÃo os principais responsÃveis por esse fenÃmeno antes conhecido apenas em Ãreas temperadas, mas jà observado em latitudes tropicais. Com a expansÃo populacional repentina e novas rotas migratÃrias, as caracterÃsticas genÃticas da espÃcie podem sofrer variaÃÃes na heterozigosidade e no fluxo genÃtico entre subpopulaÃÃes geograficamente separadas. Marcadores moleculares podem revelar a variabilidade genÃtica entre populaÃÃes, demonstrando os padrÃes de diversificaÃÃo de espÃcies, ancestralidade e movimentos migratÃrios. Esse trabalho analisou as caracterÃsticas genÃticas da populaÃÃo de ouriÃo-branco em Fernando de Noronha e Bahia atravÃs seqÃenciamento parcial do mtDNA, gene da citocromo oxidase 1 de 89 indivÃduos, com a finalidade de avaliar o fluxo gÃnico entre as populaÃÃes brasileiras e suas relaÃÃes com as demais existentes no AtlÃntico. Os resultados obtidos mostraram que os ouriÃos-brancos do arquipÃlago formam um grupo com caracterÃsticas genÃticas especÃficas e, embora haja ocorrido expansÃo da populaÃÃo, as modificaÃÃes haplotÃpicas observadas revelam que esses animais nÃo sÃo provenientes de uma migraÃÃo em massa de uma populaÃÃo. O compartilhamento de haplÃtipos com ouriÃos da Bahia à resultado de um fluxo gÃnico contÃnuo, na direÃÃo continente-ilha, e que a possÃvel rota migratÃria se daria pela contracorrente Equatorial Norte. A freqÃÃncia de haplÃtipos especÃficos da ilha demonstra alta heterozigosidade da populaÃÃo e baixa divergÃncia com relaÃÃo à populaÃÃo da Bahia.

<p>Specification and differentiation of a cell is accomplished by changing its gene expression profiles. These processes require temporally and spatially regulated transcription factors (TFs), to induce the genes that are necessary to a specific cell type. In each cell a set of TFs interact with each other or activate their targets; as development progresses, transcription factors receive regulatory inputs from other TFs and a complex gene regulatory network (GRN) is generated. Adding complexity, each TF can be regulated not only at the transcriptional level, but also by translational, and post-translational mechanisms. Thus, understanding a developmental process requires understanding the interactions between TFs, signaling molecules and target genes which establish the GRN.</p><p>In this thesis, two genes, FoxN2/3, a TF and FGFR1, a component of the FGF signaling pathway are investigated. FoxN2/3 and FGFR1 have different mechanisms that function in sea urchin development; FoxN2/3 regulates gene expression and FGFR1 changes phosphorylation of target proteins. However, their ultimate goals are the same: changing the state of an earlier GRN into the next GRN state. </p><p>First, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN. Expression of foxN2/3 begins in the descendants of micromeres at the early blastula stage; and then is lost from PMCs at the mesenchyme blastula stage. foxN2/3 expression then shifts to the secondary mesenchyme cells (SMCs) and later to the endoderm. Here we show that, Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in the descendants of micromeres. The later endomesoderm expression is independent of the earlier expression of FoxN2/3 in micromeres and independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of larval skeleton. A number of proteins are necessary for skeletogenesis, and early expression of at least several of these is dependent on FoxN2/3. Furthermore, knockdown (KD) of FoxN2/3 inhibits normal PMC ingression. PMCs lacking FoxN2/3 protein are unable to join the skeletogenic syncytium and they fail to repress the transfating of SMCs into the skeletogenic lineage. Thus, FoxN2/3 must be present for the PMC GRN to control normal ingression, expression of skeletal matrix genes, prevention of transfating, and control fusion of the PMC syncytium.</p><p>Second, we show that the FGF-FGFR1 signaling is required for the oral-aboral axis formation in the sea urchin embryos. Without FGFR1, nodal is induced in all of the cells at the early blastula stage and this ectopic expression of nodal requires active p38 MAP kinase. The loss of oral restriction of nodal expression results in the abnormal organization of PMCs and the larval skeleton; it also induces ectopic expression of oral-specific genes and represses aboral-specific genes. The abnormal oral-aboral axis formation also affected fgf and vegf expression patterns; normally these factors are expressed in two restricted areas of the ectoderm between the oral and the aboral side, but when FGFR1 is knocked down, Nodal expands, and in response the expression of the FGF and VEGF ligands expands, and this in turn affects the abnormal organization of larval skeleton.</p> / Dissertation

The role that signaling molecules play during morphogenesis and their interactions is a field of intense study and the sea urchin represents a facile system to study these aspects of development in the early embryo. In many instances, the S. purpuratus genome contains relatively simple receptor-ligand signaling systems compared to vertebrate counterparts and this provides interesting opportunities to study their diversity of function during the morphogenetic events that shape the embryo. The Eph-Ephrin signaling components are an excellent example of this and they are represented by dozens of members in the vertebrate system with developmental functions that include axon guidance, cell migration and tissue segregation. In contrast, the sea urchin genome contains a single Eph receptor and a single Ephrin ligand and by interacting with different effectors of signal transduction, this simple, bipartite system can fulfill a variety of functional roles during morphogenesis. Studying the function of Eph-Ephrin signaling in the sea urchin embryo, I have revealed two distinct morphogenetic movements in which Eph-Ephrin signaling is necessary; apical constriction of ciliary band cells and pigment cell migration. In both examples, a functionally relevant Ephrin gradient establishes spatial information in the developing tissues, producing a reaction from cells expressing the Eph receptor. In the case of pigment cells, the distribution of migrating cells is affected and in the case of ciliary band cells, apical constriction occurs. The different outcomes of Eph-Ephrin signaling in these two tissues exemplifies signaling components communicating spatial information and initiating morphogenetic programs with outcomes dependent on cellular context. Furthermore, I have identified downstream components of Eph-Ephrin signaling that have necessary functions in both models, illustrating how different cellular programs can be induced by the same signaling iii iv components. My research contributes to understanding fundamental aspects of how complex 3 dimensional tissues arise from the genes and regulatory elements encoded in metazoan genomes. / Graduate

<p>Embryonic development occurs with precisely timed morphogenetic cell movements directed by complex gene regulation. In this orchestrated series of events, some epithelial cells undergo extensive changes to become free moving mesenchymal cells. The transformation resulting in an epithelial cell becoming mesenchymal is called an epithelial to mesenchymal transition (EMT), a dramatic cell biological change that occurs throughout development, tissue repair, and disease. Extensive <italic>in vitro</italic> research has identified many EMT regulators. However, most <italic>in vitro</italic> studies often reduce the complicated phenotypic change to a binary choice between successful and failed EMT. Research utilizing models has generally been limited to a single aspect of EMT without considering the total transformation. Fully understanding EMT requires experiments that perturb the system via multiple channels and observe several individual components from the series of cellular changes, which together make a successful EMT.</p><p>In this study, we have taken a novel approach to understand how the sea urchin embryo coordinates an EMT. We use systems level methods to describe the dynamics of EMT by directly observing phenotypic changes created by shifting transcriptional network states over the course of primary mesenchyme cell (PMC) ingression, a classic example of developmental EMT. We systematically knocked down each transcription factor in the sea urchin's PMC gene regulatory network (GRN). In the first assay, one fluorescently labeled knockdown PMC precursor was transplanted onto an unperturbed host embryo and we observed the resulting phenotype <italic>in vivo</italic> from before ingression until two hours post ingression using time-lapse fluorescent microscopy. Movies were projected for computational analyses of several phenotypic changes relevant to EMT: apical constriction, apical basal polarity, motility, and de-adhesion. </p><p>A separate assay scored each transcription factor for its requirement in basement membrane invasion during EMT. Again, each transcription factor was knocked down one by one and embryos were immuno-stained for laminin, a major component of basement membrane, and scored on the presence or absence of a laminin hole at the presumptive entry site of ingression. </p><p>The measured results of both assays were subjected to rigorous unsupervised data analyses: principal component analysis, emergent self-organizing map data mining, and hierarchical clustering. This analytical approach objectively compared the various phenotypes that resulted from each knockdown. In most cases, perturbation of any one transcription factor resulted in a unique phenotype that shared characteristics with its upstream regulators and downstream targets. For example, Erg is a known regulator of both Hex and FoxN2/3 and all three shared a motility phenotype; additionally, Hex and Erg both regulated apical constriction but Hex additionally affected invasion and FoxN2/3 was the lone regulator of cell polarity. Measured phenotypic changes in conjunction with known GRN relationships were used to construct five unique subcircuits of the GRN that described how dynamic regulatory network states control five individual components of EMT: apical constriction, apical basal polarity, motility, de-adhesion, and invasion. The five subcircuits were built on top of the GRN and integrated existing fate specification control with the morphogenetic EMT control.</p><p>Early in the EMT study, we discovered one PMC gene, Erg, was alternatively spliced. We identified 22 splice variants of Erg that are expressed during ingression. Our Erg knockdown targeted the 5'UTR, present in all spliceoforms; therefore, the knockdown uniformly perturbed all native Erg transcripts (&#8721;Erg). Specific function was demonstrated for the two most abundant spliceoforms, Erg-0 and Erg-4, by knockdown of &#8721;Erg and mRNA rescue with a single spliceoform; the mRNA expression constructs contained no 5'UTR and were not affected by the knockdown. Different molecular phenotypes were observed, and both spliceoforms targeted Tbr, Tel, and FoxO, only Erg-0 targeted FoxN2/3 and only Erg-4 targeted Hex. Neither targeted Tgif, which was regulated by &#8721;Erg knockdown sans rescue. Our results suggest the embryo employs a minimum of three unique roles in the GRN for alternative splicing of Erg. </p><p>Overall, these experiments increase the completeness and descriptive power of the GRN with two additional levels of complexity. We uncovered five sub-circuits of EMT control, which integrated into the GRN provide a novel view of how a complex morphogenetic movement is controlled by the embryo. We also described a new functional role for alternative splicing in the GRN where the transcriptional targets for two splice variants of Erg are unique subsets of the total set of &#8721;Erg targets.</p> / Dissertation

The gastrula stage represents the point in development at which the three primary germ layers diverge. At this point the gene regulatory networks that specify the germ layers are established and the genes that define the differentiated states of the tissues have begun to be activated. These networks have been well characterized in sea urchins, but not in other echinoderms. Embryos of the brittle star Ophiocoma wendtii share a number of developmental features with sea urchin embryos, including the ingression of mesenchyme cells that give rise to an embryonic skeleton. Notable differences are that no micromeres are formed during cleavage divisions and no pigment cells are formed during development to the pluteus larva stage. More subtle changes in timing of developmental events also occur. To explore the molecular basis for the similarities and differences between these two echinoderms, the gastrula transcriptome of Ophiocoma wendtii was sequenced and characterized. I identified brittle star transcripts that correspond to 3385 genes in existing databases, including 1863 genes shared with the sea urchin Strongylocentrotus purpuratus gastrula transcriptome. I have characterized the functional classes of genes present in the transcriptome and compared them to those found in sea urchin. I then examined which members of the germ-layer specific gene regulatory networks (GRNs) of S. purpuratus are expressed in the O. wendtii gastrula. The results indicate that there is a shared "genetic toolkit" central to the echinoderm gastrula, a key stage in embryonic development, though there are also differences that reflect changes in developmental processes. The brittle star expresses genes representing all functional classes at the gastrula stage. Brittle stars and sea urchins have comparable numbers of each class of genes, and share many of the genes expressed at gastrula. Examination of the brittle star genes whose sea urchin orthologs are utilized in germ layer specification reveals a relatively higher level of conservation of key regulatory components compared to the overall transcriptome. I also identify genes that were either lost or whose temporal expression has diverged from that of sea urchins. Overall, the data suggest that embryonic skeleton formation in sea urchins and brittle stars represents convergent evolution by independent cooptation of a shared pathway utilized in adult skeleton formation. Transcription factors are of central importance to both development and evolution. Patterns of their expression and interactions form the gene regulatory networks which control the building of the embryonic body. Alterations in these patterns can result in the construction of altered bodies. To help increase understanding of this process, I compared the transcription factor mRNAs present in early gastrula-stage embryos of the brittle star Ophiocoma wendtii to those found in two species of sea urchins and a starfish. Brittle star homologs were found for one third of the transcription factors in the sea urchin genome and half of those that are expressed at equivalent developmental stages in sea urchins and starfish. Overall, the patterns of transcription factors found and not found in brittle star resemble those of other echinoderms, with the differences largely consistent with morphological differences. This study provides further evidence for the existence of deeply conserved developmental genetic processes, with various elements shared among echinoderms, deuterostomes, and metazoans.

microRNAs (miRNAs) are small (20-23 nt), non-coding single stranded RNA molecules that play an important role in post-transcriptional regulation of protein-coding genes. miRNAs have been found in all animal lineages, and have been implicated as critical regulators during development in multiple species. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development due to their well-characterized transcriptional gene networks, ease of working with their embryos in the laboratory and phylogenetic position as invertebrate deuterostomes. Literature on miRNAs in echinoderm embryogenesis is limited. It has been shown that RNAi genes are developmentally expressed and regulated in sea urchin embryos, but no study in the sea urchin has examined the expression of miRNAs. The goal of my work has been to study miRNA regulation in echinoderm developmental gene networks. I have identified developmentally regulated miRNAs in sea urchin and sea star embryos, using a combination of computational and wet lab experimental techniques. I developed a probabilistic model (named HHMMiR) based on hierarchical hidden Markov models (HHMMs) to classify genomic hairpins into miRNA precursors and random stem-loop structures. I then extended this model to make an efficient decoder by introduction of explicit state duration densities. We used the Illumina Genome Analyzer to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of S. purpuratus and P. miniata. We developed a computational pipeline for analysis of these miRNAseq data to reveal the miRNA populations in both species, and study their differential expression. We also used northern blots and whole mount in situ hybridization experimental techniques to study the temporal and spatial expression patterns of some of these miRNAs in sea urchin embryos. By knocking down the major components of the miRNA biogenesis pathway, we studied the global effects of miRNAs on embryo morphology and differentiation genes. The biogenesis genes selected for this purpose are the RNAse III enzyme, Dicer and Argonaute. Dicer is necessary for the processing of mature miRNAs from hairpin structures while Ago is a necessary part of the RISC (RNA interference silencing complex) assembly, which is required for the miRNA to hybridize to its target mRNA site. Knocking down these genes hinders normal development of the sea urchin embryo and leads to loss of the larval skeleton, a novel phenotype not seen in sea stars, as well as abnormal gastrulation. Comparison of differentiation gene marker expression between control and Ago knocked down sea urchin embryos shows interesting patterns of expansion and suppression of adjoining some embryonic territories, while ingression of larval skeletogenesis progenitors does not occur.

Integrins are heterodimeric transmembrane receptors composed of an α and a β subunit, that are expressed on the surface of all metazoan cells. These bidirectional signaling molecules are involved in many well-known aspects of cell function, although the role of integrins in early embryonic development remains a mystery. The purpose of this study was to characterize S. purpuratus integrins and determine if they are necessary for early embryonic development. Full length cDNA sequences for four incomplete gene predictions, αC, αD, αF, and βD, were determined by amplifying overlapping fragments and sequencing EST clones. Each cDNA has a single open reading frame predicting a protein with canonical integrin features. QPCR results show αC, αD, and βD are expressed in the embryo at relatively constant levels during the first 96 hours of development. αF is expressed in blastulae, during morphogenesis and tissue differentiation, at up to 35 times the levels of mRNA in the egg. Using a morpholino antisense oligonucleotide to block translation of αC results in a higher than normal mortality rate (57.1%) by 24 hours of development and 36.7% of embryos during this period have defects in aspects of cell division. These results indicate that αC is an essential gene for early development and that it may function in coordination of mitosis and cytokinesis. The expression of multiple subunits and the demonstration that αC has an essential role suggests that there are several non-overlapping functions for integrins in early embryonic development.

Functional and temporal-spatial studies of Netrin and its receptors have been reported in several species including, M. musculus, D. melanogaster and C. elegans. These studies indicate that Netrins are a family of evolutionarily conserved, secreted proteins that function to elicit the extension and turning responses of axons. Here, I describe the sequences for netrin and its receptors, unc5 and neogenin, in Strongylocentrotus purpuratus and show that the larval nervous system is patterned predictably with respect to cell body and axon location, early in its development. These findings led to a tentative hypothesis that Sp-Netrin functions to guide axonal growth in the larval nervous system. Quantitative PCR indicates that Sp-netrin and Sp-unc5 are expressed prior to neurogenesis, whereas Sp-neogenin is expressed close to the stage at which neurons differentiate. A polyclonal antibody to Sp-Netrin and in situ hybridizations reveal that Sp-Netrin is initially expressed in the vegetal plate, the archenteron and the protein is present on the basal surface of the oral ectoderm in early prism stage embryos. Suppression of Netrin expression, with a morpholino antisense oligonucleotide, results in loss of neurons, loss of ciliary band cells and loss of the oralectoderm markers, Chordin and Goosecoid. These findings suggest that Netrin is responsible for maintaining or differentiating oral and ciliary band ectoderm, which is necessary for neural specification or differentiation. Further study of this model is necessary to determine if Sp-Netrin retains a role in axon guidance.