Marine dissolved organic matter (DOM) comprises one of the largest carbon reservoirs on earth and has long been considered a potential energy source for marine invertebrates. The importance of DOM transport has been adequately demonstrated for unicellular organisms, where DOM can meet 100% of an organisms energy needs, but the effects of DOM uptake for marine metazoans are less well understood. In this study, three general areas involving the influence of DOM transport to marine invertebrates were explored. First, we assessed the effects of using seawater exposed to high intensity ultraviolet radiation (UVR) on the study organism; embryos of the purple sea urchin, Strongylocentrotus purpuratus. This was important because we used seawater treated in this way to create water types used in the experiments. Exposing seawater to high intensity UVR oxidizes (and functionally removes) DOM in the seawater. Second, the influence of the presence of DOM on the timing of embryonic development was examined for embryos of S. purpuratus. Specifically, the time of cell division and the time of hatching were determined for embryos in seawater with and without DOM. Finally, the ability of DOM to moderate the negative effect of UV-exposure on time of cell division was assessed. To make these comparisons experiments were performed using three water types: FSW (0.22 micron filtered seawater), DOM-depleted seawater (UV oxidized 0.22 micron filtered seawater), and DOM-enriched seawater (UV oxidized 0.22 micron filtered seawater enriched with labile DOM). In the first experiment, batches of embryos in the three water types were either exposed or not exposed to ultra-violet radiation and the time of first cell division was compared for embryos across the six treatments. In the second experiment, batches of embryos were placed in the same three water types and the time of first cell division and the time of hatching were quantified. From these experiments several results were generated. First, seawater exposed to high intensity UVR did not influence the timing of development of embryos of S. purpuratus. Embryos in water exposed to high intensity UVR (DOM-enriched and DOM-depleted seawater) hatched at similar times and completed first cell division at times similar to embryos in water not exposed to high intensity UVR (FSW). Next, we found that the influence of the presence of DOM on the development timing of S. purpuratus embryos depended on the event that was examined. The time of first cell division was not affected by the presence of DOM but the time of hatching was. Embryos in water with dissolved organic matter hatched on average 86 minutes later than embryos in water without DOM. Potentially, embryos in seawater without DOM speed up development to more quickly reach the point that they can feed on particulates. Lastly, the presence of DOM did not influence UVR-induced cleavage delay. The percent cleavage delay was not significantly different for embryos in seawater with (DOM-enriched) and without (DOM-depleted) DOM. In addition to the experiments, all studies in the literature that examine the realized effects of DOM transport were analyzed to ascertain when the manifestation of DOM uptake is most likely to occur. From these results, it appears that the effects of DOM transport are most likely to manifest after the life stage in which the majority of uptake occurred. If DOM transport has an affect within a life stage it is most likely to manifest as moderation of biomass loss or maintenance of endogenous reserves. With the addition of the experimental results from this study to the information already in the literature we begin to more fully grasp the importance of DOM transport to S. purpuratus. DOM influences the time of hatching, biomass, arm length, and stomach size of the species; results that highlight the importance of examining multiple affects of DOM transport for a single species. In conclusion, future research should look for multiple effects of the presence of DOM both within and across life stages (for a single species) to better understand the importance of DOM to marine invertebrates.

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.

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.