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.