high-throughput-microinjections-of-sea-urchin-zygotes

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

University of Delaware 

Prenylation is a post-translational protein modification process that results in correct protein localization to membranes in the cell. It is mediated by at least three prenyltransferases (PTFs) in eukaryotic cells. The RAM2 gene encodes the common alpha-subunit of two PTFs, farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I). In this study, the RAM2 gene of the prenylation pathway in the opportunistic pathogen Candida albicans was analysed. The heterozygote trisomy test was used to demonstrate that RAM2 is essential to the viability of C. albicans. RAM2 mRNA in the yeast and hyphae growth states was not detected by Northern blot analysis, but was detected by RT-PCR. Drugs that inhibit mammalian PTFs do not alter Candida cell growth, but they do inhibit FTase and GGTase I activities in cell-free enzyme assays. The data from genetic studies and cell-free enzyme assays suggest that the drugs may not have access to the Candida cytoplasm. The regulation of PTF subunits was also examined in a strain in which RAM2 was under the control of a regulable promoter. Overall, this study demonstrated that RAM2 is essential to C. albicans, indicating that protein prenylation is an indispensable cellular process in this yeast.

RAM2: an essential gene in the prenylation pathway of Candida albicans.

The sterol pathway in Candida albicans is the target for several classes of antifungal drugs. Intermediates in the sterol pathway are involved in ergosterol synthesis, prenylation and dolichol synthesis. This study examines gene expression of the sterol pathway in response to lovastatin, an inhibitor of HMG-CoA reductase (Hmg1p), and fluconazole, an inhibitor of 14 alpha-lanosterol demethylase (Erg11p). Minimum inhibitory concentration (MIC) studies indicated that lovastatin acts synergistically with fluconazole in vitro. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results indicated that genes in the early part of the sterol pathway, such as HMG1 and ERG20, did not alter expression in the presence of both lovastatin and fluconazole, whereas genes in the later part of the sterol pathway, such as ERG9 and ERG11, had increased expression in response to these drugs in mid-logarithmic growth. Genes involved in prenylation, such as RAM1 and RAM2, also respond to these drugs in mid-logarithmic growth, although another prenylation gene, CDC43, was not affected. After 24 h of growth, the relative expression of ERG20, ERG9, and ERG11 remained unchanged or increased in the presence of both drugs, while all other genes decreased in expression under all drug treatments.

Antifungal activity of fluconazole in combination with lovastatin and their effects on gene expression in the ergosterol and prenylation pathways in Candida albicans.

The azole antifungal drugs that target lanosterol 14-alpha-demethylase, encoded by the ERG11 gene, are used to treat a variety of infections caused by Candida albicans. Azoles are known to induce expression of ERG11 mRNA. The ERG11 promoter was cloned 5' of the luciferase-coding region, and the induction of ERG11 expression by azoles was monitored by luciferase assays. Maximal induction of the ERG11 promoter by azoles occurs not during logarithmic growth but after the diauxic shift and requires azoles to be present throughout logarithmic growth. The effects of pH, carbon source, and aerobic or anaerobic growth on induction of the ERG11 promoter by azoles were analyzed. Treatment with terbinafine and fenpropimorph, which target other enzymes in the ergosterol biosynthetic pathway, also resulted in a delayed induction of ERG11 promoter activity. Nascent sterol synthesis was shown to parallel ERG11 promoter activity, and total sterols were reduced coincident with the timing of ERG11 promoter activation. These results as a whole suggest that expression of the ERG11 promoter is regulated in response to sterol depletion.

The Candida albicans lanosterol 14-alpha-demethylase (ERG11) gene promoter is maximally induced after prolonged growth with antifungal drugs.

The oocyte is a highly differentiated cell. It makes organelles specialized to its unique functions and progresses through a series of developmental stages to acquire a fertilization competent phenotype. This review will integrate the biology of the oocyte with what is known about oocyte-specific gene regulation and transcription factors involved in oocyte development. We propose that oogenesis is reliant on a dynamic gene regulatory network that includes oocyte-specific transcriptional regulators.

How to make an egg: transcriptional regulation in oocytes.

Resistance of Candida albicans to azole antifungal drugs is mediated by two types of efflux pumps, encoded by the MDR1 gene and the CDR gene family. MDR1 mRNA levels in a susceptible clinical isolate are induced by benomyl (BEN) but not by other drugs previously shown to induce MDR1. To monitor MDR1 expression under several conditions, the MDR1 promoter was fused to the Renilla reniformis luciferase reporter gene (RLUC). The promoter was monitored for its responses to four oxidizing agents, five toxic hydrophobic compounds, and an alkylating agent, all shown to induce major facilitator pumps in other organisms. Deletion constructs of the MDR1 promoter were used to analyze the basal transcription of the promoter and its responses to the toxic compound BEN and the oxidizing agent tert-butyl hydrogen peroxide (T-BHP). The cis-acting elements in the MDR1 promoter responsible for induction by BEN were localized between -399 and -299 upstream of the start codon. The cis-acting elements responsible for MDR1 induction by T-BHP were localized between -601 and -500 upstream of the start codon. The T-BHP induction region contains a sequence that resembles the YAP1-responsive element (YRE) in Saccharomyces cerevisiae. This Candida YRE was placed upstream of a noninducible promoter in the luciferase construct, resulting in an inducible promoter. Inversion or mutation of the 7-bp YRE eliminated induction. Many of the drugs used in this analysis induce the MDR1 promoter at concentrations that inhibit cell growth. These analyses define cis-acting elements responsible for drug induction of the MDR1 promoter.

Drug-induced regulation of the MDR1 promoter in Candida albicans.

Oocytes express a unique set of genes that are essential for their growth, for meiotic recombination and division, for storage of nutrients, and for fertilization. We have utilized the newly sequenced genome of Strongylocentrotus purpuratus to identify genes that help the oocyte accomplish each of these tasks. This study emphasizes four classes of genes that are specialized for oocyte function: (1) Transcription factors: many of these factors are not significantly expressed in embryos, but are shared by other adult tissues, namely the ovary, testis, and gut. (2) Meiosis: A full set of meiotic genes is present in the sea urchin, including those involved in cohesion, in synaptonemal complex formation, and in meiotic recombination. (3) Yolk uptake and storage: Nutrient storage for use during early embryogenesis is essential to oocyte function in most animals; the sea urchin accomplishes this task by using the major yolk protein and a family of accessory proteins called YP30. Comparison of the YP30 family members across their conserved, tandem fasciclin domains with their intervening introns reveals an incongruence in the evolution of its major clades. (4) Fertilization: This set of genes includes many of the cell surface proteins involved in sperm interaction and in the physical block to polyspermy. The majority of these genes are active only in oocytes, and in many cases, their anatomy reflects the tandem repeating interaction domains essential for the function of these proteins. Together, the expression profile of these four gene classes highlights the transitions of the oocyte from a stem cell precursor, through stages of development, to the clearing and re-programming of gene expression necessary to transition from oocyte, to egg, to embryo.

Oogenesis: single cell development and differentiation.

Riportiamo la sequenza e l&#39;analisi del genoma 814-megabase di riccio di mare Strongylocentrotus purpuratus, un modello per lo sviluppo e la biologia dei sistemi. La strategia di sequenziamento combinato whole genome shotgun e sequenze di cromosomi artificiali batterici (BAC). Questo uso di cloni BAC, aiutato da una strategia di pooling, superato Difficolt&agrave; associate con elevata eterozigosi del genoma. Il genoma codifica circa 23.300 geni, tra cui molti pensava di essere vertebrate innovazioni o noto solo di fuori i deuterostomi. Questo genoma echinodermi fornisce un evolutivo outgroup per i cordati e rendimenti intuizioni l&#39;evoluzione dei deuterostomi.

Il genoma del riccio di mare Strongylocentrotus purpuratus.

&#x6211;&#x4EEC;&#x62A5;&#x544A;&#x7684;&#x987A;&#x5E8F;&#x548C;&#x6D77;&#x80C6;&#x6D77;&#x80C6; purpuratus&#xFF0C;&#x4E00;&#x79CD;&#x6A21;&#x578B;&#x7B2C; 814 &#x7ECF;&#x8FC7;&#x57FA;&#x56E0;&#x7EC4;&#x5206;&#x6790;&#x53D1;&#x5C55;&#x548C;&#x7CFB;&#x7EDF;&#x751F;&#x7269;&#x5B66;&#x3002;&#x6D4B;&#x5E8F;&#x6218;&#x7565;&#x8054;&#x5408;&#x5168;&#x57FA;&#x56E0;&#x7EC4;&#x9E1F;&#x67AA;&#x548C;&#x7EC6;&#x83CC;&#x4EBA;&#x5DE5;&#x67D3;&#x8272;&#x4F53; (BAC) &#x5E8F;&#x5217;&#x3002;&#x8FD9;&#x79CD;&#x5229;&#x7528;&#x6240;&#x6C47;&#x96C6;&#x7684;&#x7B56;&#x7565;&#xFF0C;&#x8F85;&#x52A9;&#x7684; BAC &#x514B;&#x9686;&#x514B;&#x670D;&#x4E86;&#x56F0;&#x96BE;&#x4E0E;&#x9AD8;&#x6742;&#x5408;&#x6027;&#x57FA;&#x56E0;&#x7EC4;&#x76F8;&#x5173;&#x8054;&#x3002;&#x57FA;&#x56E0;&#x7EC4;&#x7F16;&#x7801;&#x7EA6; 23,300 &#x7684;&#x57FA;&#x56E0;&#xFF0C;&#x5305;&#x62EC;&#x8BB8;&#x591A;&#x4EE5;&#x524D;&#x8BA4;&#x4E3A;&#x662F;&#x810A;&#x690E;&#x52A8;&#x7269;&#x521B;&#x65B0;&#x6216; deuterostomes &#x4EE5;&#x5916;&#x53EA;&#x77E5;&#x9053;&#x3002;&#x8FD9;&#x4E2A;&#x68D8;&#x76AE;&#x52A8;&#x7269;&#x57FA;&#x56E0;&#x7EC4;&#x63D0;&#x4F9B;&#x4E86;&#x8FDB;&#x5316; outgroup &#x5230; deuterostomes &#x7684;&#x6F14;&#x53D8;&#x810A;&#x7D22;&#x52A8;&#x7269;&#x548C;&#x6536;&#x76CA;&#x7387;&#x7684;&#x6DF1;&#x523B;&#x89C1;&#x89E3;&#x3002;

&#x6D77;&#x80C6;&#x6D77;&#x80C6; purpuratus &#x7684;&#x57FA;&#x56E0;&#x7EC4;&#x3002;

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

The genome of the sea urchin Strongylocentrotus purpuratus.

Nous rapportons la s&eacute;quence et l&#39;analyse du g&eacute;nome de l&#39;oursin Strongylocentrotus purpuratus, un mod&egrave;le pour 814-megabase d&eacute;veloppementale et biologie des syst&egrave;mes. La strat&eacute;gie de s&eacute;quen&ccedil;age combin&eacute; whole-genome shotgun et des s&eacute;quences des chromosomes artificiels bact&eacute;riens (BAC). Cette utilisation des clones BAC, aid&eacute; par une strat&eacute;gie de mise en commun, a surmont&eacute; les difficult&eacute;s associ&eacute;es &agrave; l&#39;h&eacute;t&eacute;rozygotie &eacute;lev&eacute; du g&eacute;nome. Le g&eacute;nome encode environ 23 300 g&egrave;nes, dont un grand nombre qu&#39;on croyait &ecirc;tre des innovations vert&eacute;br&eacute;es ou connu seulement en dehors des deut&eacute;rostomiens. Ce g&eacute;nome &eacute;chinoderme fournit un &eacute;volutionniste exogroupe pour les id&eacute;es chord&eacute;s et les rendements dans l&#39;&eacute;volution des deut&eacute;rostomiens.

Le g&eacute;nome de l&#39;oursin Strongylocentrotus purpuratus.

Wir berichten die Abfolge und die Analyse des Genoms 814-Megabase Seeigel Strongylocentrotus Purpuratus, ein Modell f&uuml;r Entwicklungsbiologie und Systembiologie. Die Sequenzierung-Strategie kombiniert ganz-Genoms Schrotflinte und bacterial artificial Chromosome (BAC) Sequenzen. Diese Verwendung von BAC Klone, unterst&uuml;tzt durch ein pooling-Strategie, &uuml;berwand die Schwierigkeiten im Zusammenhang mit hohen Heterozygosit&auml;t des Genoms. Das Genom kodiert rund 23.300 Gene, darunter viele, die vorher als Wirbeltiere Innovationen werden oder nur au&szlig;erhalb der Deuterostomiern bekannt. Diese Stachelh&auml;uter-Genom enth&auml;lt einen evolution&auml;ren Fremdgruppe f&uuml;r die Chordatiere und Renditen Einblicke in die Entwicklung der Deuterostomiern.

Das Genom der Seeigel Strongylocentrotus Purpuratus.

&#x6211;&#x3005; &#x306F;&#x3001;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3068;&#x30A6;&#x30CB; &#x30C8;&#x30E9;&#x30B6;&#x30E1; purpuratus &#x306E;&#x305F;&#x3081;&#x306E;&#x30E2;&#x30C7;&#x30EB;&#x306E; 814 &#x7D50;&#x679C;&#x30B2;&#x30CE;&#x30E0;&#x89E3;&#x6790;&#x5831;&#x544A;&#x767A;&#x9054;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x751F;&#x7269;&#x5B66;&#x3002;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306E;&#x6226;&#x7565;&#x306F;&#x3001;&#x5168;&#x30B2;&#x30CE;&#x30E0;&#x306E;&#x6563;&#x5F3E;&#x9283;&#x3068;&#x7D30;&#x83CC;&#x4EBA;&#x5DE5;&#x67D3;&#x8272;&#x4F53; &#xFF08;BAC &#x306E;&#xFF09; &#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x7D50;&#x5408;&#x3002;&#x3053;&#x306E;&#x4F7F;&#x7528;&#x30D7;&#x30FC;&#x30EA;&#x30F3;&#x30B0;&#x306E;&#x6226;&#x7565;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x652F;&#x63F4;&#x3001;BAC &#x30AF;&#x30ED;&#x30FC;&#x30F3;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x306E;&#x9AD8;&#x3044;&#x30D8;&#x30C6;&#x30ED;&#x63A5;&#x5408;&#x6027;&#x3068;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x308B;&#x96E3;&#x3057;&#x3055;&#x3092;&#x514B;&#x670D;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30B2;&#x30CE;&#x30E0;&#x4EE5;&#x524D;&#x810A;&#x690E;&#x52D5;&#x7269;&#x306E;&#x6280;&#x8853;&#x9769;&#x65B0;&#x3067;&#x3042;&#x308B;&#x8003;&#x3048;&#x307E;&#x305F;&#x306F;&#x306E;&#x307F;&#x3001;&#x5F8C;&#x53E3;&#x52D5;&#x7269;&#x5916;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x308B;&#x591A;&#x304F;&#x3092;&#x542B;&#x3080;&#x3001;&#x7D04; 23,300 &#x907A;&#x4F1D;&#x5B50;&#x3092;&#x30A8;&#x30F3;&#x30B3;&#x30FC;&#x30C9;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x306E;&#x68D8;&#x76AE;&#x52D5;&#x7269;&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x9032;&#x5316;&#x63D0;&#x4F9B;&#x5F8C;&#x53E3;&#x52D5;&#x7269;&#x306E;&#x9032;&#x5316;&#x306E;&#x810A;&#x7D22;&#x52D5;&#x7269;&#x3068;&#x5229;&#x56DE;&#x308A;&#x6D1E;&#x5BDF;&#x3092;&#x91CD;&#x8981;&#x3002;

&#x30A6;&#x30CB; &#x30C8;&#x30E9;&#x30B6;&#x30E1; purpuratus &#x306E;&#x30B2;&#x30CE;&#x30E0;&#x3002;

&#xC6B0;&#xB9AC;&#xB294; &#xC2DC;&#xD000;&#xC2A4; &#xBC0F; &#xC131; Strongylocentrotus purpuratus&#xC5D0; &#xB300; &#xD55C; &#xBAA8;&#xB378;&#xC758; 814 megabase &#xAC8C;&#xB188; &#xBD84;&#xC11D; &#xBCF4;&#xACE0;&#xC11C; &#xAC1C;&#xBC1C; &#xBC0F; &#xC2DC;&#xC2A4;&#xD15C; &#xC0DD;&#xBB3C;&#xD559;. &#xC2DC;&#xD000;&#xC2F1; &#xC804;&#xB7B5; &#xC804;&#xCCB4; &#xAC8C;&#xB188; &#xC0F7;&#xAC74; &#xD558; &#xACE0; &#xC138;&#xADE0; &#xC778;&#xACF5; &#xC5FC;&#xC0C9;&#xCCB4; (BAC) &#xC2DC;&#xD000;&#xC2A4; &#xACB0;&#xD569;. BAC &#xD074;&#xB860;, &#xD480;&#xB9C1; &#xC804;&#xB7B5;&#xC5D0; &#xC758;&#xD574; &#xC6D0;&#xC870;&#xC758;&#xC774; &#xC0AC;&#xC6A9;&#xC774; &#xAC8C;&#xB188;&#xC758; &#xB192;&#xC740; heterozygosity&#xC640; &#xAD00;&#xB828; &#xB41C; &#xC5B4;&#xB824;&#xC6C0;&#xC744; &#xADF9;&#xBCF5; &#xD588;&#xB2E4;. &#xAC8C;&#xB188;&#xC5D0;&#xB294; &#xC57D; 23,300 &#xC720;&#xC804;&#xC790;&#xB97C; &#xD3EC;&#xD568; &#xD55C; &#xB9CE;&#xC740; &#xC774;&#xC804;&#xC5D0; &#xCC99;&#xCD94; &#xD601;&#xC2E0; &#xC0DD;&#xAC01; &#xB098;&#xB294; deuterostomes &#xBC16;&#xC5D0; &#xC11C;&#xB9CC; &#xC54C;&#xB824;&#xC9C4; &#xC778;&#xCF54;&#xB529;&#xD569;&#xB2C8;&#xB2E4;. &#xADF9;&#xD53C;&#xB3D9;&#xBB3C; &#xAC8C;&#xB188;&#xC774; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xC9C4;&#xD654; outgroup chordates &#xBC0F; &#xC218;&#xC728; &#xD1B5;&#xCC30;&#xB825; deuterostomes&#xC758; &#xC9C4;&#xD654;&#xC5D0; &#xB300; &#xD55C;.

&#xBC14;&#xB2E4; &#xC131; &#xAC8C; Strongylocentrotus purpuratus&#xC758; &#xAC8C;&#xB188;&#xC785;&#xB2C8;&#xB2E4;.

Divulgamos la secuencia y el an&aacute;lisis del genoma 814-megabase del erizo de mar Strongylocentrotus purpuratus, un modelo de desarrollo y Biolog&iacute;a de sistemas. La estrategia de secuenciaci&oacute;n hab&iacute;a combinado escopeta todo el genoma y secuencias del cromosoma artificial bacteriano (BAC). Este uso de clones BAC, ayudado por una estrategia de agrupaci&oacute;n super&oacute; dificultades asociadas con el alto heterocigoto del genoma. El genoma codifica unos 23.300 genes, incluyendo muchos pensaba ser vertebradas innovaciones o conocida s&oacute;lo fuera los deuterostomes. Este genoma de equinodermos proporciona un evolutivo de grupos para las penetraciones de cordados y rendimientos en la evoluci&oacute;n de deuterostomes.

El genoma del erizo de mar Strongylocentrotus purpuratus.

RNA-mediated interference (RNAi) is a conserved gene silencing mechanism that involves double-stranded RNA as a signal to trigger the sequence-specific degradation of target mRNA, resulting in posttranscriptional silencing and/or translational repression. Bioinformatic searches in the sea urchin genome database identified homologs of Drosha, DGCR5, Dicer, TRBP, Exportin-5, and Argonautes. Quantitative, real-time polymerase chain reaction indicated that all mRNA accumulate in eggs and in variable levels throughout early development. Whole-mount in situ RNA hybridization showed that all of the important players of the RNAi silencing pathway have abundant mRNA accumulation in oocytes and eggs, but have distinct spatial and temporal expression patterns throughout development. Sequence analysis revealed that each of the four Argonautes examined contain conserved residues important for RNAseH activity within the Piwi domain. This study elucidated that genes involved in the RNAi silencing pathway have dynamic expression and, thus, may have regulatory roles during germ cell development and embryogenesis.

Genes involved in the RNA interference pathway are differentially expressed during sea urchin development.

The azole antifungal drugs are used to treat infections caused by Candida albicans and other fungi. These drugs interfere with the biosynthesis of ergosterol, the major sterol in fungal cells, by inhibiting an ergosterol biosynthetic enzyme, lanosterol 14 alpha-demethylase, encoded by the ERG11 gene. In vitro, these drugs as well as other ergosterol biosynthesis inhibitors increase ERG11 mRNA expression by activation of the ERG11 promoter. The signal for this activation most likely is the depletion of ergosterol, the end product of the pathway. To identify cis-acting regulatory elements that mediate this activation, ERG11 promoter fragments have been fused to the luciferase reporter gene from Renilla reniformis. Promoter deletions and linker scan mutations localized the region important for azole induction to a segment from bp -224 to -251 upstream of the start codon, specifically two 7-bp sequences separated by 13 bp. These sequences form an imperfect inverted repeat. The region is recognized by the transcription factor Upc2p and functions as an enhancer of transcription, as it can be placed upstream of a heterologous promoter in either direction, resulting in the azole induction of that promoter. The promoter constructs are not azole inducible in the upc2/upc2 homozygous deletion, demonstrating that Upc2p controls the azole induction of ERG11. These results identify an azole-responsive enhancer element (ARE) in the ERG11 promoter that is controlled by the Upc2p transcription factor. No other ARE is present in the promoter. Thus, this ARE and Upc2p are necessary and sufficient for azole induction of ERG11.

cis-Acting elements within the Candida albicans ERG11 promoter mediate the azole response through transcription factor Upc2p.

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.

Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development.

microRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene regulation and have emerged as essential regulators of many developmental events. The transcriptional network during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus, is well described and can serve as an excellent model to test functional contributions of miRNAs in embryogenesis. We examined the loss of function phenotypes of major components of the miRNA biogenesis pathway. Inhibition of de novo synthesis of Drosha and Dicer in the embryo led to consistent developmental defects, a failure to gastrulate, and embryonic lethality, including changes in the steady state levels of transcription factors and signaling molecules involved in germ layer specification. We annotated and profiled small RNA expression from the ovary and several early embryonic stages by deep sequencing followed by computational analysis. miRNAs as well as a large population of putative piRNAs (piwi-interacting RNAs) had dynamic accumulation profiles through early development. Defects in morphogenesis caused by loss of Drosha could be rescued with four miRNAs. Taken together our results indicate that post-transcriptional gene regulation directed by miRNAs is functionally important for early embryogenesis and is an integral part of the early embryonic gene regulatory network in S. purpuratus.

Select microRNAs are essential for early development in the sea urchin.

FoxY is a member of the forkhead transcription factor family that appeared enriched in the presumptive germ line of sea urchins (Ransick et al. Dev Biol 2002;246:132). Here, we test the hypothesis that FoxY is involved in germ line determination in this animal. We found two splice forms of FoxY that share the same DNA-binding domain, but vary in the carboxy-terminal trans-activation/repression domain. Both forms of the FoxY protein are present in the egg and in the early embryo, and their mRNAs accumulate to their highest levels in the small micromeres and adjacent non-skeletogenic mesoderm. Knockdown of FoxY resulted in a dramatic decrease in Nanos mRNA and protein levels as well as a loss of coelomic pouches in 2-week-old larvae. Our results indicate that FoxY positively regulates Nanos at the transcriptional level and is essential for reproductive potential in this organism.

The forkhead transcription factor FoxY regulates Nanos.

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells during embryogenesis. Three nanos homologs are present in the genome of the sea urchin Strongylocentrotus purpuratus (Sp), and each nanos mRNA accumulates specifically in the small micromere (sMic) lineage. We found that a highly conserved element in the 3' UTR of nanos2 is sufficient for reporter expression selectively in the sMic lineage: microinjection into a Sp fertilized egg of an RNA that contains the GFP open reading frame followed by Sp nanos2 3'UTR leads to selective reporter enrichment in the small micromeres in blastulae. The same result was seen with nanos2 from the sea urchin Hemicentrotus pulcherrimus (Hp). In both species, the 5'UTR alone is not sufficient for the sMic localization but it always increased the sMic reporter enrichment when present with the 3'UTR. We defined an element conserved between Hp and Sp in the nanos2 3'UTR which is necessary and sufficient for protein enrichment in the sMic, and refer to it as GNARLE (Global Nanos Associated RNA Lability Element). We also found that the nanos2 3'UTR is essential for the selective RNA retention in the small micromeres; GNARLE is required but not sufficient for this process. These results show that a combination of selective RNA retention and translational control mechanisms instills nanos accumulation uniquely in the sMic lineage.

The 3'UTR of nanos2 directs enrichment in the germ cell lineage of the sea urchin.

Background: Piwi proteins are essential for germ line development, stem cell maintenance, and more recently found to function in epigenetic and somatic gene regulation. In the sea urchin Strongylocentrotus purpuratus, two Piwi proteins, Seawi and Piwi-like1, have been identified, yet their functional contributions have not been reported. Result: Here we found that Seawi protein was localized uniformly in the early embryo and then became enriched in the primordial germ cells (PGCs) (the small micromere lineage) from blastula stage and thereafter. Morpholino knockdown of Sp-seawi diminished PGC-specific localization of Seawi proteins, and altered expression of other germ line markers such as Vasa and Gustavus, but had no effect on Nanos. Further, Seawi knockdown transiently resulted in Vasa positive cell proliferation in the right coelomic pouch that appear to be derived from the small micromere lineage, yet they quickly disappeared with an indication of apoptosis by larval stage. Severe Seawi knockdown resulted in an increased number of apoptotic cells in the entire gut area. Piwi proteins appear to regulate PGC proliferation perhaps through control of Vasa accumulation. In this organism, Piwi is likely regulating mRNAs, not just transposons, and is potentially functioning both inside and outside of the germ line during embryogenesis. Developmental Dynamics, 2013. © 2013 Wiley Periodicals, Inc.

Jia L. Song

Department of Biological Sciences

University of Delaware

High Throughput Microinjections of Sea Urchin Zygotes

Jia L. Song

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

University of Delaware

High Throughput Microinjections of Sea Urchin Zygotes

Department of Biological Sciences