Xenopus Zygote Arrest 2 (Xzar2) binds to the TCS in the 3′ UTRs ofthe key cell cycle mRNAs, Mos and Wee1. Dominant inhibitory Xzar2also attenuates the accumulation of Mos and Wee1 proteins duringmeiotic maturation of Xenopus oocytes. We propose that one role ofthe Zar proteins in early development may be to regulate thesynthesis of maternal cell cycle proteins in the maturing oocyte inanticipation of their roles in fertilization and embryogenesis.

doi:10.1016/j.ydbio.2011.05.523

Program/Abstract # 559Asian Sand Dust (ASD)—Particle Matter (PM) effect on overexpressof tissue Transglutaminase2You-Jin Hwanga, Gunhyun Parkb, Sung-Hun Baea, Myung-Jin Kima, Ji-Sun Kima, Jae-Hee Yoona, Dae-Young Kima

aGachon University of Medicine and Science, Incheon, Republic of KoreabGachon University of Medicine and Science Division of BiologicalScience, Incheon, Republic of Korea

During springtime in the East Asia, Asian Sand Dust (ASD)—Particular Matter (ASD-PM) from China and Mongolia desert areasover to East Asia on the westerlies and is generally thought tothreaten the East Asian health by provoking respiratory illness likebronchitis and asthma and conjunctivitis. And tissue Transglutami-nase (tTG) are enzymes that are widely used in biological systemsand can contribute to various pathophysiologies. tTG participates inposttranslational modification reactions and affect to blood coagula-tion, skin barrier formation, inflammatory, autoimmune and tissuerepair. In this study, we examined how ASD-PM associates lungfibrosis and hepatocyte. C57BL/6 mice were exposed to salinesuspensions of ASD particle 3 times a week for 4 weeks, 8 weeks,and 12 weeks. Following exposure with ASD, the liver was analyzedby immunochemistry using hematoxylin and eosin (H&E) andMasson's trichrome (MT) staining. We studied TransglutaminasemRNA (Tg mRNA) and tTG expression, using Real-Time PCR andWestern Blot in mice hepatocyte treated with ASD. Long termexposure to ASD showed significant collagen accumulation in theliver as compared with short term mice. And long term exposedsample also overexpress Tg mRNA and tTG in hepatocyte. As a result,ASD-PM accumulates collagen and causes overexpression of Tg mRNAand tTG. Our results suggest that if people or animals are exposed toASD, ASD will damage the lung and liver and result to fibrosis.

doi:10.1016/j.ydbio.2011.05.524

Program/Abstract # 560The expression of urokinase-type plasminogen activator isinduced in cultured mouse blastocyst by the highglucose concentrationAlejandra Sánchez-Santos, Alonso Vilches-Flores, María GuadalupeMartínez-Hernández, Alejandro Castillo-Trápala,Luis Arturo Baiza-GutmanFES Iztacala, UNAM, Tlalnepantla, Mexico

During embryo implantation in mammals, the blastocyst pene-trates the uterine wall at different depths by an invasive process,involving proteases that degrade the extracellular matrix (ECM),including matrix metalloproteinase 9 (MMP9) and urokinase-typeplasminogen activator (PLAU). Plasminogen is activated to plasmin byPLAU and plasmin degrades ECM and activates some MMPs, likeMMP-9. PLAU and MMP-9 are expressed in primary trophoblast cellsof mouse blastocyst in vivo and in vitro and they are secretedabundantly during embryo implantation. High concentration of

glucose affects the synthesis and degradation of the ECM in differentcell types, because it induces the formation of reactive oxygen species(ROS) that alter the expression of PLAU and MMPs. Therefore theeffect of glucose on the expression of PLAU in cultured mouseblastocysts was evaluated. Gestation fourth blastocysts were culturedin HAM-F-10, and high glucose 25 mM, was added in differentschedules, glucose 6 mM was used as a control, the expression ofPLAU was evaluated using real time RT-PCR and amidolytic assay.Glucose 25 mM inhibits hatching (−25%) and induces a higheractivity of PLAU in the conditioned medium and enhanced the level ofPLAU mRNA in embryo extracts obtained after four days of culture.Hydrogen peroxide (10 mM) induces similar increase in PLAU activityin the conditioned medium. High concentrations of glucose promoteoxidative stress, due to increased formation of ROS, which probablyincreased the expression of PLAU in trophoblast of mouse blastocysts.Supported by PAPIT, DGAPA, UNAM, grant IN230611.

doi:10.1016/j.ydbio.2011.05.525

Program/Abstract # 561Comprehensive survey and perturbation of the transcriptionalcontrol of Ptf1aEvanthia E. Pashosa, Shannon FisherbaUniversity of Pennsylvania Cell and Developmental Biology,Philadelphia, PA, USAbUniversity of Pennsylvania, Philadelphia, PA, USA

Pancreatic transcription factor 1a (Ptf1a) participates in the forma-tion of the ternary complex Ptf1, that has a critical role in pancreasspecification and is involved in cell fate choices within the pancreas andadditionally in the retina, cerebellum, hindbrain and dorsal spinal cord.The proximal promoter of mouse Ptf1a can partially recapitulatepancreatic expression; in addition, an autoregulatory element positivelyregulates Ptf1a expression in the pancreas, dorsal spinal cord andhindbrain, while a conserved immediate downstream area has dorsalspinal cord activity. Analysis of a spontaneous deletion of downstreamPtf1a non-coding sequence suggests that necessary regulatory elementsfor proper cerebellar and pancreatic development remain to beidentified. We sought to assess the role of Ptf1a autoregulation in atractable genetic system. As a first step towards this direction wecomprehensively characterized cis-regulatory elements tiling the entiresequence of a BAC that recapitulates endogenous expression inzebrafish. We discovered previously uncharacterized regulatory ele-ments with activity in the hindbrain, retina and spinal cord, andalso identified a zebrafish autoregulatory enhancer with comparableactivity to the known mouse enhancer. Using the BAC transgene thatcontains all necessary Ptf1a regulatory sequences, we mutated the Ptf1binding sites within the autoregulatory enhancer, thus perturbing theautoregulatory loop. We are currently testing the ability of the mutatedtransgene to rescue the Ptf1a null phenotype, to determine the role ofautoregulatory control in the dynamics of allocation between opposingcell fates and the stability of terminal differentiation.

doi:10.1016/j.ydbio.2011.05.526

Program/Abstract # 562Multiple cis-acting enhancers regulate temporal and spatialexpression of the human LHX3 gene in the developing pituitarySoyoung Park, Rachel Mullen, Simon RhodesIndiana Univ., Indianapolis, IN, USA

LHX3 is a LIM homeodomain transcription factor necessary forproper development of the pituitary and central nervous system.

Abstracts 267

Patients with mutations in coding regions of the LHX3 gene havecombined pituitary hormone deficiency and nervous system defectsresulting in symptoms such as dwarfism, thyroid insufficiency,infertility, and developmental delay. Although previous studies fromour group and others have identified promoter and intronic elementsof LHX3 that are important for basal gene expression in vitro, themechanisms by which the LHX3 gene is regulated in vivo were notknown. Using transgenic mouse models and bioinformatic ap-proaches, we have mapped conserved distal enhancer regions thatdirect tissue-specific expression to the pituitary gland and spinal cordin a pattern consistent with endogenous expression. Severaltransferable cis elements can individually guide nervous systemexpression; however, a 180 base pair minimal enhancer is alonesufficient to confer specific expression in the pituitary and spinalcord. Within this sequence, tandem binding sites recognized by theISL1 LIM homeodomain protein are essential for enhancer activity inthe pituitary and spine and a PITX1 bicoid class homeodomainelement is required for spatial patterning in the developing pituitary.This study establishes ISL1 as a novel transcriptional regulator ofLHX3 and describes a mechanism for regulation by PITX1. In addition,we are screening candidate patients for variations in the LHX3promoters and enhancer regions. Characterization of novel geneticdefects will facilitate patient treatment and enable genetic counsel-ing. Sources of Research Support: NIH HD42024 to SJR.

doi:10.1016/j.ydbio.2011.05.527

Program/Abstract # 563Distinct functional constraints partition sequence conservation ina cis-regulatory elementAntoine Barrierea, Kacy Gordonb, Ilya RuvinskyaaChicago, IL, USAbUniversity of Chicago, Chicago, IL, USA

Different functional constraints contribute to different evolution-ary rates across genomes. To understand why some sequences evolvefaster than others in a single cis-regulatory locus, we investigatedfunction and evolutionary dynamics of the promoter of the C. elegansunc-47 gene. We found that this promoter consists of two distinctdomains. The proximal promoter is conserved and is largely sufficientto direct appropriate spatial expression. The distal promoter displayslittle if any conservation between several closely related nematodes.Despite this divergence, sequences from all species confer robustnessof expression, arguing that this function does not require substantialsequence conservation. We showed that even unrelated sequenceshave the ability to promote robust expression. A prominent featureshared by all of these robustness-promoting sequences is an AT-enriched nucleotide composition consistent with nucleosome deple-tion. Because general sequence composition can be maintaineddespite sequence turnover, our results explain how different func-tional constraints can lead to vastly disparate rates of sequencedivergence within a promoter.

doi:10.1016/j.ydbio.2011.05.528

Program/Abstract # 564Metallothionein and cadmium toxicity in developing zebrafishAna Malone-Olivera, Stephen O'Sheaa, Kerri S. Warrenb

aRoger Williams University, Bristol, RI, USAbRoger Williams Univ Biol, Bristol, RI, USA

To determine the detoxifying role of metallothionein (MT) proteinin zebrafish, cadmium-treated embryos were analyzed to correlatephysiological response with the characteristics of expressed MT.Embryos were exposed to aqueous cadmium chloride from day 1post-fertilization until day 5 as well as short exposures starting at day3 or 4 until day 5. Cd-exposed embryos produce consistentphysiological responses; however, fish treated late experience moresevere morphological effects. Western Blotting revealed 15 kDa MT inday 1 embryos and an induction of this form in day 1–5 Cd-treatedembryos. High molecular weight MT, likely representing MT-proteincomplex formation, is the only form seen in later development andpredominates in day 4–5 cadmium-treatments. Induction of MT inthe day 1–5 treated embryos is verified by UV–Vis and HPLCfluorescence with DTNB and mBBr tags, respectively. Results suggestCd-induced production of MT protects developing zebrafish ifcadmium exposure occurs early in development.

doi:10.1016/j.ydbio.2011.05.529

Program/Abstract # 565The Dapper genes are expressed in sites of body elongation duringlater mouse developmentS. Dietricha, L.A. Sensiateb, A.V. Pedrosab, F.C. da Veigab, Lúcia AlvaresbaUniversity of Portsmouth, Portsmouth, UKbState University of Campinas, Campinas, Brazil

After the establishment of primary body axis, the outgrowth ofmany structures whose development requires extension is initiated inthe body of vertebrate embryos. In this context, Wnt5A and the four-pass transmembrane protein Vangl2, have been associated to bodyelongation by establishing PCP along the proximal–distal axis of thelimbs. Considering that the Dapper (Dpr) genes have been associatedto Wnt/PCP signaling and that Vangl2 physically interacts with Dpr1,we decided to investigate the expressionpattern of the threeDpr genes(Dpr1, Dpr2 and Dpr3) of mouse to identify a possible associationbetween their expression domains and known sites of body elonga-tion. The expression patternswere determined by in situ hybridizationin whole mount and in paraffin sections of embryos from E10 to E15.Further details were obtained by vibratome sectioning. From E10 toE11, the main sites of expression for the three Dpr genes were somites,genital tubercle, limbs and nose. The expression domains at E12 werethe same as described for the previous stages but a new area ofexpression was observed in the physiological umbilical hernia. At E13andE14 the samedomains are kept. Overall, the expression of the threeDpr genes in the developing limbs, nose and genitals suggests thatthese molecules could be involved in a pathway common to Wnt5Aand Vangl2 to regulate the outgrowth of body structures duringdevelopment. Grant sponsors: Fapesp, Capes and PIBIC/CNPq.

doi:10.1016/j.ydbio.2011.05.530

Abstracts268