Tuesday. Tissue-Specific Expression III (1957-1959) 337a

1957 1958 ANTIGEN PRESENTATION AFTER DNA VACCINATION: GENERATION DIFFERENTIAL DISPLAY RT-PCR (DDRT-PCR) AS A TOOL TO STUDY OF IMMUNE RESPONSES BY EXPRESSION OF A VIRAL IN GENE EXPRESSION: OPTIMIZATION AND APPLICATION. ((K.R. MUSCLE CELLS IN VIVO. ((effrey B. Ulmer, R. Randall Deck, Corrille M. Luehrsenl, M.G. Brubacherl, S. Cumberledge2, A. Lloyd3, and P.E. DeWitt, John J. Donnelly, and Margaret A. Liu)) Department of & MayrandI)) IPerkin Elmer Corp., Applied Biosystems Division, Foster City, Biology, Merck Reearch Laboratories, West Point, PA 19486. CA 94404; 2Dept. of Biochemistry, Univ. of Massachusetts, Amherst, MA CA 94305. Transfection of muscle cells viw has been acdhieved by intramuscular 01003; 3Dept. of Biological Sciences, Stanford Univ., Stanford, injection of naked DNA (Wolff ct al, Science 247, 1465, 1990). We gene underlies the phenotypic differences between cell utilized this technique as a novel means of vaccination and generated protective Differential expression types. is a standard tool to identify cell-mediated and humoral immune responses agaist influenza in mice (Uner cDNA library construction and screening and clone the sequences of differentially expressed mRNAs. However, c al, Science 259, 1745, 1993). Tlhse results sugested that muscle cells may be in antigen presentation leading to the generation of the immune traditional cDNA libraries that rely on plasmid or phage vectors and passage involved E. from several drawbacks including the requirement of responses observed after DNA injection. However, injected DNA may have been through coli suffer libraries intralized and expessed by other cells and, moreover, muscle cells are not large amounts of polyA+ mRNA, an intensive labor commitment and considered to be antigen presenting cells. Therefore, we addressed the that are not representative of the mRNA pool. DDRT-PCR was developed by sufficiency of muscle-specific expression of a foreign antigen to generate P. Liang and A. Pardee (Science 257: 967-971 [1992]) and promises to the protecive immunity. In the present study, we demonstrated that i) valy- revolutionize cDNA library construction and screening. In the first step of infected pnmary cultured myoblasts and C2C12 myoblasts were recognized and method, a subset of mRNAs are reverse transcribed using a set of anchored lysed by influenza-specific cytotoxic (CILs) in vitro, ii) CIL poly(dT) primers. In the second step, a subset of the first-strand cDNA is T-lymphocytes The recognition and lysis were not dependent on the differentiation state of the PCR amplified using an assortment of random sequence 10-mer primers. myoblasts, dii) NP-transfected C2C12 myoblasts presented NP epitopes to CrL PCR products from each set of primers are separated by gel electrophoresis, two cell types are compared. The differentially in vitro, iv) transplantation of NP-transfected myoblasts into syngeneic mice and the cDNAs from directly are recovered and or used as for northern resulted in the genwration of NP-specific antibodies and CIL, and protective expressed cDNAs sequenced, probes small amounts of total immunity to live virus chaUllenge, and v) these immune responses were not blots or in situ hybridization. The method requires data on the generated following transplantation of lysed transfectants. Therefore, muscle RNA and can be done in a few days. We will present optimization use with Perkin Elmer thermal We will also cells appear to be capable of viral in vivo, with of reaction conditions for cyclers. expressing subsequent construction cell lines of cell-mediated and humoral inimu DNA present data on DDRT-PCR library from Drosophila generation responses. and the maize uptake and expression of antigns by non-muscle cells do not seem to be reqed expressing the wingkss gene, Arabidopsis seedlings expressing to achieve the protecdve immune responses seen after DNA injection. R factor.

1959 BASAL VS PHORBOL-INDUCED TRANSCRIPTION OF THE COLLAGENASE GENE DEPENDS ON MULTIPLE CIS-ACTING ELEMENTS ((L.A. White and C.E. Brinckerhoff)) Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755. Degradation of the extracellular matrx is an integral step in normal physiology and in disease pathology, and is accomplished by the metalloproteinases. Collagenase (Matrix Metalloproteinase-l; MMP-1) is the only enzyme that degrades the interstitial collagens, the body's most abundant proteins. We used a series of 5' deletional fragments (1800 to 182 bp) of the rabbit collagenase promoter to study transcriptional regulation by phorbol myristate acetate (PMA) in primary fibroblasts. Previous data indicate an important role for an AP-1 element at -77 which binds FOS and JUN. We found that a mutation in the AP-1 site gready reduces basal/constitutive transcription of all of the fragments and, in the -182 bp fragment, mutations in this site virtually abolish PMA induction. However, mutant constructs containing more than 182 bp ofpromoter respond to PMA in a manner comparable to their wild-type counterparts. These data support a role for AP-1 in regulating basal transcription, and implicate upstream regions in phorbol responsiveness. At -186 there is an AP-1 site (J. Cell Biochem 52:337, 1993). We find that a T to G alteration in this site in 320 bp of promoter DNA does not affect basal transcription but reduces PMA inducibility by 50%. Mobility shifts with oligonucleotide (-212/-177) and extracts from PMA treated cells show specific binding that can be competed by an AP-1 oligonucleotide. Antibodies to Jun and Fos supershift the complex. Further 5' deletion between 320 and 182 bp of promoter uncover a 30 bp region from -212 to -182, which when deleted gives a 75% loss ofPMA induction. Our data indicate that basal and PMA-induced transcription are controlled in part by different mechanisms andupstream regions of the collagenase promoter, including a novel AP-1 site, contribute to PMA inducibilty. RNA Localization (1960-1961)

1960 1961 DETECTION OF TRINUCLEOTIDE REPEAT SEQUENCES IN HYBRIDIZATION OF POLY (dT) OLIGONUCLEOTIDES TO POLY (A) MYOTONIC DYSTROPHY PATIENT CELLS USING A SINGLE RNA IN LIVING CELLS. ((J.C. Politz, KL. Taneja and R.H. Singer)) Dept. OLIGONUCLEOTIDE PROBE. ((KL. Taneja, M. McCurrach*, M. of Cell Biology, Univ. of Mass. Med. Center, Worcester, MA 01655 Schalling*, D. Housman* and R.H. Singer)). Dept. of Cell Biology, University of Mass. Med. Center, Worcester, MA and *Dept. of Fundamental technology has been developed to evaluate the hybridization Biology, Cancer center, MIT. reaction between a synthetic antisense oligonucleotide (oligo) and its target mRNA in live muscle cells. Poly (A) RNA was used as an intracellular Myotonic Dystrophy (DM) is an autosomal dominant neuromuscular target for antisense oligo (dT) probes in vivo. Two approaches inherited human genetic disease with multisystem effects. DM is hybridization were used to detect this hybridization. First, exposure of cells to oligo (dT) in associated with the expansion of an unstable trinucleotide repeat to situ. This result would sequence (CTG)n, present at the 3'-UTR of protein kinase (Mt-Pk) vivo reduced subsequent hybridization oligo (dT) in mRNA.We have used a single oligonucleotide probe to the CIG repeat, be expected if the oligo hybridized to poly (A) RNA in vivo and blocked conjugated to either digoxigenin or fluorochromes, to detect the CTG subsequent hybridization in situ after fixation of the cell. Second, it was found repeat transcripts in the nucleus and in the cytoplasm of fibroblast that oligos taken up by cells can act as primers for reverse tanscription in situ cells and muscle biopsies of DM patients. The mRNA contining the (IST). Because reverse transcriptase elongates a DNA primer hybridized to repeat was found in a perinuclear region of the cytoplasm. It was found RNA, only hybridized oligos support incorporation of labelled nucleotides into that the transcript containing CIG repeat was present in the nucleus as a new DNA to give measurable signal in situ. Oligos intermalized by the cell but number (2-10) of discrete foci. No foci or other signal were found not hybridized to a target are not detected. Thus hybridization is evaluated as it in normal cells, or with the sense probe to the affected cells. Probe to the occurred within the living cell. Intemalization of fluorescently labelled poly 5'-end of Mt-Pk mRNA labelled directly with red fluorochrome (dT) oligo by live cells and subsequent intracellular hybridization, measured colocalized with the signal to the CIG repeat labelled with green using silver-gold enhancement after IST, were quantitated as a function of time fluorochrome indicating that the foci were from a single transcipt. In and Both normal cells, signal as two tofour spots could be detected probably at concentration using digital imaging microscopic analysis. the site of transcription. The foci therefore represents fluorochrome conjugated phosphodiester poly (dT) oligos and oligo (dT) released, post-tanscriptional RNA, and did not colocalize with SC-35 phosphorotisoates primed elongation by reverse transcriptase. These results "speckles" or poly(A) patches, therefore suggesting a separate demonstate directly that exogenously introduced antisense DNA hybridizes to compartment in the nucleoplasm for these released twanscripts. A single RNA in living cells and provide a rigorous method for evaluation of oligonucleotide probe can thus be used as a diagnostic test for DM hybridization efficiency as a ftmction of a variety of parameters. This expresson. methodology is applicable to visualization of RNA in living cells using fluorochrome conjugated oligos. 338a RNA Localization (1962-1967). Tuesday

1962 1963 ANNEIN II IS ASSOCIATED WITH CYTOSKELETAL-BOUND POLY- INTRON-DEPENDENT LOCALIZATION OF RNA IN THE SOMES IN KREBS II ASCIIES CELLS. ((A. Vedeler and V. Gerke*)) CELL NUCLEUS. ((S. Huang, S. Gunnery, M.B. Mathews and D.L. Department of Biochemistry and Molcular Biology, University of Bergen, Spector)) Cold Spnng Harbor Laboratory, Cold Spring Harbor, New York N-5009 Bergen, Norway and *Department of Biochemistry, Max-Planck- 11724. Sever models have been for the functonal oizaon of RNA Insttute for Biophysical Chemistry, D-37077 Gottingen, Germany. proposed within the eukaryouc nucleus and for the rel p of this oization to the distribution of pro-mRNA splicing factors. One model has suggested Annexin is a member of a family of calcium-dependent phospholipid bind- thatRNAs whch must be spi:ced capable of rruiong splicng facts to ing proteins and also associates with actin. It is a substrate of protein kinase the sifts of traniption from storage and/or reassembly sites (Jim6nez- C, pp60' and tyrosine kinases associated with a number of growth factor Garcia and Spector, 1993). In otler to further evaluate this model we have utilized a aansient transfction where the of RNA receptors. The polysomes in Krebs II ascites cells can be separated into at least system expression transripts containing introns, lacking inos, or containg introns with a three different populations: "free' (loosely bound), cytoskeletal-bound, and deleton in their 3' splice site were evaluated for their association with membrane-bound polysomes. The proportion of cytoskeletal-bound polysomes splicing factors. Using in sint hybridization, we have found that can be modulated by hormones; it can be increased e. g. by a one hour incu- approximately 90% of the RNA transcripts produced from constructs which bation with insulin. The cytosceletal fraction is enriched in actin, cytokeratin exprs intron-containig gems such as f-globin, tropomyosin, and HIV tat 19 and annexin II, of which annexin II is the major protein. Only polysomes are associated with splicing factors immunolabeled simultaneously with an RNA isolated from the cytoskeletal fraction were found to be associated with antibody to SC35. In contrast, only 2% (on avcragc) of transcribed from constnus encoding intron-less such as p-galactosidase, and annexin II. This associaton appears to be calcium-dependent as EGTA releases adenovirus VAj are associated with splicing factors. Furthermore, the annexin H from the polysomes. When cytoskeletal-bound polysomes were split majority of HIV Tat RNA, which contains a deletion in its intron at the 3' with TPCK (N-tosyl-L-phenylalanine chloromethyl ketone) and separated on splice site, showed little association with splicing factors. These sucrose gradients into fracdons containing the 60S and 40S ribosomal subunits observations suggest that the spatal associatn of RNA twanscrpts with as well as a peak containing mRNAs, annexin was found in the mRNA splicing factors is intron-dependent. Such an association is functionally significant as tanscripts containing mutated intrn, which are incapable of fraction. It thus seems possible that annexin H, present in the cytoskeletal- being spliced, are not associated with splicing factors. Therefore, we bound polysomes, is associated with mRNA or another, yet un-identified propos that the nization of RNA and splicing faors in the cell nucleus component and not with the ribosome. Annexin II is known to have a role in reflect the transion activity of the ceTand the locaization patterns of vesicle transport These results, however, suggest that annexin H may also facts will change to refiect changes in activity. have a role in specific mRNA transport and/or localization.

1964 135 MAPIA-CONTAINING PROTEIN COMPLEX BINDS RNA IN A SEQUENCE BEATING EFFECTS ON POST-TRANSCRIPTIONAL REGULATION OF INDEPENDENT MANNER. ((C. DeFranco, M. Chicurel, and H. Potter)). Dept. MYOSIN mRNA IN CARDIAC CULTURES. ((P.H. Goldspink, S. Anic, A.M. Neurobiology, Harvard Medical School, Boston, MA 02115. Samarel, D.B Thomason, and B. Russell)) Departments of Physiology, University of Illinois at Chicago, IL 60612, Medicine, Loyola University at Association of mANA with the cytoskeleton has been recognized for some time. Chicago, Maywood, IL 60153, and Physiology, University of Tennessee, Many cell types Including neurons, appear to use this Interaction as one means of Memphis, TN 38163. establishing and maintaining celkluar polarity by translocating some mRNAs to speciftic celHular sites for bcal . The mechanisms by which the beating of cardiac cells controls Using brain tissue and the PC12 cel line we have begun to search for the RNA translation of myosin isoforms is unknown. We use several approaches to sequences and proteins involved in RNA-cytoakeletal interactions. We have found investigate post-transcriptional mechanisms that maybe controlling that, In contrast to previous expectations, In PC12 celis treated with NGF RNA translation of heavy chain mRNA. In rat neonatal cardiac cells the arrest cytoskeletal association does not depend on the 3' untranslated region. In of beating for 48 hours by calcium charnel blockers (verapamil) causes a additon, the translational state of the mRNA is not inportant for its interaction with decrease myosin heavy chain protein synthesis and content. Differential the cytoskeleton since treatment of the cells with puromycin has no effect. In vitrc centrifugation of polysomes extracted from myocytes shows a shift experiments show that mRNA is capable of binding in a sequence independent towards heavier fractions for 1 Bs RNA with 6 hours of contractile arrest manner with a protein complex found in brain and PCI2 cal extracts. The mRNA- compared to control. However, the myosin heavy chain RNA polysome protein complex appears to contain at least two components: the microtubule- distribution profile is still the same as control at this time point. The associated protein, MAPlA, and a 170 kD species. UV cross-Inking indicates that cellular localization of the myosin mRNA was observed by in situ the 170 kD protein binds directly to the mRNA. hybridization. Initially the mRNA is uniformly distributed but by 2 - 6 The protein complex Inkig mRNA to the cytoskeleton (a putative cytosome) may hours of verapamil treatment there is a perinuclear concentration which create an RNA-protein scaffold inportant in the translocation and translation of the dissipates by 48 hours. This perinuclear concentration was also seen mRNA. when beating is blocked at the level of the cross-bridge by butanedione monoxamine for 1-2 hours. Isoproterenol-treated cells, beating at higher amplitude and frequency, have increased abundance of myosin mRNA uniformly distributed throughout the cytoplasm. From these results we conclude that the physiological state of the cell influences post- transcriptional regulation of myosin.

1966 i1 LOCALIZATION OF --ACTIN RNA TO NEURrTES AND GROWTH ISOLATION AND CHARACIERIZATION OF PROTEINS BINDING CONES OF DEVELOPING NEUONS IN CULTJRE. ((GJ. Basl*, TO THE LOCALIZATION "ZIP CODF' OF F-AC` mRNA IN K.T. T111ja, RI. SInmr* and KS. Kosik)) Cener fr Narlogic Dlseaa CHICK EMBRYO FIBROBLASTS. ((A.F. Ross, E.H. Kislausis, KL. Brighbm d Women'sW ospital ad Havad Medical School, Bost MA 02115 Taneja, and RH. Singer)) Department of Celi Biology, Univenity of and #DepLCell Biooy, Univerity of Mass Medical Ctr., Worcser, MA 01655. Massachusetts Medical School, Worcester, MA 01655. Neunl grwth cone, lalzed to the tenmini of developing proesses, are It is now well established that the mRNA for P-actin in chick embryo spilized uctur whIh rod als and cont neudte pr fibroblasts is localized to the leading edge of the cell in a region referred outgowt. Proteins which are synteiqzed within the cell body may have to be to as the lamella. This localization may a role in the consdkerable dia so rach sits of functon within gw cones. peripheral play Ev c indicas that growth cones may also possess protein synthedc establishment of a polarized actin cytoskelen, a requirement for cellular mahnery, alowing for local sysdiels of specific proteins This mechanism motility. Although the mechanism of p-actin mRNA sorting is not would rquie Xt mRNAs roeted frm the cell body into neuites and undersood, evidence suggests that the mRNA is physically associated grwth cones We atin mRNAs could be detected with actin filaments at the cell peiphery. A short (54 base) sequence has in developing and growco of tured cere -ordcal ion u been identifed in the proximal 3' untanslatd region of acin mRNA high reslin in Oine probes plmtary which localizes a reporter gene (P-alacodase) in a hetrologous untran_baed rgios of mRNA wer 3' end lsbeled with digoxigenin-l 1- expsion system (Ksls et al., JCB in press). It is possible that a dUTP WAn hybidized at time during the flrst week in ature and protein or complex of proteins interacts with this laizion "zip code" i le of hybdiz detecd utiling Cy3 majorIty wu to transport and/or link p-actin mRNA to peripheral actin filaments. To confind to the ce bod, ever, low lecvds of izin signal exended isolate such proteins we have constucted affinity resins consisting of into dcvoping d c d axon processes and their gowth cones. Actin mRNA wiIn distal neirites sd grwth cones was frquety >100 m frm the oligoribonuclotide probes modified with biotinylated bases at the 3' end cell body, ad was most abundant withn lager, moe elaborate gth cones. and linked to streptavidin-agarse. Using these resins we have isolated a Tle h nal ithin processes was highly punctate, suggestng the 70kDa protein from extracts of chick embryo fibroblasts which binds sence of cuser of n mRNA molecule Deteton of acin mRNA within specificlly to the proximal 27 bases of this l ion zip code. aopesid aft epPocesses ha diferuited IoD eltheraxon or denditeg, and Microsequence analysis and GenBank comprison to Inown proteins after most poly (A) mRNA had been compartmentalized to t1w somatodenduitc indicates that this protein has not been identified. In addition, several active of soin witiin growth cones may polymeiz other proteins of molecular mass 9OkDa, 53kDa, 35kDa, and 30kDa bind require alboaled anures iinewly syntsized actin monomrsm. The deteon of to this in a variable manner. These data suggest that a acdnmRNA withn developing axo, deidrite and wth cones has further sequence complex binds to a of the of -actin for identiy sites of locized synthesis which may of proteins specifically segment 3'UTR mRNA to be contribu to pres otgowth md asemby of cytoarhitoctue. which has been shown responsible peipheral localization. Tuesday. RNA Localization (1968-1973) 339a

168 169 LOCALIZATION OF I-ACTIN mRNA IN PREIMPLANTATION MOUSE THE ROLE OF CYTOSKELETAL COMPONENTS IN THE DENDRITIC EMBRYOS. ((A.E. Allworth, R.H. Singer*, KL. Taneja* and D.F. TRANSPORT OF RNA ((L Ugon, G. Banker and 0. Steward)) Departnent of Neuroecenoe, University of ViWa Healh Sciences Center, Charlottesville, VA Albertini.)) Tufts University School of Medicine, Boston, MA and 22908. University of Massachusetts Medical School*, Worcester, MA. RNA is trnpored kilo neuronal dendites, but itle is known about the The localization of mRNA to cellular microdomains where particular mehanisms, undelying btaporL has been hypothesized that the cyicktea po m,fimentu aciin and microtubues, may p the proteins are syntsized abundantly has been well documented in sntrctural support forthe as yet unidentiled cha rehnic process that cultured fibroblasts and neurons. In a previous study, using tnpots RNA. In order to evauate this hypothesis, we used the drugs fluorescence in situ hybridization of oligo dTs, we documented the nocodazole and cych D to sodectively cytoskeletal proteins A+ RNA to microfilaments in of cell-cell and Utn evalated the traport of recently synsized RNA into dendrites. n juxtaposition of poly regions the ntrascatlon of RNA depends upon an intact cytoskeletal polymer, then the contact in 8-cell, morula and blastocyst embryos. In the current study, disruption of that polymr should IorrOrnSe the celis abilty to transocate RNA a fluorescein conjugated antisense probe to the 3' end of mRNA for dendrites. Culured lpocana neu Vwere ncubated for three hours with 1O uWml nocodazole, 25 ugfrl cyochalasin D or vehicle alone. The transport rat l-actin was used to study the distribution of l-actin mRNA in 8- of recentl synteed RNA ino dendries was assessed by puilseabelng were cell, morula and blastocyst embryos. Samples fixed, extracted, neurons wIth 3-Ud for one hour and t ferring them to medlum without 3H- hybridized, counterstained with rhodamine phalloidin and analyzed Uddhe for an addIo six hours. Celia were then fixed and prpared for using laser scanning confocal microscopy. In all stages examined, 1- 1rlogr--hy. Under these conctlons, recently synthesized RNA moves from was seen to and coincident with microfilaments. the nus no dedres at a rate of about 11 umnhour. Ffty cels per condftion actin mRNA adjacent wer chosen for analysis. The mean distance from the nucleus to the end of This association was most pronounced in regions of cell-cell contact labeling in the dendries was caculated for each cel. Al groups exhbited and the apparent amount of mRNA seen associated with dendrtc lbeling, but the mean distance of bel In the cels incubated with microfilaments increased with embryonic age. Treatment of embryos nocodazobe was signiicantly ss than at in cels Incubated with vehicle alone of B-actin mRNA localization (p<.05). There was, however, no sinTca difference in the distance of labeling with cytochalasin D disrupted the pattern between cek Incubated with vehcle and cyochalasin D. These resuits indicate seen in control embryos. Supported by NIH grants HD 20068, HD that tanioaMon of RNA may depend upon the presence of intact mlcrotubules, 18066, P30HD28897 and the Markey Charitable Trust Foundation. but not an kWt actin netork The falure to corrletely blod trasport could reflet the continued operation of a miftubuleindependent phase of transport or may be due to the presence of drug-resistant microtubules in the relatively mature neurons used In this study. Supponed by NIH NS23094.

1970 1971 TRANSPORT OF MYELIN BASIC PROTEIN MESSENGER RNA THE SUBCELLULM LOCALIZATION OF MRNA IN NEURONAL DENDRITES IS RELATED TO THE PATrERN OF AFFERENT INNERVATlON. ((O. Steward)), IN OLIGODENDROCYTES REQUIRES INTACT Deparment of Neuroscience, Unversity of VuInra Health Sdences Center, MICROTUBULES. ((K. Worboys, K. Ainger, E. Barbarese Chadotteevlle VA 22908 and J.H. Carson)) University of Connecticut Health Center, Neuons bcale different mRNAs In derent subcelar corDpartments. Most Farmington, CT 06030. nro namNAs are bcalzed only In cel bodie but a few mRNAs are present at high In denkies. Here, we report tha dsrbution of particular mRNAs in Myelin basic protein (MBP) mRNA is localized to the dendrites corretes with the paemn of temarration of difhrent afferet systems. In hybrdizatn technIues wro usd to corpae the suboellular distributon of peripheral processes and membrane sheets of dendftic mRNA in neurons of the h4ipoccmrop hi vim. The mRNA enooding the a- oligodendrocytes in vivo and in vitro. Microinjected MBP suuni of aprtin inase (CAMII knase) was present mRNA is transported and localized to the periphery of at high tho the ys that contain the dendrites of hWpocampal pyramWod and dentate granule cal. In conrast, the mRNA enoding the high oligodendrocytes in a similar pattern as endogenous MBP mowcular nhprotn MAP2 had a more ted dstrbion. In mRNA. In the work described here oligodendrocytes in the dentate gys, lbeling for MAP2 mRNA was present a dscrete band In the lamina nta proxiial derites and decwd to bw lbvels In lainwe culture were treated with nocodazole to disrupt containdistlW dendrites. The ayer exhIIIng high abelg is the one in which microtubules, taxol to stabilize microtubules, or affernts of the comnisaura al fibe terminate; the lmina exhibiting bw cytochal-asin B to disrupt microfilaments. Digoxigenin- lbeling is ineraed by afferet from the Iorhinal cortex. The same pattem was present In the lippocaous proper a ugh the diferences in abeling of different was labelled MBP mRNA microinjected into the treated bmhne wre ts dramaic. To determine whter the differertial dirbution of cells and the extent of transport of the microinjected RNA denrdtc was led by the presence or acMty of afferenis, we evaluated was determined by confocal microscopy after appropriate mRNA diriuon afterthe kut from the eorxhnl cortex had been removed by lsbns in the cootex. The dstrbution of the two dendritic mRNAs staining. Nocodazole and taxol inhibited transport of was not dewctabl altred b such lesons. This was tu whether the lesions microinjected MBP mRNA while cytochalasin B had no wer made In adult aniuis or in devobpIn (13 day old) rats when the pattern of effect. These results indicate that intact microtubules but afferent innvation is being estabhed. These results suggest that there is a ltinship between the pattem of afferent temination and the distribution of not microfilaments are required for transport of MBP dendritic mRNAs, but that the afferents do not dircty regulate RNA distrbution. ft mRNA in oligodendrocytes. reman to be determned whether the the opposite is t-hat the differential dibutin of mRNA plays a role In determIing the pattem of termination of atferents. Supported by NIH Wrat NS12333.

192 1973 Xcat-2 RNA LOCAUZATION IN XENOPUS OOCYTE((Y.Zhou and HUMAN U7 snRNA IS TARGETED TO C SNURPOSOMES IN M.L.King))Department of Cell Biology and Anatomy,University of Miami AMPHIBIAN GERMINAL VESICLES. ((C.-H.H.Wu and J.G.Gall)) School of Medicine, Miami,FL33101. Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210 Maternally localized mRNAs in Xenopus and Drosophila are believed to initiate pattern formation in the early embryo. Vegetally localized RNAs snRNA targeting inside the nucleus was studied by injecting like Xcat-2 and Vgl are of particular developmental interest. In situ chimeric 3H-labeled human U7 snRNA into the cytoplasm of hybridization studies reveal that Xcat-2 is exclusively localized to the amphibian oocytes. The injected U7 snRNA was imported into the mitochondrial cloud (m.c.) in stage I oocytes, moves with this body into germinal vesicle (GV) of the frogs Xenous laevis, Ran ippiens, and the vegetal cortex durlng stage 11, and later, partitions into islands Rana tempgorara, and the newt Notophthalmus viridescens, where it consistent with it being a component of the germ plasm. Xcat-2 precedes was targeted specifically to C snurposomes. Deletion of the histone Vgl to the vegetal cortex by almost two stages. Vgl appears to be mRNA binding region, the 3' end stem, or the 3' end loop of the 64 translocated in stage IV oocytes in discrete particles. Xcat-2 transcripts, nucleotide human U7 snRNA does not affect C snurposome- full length or mutants containing only the 3'UTR, localize to the vegetal specific targeting. However, deletion of the Sm region abolished cortex in a pattem identical to Vgl after injection into stage IV (but not nuclear import, and hence no C snurposome targeting was stage VI) oocytes. However, these same transcripts injected into stage I observed. About 80% of the endogenous U7 snRNA in the GV was oocytes localized to the m.c. after one day in culture while Vgl did not. pelleted by centrifugation at 15,000 rpm for 15 minutes, suggesting Therefore, the Xcat-2 3'UTR is competent to correctly localize to the m.c. that it was in C snurposomes. The exogenous human U7 snRNA as well as to assemble into transport particles in stage IV oocytes. Fine introduced by injection replaced the endogenous Xenopus U7 deletion mapping of the 400 nucleotides 3'UTR will determine the minimal snRNA in the C snurposomes. Excess U7 snRNA in the GV did not element(s) required for these pattems. Intact microtubules are required for promote formation of new C snurposomes and remained in the translocation, but not for the intagrity of the transport particles nor for nucleoplasm. Our data suggest that the C snurposome plays a role maintaining their asymmetric distribution on day two and three of culture. in cycling of U7 snRNA. Cytochalasin B and nocodazole (or colchicine) completely disrupts Vg1 but not Xcat-2 cortical binding indicating that these RNAs are not anchored identically. We propose that this differential anchoring is critical to normal development. 340a RNA Localization (1974). Tuesday

1974 TWO DISTINCT PATHWAYS FOR THE LOCALIZATION OF RNAs INVOLVED IN AXIAL PATTERNING AT THE VEGETAL CORTEX OF XENOPUS OOCYTE. ((M. KIoc, and L. Etkin)) Department of Mdecular Genetics, U.T. M.D. Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030. Normal embryonic development requires the proper temporal and spatial pattems of expression and localization of matemal transcripts. The analysis of the molecular anatomy of the radially symmetrical XnoQpus oocyte reveals the presence of populations of matemal RNAs that are kocaized in the and vegetal regions. We have found two distinct pathways for the localization of RNAs at the vegetal cortex during oogenesis in Xmo=us laevis. One of these, through which Xlsirts, Xcat2 and Xwntl 1 are localized, involves transport via a specific region of i dr cloud (Balbiani body) that we call the message transport organizer or METRO. This pathway involves three steps icdudng transport from the GV to the mic d cloud, sorting of the RNAs to specific regions of the METRO, and translocation to and anchoring at the vegetal cortex. It also involves a hierarchy in the order of association of these three RNAs with the cortex, with Xcat2 associating first, followed by Xlsirts and Xwntl 1. The second pathway is used by Vg1 which in stage 1-2 oocytes is spread throughout the entire oocyte cytoplasm and is excluded from mtochondri coud. Later, Vg1 tracks toward the vegetal cortex through the path set up by the mitochondrial cloud. Vgl first associates with the cortex in the region where the Xlsirts, Xcat2, and Xwntl 1 are anchored. We have also shown, using cytoskeletal inhibitors, that anchoring of these localized RNAs is dependent in part on actin microfilaments, but is independent of microtubules. These results demonstrate a novel mechanism of translocation and sorting used by several RNAs involved in the establishment of the vertebrate embryonic axis. Proteins of the Nucleus, Nuclear Matrix, and Nucleolus (1975-1978).

1975 1976 EFFECTS OP TRANSCRITON INHBITORS ON THE SUBCELLULAR LOCA- SV40 LARGE T ANTIGEN IS MODIFIED WITH O-LINKED N- TIONS OF HIV-1 REV AND PROTEIN B23. ((M.Dundrt, D. Rekosh2, H.M. Caldot- ACETYLGLUCOSAMINL ((L. Medina-Vera and RS. Haltiwanger)) tirA, M.-L. Hammarskjld' ad M.OJ.Olouit)) tDepartmsnt of Biochemistry, University Departmnt of Biochemistry and Cell Biology, SUNY-Stony Brook, of Mississippi Medical Center, Jwaco MS 39216; 'Myles H. Thaler Center for AIDS and Stony Brook, N.Y. 11794 Hums Retrvims Resrch, University of Virginia, Charotw viLe VA 22908 SV40 large T antigen plays an essential role in the infection cycle of the Proeian B23 is a major nucloolar ptein and putaive nbosome assembly factor which virus. T antigen has multiple functions including binding of p53, shuttls between nuclbeolu ad f cytoplasm. Prvious studies denstrated fthe altering traniption patterns in host cells and replicating of viral DNA. HIV-1 Rev protein forn a spcific complex i Wro witf protein B23 which is ggested T antigen has been reported to bear multiple post-translational to be a nucleolar reeptor ansd/or carier for de Rev proten. To examine possible in wv modifications such as phosphorylation and glycosylation. Severl interactio betwe Rev ad B23 we used COS-7 cells tnienty trnfected wifa a Rev woes have reported glycosylation of T antigen, but no known expressg plsnid and sable Rev-expresng COS-7 cell lines. Protein B23 was parally function for the modification or agreement on a sugar stucture has been co-precipitaed by anti-Rev antibody from extrcts of Rev-expssing cls, uggesting att reached. We have demonstrated the presence of 0-linked N- two protein ieract Ui swo. Rev and B23 lcatios were examined by indirect acetylglucosamine (O-GlcNAc) on T antigen by metabolic labeling of immunofluorecnce using confocal aser micromscopy. In early stag after transfection Rev SV40 infected cells with [3H]-glucosamine. Allkali induced accuntlsd inofnnucloolar dense fibrilr and granular components where it colocalimed eliminaton of the radiolabeledT antigen releases [31]-N- with protein B23. Treatment of faeae cells with actinomycin D (AMD) under conditions acetylglucosauinitol. B1,4 galactosyl transferase labeling of T antigen fast blocked RNA pot I Utncriptin caused Rev to compktely redistribute from nucleoli to further confirms the presence of O-GlcNAc. The presence of other tfe cytopbsm. Simulaneously, protein B23 was paruilly relased from nucloli mosdly sugars has not yet been eliminated. The most striling obsevation is that into thf nudeoplam with detctabl amounts in tfe cytoplasm. In contrast, inhibition of the fiaction of T antigen tightly associated with chromatin seems to be RNA pot II by ae-anitin did not cause Rev to be released into the cytoplasm. I oells more highly glycosylated that the bulk T antigen. We are currently exprssn Rev at higher levels Rev and B23 accumulated both in nucloli and in pauinu- attempting to map the site of the modification. These studies should clear regios. These cells were imasitive to AMD induced redistribution of Rev. In help us to better understand the function of O-GIcNAc on this important certin ceUs, apprently expremsing high levels of Rev, both proteins wee predominanty protein. This wori was supported by NIH Grant GM 48666. located in perinuckar regions. In cells recovering from AMD trement in tfe presence of cycloheximide Rev and B23 reocatd to nucleoli. These results suggest tfat tfe nucleolar targeting of Rev depends on continuous preibosomal RNA transcription. The overaccumu- lation of the Rev protein affecta nucl ar stucture, probably remiting in nucAeolar dys- function. Supported by NIH grts AI34277 (to M..) and AI25721 (to M.L.H.).

1977 1978 MOLECULAR AND BIOCHEMICAL ANALYSIS OF A NOVEL 170 kD TRANSLOCATION OF PROTEIN KINASE CK2 TO THE NUCLEAR NUCLEAR KINASE IN DROSOPHILA W1TH HOMOLOGY TO THE raf- MATRIX IS A POTENTIAL GROWTH SIGNAL. ((S. Tawfic, and K. FAMILY OF PROTOONCOGENES. ((KM. Johansen, J. Johansen and A. Ahmed)) Cell. & Molec. Biochem. Lab., and Dept. of Lab. Med. Path., V.A. Silvers)) Department of Zoology and Genetics, Iowa State University, Ames,IA 50011. Medical Center and University ofMinnesota, Minneapolis, MN 55417. Protein kinase CK2 (casein idnase 2) is a ubiquitous multipotential We have previously reported the cloning of a novel nuclear kinase p2Abl7 in messenger-independent serine/threonine protein kinase that has been Drosophila (Johansen et al., Mol. Biol. Cell 4:240a), which by antibody amount of nuclear labeling shows a dynamic cell cycle-specific patten of distribution within the implicated in cell growth and proliferation. A significant nucleus. During interphase the labeling is localized to the chromatin and/or CK2 is associated with the nuclear matrix (NM), where it may be involved in nuclear matrix, whereas during metaphase antibody labeling becomes the phosphorylation of several intrinsic proteins (Tawfic, S. and Ah}ned, K J. redistributed and co-localizes with the mitotic spindle apparatus. We have Biol. Chem. 269: 7489-7493, 1994). Consideing the importance of the NM extended the biochemical and molecular characterization ofp2Abl7 and show in chromatin organization and cell growth, we examined whether the clone on Western blots that antibodies made to fusion proteins from the association of CK2 with the NM was regulated by a growth stimulus,. namely recognize a triplet of protein bands ranging from 170-180 kD. The detection of a triplet of bands is consistent with the obsevation that many kinases are androgen action in the rat ventral prostate. In rats, androgen deprivation leads themselves phosphorylated. These proteins are found only in nuclear fractions to a differential loss of CK2 activity and protein from the NM fraction in the and not in the cytoplasm. The size of the proteins is consistent with data from prostate. At 24 hr after androgen deprivation, the NM-associated CK2 Northern analysis which shows that 2Abl7 encodes a single transcript of activity declines by about 80%/. compared with a 37%/o decrease in total nuclear approximately 6.5 kb. Sequencing of the partial 2Abl7 cDNA clone has CK2 activity. This change is also apparent in the immunoreactive CK2 a as well as currently yielded unique C-terminal domain sequence comprising a diffnial the struture of an entire catalytic linase domain. Searches of the sequence protein in the NM. Androgen administration evokes rapid data bases with the kinase domain show that p2Abl7 is most homologous to increase in CK2 activity in the NMK so that within 1 hr following the serine-threonine kinases related to the raf-family of protooncogenes. androgenic stimulus, the CK2 activity increases by 1101% in the NM fraction However, p2Abl7 is only about 40% homologous to members of the raf- versus 45% in the total nudei. We propose that the stimulus-mediated family, whereas homology between raf-family members is greater than 80%. differential translocation ofCK2 to the NM may play a role in the transduction In addition, p2Abl7 shows intriguing lower level homologies to other kinases signal. in part by NIH grant CA-15062 and by the which appear to be directly involved in cell cycle control, such as nimA. ofthe growth [Supported Thus, p2Abl7 may define a new class of kinases related to the raf-family Medical Research Fund ofthe U.S. Dept. ofVeterans Affairs]. which plays a role in mitosis and . Suppond by NIH grantGM 50906. Tuesday. Proteins of the Nucleus, Nuclear Matrix, and Nucleolus (1979-1984) 341a

1979 1980 IMMUNOFLUORESCENT LOCALIZATION OF NUCLEOSIDE BUDDING YEAST RAN-BPI, A NEW COMPONENT OF A CONSERVED DIPHOSPHATE KINASE IN NORMAL AND TUMOR CELL NUCLEAR GTPASE MOLECULAR SWITCH AFFECTS BOTH DNA LINES ((S.K.Kraeftl, F.Traincart2, S.Mesnildrey2, M.Veron2, and METABOLISM AND MITOTIC CHROMSOME SEGREGATION. ((1.I. Ouspensk, L.B.Chenl)) 1Dana-Farber Cancer Institute, Harvard Medical U.W. Mueller,*A.Matynia,* S. Sazer,*, S.J. Elledge* and B.R. Brinkley)) Dqpmtnts of Ccl Biology and Biochemistry, Baylor College ofMeicine, Houston TX 77030. School, Boston, MA, 02115 and 2CNRS, URA 1129, Unite de Biochimie Cellulaire, Institut Pasteur, 75724, Paris, France. We have performed a systematic search for yeast genes that interfere with stability when overexpressed (CST genes). Phenotypes produced The two human isoforms of nucleoside diphosphate (NDP) by two of these genes (CST17 and CST20) are very similar, suggesting their kinases A and B are identical to the gene products of nm23-H1 involvement in the same molecular pathway. The sequence data indicate that and nm23-H2, respectively. The nm23-H1 gene was implicated CST17 is identical to GSPl/CNRl, and encodes a protein nearly identical to in cancer metastasis and the nm23-H2 gene procuct was shown Ran, a small nuclear GTP-binding protein that is thought to regulate a variety of to be a transcription factor. Here we report the localization of cellular processes, including the organization of the interphase nucleus (Dasso, NDP kinases A and B in normal and tumor cell lines using TIBS 18:96). CST20 is a new essential yeast gene with homology to mammalian mono- and polyclonal antibodies. Immunofluorescence was Ran-binding protein RanBPl (Coutavas et.al. Nature 366:585), which specifically performed by both epifluorescent and confocal laser scanning interacts with GTP-bound Ran and thus may be its effector. By using an microscopy. With antibodies specific for NDP kinase A, a epitope-tagged version of Cst20p, we have demonstrated that it interacts with cytoplasmic fluorescence without nudear staining was observed. Cstl7p, both in vitro and in vivo, indicating that Cst2Op is a functional In contrast, staining with antibodies recognizing both NDP homologue of mamm RanBPl. The overexpression of either of these kinases showed cytoplasmic as well as nudear fluorescence. The genes affects DNA metabolism (as evidenced by sensitivity to hydroxyurea and nuclear as fluorescence appeared spot-like pattern. The spots UV, appearance of DNA lesions and lethality with rad9 DNA damage were mostly found in regions that are not strongly stained with checkpoint mutation), as well as segregation of in mitosis DNA dyes. (manifested by chromosome nondisjunction, sensitivity to anti-microtubule drugs and lethality with mitotic checkpoint mutation mad2). The Ran molecular switch may control nuclear architecture or conformation of the chromatin in a way that ensures both correct functions ofthe interphase nucleus and proper folding ofthe chromosomes in mitosis.

1981 1982 THE ROLE OF RCC1 AND RAN IN NUCLEAR ASSEMBLY AND DNA A DROSOPHILA ARLl-RELATED MAMMALIAN SMALL GTP-BINDING REPLICATION: STUDIES IN XENOPUS EXTRACTS. ((H. Saitoh and M. PROTEIN (rARLl) IS UBIQUITOUSLY LOCALIZED TO THE NUCLEUS. ((S.L. Dasso)) Laboratory of Molecular Embryology, NICHD, National Institutes of Lowe, S.H. Wong, and WJ. Hong)) Institute of Molecular and Cell Biology, Health, Bldg. 18, Rm. 101, Bethesda, Maryland 20892. U.S.A. 10 Kent Ridge Crescent, Singapore, SE 0511.

RCCI is an abundant, highly conserved, chromatin-associated protein whose ADP-ribosylation factors (ARFs) are small molecular weight GTP- function is necessary for the preservation of a properly ordered cell cycle. RCC1 binding proteins which belong to the family of Ras-like p2ls and are is also necessary for numerous nuclear processes, including nuclear transport and implicated to play a role in vesicular transport. Recent studies have RNA metabolism; and it functions enzymatically as a guanine nucleotide exchange revealed a subfamily of ARF-like proteins (ARLs) which are related but factor (GEF) for a small, ras-like GTPase called Ran. By immunodepletion of distinct from the ARFs. The first member was identified in Drosophila RCC1 from Xenopus egg extracts, it has been shown that RCC1 is essential for (dARLl), but no function has been ascribed to this gene. We have cloned DNA replication of sperm chromatin templates in vitro. In order to understand and sequenced a rat homologue of dARLl (rARLl) and found it to be 79% the role of RCC1 and Ran in nuclear functions and cell cycle control, we have identical to dARLI. Northern blot analysis of eight rat tissues revealed examined nuclear formation in the absence of RCCL. Specifically, we have the presence of a ubiquitous 1.8 kb message. Biochemical studies using a examined steps in nuclear assembly from demembranated sperm chromatin in fusion protein comprising rARLI fused to GST showed that rARLI binds RCCI -depleted egg extts. These steps include: chromatin decondensation, the the GTP7S form of GTP in a dose dependent manner. Monospecific formation of pre-replication foci and the formation of a nuclear envelope with polyclonal antibodies, raised against unique rARLI , recognized actively transporting pores. Steps prior to the assembly of the nuclear envelope the rARLl-GST fusion protein as well as a 22 kDa protein in total NRK do not appear to require RCCI. However, the depletion of RCC1 strongly cell lysate by Western blot analysis. Immunofluorescence microscopy of effects later steps in assembly, and nuclei formed in the absence of RCCI have a NRK cells using the -purified antibodies revealed discrete lower nuclear import capacity than those in control exts. Studies are currently punctate nuclear labelling that was abolished specifically by the antigen being done to determine whether this inability to transport results from a failure to peptide or the recombinant rARLl fusion protein. Examination of nine assemble pores, or from the assembled pores being non-functional in the absence additional cell lines showed similar nuclear labelling. These of RCCI and its activity as a Ran-GEF. In Xenopus extracts, RCC1 and Ran observations suggest that rARLI may participate in some nucleus- behave as components of a large, multi-protein complex when analyzed by gel associated function. rARLl is the second member of the Ras-like p21 filtration chromatography or by sedimentation on sucrose gradients. In order to family to be localized to the nucleus, the first being RanlTC4. This raises understand the molecular role of this putative complex, we have examined it the possibility that other members of the ARL subfamily may also be biochemically. We have isolated at least one other protein which is a candidate localized to this organelle and may have either similar or distinct component of the complex and which has not previously been reported to functions. associate with RCC1 or Ran.

1982 1984 cDNA CLONING OF LAMINA-ASSOCIATED POLYPEPTDE (LAP) 2 AND DYNAMICS OF NUCLEAR LAMIS: EXPRESSION IN TRANSFECTED BHK-21 CELLS. IDENTIFICATION OF REGIONS FOR TARGETING TO THE NUCLEAR ((R.D. Moir, A.E.Goldnian, M.Sinensy, nd R.D. Goldman)) Dept. of Cell and Moleular ENVELOPE. ((K. Furukawal, N. Pant62, U. Aebi2 and L Geracel)) Biology, Northwetem University, Chicago, IL 60611. Eleanor Roosevelt Istitute, Denver, 1Departments of Cell & Molecular Biology, The Scripps Research CO 80206 Institute, La Jolla CA 92037. 2M.E. Miller Institute for Microscopy, Biozentmm, University of Basel. Switzerland. When prelamin A is mcronjeted ito colls it ially accumulates in nucloplasmic foci before into te amina. The foci stei an L mina-associated polypeptide (LAP) 2, which directly binds becoming iorporated perqporal with antibody for form of A. the which nuclear lamin B1 and chromosomes in a phosphorylation regulated spcific the unprocesed lamin Howver, inected protei is manner, is thought to have an important role in linking nuclear incorporad into the lanuni does not sti with this antbody, sugesn tbat some of the membras and chromatin to the nuclear lamina and in promoting proceseng seps lamin A undergoes occurs within te lamin foci. Othr resle miggest that reasembly of the nuclear envelope at the end of mitosis. In this lmin B is fone in foci duth e arsociaed with replican chromatin at S pha (Moir et al, J. Cell Bidl. We have initated trnscto expeiment human study, we cloned a cDNA encoding LAP2 to define its molecular 125:1201). using struture nd to analyze functional domains of this protein. The lamin cDNAs teged with the my epiope to fahr addrs die hfncto of die suciresc cDNA sequence revealed that LAP2 is a polypeptide of 452 residues, When a wild-ype, prelamin A cDNA is tanfected into BK21 cells, the majority of the consisting of a large hydrophilic N-terminal portion (residues 1- u_nfcted cells inorpora the expressed protein into the _ndogenous lamina as well into 409) containing several putative CDC2 kinase sites, followed by a nucleoplasmic foci. In addition, a sgnificant fraction of cells (10-20%) at early time points single hydrophobic predicted transmembrane sequence (residues post-transfection (16 bh) overexpres kmi A resel in cytplam aggrgtes of prote. 410-433), and a short hydrophilic C-terminal tail (residues 434-452). Cells conteining aggregates are not in S phss sown by the lack of costaining with the Immunoelectron microscopy of rat liver nuclear envelopes with a DNA replcaton markes BrDu sd PCNA. When overexpe o cells re ridwith LAP2 specific monoclonal antibody demonstrated that the N-terminal a 'anin B antibody, lamia B is not seen in the nuclsus but only in inteonly staning cytplasmic region is located to nucleoplasmic surface of the inner nuclear epote sggestng that the expresd bmin A hs displaced the endog s lami West membrane. Thus LAP2 in suggested to be a type U integrl membrane blots of extas of truncted culturs usng the myc antibody shw two bands:on comigae protein with a large N-terminal nucleoplamic domain and a small C- with thde ndogenon, procesd l-in A and thde soondho a moleular weight terminal lu1enal tail Regions of LAP2 involved in targeting to the migetng it is unproced. Thee reslts gge tat ie ovrexpreo of preamin A and nuclea envelope were analyzed by expressing deletion mutants of the inabiliq to proce the protein resslts in a pathoogical phenotype. We also tanfected tde LAP2 in cultured cells. Two different regions of LAP2 sufficient for procesd form of l1mi A. In contr to the reslts with the unresd form, few cel have concentration at the nuclear envelope were identified within the N- cytoplasmic accuulo ad in most cells tde exprosd protein appe large terminal nucleoplasmic domain. This suggests that the N-terminal mcleoplamic foci and cabb". The lain B sining relatively un cted by this region of LAP2 contains multiple functional domains that phenotp and a highe peenteg of cel replica DNA. The obserVaton lead spport to cooperatively bind to nucleoplasmic components, and thereby di idea that the uclear lemma, especially bum B, ae asociased with ropliycat chmatin mediate sorting of LAP2 to the nuclar lamina. (Moir at al, J. Cell Biol. 125:1201). Supported by NCI grant 31760 342a Proteins of the Nucleus, Nuclear Matrix, and Nucleolus (1985-1990). Tuesday

1985 1986 SUBNUCLEAR LOCALIZATION OF PRELAMIN A CAAX BOX IDENTIFICATION OF PROTEINS WHICH INTERACT WITH NuMA MUTANTS ((M. Sinensky', M. Dalton', R. Khosravi-Far2, P. Kirschmeier3 USING THE YEAST TWO HYBRID SYSTEM. ((S.M. McPherson, N. and K. Fantle')) 1Elenor Roosevelt Institute, Denver, CO 80206, Matova, and M. Snyder)) Dept. of Biology, Yale University, New Haven, CT 2University of North Carolina, Chapel Hill, NC 27599, 3Schering-Plough, 06520-8103. Kenilworth, New Jersey 07033. The .haclear Mitotic Apparatus protein, NuMA, is a large (-240 kDa) coiled- coil protein which localizes to the nucleus during interphase and to the mitotic Mature 72 kD lamin A arises from its 74 kD precursor through four serial apparatus during metaphase in mammalian and amphibian cells. NuMA is post-translational modifications: farnesylation, removal of the carboxyl resistant to high salt, detergent and nuclease extractions, suggesting that it is a of the now terminal component of the nucleoskeleton. Microinjection of NuMA polyclonal terminal 3 amino acids, carboxymethylation cysteine antibodies into interphase, anaphase and metaphase cells has demonstrated that and end eolytic cleavage of the 15 carboxyl terminal, NuMA is important for the formation and maintenance of the mitotic spindle. farnesylated peptide. We have generated mutants in the CAAX box of In order to gain further insight into the role of NuMA in the structure and prelamin A which result in production of proteins that are defective in the function of the nucleus and the mitotic spindle apparatus, we have used the two proteolysis steps. We also have produced a mutant that is two hybrid system to identify proteins which interact with NuMA. In this geranylgeranylated rather than farnesylated. Examination of the subnuclear analysis, a fusion protein between the Gal4 DNA binding domain and NuMA localization of these mutants indicates that, in contrast to observations on was generated and used to screen a human cDNA library fused to the Gal4 activation domain. Of approximately 5.2 x 106 transformants screened, seven non-farnesylated mutants, these mutant proteins appear as intranuclear foci. clones representing a maximum of five genes were found. The proteins The geranylgeranylated mutant does not appear to undergo removal of the produced from these clones interact specifically with NuMA and not with carboxyl terminal peptide and localizes to the nuclear penphery. We control yeast proteins fused to the Gal4 DNA binding domain. Sequence conclude from these observations that: a) the carboxyl terminal tripeptide analysis indicates that two of the interacting proteins are novel, a third has proteolysis is required for the subsequent endoproteolytic cleavage; b) similarity to a nucleotide excision repair protein, a fourth has homology to a famesylation targets preamin A to specific subnuclear sites where yeast protein important for nuclear integrity, and the fifth gene encodes histone H1.2. The finding that NuMA interacts with histone H1 raises the possibility subsequent processing takes place and c) recognition of prelamin A at these that NuMA participates in assembly and/or disassembly of chromatin. These sites is specific for the farnesyl substituent. different NuMA binding proteins are being further characterized in order to understand the role of NuMA in vivo and the function of the nucleoskeleton in various nuclear processes.

1987 1988

STRUCTURAL AND MOLECULAR STUDIES OF A LIKELY DROSOPHILA THE YEAST NUCILOLUS IPLICATED IN mRNA EXPORT NUCLEAR SKELETAL COMPONENT SHOWING BROAD SEQUENCE ((R Schneitert, T. Kadowald2 and A. Tartakoff)), 'Pathology Institute, HOMOLOGY TO HUMAN TPR ((G. Zimowska-Handler'2, J.P. Aris?, H. Case Western Reserve University School of Medicine, Cleveland, OH Saumweber', and M.R. Paddy'2)) 'Center for Structural Biology and 2Department 4106; 2Department of Genetics, Harvard Medical School, Boston, MA of Anatomy & Cell Biology, University of Florida, and 'Biologische Institute, Abt. 02115. Zytogenetik, Humboldt Universitaet zu Berlin. While temperature sensitive mRNA Iansport (m*t) mutants of We have previously described an -180 kD antigen in Drosophila melanogaster which is anayzing localized to the nuclear periphery in interphase, remains concentrated in the chromosomal Sacchwumyces cereviiae (1) we have made several observations which region during metaphase and early anaphase, and isolates biochemically in the nuclear suggest that the nucleolus is on the path of transport of mRNA from the pore complex-lamina fraction'. We have recently extended these observations to include nucleus to the cytoplasm. 1) In wild type yeast cells, polyA+RNA is 3-D immunofluorescence localizations at higher resolution and cloning and sequencing detected in the nucleolus, where it is more concentrated than in the of Bx34 cDNA. CCD-based deconvolution fluorescence microscopy of Drosophila chroman-rich nudceoplasm Control bybridizations demonstrate that the embryonic and salivary gland nuclei clearly shows a nuclear interior localization for in situ hybridization procedure used is reliable and specific. 2) In several Bx34 in addition to the nuclear periphery. We have cloned and sequenced 5.5 kb of mir strains - unlike a strain which is ts for RNA polymerase I - the Bx34 cDNA and have identified an open reading frame that spans the entire 5.5 kb. The nucleolus either fragments or enlarges at 37C 3) In mtr2-1 polyA+RNA 1800 amino acids encoded by this stretch constitute a biphasic protein with the N- terminal 60% containing extensive regions of heptad repeals characteristic of coiled-coil in association with nucleolar fragmentL 4) In ntr2-1, forming proteins. The C-terminal 40% is markedly less structured and shows low to fragentation of the nucleolus occurs only if RNA polymerase II is moderate homology with a number of nuclear proteins. Most of the Bx34 sequence active. 5) In mtr3-1 po}yA+RNA accumulates in association with (1500 of 1800 amino acids) shows significant homology to the human tpr protein. The nucleolar antigens, although the nucleolus does not fragment 6) Bx34 predicted protein secondary structure, interphase and cell cycle localization, and Conditional mutations in the nucleolar proteins MTR3 or RNA sub-nuclear biochemical fractionation pattem are consistent with a nuclear skeletal polymerase I inhibit export of polyA+RNA at 37°C We therefore suspect We are currently pursuing in vivo fluorescence imaging, molecular genetic, and protein. that tramnsport from the sites of tanscition to the nucleolus is rapid and biochemical to Bx34 in Drosophila. approaches that export from the nucleolus is rate-limiting in yeast IPN4S, 89, 2312; EMBO J., 12, 2929; J. Cell BioL (in press) 'Frasch, Paddy, and Saumweber (1988): J. Cell Science 290 247-263.

1989* 199@ PML-CONTAINING NUCLEAR BODIES ARE TIGHTLY ASSOCIATED ELIMINATION OF PML FROM NUCLEAR BODIES BY THE WITH DNA REPLICATED LATE IN S PHASE IMMEDIATE EARLY PROTEIN 1 (VMW 110) OF HERPES ((Ma4jolein Grande, Nico Stuurman, Bas van Steensd, Ieke van der SIMPLEX VIRUS TYPE L. ((G.G. Maul)) The Wistar Institute, Kraan, Roel van Driel and Luitzen de Jong)) E.C. Slater Institute, 3601 Spruce Street, Philadelphia, PA 19104. University of Anmterdam, Plantage Muidergracht 12, 1018 TV Amstrdam, The Netherlands Specific circumscribed nuclear domains (ND10) have been shown to contain several nuclear matrix proteins, among them In the nucleu several compartments can be recognized. The nucleo- PML. Using quantitative evaluation and immunoelectron lus is the most conspicuous one. Other morphologically discenable nuclear microscopy, some of the immunofluorescently defined ND10 are compartments are (i) clusters of interchromatin granules, which are en- shown to be nuclear bodies. Since their number increases riched in several snRNPs and other splicing factors, (ii) coiled bodies, substantially upon interferon exposure of the cells, we asked likewise enriched in snRNPs and (iii) PML-containing nuclear bodies. PML whether virus infection would have an opposing effect. Herpes is encoded by a gene of unknown function involved in the t(15;17) trans- virus type I eliminated ND10 and nuclear bodies, an effect of the location of acute promyelocytic leukaemia. We are intersted in the struct- immediate early set of viral proteins. Using the appropriate ural basis of nuclear organizaon and in the role of nuclear compartment- viral mutants and transfection, we showed that the Vmw 110 ation in replication and the synthesis and processing of (pre)mRNA. Here alone was sufficient to eliminate these nuclear structures. we show by dual label immunofluorescence mioscy that PML bodies in Vmw 110 has a zinc finger motif that is similar to PML, and this T24 (human bladder carcinoma) cells are located close to DNA that is molecular domain is essential for the removal of PML from replicated only at a particular stage in the last half of S-phase. PML bodies ND10; however, a specific area at the COOH terminal of Vmw may also contain RNA. When injected in the cytosol monoclonal antibodies 110 is necssary for targeting the viral protein to the host site. against PML rapidly label the nuclear bodies. This suggests that PML Vmw 110 is not necessary for HSV replication in cultured cells, shuttles between the nucleus and the cytosol. Results are compatible with but is essential for recovery from latency. Its interaction with a the hypothesis that PML bodies are dynamic structures involved in the host nuclear structure that is interferon upregulated suggests metabolism or transport of a special type of RNA encoded by DNA that is that proteins accumulated in nuclear bodies may be part of a replicated in a small time window late in S phase. cellular defense mechanism that HSV-1 can circumvent. *Author(s) and/or presenter(s) have noted that there is a potential conflict of interest. Tuesday. Proteins of the Nucleus, Nuclear Matrix, and Nucleolus (1991-1996) 343a

1991 1"2 CHARACTERIZATION OF NUCLEAR MATRIX-INTERMEDIATE PROTEIN 4.1 IS A COMPONENT OF THE FIBROBLAST NUCLEAR FILAMENT PROTEINS FROM PRIMARY HUMAN OSTEOBLASTS. MATRIX((S.W. Krauss, J.A. Chasis, S. Lockett*, R. Blaschke and N. ((H. Feister, J. Onyia, J. Hock and J. Bidwell)) Departments of Mohandas)) Departments of Cell and Molecular Biology and *Molecular Anatomy and Periodontics, Indiana University Schools of Medicine and Nuclear Medicine, University of California, Lawrence Berkeley and Dentistry, and Endocrinology, Lilly Research Labs, Indianapolis, Laboratory, Berkeley, CA 94720 IN 46202. Protein 4.1 is a multifunctional 80 kD structural protein of the human erythrocyte plasma membrane skeleton where its protein-protein The nuclear matrix (NM) plays a significant role in mediating gene interactions are critical determinants of membrane mechanical stability. expression. The protein composition and organization of the NM are Erythroid protein 4.1 contains binding sites for glycophorin, calmodulin, specific to cell type, stage of development, and may serve as band 3, actin and spectrin. Protein 4.1 is also present in many non- markers for pathologic states. Our objective was to characterize the erythroid cells where multiple isoforms generated by alternative splicing NM and intermediate filament (IF) protein composition of human are tissue-and differentiation-specific but have as yet undefined roles. We osteoblasts. Bone chips were obtained from different parts of the are investigating the structure/function of 4.1 isoforms in human diploid skeleton from heaithy donors of varying age (16 yr-43 yr). fibroblasts. Whole-cell human fibroblast extracts from early passage, Osteoblasts were derived from trabecular explants and grown to replicating cells contain multiple 4.1 isoforms ranging from 45kD to detected by Western blotting. Expression of some 4.1 species first cells were used. The osteoblast 230kD confluence. Only passage differs in extracts of fibroblasts in varying proliferative states. PCR phenotype was monitored by demonstrating alkaline phosphatase analysis of fibroblast protein 4.1 cDNA shows expression of a translation activity. Cells were labelled with 35S-methionine for 1 hr or 24 hrs initiation site capable of encoding >8OkD 4.1 isoforms. A novel fetal just prior to harvest. NM-IF proteins were obtained by a standard fibroblast-specific PCR band also was detected. Immunofluorescence of sequential extraction protocol. 1-D SDS/PAGE profiles of 35S- fibroblasts using IgG's against 8OkD erythroid protein 4.1 and against methionine-labelled, NM-IF proteins were remarkably similar from defined N- and C-terminal 4.1 domains produces "speckled" staining of osteoblasts isolated from rib, mandible and hip. Osteoblast NM-IF the nucleus. In some cases, cytoplasmic staining is also noted. By protein composition was not dependent on the age or sex of the microscopic optical sectioning of the nucleus, immunofluorescent 4.1 that the NM-IF of the foci localize at intranuclear sites and are retained after serial steps of cell donor. We conclude protein composition permeabilization and salt extraction. After the final step of nuclear matrix healthy human osteoblast is independent of skeletal site, age and preparation, DNase/RNase digestion and re-extraction, nuclear 4.1 sex of the donor. This provides a molecular baseline for comparing signals persist. We conclude that some 4.1 isoforms appear to be bone cells from skeletal pathologies. components of the nuclear skeleton in human fibroblasts.

193 199 EXTCRA-CHROMOSOMAL PEREPHERAL MATERLL IN WHEAT PROTEOLYTIC PROCESSING OF Ca/Mg-DEPENDENT ENDOSPERM. ((Burakov, V. V., and Y.S. Chenstov)) Dept of Biol.Cyt. ENDONUCLEASE IN RAT LIVER NUCLEI EXTRACTS IN VITRO. and Hist, Moscow State Univ. Moscow 119899, Russia. (Spon. by P. ((LG.Lebedeva, S.S.Alexandrova, I.I.Votrin, and ANG.Basnakian')) Haris.) Institute of Biological and Medical Chemistry, Russian Academy of Medical Sciences, Moscow 119832, Russia; and 'National Center for Extra chromosomal peripheral material (EPM), or matix, is revealed by Toxicological Research, Jefferson, AR 72079. (Spon. by C.A.Carter.) eectron microscopy wheat endosperm chromosomes at all stages of mitosis. Unlike other objects, in wheat endosperm this thick EPM Calcium and magnesium-dependent endonuclease (CME) is known to layer is most distinct in metaphase and aphae. EPM srrounds each present in cell nuclei of the most of eucariotic species. CME activation chromosome suface and does not penetrate into the chromosome body. seems to be the major event in apoptosis, although the mechanism of the EPM has a loose sponge-like stucture and consists mainly ofribosome-lIke regulation of the CME activity is not yet identified. We examined DNA granules and diff fibers positively stained by the Banhard method. endonucleases in rat liver nuclei extract by SDS-polyacrylamide gel There is a strict paralleism between change in pophase mcleo and EPM electrophoresis followed by the zymogram method. Four polypeptides of development. This sggests that the structure of EPM incorporates RNP 120, 54, 31 and 28 kDa, which have the endonuclease activity, were shown material ofthe nucleolar and extra-chromosomal origin In the telophase the to occur in the extract isolated in the presence of proteinase inhibitors. as well opposite incorporaion of EPM into the stwuce of newly organized Isolation without proteinase inhibitors, as autodigestion at 37°C, nucleoli i obseved. or long storage, resulted in the multiplication of active polypeptides in extract. The autodigestion was also associated with the increase of CME activity. Limited trypsin pretreatment of nuclear extract led to the appearance of DNase > 140 kDa, indicating the existence of a potential endonuclease preacrsor in the nuclear extract. All CME isoforms require Ca, Mg2e or Mn2+ ions alone, but the highest activity can be achieved by the combination of Ca2+ Mg2. Basing on the data, limited proteolysis is proposed as a possible mechanism for the regulation of the CME activity in apoptosis.

1995 1996 PURIFICATION AND CHARACTERIZATION OF AN ENDONUCLEASE THE GYLCINE/ARGININE-RICH (GAR) DOMAIN OF HUMAN FIBRILLARIN ACTMTY WHICH PREFERENTIALLY CLEAVES THE G-RICH CONTAINS A NUCLEAR TARGETING SIGNAL. ((E.A. Williams, M. IMMUNOGLOBUUN SVITCH REPEAT SEQUENCES. ((C. Lyon-, G. A. Swindler, and N.E. Christensen)) School of Biological Sciences, NO 64110. Miranda, and R. J. Agulira-)) DePartment of Biology and Molcular University of Missouri-Kansas City, Kansas City, Biology Insitut, Univesty of Cirnia, Los Angeles, CA 90024-1606. The nucleolar protein fibrillarin plays a key role in the processing of ribosomal RNA and the assembly of ribosomes in Immunol ob-uli (Ig) witch (S)regin ae compoe of highiy repet ve eukaryotic cells. Fibrillarin has an amino terminal squences which lI upsbemn of the 1g conant region genes. The glycine/arginine-rich (GAR) domain in which all of the arginine murine rgion, which is approximadty 2 kb in length, is residues are methylated. Similar domains have been observed in composed of ta nm p a repet of many other nuclear proteins, most of which localize to the TGGGG(TGAGC)23. Thes rpeat ae frequently obsered at switch nucleolus. In order to test whether this domain functions in and ae belived to be the nuclear and/or nucleolar localization, the GAR domain of human

dand ated an fibrillarin has been cloned in frame onto the amino terminus of and in HeLa cells. acyt which preferentially cleve at switch G-rich peners. We hae £. coli p-galactosidase transiently expressed a combination of a enzyme assay determined don g2+ Using p-galactosidase .iD gtu that uclease M for fu specific actit. and anti-p-galactosidase iiunofluorescence, the localization of and alterate dvalent colionis are unable to restor fuli specific ,-galactosidase versus the GAR/p-galactosidase fusion protein act. We hae chaacterizd the specf of ths nuclease has been examined. Results show that the GAR domain of human and ha found ht k can produce both single nick as wel as fibrillarin contains a signal sufficient for the localization of doublesrnded cuts at witch ptms. This ndonu sdact P-galactosidase to the nucleus. However, the fusion did not wa purified close to hoimogeneIt by column o y and w localize to the nucleolus indicating that information present in found to exhibit a mol r manss of approxitely 20 kDa. Due to the the C-terminal region of fibrillarin is required for nucleolar specificit of dth enzyme for switch repeats, w beliv this nzyme localization. These findings support the idea that the GAR is itimy asoaed with the switch ecomination proess. domain functions, at least in part, in nuclear localization. 344a Proteins of the Nucleus, Nuclear Matrix, and Nucleolus (1997-2002). Tuesday

1997 1998 OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE DNA AND ATP BINDING PROPERTIES OF PROLIFERATION-ASSOCIATED THE MURINE NUCLEOLIN PROTEIN. ((G. A. Miranda, I. Chokler, and R. J. CHARACTERIZATION OF Aguilera)) Department of Biology, Division of Molecular, Cell, and NUCLEOLAR PROTEIN P120. ((Ren, Y, E. Durban, C.W Taylor, Developmental Biology, University of Califomia at Los Angeles, Los Angeles, C. W. Gustafsort B. Valdez, R. Busch, and H. Busch)) Dept. of CA, 90024-1606. Phamacology, Baylor College ofMedicine, Houston, TX 77030) A 100 kDa DNA binding protein was found to be dramatically up-regulated upon of B is a nucleolar detectable in the mitogenic stimulation murine splenic lymphocytes with bacterial Protein p120 proliferation-associated protein lipopolysaccharide. In vitro phosphorylation experiments revealed that this early G, phase of the cell cycle and peaking in S phase. A variety of human DNA binding protein was one of the most heavily phosphorylated proteins in malignant tumor cells contain a much higher level of p120 than normal both lymphoid and non-lymphoid nuclear extracts. Although this in vitro resting cells. A full length p120 cDNA clone was inserted into a phosphorylation initially appeared to be mediated by a potent nuclear kinase baculovirus vector to overexpress p120 protein in Sf9 insect cells; the activity, it was later determined that a significant part of the detected labeling recombinant p120 comprised about 20% of the total cellular protein by was due to the direct binding of ATP by the 100 kDa protein. Anfibodies raised to the 100 kDa DNA binding protein were used to isolate cDNA clones from a mM Tris mM 0.5 mM densitometry. By DOC (10 (pH8.0)/10 KCVI MgCWI lymphocyte cDNA kgtl 1 expression library. Nucleotide sequence analysis 0.15% deoxycholate) extraction of nucleoli, the recombinant p120 was revealed that the cloned cDNAs were identical to the mouse nucleolin gene. 90% pure. By indirect immunofluorescence, most of the recombinant p120 The 0 galactosidase fusion proteins (encoded by exons 3-14 of nucleolin) and was localized in a large nucleolar mass which was also stained by Azure-C a more severely truncated 45 kDa protein (encoded by exons 5-14 of Like endogenous p120 in HeLa cells, recombinant p120 expressed in insect nucleolin) were both found to bind strongly to both DNA and dATP. Furthermore, the native and recombinant nucleolin proteins were found to bind cells was phosphorylated in vivo. On sucrose density gradients, both ATP, dATP, GTP, and dGTP but not dCTP, dTTP, or dUTP. Nucleolin was endogenous p120 from HeLa cells and recombinant p120 from insect cells also found to bind preferentially to single stranded G- and C-rich sedimented in the 60-80S region, in which, preribosomal particles oligonucleotides (18-mers) but bound very poorly to oligo dA and oligo dT (18 sedimented using similar extraction and centrifugation procedures. The mers). Computer sequence analysis of the nucleolin gene revealed that the sedimentation of p120 shifted to 5-lOS by treatment with high salt or with putative ATP binding domains may fall within two of the phylogenetically conserved RNA binding domains of nucleolin. RNAse suggesting p120 is linked to an RNA. A study of interactions of p120 with other macromolecules is in progress.

1999 2000 YEAST NP146 ENCODES A NOVEL PROLYL CIS-TRANS CHARACTERIZATION OF A NUCLEAR MATRIX PROTEIN ISOMERASE THAT IS LOCATED IN THE NUCLEOLUS. ((X. Shan, DETECTED IN THE URINE OF PATIENTS WITH BLADDER Z. Xue and T. Melese)) Dept. of Biological Sciences, Columbia CANCER. University, Fairchild Bldg. New York, N.Y. 10027; (212)854-5443. ((R.Szaro, M.Halvorsen, H.Yankelev, AOreper, B.Lentrichia, Y.-J.Wu, We have identified a gene encoding a novel Nucleolar Proline Isomerase J.Briggman)) Matritech, 763 Concord Ave., Cambridge, MA 02138 (NPI46). Sequence analysis shows that NPI46 has a highly acidic N- terminus and is homologous to the yeast nucleolar protein NSRl and, to A two-site immunoassay, employing two monoclonal antibodies two other mammalian nucleolar proteins; nucleolin and Noppl4O. Like prepared against a fraction of nuclear matrix proteins (NMPs) from a NSRI, nucleolin, and Noppl40, NPI46 binds in vitro to an affinity human line, detects a NMP in the urine of patients with column containing a wild-type nuclear localization sequence (NLS), but bladder cancer. The objective of this study was to characterize the shows little or no affinity for a mutant NLS affinity column. The C- antigen detected in urine and compare it to antigen purified from a terminal domain of NPI46 is homologous to a family of proteins known human bladder cancer cell line. Western blots of the antigen purified as FK506 binding proteins or prolyl cis-trans isomerases. NPI46 is fom the J82 bladder cell line revealed a doublet at 99 kD, pl 5.7 and capable of catalyzing the prolyl cis-trans isomerization of two small 104 kD, pi 5.8. Immunoassay of FPLC fractions of urine detected synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinyl- antigen activity at >300 kD and 19 kD. FPLC of antigen purified from Ala-Ala-Pro-Phe-p-nitroanilide as measured by a chymotrypsin-coupled J82 showed only the high (>300 kD)molecular weight component, spectrophotometric assay. The diversity of the functional domains of the whereas FPLC of a freeze/thaw preparation of J82 cells produced a nucleolar NLS-binding proteins makes it unlikely that they are a family high and low molecular weight profile equivalent to that found in urine. of general nuclear import receptors. However, these proteins could be The epitopes are sensitive to both periodate and trypsin treatment. An involved in the process of ribosome biogenesis. NPI46 is phosphorylated immunohistochemical survey of frozen human tissues revealed a by a kinase activity in the yeast nucleus. The phosphorylation of the nuclear localization of antigen in normal urothelium and bladder tumor protein is required for its tight association with nucleolus. We are as well as other tissues. The smaller (19 kD) form of the antigen currently searching for proteins that interact with NP146 by both detected in the urine of patients with bladder cancer may be a fragment biochemical and genetic studies. of the protein purified from the J82 cell line.

2001 2002 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL NUCLEOLAR PARTIAL CHARACTERIZATION OF A HUMAN NUCLEAR SHUTTLING PROTEIN. ((R.L. Ochs, T.W. Stein, Jr., M. Ruutu., and E.M. Tan)) Dept. 85 KD PROTEIN. ((M. Paulin-Levasseur and M. Julien)) of Department of Biology, University of Ottawa, Ottawa, Molecular and Experimental Medicine, The Scripps Research Institute, Ontario, Canada, KlN 6N5. 10666 N. Torrey Pines Rd., La Jolla, CA 92037 and Fourth Dept. Surgery, University of Helsinki, Helsinki, Finland. The karyoskeleton comprises an intricate network of scaffolding proteins which have been suggested to play In an initial study of antinuclear antibodies in the chronic inflammatory major roles in the functional organization of the bladder disease, interstitial cystitis (IC), we reported that 7/96 (7%) IC nuclear compartment. We report here on the partial patients studied had autoantibodies to the nucleolus (J. Urol. 151:587- characterization of a human nuclear antigen using a monoclonal antibody, referred to as 2A7, that has been We now an serum a 592, 1994). report that using autoimmune from produced against the urea extract of Triton X-100 patient with IC, we have identified and partially characterized a novel skeletons from HeLa S3 cells. By immunoblotting and protein doublet of approximately 55 kDa M.W. (hereafter referred to as immunofluorescence, this antibody was found to react No55) localized to the granular component of the nucleolus. No55 was with a basic 85 kD human protein which, during inter- initially characterized by a diffuse nucleolar immunofluorescence in phase, is distributed throughout the nucleoplasm and interphase cells and by Western blotting as a 55 kDa doublet on whole- can be observed also in nucleoli. No reactivity was cell extracts. Affinity purification of antibody from the 55 kDa bands detected in cells from other species. The targeted protein was seen in close association with the chro- confirmed its nucleolar localization. During mitosis, No55 was mosomes during mitosis. Immunofluorescence monitoring associated with the chromosomes and appeared in prenucleolar bodies of the 2A7 antigen during in situ isolation of nuclear during telophase. Immunoelectron microscopy revealed that No55 was matrices from HeLa cells demonstrated that the nucleo- localized uniformly throughout the granular component of the nucleolus, plasmic fraction of the protein is rendered labile compared to a more peripheral localization of nucleolar protein B23. after treatment with detergent and salts while the Upon segregation of the nucleolus with 1 jg/ml actinomycin D for 4 hrs., nucleolar fraction resists detergent, salt extraction No55 remained with the granular component of the segregated and DNase digestion, being released only upon RNase digestion. Using the technology of interspecies hete- nucleolus, while protein B23 was found dispersed throughout the rocaryons, the 2A7 antigen was shown to shuttle bet- nucleoplasm. Therefore, based on molecular weight. nucleolar ween the nucleus and the cytoplasm. Our data suggest sublocalization and response to actinomycin D, No55 is a novel protein an involvement of the 2A7 protein in the process of of the interphase nucleolus. Supported by NIH grant P01-DK42717. RNA transport.(Supported by MRCC and NSERCC) Tuesday. Proteins of the Nucleus, Nuclear Matrix, and Nucleolus (2003-2004) 345a

2003 2004 BIOCHEMICAL CHARACTERIZATION OF THE INTRANUCLEAR SPECIFIC DNA SEQUENCES ASSOCIATED WITH THE SPERM STRUCTURE THAT RESPONDS TO MACROMOLECULAR NUCLEAR ANNULUS. ((Jocelyn de Lara and W. Steven Ward)) Robert CROWDING. ((G.R. Rosania and J. Swanson)) Department of Cell Wood Johnson Medical School. (Spon. by W. S. Ward) Biology, Harvard Medical School, Boston, MA 02115. Nuclei of saponin-permeabilized macrophages expand and We have previously described the identification of a novel nuclear contract reversibly in response to changes in the size and concentration structure in hamster sperm nuclei termed the nuclear annulus, which of surrounding uncharged solutes. We have characterized the anchors the entire to the base of the tail when the nucleus is intranuclear structure responsible for these changes in nuclear volume. decondensed. The function of the sperm nuclear annulus is unknown, Nuclei extracted with 0.5M NaCl or 1% Triton X-100 shrank in the but one possibility is that it is an organizing center for the sperm presence of a variety soluble polymers of 200 mol. wt. or greater, but chromatin during the reorganization of the nucleus after penetratration unlike unextracted nuclei, they did not swell in the presence of these of the egg. Efforts to isolate the sequences of DNA associated with the solutes at lower concentration. This observation implicates at least one extractable structural component involved in nuclear expansion. To annulus have failed because the annulus is resistant to proteoloysis and test whether chromatin, DNA, or the nuclear matrix were involved in SDS solubilization, preventing standard DNA purification techniques. nuclear swelling or shrinkn histones and other soluble nuclear proteins We have overcome this problem using degenerate oligonucleotide were extracted with 2M NaCl, and the remaining nucleoids were primed PCR (DOP-PCR). Isolated nuclear annuli were used as digested with DNAse I or micrococcal nuclease. After these digestions, templates for DOP-PCR, a technique which amplifies random fragments the residual nuclear matrix shrank in high solute concentrations. of genomic DNA using degenerate primers and low stringency nudeoids failed to shrink. naked DNA the Undigested Thus, expands conditions. This DNA was then labelled with nucleus and prevents nucleoids from shrinidng. Shrinkage of undigested hybridization amplified nucleoids could be restored by pre-incubating them with high speed biotin and used as a probe for fluorescent in situ hybridization (FISH) supernatant of Xenopus egg extract, purified histones, or by including with decondensed sperm nuclei. DNA amplified from nuclear annuli polyamines in the test solutions. These experiments suggest that exhibited a background of fluorescence throughout the sperm nuclei, changes in nuclear volume in response to changes in the size and but also showed a high degree of reactivity with the annuli. Control concentration of surrounding solutes depend on the contractile DNA that did not contain nuclear annuli showed no reactivity. These of the nuclear matrix. Histones and properties other fixed positively data suggest that there are specific sequences associated with the charged molecules may facilitate nuclear shrinkage by counteracting the electrostatic repulsion of negative charges along the DNA backbone. sperm nuclear annulus. (Suppd. by NICHD HD28501, and the Edwin A. Beer Award) Fertilization II: Sperm Activation (2005-2008)

2005 2006 CAPACITATION OF MOUSE SPERMATOZOA I: CORRELATION BETWEEN THE CAPACITATION OF MOUSE SPERMATOZOA II TYROSINE PHOSPHORYLATION CAPACITATION STATE AND TYROSINE PHOSPHORYLATION. ((P. E. Viscont, J. AND CAPACrrATION ARE REGULATED BY A cAMP-DEPENDENT PATHWAY. Bailey, G.D. Moore, D. Pan, P. Ods-Cwkel and GS. Kopf)) Div. of Reprod. Biol., Dept ((PE. Visconti, GD.Moore, J.Bailey, PLeclerc, D1Pan, P.Olds-Clarkel and G.S.Kopf)) OB/BYN, Univ. Penn., Phila, PA 19104 and IDqeL AnatomyUCel Biol. Temple Div. ofReprod. Biol., DepL OB/GYN, Univ. Penn, Phila., PA 19104 and 1DepL Univ.,PhihL, PA. 19140. Anatomy/Cell Biology, Temple Univ.,Phila, PA 19140 (Spon.by B.Storey)

TIlnmocularbasis of mammali_ sperm capciation, defined functiony as In the report (Viconti et al., these abstacs) we denstrated that tdose poceaes that conferon the spm the acqusio of frilizaon-competen either in the etyi phophorytion of a subset of mouse sperm protim appeared highly vivo in te female reproducv twact or in viro, is poorly tndestood We demonsta her correlied with the capactatn stae of the sperm. Te mochnism by which this protein ttat capyation of cautal epididymal mouse sperm insitro is ied by a tme- tyroie phosphoryation is regulated during this process is not clear. Caida epididymal dqpedent increase in the psein ensine phohylation of a subset of proteins ranging sperm, when inubated in media devoid of NaHCO3, CaC12 or bovine serum albumin from Mr 40,000 to Mr 120,000. Incuba1o of sperm in media devoid of bovine semm (BSA) do not display theated incrases in protein tyrsine lbumin (BSA), CaC12 or NaHCO3, components which individually ar requred for phospherybt TIis NaHCO3, CaC12 or BSA requirement for protein tyrosine capacitmion, prevent the sperm from underging capacitation as assessed by the ability of posphorylation can be completely overcome by the addition of biologically active, but not the cdls to acquire the patern B chltetracycline fluorescence, to undergo the zona inactive, cAMP analogues, suggesting that cAMP, in sone manner can up-regulate the pellucida-induced aosome reacton and, i some cases, to frilize metaphase II-arrested tyroasne phosporylatin of this subset of sperm proteins. Likewise, addition of the active egg in vitro. In each of tese cases the proin tyrosine phosphorylation of the subset of cAMPanagus to sperm incubated in modia devoid of NaHCO3, CaC12 or BSA capactation-ssociated proteins does not occur. Pin tyroe plosphorylation of dtese ovecame the inability of these media to support capacitation, as assessed by the ability of pwtcua rpromsa, as well as sperm capwacit , can be recovad in modia devoid ofeach the cells to acquire the panta B chlotetracycine fluorescence and to undergo the zona of these the consuents (BSA, CaC12 or NaHCO3) by adding back the approprate pelacida-induced acroaome reaction. The effects of the cAMP analogues to enhance in a concenton-dependn maimer. Th requiementofNaHCO3 fr thes proin tyrsine phosphorylaio and to promote capacitation appeared to be at the level of phoaphoy ations is not due to an akainiato of intr lael sperm pH or to an i the cAMP-dependentprotein kinase, since a specific inhibitor of this enzyme (H-89) in meds pIH Capatepdymal spem, which lak the ability to undergo capaciaon in bbcked capacitation, as aessed by the aforementioned endpoints, and the capacitation- in trne is iro, do nt display te subset of tyrotine phosphorylaled proteins dependent nreaes protin phosphrylaton a concnttion-dependent in complete media even afer extended incubaton peiods. These data suggest that protein manner sperm incubated media suorting capacitaton. eserults provide furher tyosine pho oybaion in sprm, may reesnmtan important regulaty pathway that may evienc for the intrreltionship between prtei tyrne phosphorylation in mouse sperm uitimately modulate events assoated with capacitai. Suppored by NIH (GSK and the appeatnce of the capacised state. They also demostrate that both prtein POC) and The Rockefell Foundion (GDM and PEV). tyroine phosphorylation and capacitation appear to be regulated by cAMP/protein kmase A. Te tp-regulation of protein tyrosine phosphorylation by cAMP/protein kinase A in sprm is, to our knowledge, the first demonsmton of such an interelationship between trosne and protein kinas A signaling pathways. Supported by NIH (GSK and POC), NSERC (PL) and Rockefelle Foundation (GDM and PEV).

2007 2008 LOCALIATION OF PROGESTERONE-MEDIATED CALCIUM INFLUX DIFFERENT CHLORIDE CHANNELS ARE INVOLVED IN THE AND DETETION OF PROGESTERONE-MEDIATED CHLORIDE PROGESTERONE- AND ZONA-INITIATED PORCINE SPERM EFFLUX IN HUMAN SPERM. ((S. Meizell, K.O. Turnerl, and R. ACROSOME REACTION ((C.S. Melendrez and S. Meizel)) Department of Nuccitelli2)) Department. of Cell Biology and Human Anatomyl and Section of Cell Biology and Human Anatomy, University of Califomia, Davis, CA 95616. Molecular and Cell Biology 2, University of California, Davis CA 95616. C1- is essential for the mammalian sperm acrosome reaction (AR). Previous The progesterone (P)-mediated human sperm acrosome reaction (AR) requires studies by this lab indicated that a unique steroid receptor/Cl- channel, Ca2+ influx and Cl- flux (whether influx or efflux is not known but a steroid resembling but not identical to a GABAA receptor/CI- channel, is involved in the receptor/Cl- channel resembling a GABAA receptor/Cl-channel appears to be progesterone-initiated human AR. Here, we investigated: 1) whether CE- is involved). This report describes two studies with human sperm capacitated in essential for the porcine sperm AR initiated by progesterone (P) and heat vitro that investigate: 1) whether P increases the cytosolic [Ca2+] in the sperm solubilized porcine zona pellucida (Z); 2) the effects of Cl- channel antagonists head during AR initiation and, if so, the initial site of the sperm head CaZ+ on the P- and Z-initiated AR. Percoll gradient-washed sperm from boar increase; 2) the effect of P on sperm cytosolic [Cl-] and whether any change in ejaculates were diluted to 12 x 106 sperm/ml and capacitated for 4 hr at 390C in that [C1-] involves the Cl- channel mentioned above. l)We have used a Hepes-buffered medium. Capacitated porcine sperm were washed in a C1-- fluorescence ratio-imaging of fura-2 loaded sperm to study the P-mediated deficient medium, and resuspended in that medium or in a Cl-containing increase of intracellular Ca2+. After addition of 1 pM P, Ca2+ in acrosome- version, followed by 10 min treatment with P (1 pg/ml), Z (150 ±g/ml) or reacting sperm increases transiently beginning in the mid-head region and solvents. AR were assayed in viable sperm only (FITC-Pea lectin/Hoechst rapidly spreading anteriorly over the head within 1 second (n=2, total stain method). Neither P nor Z initiated the AR in the C1-deficient medium but sperm=5). Sperm which do not undergo the AR exhibit a slower Ca2+ both did so in the CE--containing medium. Moreover, capacitated sperm increase. 2) We have also loaded capacitated human sperm with the fluorescent preincubated for 5 min with one of two GABAA- and glycine receptor/Cl- Cl- probe MEQ to investigate P-mediated Cl- flux by spectrofluorometry. At channel blockers, picrotoxin and pregnenolone sulfate (200 pIM), failed to least 80% of the total MEQ in sperm was cytosolic. P (1 pM) decreased sperm undergo the P- or Z-initiated AR. Preincubation with strychnine, a drug known [Cl-]i suggesting that the steroid caused an efflux of Cl- (n=4). Preincubation to antagonize activation of neuronal glycine receptor/ClE channels at [nM] and of sperm with picrotoxin (200 pM), an antagonist of GABAA receptor/Cl- neuronal GABAA receptor/ClE channels at [gKM], inhibited the Z-initiated AR at channels and an inhibitor of the P-initiated AR, completely inhibited the P- 50 nM, 100 nM, 250 nM and 1 pM but required 1 pLM to inhibit the P-initiated mediated efflux (n=3). These results suggest that P-mediated Cl- efflux is AR. Glycine (225 IIM) also initiated the AR and, like Z, was inhibited by all involved in the AR and that the efflux occurs via a sperm steroid receptor/Cl- four levels of strychnine tested. These results are the first to suggest that P- and channel resembling a GABAA receptorCl-channel. Z-initiation of the AR involve different CE- channels and that the latter requires a (Supported by NIH grants HD- 19966 to RN and HD-23098 to SM). putative glycine receptor/CEl channel. (Supported by an NIH-MARC predoctoral fellowship to CM and NIH grant HD-23098 to SM). 346a Fertilization II: Sperm Activation (2009-2014). Tuesday

2009 2010 HAMSTER SPERM MOTILITY HYPERACTIVATION IS SUPPRESSED IDENTIFICATION AND LOCALIZATION OF THE INOSITOL BY INORGANIC CALCIUM CHANNEL BLOCKERS. ((S.S. Suarez and TRISPHOSPHATE RECEPTOR IN MAMMALIAN SPERM. X.B. Dai)) Department of Anatomy, College of Veterinary Medicine, ((L.D. Walensky and S.H. Snyder)) Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD, Cornell University, Ithaca, NY 14853. (Spon. by C. Fewtrell.) 21205. In mammalian sperm, hyperactivation can facilitate passage through viscoel- Calcium flux is central to sperm motility, hyperactivation, and the astic substances and the zona pellucida. It is characterized by high-ampli- acrosome reaction. We investigated whether mammalian sperm tude, asymmetrical flagellar beating. In hamster sperm, the levels of possess IP3-gated calcium channels that may participate in calcium intracellular Ca2+ (Ca.) for different states are: acrosome-reacted > hyperac- dynamics. Scatchard analysis of 3H-IP3 binding to rat sperm tivated > activated. While it has been membranes revealed a curvilinear plot with high affinity (Kd=26 shown that L-type calcium channels nM, Bmex=30 pmoUmg) and low affinity (Kd=1.6 pM, Bm.=550 are involved in raising Ca, during the acrosome reaction (Florman et al, Dev pmoUmg) sites. Western blots using polyclonal antibodies directed Biol 152:304;1992), the role of Ca2+ channels in regulating hyperactivation against purified brain IP3 receptor and type- I P3 receptor peptide is not known. The effects of various channel blockers were tested. Inorg- sequence both identified a 260 kD band in 1% Triton X-100 extracts anic blockers dampened the flagellar bend, measured as a significant in- of rat sperm membranes. Indirect immunofluorescence confocal crease (*P< 0.001) in flagellar curvature ratio from the hyperactivated state microscopy localized the IP3 receptor to the proximal middle piece and the acrosome cap of rat sperm. Immunoelectron microscopy (activated: 0.81+0.02, hyperactivated: 0.40+0.03, hyper+lmM CdCl2: confirmed the acrosomal localization. Immunofluorescent staining 0.75+0.02*, hyper+lmM NiCl2: 0.75±0.04*, hyper+lmM CoCl2: of the anterior acrosome was additionally observed in hamster 0.45±0.06; n =3, 20 sperm/treatment). The effect of CdCl2 was reversible. sperm. The distinct localizations of the IP3 receptor in rat sperm The organic L-channel blockers diltiazem, isradipine, and verapamil were suggest a role for this channel in regulating motility and mediating tested at doses up to 100uM. Only verapamil at 100 1sM depressed hyperac- calcium flux required for the acrosome reaction. tivation, and did so in a reversible manner; however, this dose can affect other ion channels. Thus, although blocking entry of Ca2+ suppresses hyperactivation, there is as yet no evidence that L channels are involved as they are in the acrosome reaction. This may explain why Ca, is higher in acrosome-reacted sperm than in hyperactivated sperm. NIH HD19584.

2011 2012 LOCALIZATION AND QUANTITATION OF INTRACELLULAR PROTEIN K[NASE C-STIMULATION OF NA/H ANTIPORTERS IN ASCIDLAN CALCIUM AND pH IN ACROSOME INTACT MOUSE SPERM. ((S.B. SPERM ACIIVATION. ((L.T. Woods, KM Allen, and RA Koch)) Depaitment of Herick and R. A. Cardullo)) Department ofBiology, University ofCalifornia, Biological Science, California State University, Fullerton 92634-9480. Riverside, CA 92521. In the sea squirt Ascidia ceratodes, sperm activation is characterized by mitohondrial translocation (MM), a process dependent on a rise in intracellular Ca Changes in intracellular [Ca2+] and pH have been postulated to play a role in ions We hypothesize that sperm-egg inteaction stimulates MTL via a acrosomal exocytosis (the acrosome reaction). In this study we used the pH and diacylglycerol(DAG)-activated protein kinase C (PKC) that stimuates Na/H Ca2' specific fluorescent indicator probes, BCECF and FURA-2, to determine the antporters. Exchange of Hi for Nao cus pHi to rise which is linked by an localization of these dyes in acrosome intact and acrosome reacted sperm as well unknownmechanim to extern Ca entiy via Ttype Ca channels, Activation can be artificially stimulated by pladng sperm into pH seawaer. The function of as the levels ofpH and Ca2" in these cells. Populations of mouse sperm were high Na/H antiportes was establhe by exprim with the aniloride derivatives, greater than 85% acrosome intact as determined by a histological assay using HMAandMA,opo knwntoinoibit the antiporter. Sperm were exposed to Coomassie Brilliant Blue. Sperm, at a concnttion of 10'sperm/ml, were HMA (25 pM or MIA (12.5 in pH 9.4 artificial seawater (9.4ASW) for 3 min. incubated in capacitating media in the presence ofthe AM forms ofthe probes HMA and MIA inhied 9.4ASW-triggered sperm acivation by 60-90%. In sodium- for intracellular loading. Extracea fluorescence was quenched using 150 pM free, lithium-eubstituted ASW (LIASW), 9.4ASW-activation was 50% of normal, an effect MnCl2. Under these conditions, video enhanced fluorescence microscopy completely bbdced by HMA and M[L To test the role of PKC n sperm activation, sperm waee iuubated with the phorbol ester PMA (5-20 pM, a revealed that the dyes were locahzed within the acrosomal crescent and midpiece compound known to stimulate PKC, for 3 am. In nonmal Ca ASW (10 mM Ca), ofviable mean sperm. The intracellular levels ofpH and Ca2e were quantified in peta sperm activation increased 3.6-fold over control, whereas in lw Ca sea a fluorimeter at 37°C. Initial estimates using BCECF revealed that the (1 mM Ca), the increase was reduced to 1.6-fold. The PKC inhibitor H-7 (10 intracellular pH of acrosome intact sperm was 6.5. However, we have and M1A each compltely blocded PMA (20 #M) activation. Finally, the DAG OAG pM, determined that even slight increases in viscosity decreases the fluorescence analog, (100 caused sperm activation up to 4fold over controls Tsken excitation ratio (495/430) resulting in artlactually low values ofcytoplasic pH. toether, these data support our model by e the preme of both PKC and Na/H "ntiporters as well as a role in sperm activation. The fact that removal of Ca Similarly, intracelhlular levels offree calcium in acrosome intact sperm using reduced the percent sperm activation by PMA supports the idea that PKC stimulates were + FURA-2 found to be 135 25 nM (n=6) using the published K, of224 nM. Na/H antiporter opertion causing a in pHi, the opening of Ca channes, and This study establishes baseline levels for pH and [Ca2el and will allow us to entry of extmn Ca. And, PKC simulation byOAG supports its position in a DAG- quantiflj changes in these levels in response to vaious sacretagogues and mediated cell igunling cawcade. (Supported by CSUF DAC Grant to LIW, ASCB pharmacological agents. Supported by the Whitaker Foundation (to RAC). Sunun Research Feowsips to KMA, and NIH R15 HD28229 to RAK)

2013 2014 CHARACTERIZATION OF MOUSE SPERM GPI-LN4KED ENERGY SUBSTRATE REQUIREMENTS FOR HAMSTER SPERM HYALURONIDASE. ((C.D. Thaler and RA. Cardullo)) University of CAPACITATION AND ACROSOME REACTION. ((J. Stewart-Savage)) California, Dept. ofBiology, Riverside, CA 92521. Departnt of Biolgical Sciences, University of New Orleans, New Orleans, LA 70148. Glycosyl-phosphatidyl-inositiol-linked (GPI-linked) proteins are widely The energy distributed among ukaryotic cells inluding mammalian sperm where they have requirement for the acrosome reaction (AR) was determined by coincubating eggs and fully capacitated sperm (4 hr In TALP with 9 a putative role in filizaon (Lin, et aL, PNAS 90:10071(1994)). We have mg/ml BSA) for 1 hr in various media, all lacidng BSA, and then determining the characterized a number ofmouse sperm surface proteins that are released by penetration rate. Sperm were able to penetrate 30-45% of the eggs in PI-PLC, including a GPI-linked hyahuronidase (GPI-Hyase) on acrosome intact medium without any sugars (NO) or with only 5 mM glucose (GLU) present; acrosome and reacted sperm. The GPI-Hyase has an apparent molecular 90% penetraffon occurred in medium containing > 1 mM lactate (LAC). weight of 54 kD and appears to be a ingle polypeptide as analzed by size Hyperactivated motility (HA) of fully capacitated sperm disappeared within exclusion HPLC under reducing and non-reducing conditions. The GPI-Hyase 30 min of transfer to either NO or GLU, but not in LAC. The energy ia inhibited by the flavone deivative apigemn, a competitive inhibitor ofHyases. requirements for capacitation was determined by preincubafing sperm in Inhibition ofthe GPI-Hyase was linear over the soluble concenration range of various media, all containing 9 mgfmI BSA, and then determining the motility over apigenin (up to 250 pM). Incubation ofthe GPI-Hyase with solubilized zonae index 6 hr and their ability at 4 hr to penetrate eggs within 1 hr in 1 mM LAC. Sperm moflity was always in NO, and they were In pellucidae (ZPs) minished the enzyme activity of the Hyase. sluggish sluggish GLU for the first 4 hr and then motility picked up. Intact and acrosome reacted (AR) fixed sperm bind equal amounts of Sperm did not develop HA motility in GLU or LAC, but did so In GLU-LAC. Sperm head association solubilized i2I-ZPs. suprisly, to But, equihbrium binding of i251-ZPs either (comets) occurred in all media containing BSA, but comet breakup did not intact or AR fixed sperm was increased by when sperm were -50°/. occur in NO and was delayed 1 hr in both GLU and LAC, as compared to preincubated with 200 pM apigenin. Increased binding of'2I-ZPs in the GLU-LAC. When assaying capadtation, no penetration occurred In NO or presence ofapigenin may suggest that this compound aters the conformation LAC, 65% penetration occurred GLU, and 90% penetration occurred in ofthe GPI-Hyase such that ZPs have greater access to binding sites in the GLU-LAC. Thus hamster sperm capacitaton requires GLU and is faciltated presence ofapigenin. These data suggest that the GPI-Hyase may play a role by LAC. The AR of fully capacitated sperm does not require any exogenous in both pe n ofthe cumuhls mas as well as in sperm-zona binding. sugars, but LAC is required for maintenance of HA motility needed for zona Supported by NIH HD27244 and The Whitaker Foundation. penetraton. Tuesday. Fertilization II: Sperm Activation (2015-2019) 347a

2015 2016 TEE ACROSOME REACTION IN DIGITONIN-PERMEABILIZED SEA PORCINE SEMINAL PLASMA CONTAINS A HIGH MOLECULAR URCHIN SPERM IN THE ABSENCE OF THE NATURAL INDUCER. WEIGHT FACTOR THAT INHIBITS THE PORCINE SPERM ((J. L6pez-Godinez, L. Castellano, G. Aldana, L. ACROSOME REACTION. (( E. Bonilla, R. Velasco, E. Garcia de De la Torre, A. Darszon and J. Garcia- Casas, Y. Ducolomb, and M. Betancourt )). Soto)) IIBE, Fac. Quimica, Univ. de Guanajuato and Universidad Aut6noma Metropolitana-Iztapalapa. Dep. Inst. de Biotecnologia, UNAM, M6xico (Spon. by Eva Ciencias de la Salud. Mexico, D.F., Mexico. Edilia Avila). Seminal plasma contains decapacitating factors The egg jelly-induced acrosome reaction (AR) of sea (DF) that prevent sperm fertilizing capacity. In urchin sperm is accompanied by increases in (Ca2+] i this study, we report the inhibition of the porcine and pHi and production of cAMP and IP The sperm acrosome reaction by a high molecular weight signaling mechanisms involved are unknown. 4e used DF from porcine seminal plasma. Sperm were washed digitonin to permeabilize the plasma membrane of sea by centrifugation to remove seminal plasma, and urchin sperm suspended in a medium that mimics the were incubated during 4 h at 37 C in a capacitating cytosolic ion composition. Within 8-10 min, 0.004- medium. Seminal plasma was ultracentrifuged, 0.006% digitonin allowed incorporation of Hoechst filtered, fractionated by gel filtration, and 33258 into the sperm. Permeabilized sa erm retained maintained frozen until use. Five fractions were lactate dehydrogenase and actin. In Ca -containing obtained: <10, 10-30, 30-50, 50-100, and >100 kDa. permeabilization buffer (pH 7.8), sperm underwent These fractions were added to capacitated sperm sp2ntaneously the AR; this reaction was pH- and and, after 1 h of incubation, solubilized zona Ca +-dependent. Electron microscopy indicates that pellucida was added to induce the acrosome the AR undergone by permeabilized sperm resembles reaction. Sperm cells were stained with the that induced physiologically. Additionally, viability stains Hoechst 33258 in combination with rhodaminyl-phalloidin staining of sperm reacted under a fluoresceinated lectin for assessment of acrosome permeabilizing conditions revealed a fluorescent status as an index of sperm capacitation. The filament in the acrosomal tubule region, results show that only the seminal plasma fraction demonstrating the occurrence of actin polymerization. >100 kDa inhibits the zona pellucida-induced Thus, permeabilized sperm offer new avenues to study acrosome reaction. The inhibition showed a linear the molecular bases of the sea urchin sperm AR. dose response. (Supported by CONACYT 1505-M9207 Supported by CONACYT. grant awarded to M.B.)

201T 2018 STEROLS INHIBIT THE DEVELOPMENT OF ACROSOMAL PROTEIN KINASE C (PKC) MAY BE INVOLVED IN THE PLATELET ACTIVATING RESPONSIVENESS OF HUMAN SPERM. ((N.L. Cross)) Department FACTOR (PAF) INDUCED HUMAN SPERM ACROSOME REACTION (AR). ((RA of Physiological Sciences, Oklahoma State University, Stillwater Tom1 and M.J. Angle2)) 1Dept of Urology, UCSF, San Francisco, CA 94143 and OK 74078. 2Virginia Mason Clinic, IVF Program, Seattle, WA 98101. (Spon. by R. Richardson.) The sterol content of the sperm plasma membrane may control the ability of sperm to fertilize eggs, or to have spontaneous This study examined the role of PKC in the PAF-induced AR. The PKC inhibitor acrosome reactions. Fertilization is believed to require an induced 1-(5-isoquinolinlylsulfonyl)-2-methyl piperazine (H-7), and the PKC inducer acrosome reaction. We tested whether exposure of sperm to 40-phorbol 12,13didecanoate (APDD) have been used in preliminary studies to sterols affects the development of sperm responsiveness to demonstrate PKC involvement in the AR. In these two experiments, Percoll- progesterone or ionomycin. Motile human sperm were selected on separated sperm were capacitated using 0.6% HSA in BWW and the effect of a Percoll gradient and incubated 24 hr in a modified Tyrode's various inducers and inhibitors of the AR examined using Pissum sativum lectin. saline containing 26 mg/ml bovine serum albumin and, in the test In Exp. 1, sperm were pre-incubated with H-7 (10km) followed by a challenge samples, serial dilutions of sterols. Cholesterol, cholesterol 3- with PAF (1pM), PPDD (0.1 uM) or aPDD (0.1uM; negative control). PAF sulfate, and desmosterol prevented the response to progesterone challenge of sperm not preincubated with H-7 (positive control) resulted in an (1 Mg/ml, 10 min), with no apparent deleterious effects on the AR of 20%; after H-7 exposure the AR was <6% in all groups following were 12.3 and 0.338 pM, cells. The IC50's 0.181 pM, pM, challenge. In Exp. II, sperm were pre-incubated with PAF receptor antagonists, as respectively. Cholesteryl palmitate was not effective when used CV 3988 (0.11&M) or L-125 (0.11&M; Meizel and Tumor, unpublished data) then high as 533 FM. Incubating sperm 24 hr with 50 FM cholesterol challenged with PAF or PPDD, with cxPDD as a negative control. PAF challenge development of responsiveness to ionomycin (4.5 pM, prevented of sperm not pretreated with antagonist was used as a positive control. Pre- percent control sperm reacting (mean [95% C.L.J: 35 (6- 20 min); incubation of sperm with PAF antagonist reduced the AR from 20% in the 71 percent treated sperm reacting: 0.3 (0.2-3%), n = 4. %); antagonist-untreated group to less than 4% after antagonist exposure in all Cholesterol also reversed sperm Addition of responsiveness. groups. These results implicate PKC in the PAF-induced AR, but also cholesterol (160 FM) to 24-hr incubated control sperm reduced the demonstrate that PAF-induced AR cannot be circumvented by PPDD. percent of sperm responding to progesterone from 19% (11-30%) to 3% (1-12%), 4 hr later. We conclude that sterols can regulate induced acrosome reactions. The sterol-sensitive event may lie at or downstream from the rise in intracellular calcium that occurs during induction of the acrosome reaction. Supported by OCAST.

2019 EXTENDING THE VIABILITY OF SEA URCHIN GAMETES. (M.A. Spiegler and S.B. Oppenheimer) Department of Biology and Center for Cancer and Developmental Biology, California State University, North- ridge, Northridge, CA 91330-8303. (Spon. by E.G. Pollock.)

The sea urchin is the material of choice for studying many develop- mental events. Methods to extend the viability of sea urchin gametes have not received much attention, but it is well known that the eggs are easily damaged by freezing. This study investigated methods to extend the viability of Lytechinus pictus eggs and sperm without freezing. Gamete viability was measured as relative numbers of fertilized versus unfertilized eggs and on observations of normalcy of development. The results indicated that gametes could be stored longer and at lower temperatures than previously described. Sperm were consistently viable for at least 11 days when stored in glass test tubes or_plastic petri dishes and submerged in ice inside a refrigerator at 0 C ± 1 C. In one exper- iment, sperm stored in glass test tubes on ice remained viable up to 20 days after extraction. Eggs were maintained from 1-7 days, rather than the one day or so previously reported, when stored in glass test tubes submerged in ice in a refrigerator at 0 C ± 1 C. Results of egg and sperm experiments varied at different times in the season. Such variations may be caused by seasonal cytoplasmic changes, population differences, or the time mature individuals were maintained unfed in refrigerated aquaria prior to use. Results from this study should be useful for a variety of research. mariculture, and teaching applications, where sea urchin supplies are limited or when the same gamete population is desired for subsequent experiments.(Supported by grants from NASA (NGT 70274), NIH MARC, NIH MBRS, NSF, Joseph Drown Foundation and Thomas Eckltrom Trust.) 348a Organogenesis (2020-2025). Tuesday

2020 2021 ALTERNATIVELY SPLICED BASIC FIBROBLAST GROWTH FACTOR IS THE LATENT TGF-B EXISTS IN EMBRYONIC-HEART EXTRACELLULAR MAJOR ISOFORM EXPRESSED IN THE DEVELOPING HEART ((X. Zhu and MATRIX (ECM) IN VIVO. (S. Ghosh and P.R. Brauer) DepL Biomedical J. Lough)) Department of Cellular Biology and Anatomy, Medical College of Sciences, Creighton University, Omaha, NE 68178 (Spon. by T. Vollberg) Wisconsin, Milwaukee, WI 53226. Transforming growth factor-B (TGF-B) is an important regulator of form and can be We have previously shown (i) that basic fibroblast growth factor (bFGF; FF-2) development. In vitro, TGF-B is secreted in a latent, inactive activated by pH extremes, chaotropic agents, or cell-surface proteases. protein is present during early chicken heart development, (ii) that an autocrine However, there is little evidence for the existence of latent TGF-B in vivo. In supply of bFGF is required for myocardial cell development and (iii) that exogenous this study, we determined if 1) cultured embryonic cardiac segments secrete bFGF protein is sufficient to support cardiogenesis in vitro. Recently, an altematively latent or active TGF-B, 2) TGF-B antibody binding to TGF-B was conformation spliced isoform of bFGF, termed alt-bFGF, was described during later stages (16-25) dependent (i.e., active vs. latent), and 3) immunostaining of embryonic hearts ofchicken embryogenesis. Because the antibody and probes used in our changed after exposure to activating conditions. Only latentTGF-B3 (acid previous studies would have recognizedalt-bFGF as well as bFGF, we have been activatable) was detected in conditioned medium of stage 14-16 chick cardiac examining the expression and role of alt-bFGF during early stages (3-24) of segments as measured by a growth inhibition biosasay. No growth inhibitory development. To date, RTIPCR has detected the presence of both bFGF and activity was present in non-acidified medium. When media was blotted onto a a1t-bFGF mRNAs as early as stage 3. Both mRNAs are detected in the developing membrane, only transiently-acidified medium bound antibody. These data it binds heart from the time of its appearance at stage 9. These findings have been show that cardiac segments secrete latentTGF-P3 and if activated, corroborated by RNaseprotection analysis of stage 5-18 total RNA using an alt-bFGF antibody. To determine if antibody binding to tissues sections required exposure to acidic pH, stage 14-16 embryos were fixed and sectioned under specific probe. This assessmentrevealed that increased amounts of alt-bFGF RNA are conditions that retained Under these conditions, expressed during early development, suggestingthat alt-bFGF is under developmental maximally E;CM. was found in the myocardium but not in the endocardium or And, using a RNase which to both immunostaining regulation. protection probe hybridizes isoforms, cardiac ECM. limited immunostaining was found in other areas of the embryo yielding protected fragments of predicted size for each, it was revealed that alt-bFGF and was always cell associated. Sections, when exposed to acidic conditions, is the major isoform expressed in the developing heart. We are now evaluating the showed immunostaining in most of the cardiac and embryonic ECM in effects of deleting alt-bFGF with antisense-oligodeoxynucleotide during in vitro addition to that above. All immunostaining was blocked by preabsorption with cardiogenesis, as well as the effects of over- and under-expressing this isoprotein TGF-B3 antigen. These data suggest that active TGF-B baa a very limited during heart development in vivo. (Supported by NIH grant HL 39829.) distribution while latentTGF-B is more abundantembryonicin ECM. Therefore, in vivo activation of TGF-B may play an important role in mediating the expression of TGF-8 function. Supported by NIH #HL-50397.

2022 2023 FOLLISTATIN INHIBITS ENDODERM-INDUCED CARDIOGENESIS ESTABLISHMNTT OFCELLPOLARTYDURING PREFCARDIAC IN VITRO((K.M. McCormick and J. Lough)) Department of Cellular MESODERMEPrITELALXZATION COINCIDESWI1T and Anatomy, Medical College of Wisconsin, Milwaukee, WI, CARDIOMYOCYTE D IFFrEREN TIATN. ((LLinak and YHL Gui)) Biology he i and Dept. o 53226. (Spon. by D.L. Bolender.) Division of Cardiology, Children's Hospitalof lphia Pediatrics, University of Pennsylvania School of Medicine, Phla PA 19104-4399. This laboratory recently reported that anterior endoderm induces pre- cardiocytes in explanted mesoderm to differentiate into a cellular multilayer Stbilizaton of cardiacc cel conmitment and differentdiat coincides with that expresses carcho-specific proteins and contracts rhythmically. Having pitlialization of the precarda mesoderm in the early chick embryo. In that one of the of anterior early stage 6-8 embryos a rosrocaudal pae of N-cadhern-cateni previously established secretory products in the heart forming region. endoderm is we that this member of the TGF-beta expression precedes epithelialization activin-A, hypothesized Observations on the cardiac polar expression of NA/K-ATPase (sodium superfamily is involved in endoderm-induced cardiogenesis. RTIPCR pump) afterepitheliation betwem stages 6-8 and its involvetm in analysis confirmed that activin-A is expressed by anterior endoderm; activin- cavitation in systems, suggested that Na/K-ATPase may be involved B was not detected. After determining that exogenous activin-A promoted in pericria coelom formaton and early cardi yog sis Experim cardiogenesis in vitro, the cardiogenic potential of endoderm-produced to pertub sodium pump acivity withthe highly selectie NK-ATPase was evaluated endoderm and mesoderm the inhsbito ouabain wer underakn to determine oubahin will affect activin-A by co-culturing cardiac cell co-mmitment. Ouabain exposu an activin Anterior lateral plate formto an/or presence of follistatin, binding protein. whole chick between stages 5-8 demonsta thatheat were grown medium early embryos endoderm and mesoderm in defined (M199) plus development and precardiomyocyte differentiation aresensidveto ion follistatin (10 1000 ng/ml). A physiological level of follistatin (10 ng/ml) ptation during a narrow, developmental window. Ouabain sentivity was sufficient to significantly inhibit cardiogenesis. After 48 hours,60% of expressed in a rotocaudal gradient withde let diff tiaed cells, control cultures (n = 10) were contractile while only 20% of the follistatin- more posteriorly, being mostsensitive to inhibition.The rostrcadal is both doe-, developmental t, and is revaesible explants (n = 8) contracted. Follistatin did not inhibit insulin's inhibidon treated by elevating outside potassium ionconcntrto in the culture medium. cardiogenic effect, indicating that follistatin itself does not suppress thethree anterior The results suggst that the regulation of the formation of cardiogenesis. These results suggest that activin-A, secreted by dimensional organ is independent from the regulation of cardiac endoderm, is animportant effector of cardiogenesis. Supported by NIH myogenesis. Cell adhesion moleculs may seveto interlatethee two grant HL 39839 processes.

2024 2025 BIOCHEMICAL AND IMMUNOHISTOCHEMICAL STUDIES PAITERNING THE HEART TUBE, A GENETIC ANALYSIS IN THE ON A NOVEL PROTEIN. ((N. Eginel-Unaltuna, D.KLDube and LF. ZEBRAFISIL ((D.Y.R. Stanier, B. Weinstein, F. Zwauis, A. Schier, Lman) D qrtmtofAnanmy and Cell Biolo, SUNY He W. Driever, and bC. Fsshsan)) Cardioaular Reseach Center, Ma. Science Center, 750 East Adams Street, Syracuse, New York 13210. General Hospital, and Havard Medical SchooL Charstown MA 02129. Recessive gene c axolodscas amutao which relsm afaile of The embryonic heart is a simple stuctr that consists of2 concentric in wall of the acfed ha ts conat. Tis can be coreciad by culuring epithelial tubes, the outer myocadial tube which forms the muscle the hear in amedismrondidoned by IanteriorRNA&om thc d the heat, and the inner endocadial tube which forms its ia anhdby RNAKisd-fzdfFm _---fsdr nWned lining. Although heartde t has been wel described insveal . was isolaed acDNA lbray was consaucted fom which 32 species,litte is ownabout the underlying cellular andmolecular medium md S clones were seced One ofthe non- randomly andprtally sequened. m ,ibosomal RNAs tha showed no gifithomologywith ndwr n We have taken agenetc arh tothe stdy of cardiac seqaec in the Genehak ws fu An and have elected to study question in the wbrfish because ofits sythesized and anaffinity-puriied polycinal antibody was pced unique advntages as a vertebrate genetc and embryological system. t it RT-PCRanali showed that the RNA is in slal From alare scale mutagenes sen wich aimd at sating the musc, brain and heat insiificant amouits,lss in long and not atall in zebrafish genome forembryonic letial mutations (Solnica-Krezel et al., liver ofjuvenileaxlod. NI-proein was further inestigalin norml Genetics136, 1401 (1994)), we have isolated more than 100 mutdons embryonic whole heat ssue (stas 35,38 and 41) and erons-secdons that affect heart formato and funon and these are curenty being put and intocompme n group through th bet regios of nomal embryos at sages 16, 33-34,37-38 ealy 41-42. At stage 16, te protein is found in thc form of sat dhe bordline Specifically, we havefound a handful of mutations that affect between noderm md endodem Stag 33, at which heart tubes ae not cardiac mOp For example, the cloche muution block the heat yet fusedconpleely, dhe proatinisnmtprominltlin the myosome differentiationafthe endocdiacells. In this mutant, the forms and heart Atlater stages a mi le distibuton of the normally even in the abence of theenIcrd layer but it exhibits a endoderm tubes. the protein is nodceabl at or the in the heart clls, weak contraclity. Two other mutaions, ulesqparr and a#f, block it cells and cardiac jely. (NIH grants HL-32184, HL-3770 and an fusion of fth primitive myocardial tubes. This results in the difretaion AHA grant toLFL.) of two hearts one onecithe sde of the midline, a sitation known as' cardia byida Athorogh cer ti of the ad other mutations dt affect the ely pattring ofthe heat tube will be prented. Tuesday. Organogenesis (2026-2031) 349a

2026 2027 INDUCTION OF EPMIELIAL MORPHOGENESIS BY WNT-1. CHANGES IN THE ACTIN CYTOSKELETON DURING CUPPING OF ((D. Herzlinger', J. Qiao', R Bradley2, S. Jue2, and AM.C. Brown2-)) THE AVIAN OPTIC VESICLE. ((J.F. Sanzo and S.R. Hilfer.)) Department Department of Physiology and Biophysics', Department of Cell Biology of Biology, Temple University, Philadelphia, PA 19122 and Anatomy2, and Strang-Comell Cancer Research Laboratory3, Cornell University Medical College, New York, NY 10021. This study was undertaken to evaluate changes that occur in F-actin in cells of the optic vesicle during invagination. Retinal discs from chick optic vesicles of stages 13 to 14 were examined by several methods. Electron microscopy The Wnt gene constitutes a set of 15 or more related genes family has revealed that organizational patterns ofactin bundles change during that act as intercellular in a wide encoding secreted proteins signals variety invagination. The changes follow one of two basic patterns according to of developmental processes. The best characterzed member of this family location in the vesicle. The patterns were studied in detail with the aid of a is the proto-oncogene Wnt-l, which normally functions in the embryonic confocal laser scanning microscope. It was found that prior to the onset of neural tube but causes hyperplasa when ectopically expressed in the mouse cupping, cells ofthe entire retinal disc are highly regular and symmetrical in nmmmay gland. Recent studies have shown that Wnt-l can cause up- shape, with a correspondingly well ordered actin cytoskeleton. After cupping regulation of cadherin-based adhesion mechanisms in certain responsive has begun, center cells that are now at the "bottom" ofthe cup, are still cells. During kidney development, the epithelia of nephrons are formed regularly and symmetrically organized, but EM reveals filament bundles are from metanephric mesenchymal cells in response to unknown signals from less organized. Cells that were more marginally located prior to cupping are the ureteric bud. This phenotypic conversion, which is accompanied by now at the "lip" ofthe cup. Some ofthese margin cells are seen to have changed shape. Their organization changes from regular and symmetrical, elevated expression ofE-cadhrin, can be recapitulated in an organ culture with symmetrical bundles, to an organization where there are clearly two system in which embryonic spinal cord acts as a heterologous inducer of populations ofcells mixed together. One population retains the regular nephron formation. We have found that Wnt-1 is a potent inducer of symmetry ofthe pre-cupped vesicle, the other population becomes laterally nephrogenesis in this system. When co-cultured with cell lines expressing flattened. As cupping progresses, the pattens shift so that a wave of cell Wnt-1, metanephric mesenchymal cells differentiated into glomeruli and shape changes is seen to migrate beginning just outside the center ofthe renal tubules. Co-culture with control cell lines had no such effect. These retinal disc (at the original lip) moving towards the periphery. As the wave data suggest that a member of the Wnt gene family may be a mediator of arrives, new dual-population margin cells are formed. As the wave passes, the renal epithelial morphogenesis in vivo. population reverts to its original homogenous nature, but with apical filament bundles that are less ordered.

2028 2029 TENASCIN MAY BE A NEGATIVE REGULATOR OF AVIAN INNER GLUCOCORTICOIDS, TGF-,, AND EMBRYONIC MOUSE EAR MORPHOGENESIS. ((S.R. Hilfer, E.D. Beck, and.J.W. Brown)) SALIVARY GLAND MORPHOGENESIS ((T. Jaskoll, H. A. Choy, M. Department of Biology, Temple University, Philadelphia, PA 19122 Melnick)) Laboratory for Developmental Genetics, University of Southern California, Los Angeles, CA 90089-0641. During inner ear development, the otic placode folds to form a box-like Glucocorticoids (CORT) are known to regulate morphogenesis in otic vesicle. Folding (invagination) of the otic primordium is not affected several branching organs (e.g. lung). We hypothesize that CORT may be by agents which either interfere with cytoskeletal function or stimulate involved in embryonic mouse salivary gland branching morphogenesis. precocious invagination of other organ primordia. In contrast, otic We studied the CORT-glucocorticoid receptor (GR) signal transduction invagination appears to be driven by changes in extracelhlular matrix under pathway during embryonic mouse submandibular gland (SG) the lateral region of the placode (paraxial mesoderm) and between the otic development. Western analysis demonstrate that the 96-kDa GR is epithelium and the neural tube. Microinjection of antibody to either present in E14-E18 SGs. The embryonic GR is functional, as defined by laminin, integrin, or NCAM inhibits folding by detaching the otic/neural its ability to bind to a DNA CORT response element, for all gestational adhesion, while enzymes which degrade chondroitin sulfate or hyaluronan ages evaluated (E14-E18); increasing GR levels are observed with inhibit by distorting paraxial mesoderm. Microinjection of anti-tenascin progressive development. Maternal injection with CORT increases (from the Developmental Studies Hybridoma Bank) with a picospritzer embryonic SG total protein and DNA in vivo. In vitro studies in accelerated undeneath the ectoderm of intact chick embryos resulted demonstrate that when E13 mouse SGs are cultured under serum-free, invaginaton relative to the control side. Folding occurred uninjected chemically-defined conditions, a significant enhancement of mean when were made into stage 9 to stage 11- embryos precociously injections branching ratios (72h/Oh) is detected in CORT-supplemented explants but at 12 or later had no effect. injections stage Photographs of living (105M to 10-1M) compared to control explants (P<0.001); 10'M CORT embryos and scanning electron micrographs showed a range from slightly consistently gives the greatest induction of branching (t:= 55.28; to moderately accelerated folding on the injected side. Tenascin has been P<0.001). These data support the importance of the CORT-GR signal suggested as an inhibitor of cell movement during other developmental transduction pathway during salivary gland branching morphogenesis. processes. The actual site of antibody blockage and the relationship to Northern analysis suggests that the CORT-GR signal transduction other components of the basal lamina are under investigation. Supported in pathway may modulate the rate of branching morphogenesis by down- part a grant from the Deafness Research Foundation. by regulatingTGF-,B1 mRNA and up-regulating TGF-#2mRNA expression.

2030 2031 TGF-a: AS A PUTATIVE AUTOCRINE/PARACRINE GROWTH FACTOR IN THE SPATIAL AND TEMPORAL EXPRESSION OF CELL SURFACE MOLECULES MOUSE EMBRYO SALIVARY GLAND. (( M. Luquette and G. Steele )) DURING NEPHROGENESIS. (CM. R. Goldberg and Q. Al-Awqati)) College of Department of Pathology, Ohio State University & Children's Physicans and Surgeons of Columbia University, New York, New York Hospital, Columbus, OH 43205. 10032.

Members of the epidermal growth factor (EGF) family including Nephrogenesis involves a complex series of morphological events that EGF, HB-EGF, TGF-a, and amphiregulin all bind to and activate requires temporal and spatial cues among the ureteric bud, mesenchyme and the EGF receptor (EGFR). While it has been shown that the EGFR invading blood vessels. To study the development of the three-dimensional is in the of the mouse present epithelium embryonic salivary structure dunng nephrogenesis we generated monoclonal antbodies (mAB) gland, definitive evidence as to which ligands are functional against cell surface molecules in the developing kidney. Dissociated In vivo has not yet been reported. We have used RT-PCR to 15 was chosen as the since it contains all total RNA extracted from mouse embryonic day kidney immunogen analyze embryo salivary glands the of ureteric uninduced dissected on days 13 and 15 of gestation. Primers for mouse early stages development including bud, renal with or TGF-a were used and showed an appropriate size product on mesenchyme, veside and S-shaped body without vascularized control tissue (mouse skin) and embryo salivary glands. No glomeruli. The mABs were dharacterized by the specific segments to which product was detected in either tissue when RT was not added. they bind and whether their antigens are transiently or continuously TGF-e mRNA was identified in salivary glands obtained on days expressed using immunofluorescence. One mAB 5B6-E4.2 stains most 13 (n-2) and 15 (n-3) of gestation. Further evidence for the intensely in mesenchymal cells that surround the branching ureteric bud in role of TGF-e was observed in cultures in which TGF-a embryonic day 13 (the critical site at which induction occurs), but stains only antiserum, but not non-immune serum, inhibited the growth of glomeruli and blood vessels in the adult The 1B3-G9 mAB is diffusely 12- and 13-day salivary explants. The TGF-ex antiserum also expressed in the embryonic mesenchyme, but its expression becomes inhibited the ability of TGF-ce to displace radiolabeled EGF restricted to the glomerular crevice in the S-shaped body and to the from the EGFR in a radioreceptor assay. These results suggest glomerulus in the adult kidney. A third mAB 3B2-F6 reveals a gradient of autocrine/paracrine functions for TGF-a in the embryo salivary expression in the neonatal kidney staining most prominently in the uninduced The site of TGF-ce and the regulation of its gland. production mesenchyme. The 3B3-A8.1 mAB is differentiation specific. It sbins only secretion remain to be determined. vascular smooth muscle cells of mature blood vessels. The mABs isolated have been classified by their immunofluorescent pattern as to whether they reognized either developmental stage, lineage, differentation or structural specific antigens. They should prove useful to study the process of induction and anglogenesis in the kidney. 350a Organogenesis (2032-2034). Tuesday

2032 2033 ISOLATION, GROWVTn DlFRENIATON. AND TRANSPLANTAION OF PIG ROLE OF KGF AND TGF-ot IN ANDROGEN-INDUCED BRANCH- FETAL PANCREATIc CES.(a . H. Dinsmore and J. Ratlif)) Diacrin, ING MORPHOGENESIS OF THE DEVELPOING RAT VENTRAL Inc., Building 96, Thirteenth Street, Charlestown, MA 02129. PROSTATE. ((B. A. Foster and G. R. Cunha)) Dept of Anatomy, UCSF, San Fran- cisco CA 94143. We report methods for the isolation, growth, and differentiation of fetal pig pancreatic cells. Fetal pancreatic dssue was dissected from pig fetuses. The Keratinocyte growth factor (KGF) and transforming growth factor-alpha (TGF-a) play dssue was dissociated into a single cell suspension and placed in culture. At a rote in androgen-dependent development of the rat ventral prostate. During the first this stage ofdevelopment, the panreatic cells have not formed insulin week postnatally the epithelium proliferates and branches in response to androgens. A secreting Idets ofLangerhans and were stilc apable ofproliferation. We serum-free culture system was developed to determine the factors involved in androgen- dependent branching morphogenesis in the prostate. When ventral prostates from aintined proliferation offetal pancreatic cells for several months in media (VP) 0 day Fisher-344 rats were isolated and cultured in serum-free medium for six days with containing bovine senun and growth factors. The pig fetal pancreadc cells continued to proliferate as long as they were kept sub-confluent. When testosterone the epithelium exhibited ductal brandcing morphogenesis. This effect was not observed when glands were similarly cultured in the absence of exogenous testos- cells were allowed to each confluence, they formed pseudo islet-like cell aggregates spontaneously and stained positive for insulin, glucagon, and terone. Remarkably, exogenous KGF was capable of eliciting growth and epithelial somatostatin. Prior to formation of islet-like aggregates, there was no ductal branching morphogenesis comparable to that elicited by testosterone. When a KGF-specific neutralizing antibody was added to the cultures testosterone-induced detectable insulin stainin& The formation ofislet-like aggregates was necessary for the expression ofinsulin, and cells did not proliferate well epithelial ductal branching morphogenesis was blocked. Furthermore, a TGFo-specific after they were allowed to aggregate. Theedc, fetal pig pancreadc cells in neutralizing antibody also blocked both testosterone-induced and KGF-induced ep- ithelial ductal branching morphogenesis. RT-PCR analysis indicated that KGF, its vitrorecapitulated aspects of diferentiation and cell prliferaton that occur receptor and TGF-a were expressed in the developing VP. These observations strongly normally m vivo. Further experiments with the fetal pancreatic cells were suggest that a cascade of signaling events occurs in the developing rat ventral prostate performed to determine ifcells would differentiatein vivo after to mediate epithelial ductal branching morphogenesis in response to testosterone. One transplantaton into nude mice hosts. For transplantation, approximately 106 undifferentiated, proliferating cells were transplanted under the renal possible sequence of events leading to prostatic development is that circulating testos- terone acts through its cognate receptor in the prostatic mesenchyme to elicit capsule of several mice. At tmes after transplantation, mice were sacrificed KGF in mediates testosterone effects to and examined for the presence ofgrafts. All mice transplanted showed production. KGF, turn, by signaling the epithe- lium via the epithelial KGFR. The KGF induced response of the is then viable grfts with insulin containing cells as shown by immunostaining for epithelium mediated, at least in part, by TGF-a via an autocrine or paracrine mechanism. a insulin. Therefore, fetal pig pancratic cells were propagated in vitro and maintained the ability to differentiate in vivo. Cunendy, we are examning whether such cells will be effective for the teatment ofdiabetes in mouse models.

2034 DIFFERENTIATION OF SMOOTH MUSCLE CELLS FROM EMBRYONIC GIZZARD CELLS MIGRATED FROM TRANS- PLANTED ORGAN. ((J.-A. Ko, S. Murahashi, T. Arata, and A. Inoue)) Department of Biology, Faculty of Science, Osaka University, Toyona- ka, Osaka 560, Japan. We examined the diffcrentiation of smooth muscle cell by culturing the dissects of embryonic chicken gizzard. It has been indicated from the morphological stdies that a masive prolification of mesenchymal cells precedes morphological differentiation and expression of muscle pro- teins However, the differentiation of smooth muscle cell in the culture had not been succeeded. At 7-day-old cmbryo (at stages 26-28) the gizzard had not an organized structure, and its cryosection was not stined by anti-chicklen gizzard smooth muscle myosin antibody. Fur- thermore, the expression of myosin determined by Western immunobiot- ting was extremely low at this stage. Therefore, we concluded that the smooth musclc cells were undifferentiated at 7-day old embryo. Then, we used the gizzard from this stage as a source of the tissue culture. When the dissects of gizard at this stage was unsplanted to the culture dish fibroblast celis were first migratec[ from the explanted cubes within 4 days aftcr culture. Then, round cells werc migrated from the explant over the layer of fibroblast cells. At 12-14 days after culture, smooth musclc cells, which were ribbon-shaped and stained by anti-myosin antibody, apared in the layer of round cells. Thc thin section of dis- sectedexp ant at day 15 culture was not stained by anti-myosin anti- body. Thereforc, we concluded that smooth muscle cells are differentiat- ed from the round cells which spread over fibroblast layer. This result suggested that fibroblast cells play an important role for the differentia- tion of smooth muscle cells. The system presented here may bc useful for studying the mechanism of differentiation of smooth musce cell. Mammalian Development (2035-2036).

2035 2636 ANALYSIS OF Na/K-ATPase SUBUNIT mRNA PHENOTYPE DURING LOCALIZATION OF IGF-I AND IGF-fl POLYPEPIIDES AND mRNAs IN BOVINE PREIMPLANTATION DEVELOPMENT. ((D.H. Betts', D.J. BOVINE OVIDUCTAL EPITHELIAL CELL PRIMARY CULTURES. ((P. MacPhee2, G.M. Kidder2, and A.J. Watson'3)) Departments of Obstetrics and Xia', V. Han3, D.T. Armstrong2, and A.J. Watsonl2)) Departments of Gynaecology', Physiology3, and Zoology2 The University ofWestern Ontario, Obstetrics and Gynaecologyi, Physiology2, and Paediatrics3, The University London, Ontario, Canada, N6A 5A5 of Western Ontario, London, Ontario, Canada, N6A 5A5

The polarized distribution of murine trophectodermal NalK-ATPase Embryo co-culture on prinmary oviductal epithelial cells is an effective method contributes to the transport of water across this epithelium to form the of supporting development through the preimplantation interval. Somatic cells blastocyst fluid. Previous studies reported the presence of mRNAs encoding the may secrete 'embryotrophic' factors that may stimulate early development or aI-subunit isoform throughout murinepreimplantation development. However, alternatively may remove 'embryotoxic' factors. We have explored this Pl-isoform mRNAs only become abundant in the late morulae stage just prior positive influence of co-culture on development by investigating the role of to the onset of blastocyst formation. We have extended our analysis to include specific growth factors in two distinct oviductal cell primary culture systems. bovine preimplantation embryos to explore the general role that Na/K-ATPase Oviductal monolayers are capable of supporting early development for up to expression may play in blastocyst formation. mRNA phenotyping via the 72 h before the zygotes must be moved to fresh cultures. Microdrops (50;l) reverse transcription-polymerase chain reaction (RT-PCR) was conducted contining non-attached, ciliated oviductal 'worms' support development employing PCR primers specific for the rat a and 1l cDNAs and the murine throughout the preimplantation interval without a tansfer to fresh cultures. P2 cDNA. Transcripts encoding the aI-isoform were detected throughout Transcripts encoding IGF-I and IGF-II were detected in both oviductal bovine preimplantation development. In variance to the mouse, no transcripts monolayers and also non-attached 'worms' on day 2, 4 and day 8 of the encoding the Pl-isoform were detected during bovine early development even culture interval. Immunocytochemistry demonstrated the presence ofIGF-I and though the rat primers amplified a p1 PCR product in bovine kidney samples. IGF-II in both oviductal cel primary cultures for the 8 day culture interval. P2 mRNAs were detected in bovine blastocysts and also throughout murine The signal was reduced in one cel type composing the day 4 and day 8 preimplantation development. Day 8 "blocked" bovine moulae did not express monolayer cultures. The localization of IGF mRNAs in both cultures was the P-subunit. These results have linked the timing of expression of the p- confirmed by in situ hybridization using 35S-labelled riboprobes. The results subunit with bovine blastocyst formation and also suggest that additional suggest that maternal growth factor circuits are likely weUl established in the isoforms of the P-subunit are expressed during murine early development. bovine oviduct and may influence bovine early development. (Supported by (Supported by NSERC and MRC). NSERC). Tuesday. Mammalian Development (2037-2042) 351a

2037 2038 EXPRESSION OF ANTIOXIDANT mRNAs DURING MURINE AND MURINE EMBRYOS EXPRESS AROMATIC HYDROCARBON BOVINEPREIMPLANTATIONDEVELOPMENT. ((M. Harvey', X.Zhang2, RECEPTOR (Ahr) mRNA DURING PREIMPLANTATION M. Arcellana-Panliliol, G.A. Schultz' and A.J. Watson3"4)) Departments of DEVELOPMENT. ((J.M. Peters, A.L. Blankenship, and LM. Wiley)) and Obstetrics and of Departments of Obstetrics and Gynecology, Division of Reproductive Medical Biochemistry', Gynaecology2, University Biology and Medicine and Environmental Toxicology, University of Calgary, Calgary, Canada; Departments of Obstetrics and Gynaecology3, and Califomia, Davis, CA 95616. (Spon. by LM. Wiley) Physiology4, The University of Westem Ontario, London, Ontario, Canada There is a substantial body of evidence suggesting that the biological effect Bovine and ovine preimplantation embryos must be co-cultured to support of 2, 3, 7, 8-Tetrachlorodibenzo-p -dioxin (TCDD) exposure is primarily reasonable rates of development through the preimplantation interval under a mediated by an aromatic hydrocarbon receptor (Ahr). Upon ligand receptor 5% CO2 in air atmosphere. Co-culture is not necessary when a reduced 02, binding, gene transcription for several enzymes is induced. The role of (5% C02, 5%02, and 90%N2) atmosphere is employed. Murine embryos these Ahr-mediated changes in gene expression during abnormal is undear. we that TCDD develop at high rates in simple media (CZB) under a 5% CO2 atmosphere. We embryo/fetal development Recently, reported for Cu/Zn dismutase directly affects preimplantation embryo development in vitro by accelerating examined the expression pattems catalase, superoxide differentiation of the blastocyst. While it is known that TCDD can influence (SOD), Mn SOD, glutathione peroxidase and glutathione synthetase (GCS) embryonic development in vitro it is not known whether this effect is an during murine and bovine preimplantation development and in two primary Ahr-mediated event. In the work reported here, we show the presence of bovine oviductal cell cultures by reverse transcription-polymerase chain Ahr mRNA in early mouse embryos with the use of reverse trancription- reaction (RT-PCR) methods. The mRNAs encoding all five enzymes were polymerase chain reaction (RT-PCR). Ahr mRNA was not detected in 4-cell constitutively expressed throughout murine early development. Bovineembryos embryos. However, at the compacted 8-cell stage and blastocyst stage, Ahr displayed a similar pattem although no Mn SOD mRNAs were detected and mRNA was present. These results suggest that the embryo transcribes, GCS transcripts were absent in blastocysts. Both oviductal primary cultures rather than matemally-inherits this message. We are currently further constitutively expressed transcripts for catalase, Cu/Zn SOD, glutathione characterizing the developmental presence of Ahr mRNA in unfertilized oocytes and other stages of preimplantation development in addition to peroxidase and GCS for an 8 day interval. mRNAs encoding Mn SOD were investigating the functional significance of Ahr during this period of early absent in fresh oviduct cell samples. These experiments suggest that differences mammalian development. (Work supported by NIH ES05409 to LMW. in gene expression may underlie variation in the tolerance of elevated 02 levels and NIH ES05707 and ES03575 to F. Matsumura.) and that co-culture with somatic cells could alleviate some of these deleterious effects. (Supported by NSERC and MRC).

2039 2040 PREIMPLANTATION EXPRESSION OF MOUSE CELL CYCLE CHARACTERIZATION OF THE HUMAN GLI GENE ((C.Z. Liu, J.T. Yang, J.W. MOLECULES and Z. of Yoon, D.O. Walterhouse, and P.M. lannaccone)) Department of Pathology, REGULATORY ((A. MacAuley Werb)) Laboratory Department of Pediatrics, and the Markey Program in Developmental Biology, Radiobiology and Environmental Health, University of California, San Norftwestem University Medical School, Chicago, IL 60611 Francisco, CA 94143-0750. The human G/i gene is the prototype of the Gli-Kruppel gene family characterized As a first step toward determining the molecular basis of cell cycle regulation by a five zinc finger motif with sequence specific DNA-binding ability. The human in mouse was G/i gene is an oncogene and disruption of another family member (Gli 3) results in during preimplantation growth embryos, cyclin expression bony dysmorphogenesis in humans. There is remarkable similarity to mouse g/i analyzed at the mRNA level by RT-PCR. Cyclins A and B1 were expressed whose expression in embryos and adult tissues is restricted temporally and throughout preimplantation development. Cyclin B2 expression was not spatially. The zinc finger DNA binding region of human and mouse Gli show a high detectable up to and including the early 4-cell stage, but was detected in degree of homology to the Drosophila segmentation gene CP. To provide a compacted 8-cell enbryos. No expression of the G1 cyclins (cyclins D1-3 and framework for understanding the function and regulation of G/i, the genomic and of the human was characterized. E) was discenible in the early 1-cell embryo, but expression of cyclin E was organization 5'-flanking sequence G/i gene The human is encoded 12 exons 11 introns and it coincident with the G/i transcript by separated by detectable at the late 1-cell stage, apparently earliest spans 13 Kb of genoranc DNA. The splicing junctions are in good agreement with zygotic transcription. The role of this early transcription is unknown. consensus sequences. The largest exon is about 1946 base pairs (bp) located in Expression of the D-type cyclins could not be detected reliably until the 3' end of the gene and the smallest exon is 90 bp. The first exon consists of a compacted 8-cell stage at which dme DI expression appeared. Cyclin D3 (C+G)-nch region. The five zinc-fingers are dispersed in four exons with finger 1 expression appeared by the 32-cell stage. Cyclin D2 did not appear to be and 3 encoded by two exons. Intronic separation of zinc-fingers has been reported in other members of the Gli-Kruppel gene family. The length of individual introns expressed to a significant level durng this period of developmnt, although ranges from 91 bp to 3.8 Kb and the first intron is the largest. Primer extension weak expression was detectable in the blastocyst. I have begun to characterize analysis of both human Tera-1 and D259MG cell lines identified a major the protein expression of these molecules and will also characterize the transcription initiation site at about 160 bp upstream of the translation starting site. regulation of the associated kinase activities. (his work was supported by There is a cluster of SPI binding sites upstream of the initiation site and a typical NIH grant HD 26732, and U.S. Deparment of Energy, Office of Health and TATA box was not found in the vicinity of this region. Transfection experiments with a fragment containing from about -900 bp to +600 bp showed strong promoter Environmental Research, contract no. DE-AC03-76-SF01012). activity in the Tera-1 cell line. Assay of 5' and 3 partial deltion mutants is under way to oialize critical promoter eleents. This work was supported in part by HD28992 (PMI) and ACS (IL) 93-32 (DW).

2041 2042 EFFECTS OF TRANSCRIPTION INHIBITORS ON THE SUBCELLULAR HOMEOBOX GENE EXPRESSION DURING EARLY DEVELOPMENT DISTRIBUTION OF POLY A+ RNA IN MURINE PREIMPLANTATION OF THE MURINE YOLK SAC. ((IRE. McGrath, P.D. Kingsley and J. EMBRYOS. ((A.E. Alworth, R.H. Singer, K.L. Taneja* and D.F. Albertini.)) Palis)) Department of Pediatrics and Cancer Center, University of Rochester, Tufts University School of Medicine, Boston, MA and University of Rochester, NY 14642. (Spon. by R.C. Angerer) Massachusetts Medical Schoor, Worcester,MA. At the timr of murine gastrulation (E6.5) mesoderm cells migrate to appose the visceal endoderm the sac the next three we have used fluorescence in situ to cemating yolk (YS). During days Previously hybridization quantifitively this simple bilayer sac becomes multifunctional containing germ cells, and qualitatively assess the distribution of poly A+ RNA in mouse oocytes hematopoietic and vasculogenic islands, and providing gut- and liver-like and preimplantation embryos. Importantly, a high concentraion of poly A+ nutritive and metabolic functions. Nothing is Inown about the underlying RNA was observed surrunding nucleoli in the nuclei of 4 and 8 cell developmental mechanisms which establish the YS. We have examined the embryos. In the present sudy, fertilized eggs, 2-cell, 4-cell and 8-cell early murine YS for expression of the homeobox family of developmental embryos were tred with Actinomycln D or oa-amanitin to determine the transcription regulators. Amplified E7.5 and E8.5 YS cDNAs were used as effects of these transcipbon inhibitors on the subcellular iocalization of poly targets in PCR reations with degenerate primers to conserved regions in A+ RNA. Samples were exposed to inhibitor for 90m, then fixed and homeobox domains. A subset of known homeobox sequences was found extcted, hybridized with Texas Red or Cy3 conugated poly dT or poly dA (Hox al, bl, a9, c9, alO, a7, b7, b8, c8 and cdxl). Two novel sequences (control) oigonucloides and subsequently counterstined with fluoresin were also isolated, including a fourth member of the labial class of Hox genes (which is a candidatefor Hox c1) and one which has since been identified by phalloidin and Hoechst 33258. Both inhibito produced a reduction of he other groups to be a pancreatic specific gene (called IPF 1, STF-l and total nucear poly A+ RNA in 2, 4 and 8 cell embryos, while no significant IDX-1). The rodent YS is Inown to produce inmlin from mid- to late- difference in signal was observed in fertilized eggs. Actinomycin D bteabrent gestation, but the panceatic functions of the early YS have not been induced the fomabton of aggregatns of poly A+ RNA in the nucei of 2, 4 examined We are explonng furthe the developmental exprtssion of these and 8 coe embryos and abolished the concentrai of signal surrounding homeobox genes as well as pancreatic homones by semi-quantitative RT- the nuleoli of 4 and 8 oell control embryos. Treatment with a-amanifin PCR and in situ analyses. RT-PCR results indicate that the two most resultd in a deceaw in the nucear and cytopasmic signal in 2, 4 and 8 cell frequently isolated YS Hox genes, a9 and c9, are seen in the YS but not the embryos. Significantly, this reaWnent had no effect on the high conc ra embryo at E7.5. By E8.5 both a9 and c9 are expressed in the embryo and of poly A+ RNA surrounding the nucloli in 4 and 8 cell embryos, suggesting YS. Preliminary data indicate hat at pre-pancreas stages of development, in the YS while somaostatin this poly A+ RNA may be not be mRNA. Supported by NIH grants HD 20088, IDX-1 and insulin ae exprssed predominatly and are not in the YS from E7.5 to E14.5. HD 18068, P30HD28897 and th Markey Chaitble Trust Foundaion. glucagon expressed 352a Mammalian Development (2043-2048). Tuesday

2043 2044 DIFFERENTIAL GENE EXPRESSION DURING MURINE EARLY YOLK Ph AND Rw MUTATIONS IN MICE: DEVELOPMENTAL DEFECTS SAC DEVELOPMENT. ((P.D. Kingsley and J. Palis)) Department of ARISING FROM CHROMOSOMAL REARRANGEMENTS ((M. Pediatrics and Cancer Center, University of Rochester, Rochester, NY 14642 Bucan1, R. B. Hough1, B. W. Nieuwenhuijsen1, D. L. Nagle1, C.W. Lo2)) 1 Depts. of Psychiatry and Biology2, University of Pennsylvania, The mammalan visceral yolk sac (VYS) performs a diverse array of functions Philadelphia, PA 19104, USA prior to organogenesis including hematopoiesis in the VYS mesoderm and absorption and secretion by the VYS endoderm. We used the strategies of A cluster of mouse mutations, white spotting (W), patch (Ph) and differential hybridization and subtractive hybridization to isolate gene products rump white (Rw) are associated with perturbations in pigmentation and upregulated during the specification and maintenance of early yolk sac tissues. other developmental defects. Characterization of the genomic organization Late primitive streak to neural plate stage (E7.5) mouse embryos were of this gene cluster has provided a set of molecular tools for further transected into the embryo proper and the VYS, which was enzymatically analyzing the observed developmental defects. W is caused by mutations separated into VYS mesoderm and VYS endoderm fractions. mRNA from in the Kit receptor tyrosine kinase (RTK) (Chabot et al., 1988; Geissler, et was to and PCR selected these tissues converted cDNA amplified. Randomly al., 1988), whereas a homologous RTK, Pdgfra , is deleted in the Ph VYS mesoderm cDNA inserts were screened with complex VYS mesoderm mutation (Stephenson et al., 1988). The synergistic action of the W, Rw and endoderm cDNA probes. Sequence analysis identified several messages and Ph pigmentation defects, and the clustering of the breakpoints of these upregulated in VYS mesoderm compared to VYS endoderm, including hsc70, mutations in a small genomic region, argues that these pigmentation hsp86, ribosomal protein L13a and alpha-enolase. While in situ hybridization deficits may be due to mutations in the same or similar regulatory elements showed that these messages were expressed throughout the embryo, their and/or gene sequences. Recently we have identified yet another gene differential accumulation in E7.5 VYS tissues was also confinned. To enrich within the Ph deletion. The developmentally regulated expression of this for cDNAs related to the differentiated functions rather than metabolic states of gene suggests its possible involvement in the Ph/Ph mutant phenotype. the yolk sac, sequences shared between the three germ layers of the embryo Further analysis showed that Rw is associated with a large inversion which proper and the VYS tissues were eliminated by subtractive hybridization. disrupts the same cluster of RTK genes. However, complementation Subtraction enriched VYS endoderm specific messages include trawsthyretin analysis suggests that the Rw mutation is positioned more centromerically. and apolipoprotein C2. Of the clones enriched in the VYS mesoderm, only Using molecular marker situated at the observed inversion, we can ,BHl-globin was identified in sequence database searches. Future work will genotype Rw embryos. This enabled us to show that Rw homozygote include further characterization of unidentified VYS enriched mRNAs and embryos die at 9.5 days. Histological analysis revealed that these mutant isolation of additional early yolk-sac specific messages. embryos undergo normal gastrulation, with developmental arrest occuring around the time of neuralation. (Supported by NIH HD29573 & NSF DCB 6886 (CWL) and NIH HD 28410 (MB)).

2045 2046 DEVELOPMENTAL POTENTIAL OF CHICK EMBRYO EPIBLAST THE SPATIAL AND TEMPORAL LOCALIZATION OF MEF2 PROTEINS IN CELLS: DIFFERENTIATION IN THE ABSENCE OF DEVELOPING MOUSE EMBRYOS.((S.V. bain md B. Nadal-Ghnard)) GASTRULATION AND TISSUE INTERACTIONS. ((J.V. Gerhart, R.A. Deparment of Cardology, Childron's HospIta, Harvard Medical School, Boson, Reed, J.N. Capparella, J. Flynn, and M. George-Weinstein)) Department of MA 02115 Anatomy, Philadelphia College of Osteopathic Medicine, Philadelphia, PA The dIfer on of skeletal musle depends on a family of musce spec 19131. btospn factors, the baslc-hellx-loop-hell (bHLH) pbtns, whIch are potent Inducro of the myogenlc progruni. Recety, Vie MEF2 (musce enhancer factor) The cells of the embryo arise from the dorsal layer, the epiblast. The proteins have also been Implicated In thIs procesa. These prteIs have muscle- developmental potential of primitive streak stage chick embryo epiblast specfc DNA bIndIng actIvIty mid bind to cnrved A/T dch elment in the cells was analyzed in vitro. Stages 3-5 epiblasts were isolated from the roguatory regions of numrwous musce spedcc genes. Thoug theIr role hI primitive streak, mesoderm, hypoblast, and area opaca. Scanning electron myogenesl Is documented, heIr relaonhp to the bHLH proteIns and theIr microscopy demonstrated that few, if any, mesoderm cells remained h chy Inte reguSlon of yogeness is qu . To gan a clerer pe attached to the epiblast. Immunofluorescence microscopy using cell type of the hlrarchy of events In skeletal myogene, ie spati ad temporal ptern specific antibodies was used to determine whether epiblast cells of MEF2 protein expression was examined at varous stages of mouse differentiated along multiple pathways in vitro. Skeletal muscle celis were develpment by whole mount In shiu hNmunolsocerls usIng polyclonal detected 24 hours after plating. These cells were labeled with antibodies anIKbodIes specIfIc for the MEF2A, MEF2C and MEF2D bsofrnms. The MEF2 against myosin heavy chain, troponin I, a-actinin, titin, and myoD. proteins are expressed eary In devopment (day 8.5-9 p.c), locang to regIon Neurons, cells synthesizing type II collagen, and a small number of wIth the formatIon of skelet muse (smtes) and heart. In the notochord cells also developed in these cultures. The lack of detection of somites expressIon proceeds hI a rosta-caudal dIrcton concurent with som*te cardiac muscle cells further supports the absence of contaminating maIon. In additon MEF2A Is detected hI cols of embryonic vascuature. mesoderm in epiblast cultures. Regions of the epiblast which do not form These results showing Ve eary appearance of MEF2 proteins and their dIsnct muscle in ovo gave rise to skeletal muscle by the second day after plating. patterns of expressn durI mouse embryogenesl support tVe vlew that MEF2 These results suggest that myogenic potential is wide-spread in the epiblast, proteins are Indeed key players hI the geneaIon and malnenance of the its cells are heterogeneous, and differentiation can occur in the absence of myogenlc program hi earty de . gastrulation and tissue interactions.

2047 2048 NOVEL EFFECTS OF RECOMBINANT HUMAN PREGNANCY- OVEREXPIESSION OF GOOSECOID IN ES CELLS INDUCES THE SPECIFIC l1-GLYCOPROTEIN ON THE PREIMPLANTATION EXPRESSION OF GENES REQUIREDFOR MESODERM EMBRYO. ((S.M. Wu, L.L. Amold, J. Rone, L.A. Blomberg, M. Trivedi DIFFERENTIATION. ((J. D. Goldl, C. A. Burdsall, J. J. Menesesl, and W.Y. Chan)) Departments of Pediatrics and Obstetrics & Gynecology, and R. A. Pedersenl,2)) tLaboratory of Radiobiology and Georgetown University Medical Center, Washington, DC 20007. Environmental Health, 2Departments of Anatomy, Radiology and Obstetrics, Gynecology and Reproductive Sciences, Pregnancy-specific l1-glycoproteins (PSG) comprise a family of proteins University of California, San Francisco, CA 94143. synthesized by the syncytiotrophoblasts during pregnancy. They have also been detected in the culture media of preimplantation embryos. The The homeobox gene goosecoid is expressed in Spemann's objective of this study is to examine the effects of these proteins on the organizer in Xenopus, Hensen's node in the chick, and in the growth and maturation of embryos. Two cell mouse embryos were anterior region of the primitive streak in the mouse. Micro- cocultured in a two chamber system with Chinese Hamster Ovary cells injection of goosecoid mRNA into Xenopus embryos induces a expressing recombinant hPSl (rhPSl 1) (product of human PSGl gene) in secondary body axis and the induction of mesodermal genes. the presence and absence of neutralizing anti-PSG antibodies. Cleavage and To investigate the role of goosecoid in the mouse, we have maturation rate of the embryos were assessed at 12 hr intervals. Embryos created stable lines of ES cells which overexpress the cocultured with transfectants expressing rhPSl showed a significant murine goosecoid gene. Goosecoid-expressing cells show enhancement of cleavage and maturation rate compared to controls from 24 induction of brachyury and nodal, two genes that have hrs on. At 24 hr, all embryos were at the 8-16 cell stage when cultured in been demonstrated to be essential for proper mesoderm transfectants the presence of rhPSl 1, while those cocultured with control formation in the mouse. We have also examined the 76 and those cultured in the presence of neutralizing antibodies had only to expression of a series of other genes characteristic of both 89% reaching similar stage of development. After 60 hrs, 100% of the the primitive streak and differentiated mesoderm, and have to embryos cocultured with transfectants expressing rhPSl developed looked at the in vivo effects of these cells upon the develop- blastocysts, whereas only 41 to 56 % of the embryos in the control cultures ment of chimeric embryos. (Supported by NIH Grant reached the blastocyst stage. These results suggest that at least one of the #HD26732 and USDOE Contract#DE-AC03-76-SF01012). human PSGs exhibits embryotrophic activity in the mouse model. Tuesday. Mammalian Development (2049-2054) 353a

2049 2050 EXPRESSION AND HORMONAL REGULATION OF Muc-I AT THE CYTOSKELETAL SHEETS IN MAMMALIAN EGGS AND EARLY APICAL CELL SURFACE OF MOUSE UTERINE EPITHELIAL CELLS. EMBRYOS PROVIDE AN ASSEMBLED FORM OF INTERMEDIATE ((G.A.Surveyor', S.K.Dey2, S.Das5, I. Chakraborty2 and D.D.Carson')) 'The FILAMENTS USED LATER IN DEVELOPMENT. ((G.I. Gallicano, S.M. University of Texas M.D.Anderson Cancer Center, Houston, TX 77030 and Schwarz, R.W. McGaughey, and D.G. Capco)) Molecular and Cellular 'University of Kansas Medical Center, Kansas City, KS 66160. Biology/Zoology, Arizona State University, Tempe, AZ 85287-1501. Mucins are large molecular weight 0-linked glycoproteins that coat and protect Mammalian eggs and embryos contain a highly crosslinked network of the apical surface of epithelial cells, including those of the uterus. The apical intermediate filaments that undergo massive spatial rearrangements at major surface of mouse uterine epithelial cells (UEC) mediates the initial phase of developmental transitions including fertilization, embryonic compaction, embryo attachment during early pregnancy. The murine mucin, Muc- I, is abundantly expressed at the apical surface of UEC. We hypothesize that Muc- blastocyst formation and at the time of embryo implantation. The elements, besides serving a protective function, also inhibits embryo attachment. referred to as sheets, are a stored form of assembled intermediate filaments which Removal of mucins from the apical surface is requisite for embryo attachment. are apparently prevented from occupying their normal cellular location until the Muc- I expression appears to be hormonally regulated since it is differentially crosslinked network begins to disassemble starting at embryonic compaction. At expressed through the estrous cycle and disappears prior to the time of embryo this time the intermediate filaments associate both with the nuclear envelope and attachment to UEC. Studies on hormonal regulation show that estrogen, but not with the plasma membrane at forming intercellular junctions. These specialized progesterone, induces Muc- expression in overiectomized mice. In fact, intermediate filament networks are composed of four prominent cytokeratins, progesterone inhibits the up-regulation of Muc- by estrogen. Injection of type 5, 6, 16, and a form referred to as "Z," but probably do not contain much, pregnant mice with the progesterone antagonist, RU486, not only inhibits if any, cytokeratin 8. Both electron microscopy and immunodetection using but also maintains Muc- I at levels. Muc- embryo implantation, expression high scanning laser confocal microscopy show a massive build up of tonofilaments as I expression also was examined in a delayed implanting mouse model. In this the embryo "hatches" from its zona pellucida, increase in the number of case, Muc- I decreased to low but detectable levels when mice were maintained derived cells as well as ectoderm and endoderm cells. on progesterone alone, a condition that maintains implantation delay. Injection trophoblastic primitive to of nidatory estrogen, a treatment that triggers implantation, did not change Tonofilaments then suddenly disappear immediately prior gastrulation. Muc- I expression. Collectively, these data indicate that estrogen is stimulatory Immunodetection demonstrates that the tonofilaments contain large amounts of for uterine Muc- I expression, while progesterone is strongly inhibitory. The cytokeratin 5, and 6. These results suggest early and late roles for the observations are consistent with a reduction of Muc- I being required prior to intermediate filaments within sheets in mammalian embryos. At early stages, initiation of embryo attachment to UEC. (These studies were supported by NIH sheets contribute individual intermediate filaments to cells of the embryo and at grants HD 29963 and HD 29968 awarded to D.D.C. and S.K.D. respectively.) later stages they contribute to tonofilament formation. This work was supported by NIH HD27151.

2051 2052 TARGETED MUTAGENESIS OF THE GATA4 GENE IN MOUSE ANTSENSE INHIB1TON OF ENGRAJLED- 1 CAUSES ABNORMAL PATTERNING OF EMBRYONIC STEM CELLS DISRUPTS IN VlTRO DIFFERENTATION INTO THE BRAIN AND SPINAL CORD OF MOUSE EMBRYOS. ((T.W. Sadlerl E.T. Lu2, PRIMITIVE ENDODERM AND CARDIAC TISSUE. ((C. Soudais, M.C. Simon, and K.A. Augustine)) Departments of Cell BIogy and Anotomy1 and M.S., M. Bielinska, J. Saffitz, J.M. Leiden, and D.B. Wilson)) University of Medcne2, Univesity of North Carolna, Chopel Hi, North Caroina 27599-7090 Chicago School of Medicine, Chicago, IL 60637 and Washington University School of Medicine, St. Louis, MO 63110. The role of enroEed- 1 (En-1) wos Investigated durhg the period of neuunatlon In mouse embryos usng a combnatIon of antWense olgorncleotides and whale embryo culture. Phosphorothkoated Transcription factor GATA-4 belongs to a family of zinc finger DNA-binding olgonucleotdes (20 boses) were krlected (100nt 15-80;sm) nto the omnotc proteins that are critical regulators of differentiation and development. Expression cavity at the 4-6 somlte stoge and embryos were examlned from 4-48h later. of GATA-4 is restricted to the heart, primitive (yolk sac) endodemn, gut epithelium, Cranial abnorialtles (narrowin and decreased expansion In the mid- and and gonads. In vitro studies demonstate that GATA4 can bind and trans-activate hNndorin regions) were observed In 43% (N=83) of antense treated embryos (versus 12% of sense controls, N-76). Neural tube abnrormaxtes, conistIng of the promoters of genes expressed preferentially in cardiac tissue (BNP, aMHC, laterol outpocketngs and undulations, occurred in 39% (N=83) of ontisense To assess the role of GATA4 in cTnC) and primitive endoderm (J6 serpin). treated embryos (versus 1% of sense controls) and 75% of the affected development, we have utilized targeted mutagenesis to disrupt both copies of the embryos exhbIted caudal dysgenesls. Embryos gIven double Irjections of GATA-4 gene in mouse embryonic stem cells. When differentiated in vitro into antbense oligonuclotides at 0 and 12h showed increosed incidences of cystic embryoid bodies, the homozygous deficient cells display specific defects in caudal dysgenesis compared to those given single injectins (100% at primitive endoderm and cardiac development. Light and electron microscopic 40/60Wm). Hlstologkcd analysIs of embryos with neural tube abnormolIties showed doanIzatlon of the neuroeplthekjm, cel death and apparent examination reveals a complete absence of visceral and parietal endoderm on the dupIcations of the tube and notochord. At early tImes after exposure (4-12h) surface of the GATA4 deficient embryoid bodies. Immunofluorescence studies affected embryos also exhibited lass of poraxbla mesoderm and show that expression of type IV collagen and laminin B is markedly diminished in disogarizatIon of somltes. Inunoht ochemistry showed that embryos the GATA4 deficient embryoid bodies. RT-PCR and in situ hybridization studies treated with ants algos hod reduced levels of En-I protein In the of the GATA4 deficient embryoid bodies indicate that transcripts encoding heodfolds 4h after exposure, In the mId- Nndbroin region after 24h, and in the neurd tube, somates, and foreImb buds after 48h. The prmIlve streak reglan primitive endoderm markers (AFP, type IV collagen, J6 serpin) and cardiac markers of antbense treated embryos wos wider and abnormoily curved compared to (aMHC, cTnC) are absent or significantly reduced, whereas other markers of controls. PCR analysis demonstrated En-I expression In the streak from the 7- differentiation (e.g., Brachyury) are expressed normally. We conclude that GATA- 12 somite stoge of development. These results confirm a rde for En-I In 4 plays an essential role in differentiation of cardiac tissue and yolk sac endodern. cranial development and suggest a nove rde for the gene In patteming of the neural tube and caudal segments of the embryonic axls due to its expression in the primitive streak.

2053 2054

TIMP-3 PRODUCIJCON IS U ATD BY IIUMAN INDUCIION OF A GENERAL (CPG)-DNA DEMETHYLATING ACTIVITY BY CYTOTROPHOBLASTS DURING INVASION IN VTrO((K. E. BassM, THE RAS SIGNALING PATHWAY IN MOUSE EMBRYONAL CILLS. H. LiM), S. P. HawkesO), and S. J. Fisher(U))) Depts. of Stomatology (1), and ((Moshe Szyf)) Department of Pharmacology, McGill University Montreal PQ Pharmacy and Pharmaceutical ChemistryO2, UCSF. Canada H30 1Y6 The pattern of DNA methylation is formed during development by de novo of the human Formation placenta requires differentiation of placental methylation and demethylation processes. The biochemical mechanisms that cytotrophoblasts (CTBs) into cells that aggregate into multilayered columns are responsible for demethylation are sill unkmown. Uaing a plasmid that and the uterus. The CTBs tumor- penetrate differentiating transiently express is in vitro methylated in all its CpO sequencea with the methylae Sul as a like of activated 92 kD IV collagenase properties, including production type substate, we pally purified an activity from the murine embryonic cell which is for invasion. The mechanisms (MMP-9), rate-limiting controlling line P19 that can convert methylated cytosines in the CpO dinuceotide invasion are understood but include inhibitors. CTB poorly may proteinase sequence to nonmethylated cytosines. The partially purified demethylase is The tissue inhibitors of and are metalloproteinases (IM -1, -2, -3) naturally active in the absence of exogenous dCTP suggesting that the activity that we occurring polypeptides. Since CTBs synthesize low levels of TIMP-1 and had identified does not excise methylated bases and replace them with -2, and both inhibit CIB invasion in culture, it is possible that maternally bases but is a derived TIMPs CTB invasion in vivo. We examined TIMP-3 nonmethylated bonafide demethylse activity. General regulate demethylation is one of the processes that is associated with cellular expression by invading CTBs in vitro. Purified CTBs from 1st, 2nd, and differentiation. To test the hypothesis that the activity of the demethylase 3rd trimester placentas were plated on Matrigel-coated filters for 48 h, during the collected is regulated by ceular signaling pathways we introduced Ha-r into P19 which they differentiated along invasive pathway. Cell lysates cells. Induction of Ras signaling pathway has been previously shown to were every 12 h analyzed by protease substrate gels for TIMP-3 production induce cell and for MMP-9 We found that CTBs differentiation of different embryonal tines. Stable Ha-ras P19 by immunoblotting synthesis. transfects a of their genome both TIMP-3 and MMP-9 invasion and that the undergo general demethylation as judged by upregulate expression during nearest neighbor and gene specific HpaII/MspI restriction enzyme analyses. more invasive 1st trimester CTBs express the most TIMP-3 and MMP-9. These cells expres an enhanced demethylase activity as judged by their we examined how TIMP-3 is induced invasion. Next, during Adding ability to demethylate transiently transfected ix vitro methylated plasmid or D to the culue medium trimester CTBs cycloheximide actinomycin for 1st DNA. These that that of TIMP-3 like that of MMP-9, experiments show demethylase ctivity is regulated by showed upregulation CTB expression, cellular signaling pathways. We sugest that induction of this general nascent RNA and these results requires protein synthesis. Together, suggest demethylase activity by Ras plays an important role in cellular that of TIMP-3 contribute to the autoine of CTB production may regulation differentiation. (Supported by the MRC Canada M. S. is a scientist of the NCI trophoblast invasiveness; thus TIMP-3 expression may be coordinately (Canada). regulated with MMP-9 production during invasion. Supported by NIH Grants HD 07659 (KEB), HD 30367 (SJF), and CA 39919 (SPH). 354a Mammalian Development (2055-2060). Tuesday

2055 2056 A NOVEL MHC CLASS I GENE EXPRESSED IN THE MURINE CYSTATIN C REGULATION BY MOUSE DECIDUAL CELLS IN VITRO. BLASTOCYST. ((S.L. Sipes, M.V. Medaglia, D.A. Stabley, C.S. ((S. Afonso and B. Babiarz)) Department of Biological Sciences, DeBruyn, C.P. Landel)) Department of Medical Cell Biology, Nemours Rutgers University, Piscataway, NJ 08855-1059. Research Programs, Alfred I. duPont Institute, Wilmington, DE 19803. The mammalian conceptus is a semi-allograft and thus should be Differentiation of uterine stroma to decidua is thought to provide the rejected by the matemal immune system. The mechanisms by which it barrier to trophoblast invasion during mouse embryo implantation. It escapes from immune surveillance are not well understood, but it is has been suggested that the decidua impedes excessive trophoblast clear that the process is mediated by the trophoblast. Unique (TB) invasion by forming a cohesive tissue barrier with an abundant nonpolymorphic major histocompatibility complex (MHC) class I basement membrane matrix. In addition, decidua also contributes to molecules have been isolated from trophoblast in humans. Similar molecules could play a role in this process in all mammalian species. the control of TB invasion by secreting proteinase inhibitors. We have We have isolated a novel nonclassical MHC Class I gene transcript reported that exogenous cystatin, a cysteine proteinase inhibitor, was from a mouse blastocyst .gt1O cDNA library which we constructed in one of the most effective inhibitors of TB invasion in vitro. We have our laboratory. The translated product of this gene appears similar to begun an analysis of cystatin C synthesis in cultures of mouse certain members of the Qa and TIa families, with a truncated decidua prepared from day 7.5 decidual capsules. Using a specific cytoplasmic portion and a unusual hydrophilic amino acid in the cDNA probe, Northern blotting, and densitometry, cystatin C mRNA transmembrane domain. However, the sequence of this gene does not correspond to any sequences present in Genbank. We have used was easily detectable after 48 hrs in vitro, with message levels multiple primer pairs specific for our cDNA sequence to identify this remaining constant for 96 hrs. Upregulation of message expression gene by PCR in genomic DNA from 6 different mouse strains. The was observed with culture in medium containing serum or at high cell sequences of the PCR products were identical, proving that every density in the absence of serum. When EPCs were co-cultured on strain tested to date contains this gene. We have isolated a decidual cells for 72 hrs, decidual cystatin levels were induced 3.5-fold containing this gene from a mouse (129SV) cosmid library over culture on plastic. Cystatin C protein synthesis is currently under (Stratagene, La Jolla, CA). The gene resides within a cluster of Class I similar genes. Sequence analysis of this gene confirms its identity with our study using culture systems. This work will employ a specific cDNA clone. Thus, this new gene, which we have named Blast MHC, antibody with immunofluorescence and immunoprecipitation tech- is a novel non-classical MHC class I gene which is expressed as niques. This work was supported by NIH grant HD20581 and the mRNA in the day 3.5 mouse blastocyst and is present in the genome Charles and Johanna Busch Memorial Fund. of multiple murine strains.

2057 2058 THE EFFECT OF NO+ IN OVERCOMING THE 2-CELL BLOCK IN THE EPINEPHiRINE SYNT'HESIS IN THE DEVELOPING HEART. MOUSE EMBRYO. ((l.-H. Bac)) Department of Biology, Sungshin Women's ((S.N. Ebert, M. FUjinaga, J. Baden, B. Siddall and D.L. Wong)) University, Sungbuk-Ku, Seoul 136-742, Korea. Department of Psychiatry, Stanford University School of Medicine, Stanford, CA 94305-5485. (Spon. by S.N. Ebert) in our Previous, unpublished studies laboratory have shown that Nib is as Epinephrine is ani imilportant stress hormone which exerts a wide range of effective as EDTA in overcoming the 2-cell block to development in the physiological effects, incitiding stimuilation of the rate and force of cardiac mouse. Ni? is a calcium channel blocker which, like EDTA, does not contractions. The ability of epinephrine to increase cardiac output becomes penetrate the plasma membrane. These previous studies suggest that Nib apparent at the begininig)ey of cardiogenesis, yet a source of embryonic interact with the membrane of mouse and some epitiephrine produiction has not been identified during such early developmental might plasma embryos cause Since signal to be conveyed to the cytoplasm To determine what this signalmight stages. phenylethanolamine N-methyltransferase (PNMT) is the final enzyme in the pathway for- epinephrine synthesis, it serves as a useful marker be, the effect of NiW on intracellular Cae was examined. Thirty day old for cells pr(xdticing this nietirohomione/neurotransmitter. We have therefore female ICR mice were injected with 5 IU PMSG, followed 48 h later with examined the expression of PNMT in developing rat enmbryos as a means of hCG, and were mated immedlately with fertle male mice. One- and two-cell identifyinig wherc and whenl epinephrine synthesis occurs. We demonstrate embryos were collected 25-35 h post-hCG by flushing the oviducts of mice that PNMT imiRNA is present in rat embryos as early as gestational day 9.5. which had vaginal plugs. The mucin layers were removed with 0.1% By gestational day 1.0, PNMT mRNA and active enzyme can be specifically localized to the heart. Immuinocytochemical analysis further reveals that hyaluronidase. Embryos were incubated in M16 medium supplemented with PNMT protein is predominantly contained within embryonic cardiac cells 0.4% were BSA and microinjected with the fluorescent Cae indicator dye, themselves. After gestational day 12.0, there is a marked decline in cardiac Fura-2 dextran, to monitor intracellular [Ca+]. Ni2+, dissolved in M16, was PNMT mRNA expression. Thuis, the expression of PNMT within the added to the incubation medium after a 10 min baseline recording; the final developing heart appears to increase transiently following its onset, with peak concentration of Ni2+ added ranged from 10-100 pM. Nib tratnent produced levels attained between gestational days 9.0 and 13.0. These results suggest an immete, transient increase in cytoplasmic Cab which returned to basal that the embryonic heart may be capable of producing epinephrine. Since levels after 2-8 min. In epinephrine can increase cardiac activity shortly after the initiation of cardiac contrast, SrP and sodium EDTA did not induce an contractions, otir findings fuirther suggest that epinephrine produced within the increase in cytosolic Ca?+. Further studies are needed to elucidate the enmbryonic heart may be important to the functioning of the developing heart mechanism by which Nibexerts its effect on intracellular Ca' and the two-cell during early cardiogenesis, before cardiac innervation is achieved. block.

2059 POSTERIOR DEVELOPMENT IN THE MOUSE. CHARACTERIZATION AND EXPRESSION OF ZNF74, A ZINC FINGER GENE ((JA. Cebra-Thomas, GS. Lin, J.L Halpern, J.S. Kim, and L.M. Silver)) DELETED IN DIGEORGE SYNDROME. ((M. Aubry, M. Bazinet, B. Grondin)) Department of Molecular Biology, Prnceton Univerasity, Princeton, NJ 0844. Clinical Research Institut of Montreal, University of Monteal, Monteal, H2W 1R7, Canada. The body axis of the mouse develops in a series of distinct stages (gastrulation, posterior elongation, tallbud formation) in which a miultipotent precursor DiGeorge syndrome is a developmental disorder affecting 1/20000 newboms and population gives rise to a variety of cell lineages. The preuror populations resulting in hypoplasia of thymus and parathyroids and conotruncal heart defects. We are believed to be related, but they exhibit distinct differentiation potentals. previously reported the consistent hemizygous deletion of ZNF74 zinc finger gene in Mol. We thus initiated a biochemical During the first stage (gasslation), the six mesodermal lineages anse from DiGeorge syndrome patients (Hum. Gen., 1993). characterization of this candidate gene and of its gene product Two mRNA species cells of the streak, while a portion of the remaining primitive primitive were detected by Northern blot in human and mouse embryos but not in adult tissues ectoderm is induced to form the anterior tube. the final neural During phase or in any human cell lines tested. A 3.6kb cDNA clone presumably corresponding to (talbud fomiation) derivatives of what are dassically considered the three the smaller mRNA species has been obtained and sequenced. This ZNF74 cDNA germ layers (gut, notochord and somites, and neurl tube) are all generated from revealed a 1.5kb open reading frame encoding 12 zinc finger motifs of the C2H2 type a mesenchymal, Brachyury-positive group of cells at the tip of the tailbud. and a long 1.6kb untanated region located 5' to the site of translation initiation. This region is beLieved to be derived from the posterior end of the prinmitive Structural analysis of the gene reveals the presence of at least two introns in the 5' streak. The alteration in the differentiation pattern may be intrisic to the untranslated region and no intron in the coding region. The exonf/ntron junctions have cells themsves, and/ordue to a change in the signaling envirnment. To look been sequenced. A 59Kdal protein was obtained by in vitro tanscription ttranslation of the cDNA. or at these issues, we compared the expresson pattens of twacription factors and Various constcs have been generated using the complete partial cDNA coding region fused to GST (pGEX), His epitote(pQE) and maltose binding signaling molecules in the tailbud with those seen during gastrslation. protein(pMAL). Only pMAL constructs alowed us to obtain bacterially expressed results suggest that there are differences in the spectrum of Preliminary soluble proteins. The expressed zinc finger protein is able to bind zinc as revealed by trascription factors tailbud and expressed by primitive streak cells that might zinc blot and DNA as shown by Southwestem. The specific sequences recognzed by suggest an intinsic difference in potential. Finally, the development of the ZNF74 protein are presently under investigation. Beside the biochemical posterior portion of the primary body axis (forelimb to hindlimb) exhibits characteization of ZNF74 protein as atranscription regulator, the disruption of ZNF74 milarities with tailbud formation. During both phases of growth, the gene by homologous recombination will be needed to investigate its potential Brechyury gene is expressed in the developing neual tube. involvement in the DiGeorge syndrome phenotype. Tuesday. Mammalian Development (2061-2065) 355a

2061 2062 ALKALINE PHOSPHATASES OF BOVINE PREATTACH- TRANSIENT EXPRESSION OF THE a8 INTEGRIN SUBUNIT DURING MENT EMBRYOS. ((J. Beecroft, C. Wasnidge, A. Hahnel)) Depart- MESENCHYMAL-EPITHELIAL TRANSITION IN FETAL KIDNEY. ((J.M. ment of Biomedical of Breuss. L.M. Schnapp, S.L. Nishimura, D. Sheppard, and R. Pytela)) Lung Sciences, University Guelph, Guelph, ON, Canada Biology Center, Dcpt. of Medicine, UCSF, San Francisco, CA 94143-0854. NlG 2W1.

Renal morphogcnesis is a commonly used model for studies on mesenchymal - Bovine blastocysts were examined for the presence of the different isoforms epithelial transition. Unlike most epithelia, which form by extension of pre- of alkaline phosphatase (AP). Preliminary protein gels indicated both tis- existing epithelial sheets, the nephrons form by a direct conversion of sue non-specific (TNAP) and tissue specific (TSAP) alkaline phosphatases mesenchyme to epithelium. This conversion is induced by the ingrowing ureteric are present in bovine blastocysts. Two primer pairs were designed from bud, which stimulates metancphrie mesenchymal cells to condense, polarize and TSAP sequences from other mammals and were used with reverse tran- form primary epiLhelial vesicles. Invagination, extension and further chain reaction to isolate differentiation of a primary vesicie Ieads to the formation of a nephron, via scription polymerase (PCR) bovine TSAP. The intermediate stages termed comma-shaped bodies and S-shaped bodies. It has exons 4-11 and were PCR products together span cloned, sequenced and been previously shown that the 31 -associated integrin at subunits, al through a6, found to contain three different bovine TSAP sequences. One is identical are expressed in ceil type speciric paterms during renal morphogenesis. For to bovine intestinal AP (bAP), but the other two are novel sequences. example, the al subunit is expressed only in the undifferenLiated mesenchyme, while the a4 subunit is expre.ssed by both undifferentiated and condensing The two new bovine TSAP are 83% and 84% identical to the bIAP nu- mesenchymal cells. We have now used immunohistochemistry to determine the cleic acid sequence and 89% identical to each other. Neither of the new distribution in human feial kidney of the more recently discovered 131-associated sequences contains introns or stop codons within the sequenced portion. a subunits, a8 and ax9, as well as the av inlegrins. Our results show that a9 is Primers specific to each bTSAP have been constructed and are being expressed by the undifferentiated mesenchyme. and absent from the condensing used to characterize the expression pattern of each TSAP isoform through mesenchyme and primary vesicles. In contrast, a8 displays a unique pattern of regulation: Il is absent from uninduced mesenchymal cells, strongly expressed in bovine preattachment development. condensing mesenchymal ceils, and again undetectable in S-shaped bodies. The av subunit and its panners 13, 15, 16, and 18 are not detectable at early stages of nephrogenesis. Thus, a8 displays a transient expression pattern that is unique among integrin subunits. These results suggest that a8 may play a unique role in the condensation process and/or the differentiation events leading to mesenchymal-epithelial conversion.

2063 2064 cDNA CLONING OF MURINE I ANTIGEN FORMING 5-1,6-N- PREF-1, A NOVEL MEMBER OFTHE EGFIKE FAMLY OF PROTEINS, INHiBITS ACETYLGLUCOSAMINYLTRANSFERASE (IGnT). ((A.D. Magnet and ADIPOCYTE DIFFERENTIATION. Cyntha M. Smaa and Hei Sook Sul, M. Fukuda)) Department of Pathology, University of California. San Diego, Univermity of CalifornIa, Berkeley, Berkeley CA 94720 La Jolla, CA 92093 and La Jolla Cancer Research Foundation, La Jolla, CA With Fe aim of idtying novel reguator of adipocyte 92037. differentiation, by dIfferentIal screening, we olated a cDNA sequence coding for a novel member of the epXdermal growth factor (EGF)like In human erythrocytes during embryonic development, the fetal (i) antigen family of proteins, which we deaigated prof-1. Pref-1 is syntheszed as is replaced by the adult (I) antigen as a result of the appearance of 8-1,6-N- a tranamembrane protein with aix tuxndm EGF.llke repeats at the putative acetylglucosaminylwransferase (the I-branching enzyme or IGnT). Previous extracellular domain. Murine pref-1 gwne Is a sngle copy gene spanning studics have shown that during mouse embryogenesis, the I antigen is 7.3 kb with 5 exons. However, mulIpl discrete forms of pref-1 protein exprssed throughout the preimplantation peiod, whereas the i antigen is of 45-60 kDa are preent in to N-linked first detected in the 5-day embryo (Kapadia et. al. (1981) Eip. Cell Res. prdpocytee, owing 131:185). However, the systematic studies on the detection of I antigen glycosylation and alterative spHlcing. RT-PCR and cDNA cloning were not caried out throughout embryonic development. A murine cDNA demonstrate at least 5 altrnatively spliced pref-1 transcripts. Pref-1 clone for IGnT would be critical in determining expession pattems of IGnT mRNA is highly retrkctd in adult tbueo and present only in adrenal during mouse development. We have isolated a cDNA encoding the murine gland. Pref-1, howover, in preent in varkity of tissues in developing IGnT by screening a mouse teratocainoma cell line cDNA library with a embryo, most prominent in pituitay and In developing vertebra. Pref-1 is human IGnT cDNA (Bierhuizen et. al. (1993) Genes & Dcv. 7:468). The first detected in E.8.5 mouse enbryo and continues until E. 18.5. While murine cDNA sequence has a 1299 bp open reading frame beginning at pref-1 mRNA is abundant in pre _oye, it expresslon is completely nucleotide and a 954 3'-untranslated A 201, bp region. Kyte-Doolittle abolished, by decraae In pref-1 transacpto, during differentiation hydrophibicity plot of the deduced amino acid sequence predicts that the gene of 3T3-L1 preadipocytes to adipoct. Differentation defective 3T3-C2 murine IGnT protein has a type II membrane topology, as does the human IGnT protein, as well as all mammalian glycosyltransferases cloned to date. cells express 3-fold higher pref-1 mRNA velb As compared to 3T3-L1 Computer searches of the Genbank and EMBL nucleotide sequence cells. Fetal calf saeum decrease prof-1 mRNA leei by 70%. Moreover, datbases revealed no homologous sequences to this cDNA, other than to constitutive expression of pref-1 in pdpoct, which in effect blocks human IGnT and human C2GnT. The murine cDNA clone will be used to ats down-regulation, dratically Inhibits adipose differentiation. This perform in situ hybridization on mouse embryos, in order to establish the indicates that pref-1 functior as a negative regulator of adipocyte developmental expression pattern of IGnT. Supported by NIH PHS T2 EY- differentiation, possibly in a manner alogous to EGF4-ke proteins that 07109-0SSI (ADM) and ROI CA33895 (MF). govem cell fate decisions in invertbrates.

2065 MULTIPLEX PCR AMPLIFICATION IN MOUSE AS A MODEL FOR PREEMPLANTATION GENETIC DIAGNOSIS. ((N. Steuerwalde, H. Lambet, RJ. Herrera3, M. Lareau2, and AJ. Steinleitnert)) 'Astarte, San Francisco, CA 94108, 2Califomia Pacific Medical Research Institute, San Francisco, CA 94115, 3Depatment of Biological Sciences, Florida Intemational University, Miami, FL 33199.

Preimplantation screening of embryos obtained as a result of in vitro fertilization (IVF) can be accomplished by means of gene amplification using polymerase chain reaction (PCR). This approach provides couples at risk for transmitting genetic disorders with an altemative to the termination of an established pregnancy following chorionic villi sampling or amniocentesis. The technique involves the removal of a blastomere from the embryo by micromanipulation for analysis. PCR is then performed to permit genetic diagnosis. Simultaneous amplification of a mouse Y-specific repeat sequence and of the ovary-specific gene which encodes ZP3 was accomplished using primers which bind to the regions which flank these sequences on the mouse Y chromosome and chromosome 5, respectively. Doing so permits screening for X-linked disorders for which a specific diagnosis is not know and also provides a control for failed PCR. Typically following amplification, the Y-specific fragment and the fragment from ZP3 in mouse were coamplified in male cells. Female cells produced only a single band. Positive and negative controls responded opriately. Such a multiplex system in a mouse model can be used for the preclinical development of methods for embryo biopsy and to demonstrate the clinical applicability of the procedure for preimplantation genetic diagnosis. 356a Cell Polarity (2066-2071). Tuesday

2066 2067 LOCAL1ZAT1ON OF TOTAL AND ACrIN MRNA DURING POLAR ACTIN DEPENDENT LOCALIZATION OF PLASMA DEVELOP1VENTOFPTE UCUS EMBRYO. (F-Y. Bouge S. Gaetula, and R. S. MEMBRANE MARKER DURING POLAR AXIS FIXATION IN Quatrano)) Department of Biology, Univesity of North Colina, Chpel Hi, NC Fucus 27599-3280. ((S.L. Shaw and R.S. Quatrano)) University of North Carolina at Becase of the uique tenporal and spatial localization patten of act#i protein dlUrng Chapel Hill, Department of Biology, Chapel Hill, NC 27599-3280. polaraxis fixation, polta growth of therhizold and MOpOes of the young Fiwu (Spon. A. Harris) embryo, we ae interetod in detemining te disufton of total and actin mRNA dwring Wedemosnaethe1hdarelo pant of total and ahtn mRNA The fluorescently-labeled dihydropyridine DM-BODIPY vitally stains in the devoingemryoby ins hybridization uin a nos-rsdioactrve, whole-ount a membrane in Fucus of ediquc Wefindnouynasnoryoftot or ctin mRNA in theegg and througb 8 hours plasma component zygotes. Labeling of zygose e k_lepso. At dhe tieof polar axis fixao (12 hour), a gradient of total ferfilized zygotes is initially uniform but begins to localize at 7 hours mRNAist*ishedwhich pasist tho a least the 4-cel stage of to the future site of polar outgrowth. Time course observations of wih the higaest concentration in the ths wea Althougb total mRNA contnues to DM-BODIPY labeled cells and FITC-phalloidin stained cells indicate bea)n -----disbeduxing thecompletion ofthefurst and wcond cel divisiou that DM-BODIPY localizes prior to polar axis fixation and at or before of the zygote, there is a gradual nd specifIc localiation of most of the acD mRNA polar localization of cortical actin filaments. Treatment of cells with around both of thene boDztal cel plan. No localion of atDn mRNA was obseved at the tip of the rhizoid were a lae concenftaon of aCtiDn protiDn 2OpM cytochalasin B prior to axis fixation reversibly prevents DM- sales Actn mRNA arnp to co-ocaincwlth the deposition ofan actin coBar' BODIPY localization and pulsing zygotes with CB after DM-BODIPY araond the first two division platen of the rhizold. Ihe function of the actin protein localization but before germination results in eventual delocalization collar is unknown, but could play a sutural role in stengtdhing the filamnntou of the DM-BODIPY label. We conclude that a plasma membrane rhizoid which serves as an alahmnt smucure for the developing cmbryo. Ibe component localizes to the site of future polar growth in an actin requs Iead"ndmcisns)sreponsblle or IheIocazatlon and sablization of attn mRNAwl bediscussed. Supported by a grant from NSF (MCB 9318757) to RS.Q. dependent manner just prior to polar axis fixation. Work supported by Grants from NSF #MCB9318757 and ONR #N00014-93-1-0888 to RS.Q.

2068 2069 GENETIC ANALYSIS OF THE BIPOLAR BUD-SITE-SELECTION PATHWAY IN BIPOLAR-SPECIFIC BUDDING MUTANTS IN BUDDING YEAST. ((J.E. Zahner, H.A. Harkins, and J.R. Pringle)) Department of SA_C-HAROMYCF-S CEREVISIAE, ((T. Freedman, J. S. Chant)) Biolgy, University of North Carolina, Chapel Hill, N.C. 27599. Department of Biochemistry, Harvard University, Cambridge, Saccharomyces cerevisiae cells select sites for bud formation MA 02138 according to two patterns based on cell type. MAT a or axcells bud axially, i.e., each new bud forms adjacent to the most recent previous bud site near The objective of this research is to identify and analyze gene the proximal pole of the cell (as marked by the birth scar). MAT a/a cells bud products required for bud-site selection and pseudohyphal in a bipolar manner, i.e., each bud can form either near the proximal pole or development (PHD) in the yeast Saccharmyces cerevisiae. near the distal pole, irrespective of the position of the most recent bud site. Since PHD requires bipolar budding, mutants which affect the We have undertaken a screen for mutants defective specifically in the bipolar bud-site-selection pathway. We have identified mutants in eight genes: bipolar budding pattern were isolated by using pseudohyphal mutants in five genes bud randomly, a mutant in one gene buds in growth as a screen to identify new bud-site-selection mutants. heterogeneous patterns (BUD7), mutants in one gene bud only at the Two haploid, bipolar PHD-competent strains were mutagenized, proximal pole (BUD8), and mutants in one gene bud only at the distal pole and PHD-defective strains were isolated. Strains were examined (BUD9). None of the mutations detectably affects the axial pathway. Of the for budding pattern defects and were crossed to previously five genes in which mutations cause random budding, four were previously known mutants which cause budding pattern defects for known (BUD2, BUD5, SPA2, and and one to be novel BNII), appears complementation analysis. Several new mutants which (BUD6). BUD2 and BUD5 encode a GTPase activating protein (GAP) and exhibit guanine-nucleotide exchange factor (GEF), respectively, that regulate the impaired bipolar budding patterns have been isolated. Work is small GTPase Rsrlp. This GTPase module is necessary for normal bud-site in progress to define complementation groups. This research selection in either the axial or bipolar pathway. The existence of bipolar- should contribute to a better understanding of how spatial specific alleles of BUD2 and BUD5 suggests that the products of both genes patterns are programmed and controlled as well as how function specifically at the presumptive bud site by interacting with axial- pseudohyphal growth is produced in the simple yeast cell. specific and bipolar-specific spatial signals.

2070 2071 THE ROLE OF BUD3 PROTEIN IN CELL DIVISION PATTERNS. ((M.D. Mischke1, USE OF PCR TO GENERATE TEMPERATURE-CONDITIONAL AL- J.R. Pringle2, and J.S. Chant1)) of Molecular and Cellular LELES IN THE SCHIZOSACCHAROMYCES POMBE CDC42 CELL PO- 1Dept. Biology, LARITY GENE. ((P.J. Miller and D.L Johnson)) Department of Microbiology and Harvard University, Cambridge, MA 02138; 2Dept. of Biology, Univ. of North Molecular Genetics, University of Vemont, Burlington, VT 05405. Carolina, Chapel Hill, NC 27599 (Spons. by S. Beckwith) The ability to direct growth to specific regions of the cell surface at appropriate times Determination of the division pattems In S. cerevisiae are controlled by in the cell cycle is essential for celular morphogenesis. The distantly related yeasts S. cell type. a and a cells bud In an axial pattem where the mother and daughter cerevsiae and S. pombe exhibit patterns of polarized grawth during their cell cycles. cells bud to their adjacent prior mother-bud junction. The BUD3 gene Is One of the genes involved in controlling morphogenesis in yeast is CDC4S , which specIcally required for axial budding. Bud3p localizes as an apparent double encodes a highly conserved GTPas of the Ran superfaznily Los of Cdc42 function ring which encircles the mother-bud neck at about the time of the G2 to mitosis bloces bud formation in S. cereiaesa but cell growth continue, resulting in large, round transition. This double ring persists through cytokinesis at which time It splits cells. Activated ofc42 mutants analogous to oncogenic roe mutants have a dominant- two one into single rings, on each cell. The Bud3p staining then disappears as lethal effect. Previously, we isolated the S. pomrbe CDC42 homolog and performed the cell of begins assembly the next axis of polarity. This staining pattem is similar mutational analyses. In contrast to results in S. cerevssiae, the activated allele identical to that seen for the of the 10-nm likely components neck filaments. of S. pombe odc42+ are not lethal and a null mutant exhibits a phenotype of small, We propose that Bud3p assembles on the neck filaments in one cell cycle and round, dense cells. To further study the null phenotype and to generate useful allels then remains to mark that site for axial budding and neck filament assembly in for genetic screens, we have taken advantage of misincorporation of nucleotides by Taq the next cellcycle. Our working hypothesis is that Bud3p assembles by directly polymerase in a PCR approach to generate temperature-cnditional-lethal mutations to the binding neck filaments but that following cytokinesis Bud3p dictates the in the S. pombe cdc42+ coding region. We isolated 13 temperabtu-enstive alleles position of the new neck filament ring Indirectly through the general bud site and three cold-ensitive alleles uSiDng this approach. To date, we have sequenced four selection proteins (Budlp/Rsr p, Bud2p, and Bud5p) and the polarity of thee alleles and found them to contain single mutations in conserved residues of establishment functions (Cdc24p, Cdc42p, and Bemlp). An important facet of the GTPase. We are currently charctersing the phenotypes of thee mutations in this model the is temporal control of Bud3p assembly and the proteins acting both S. pombe and S. crevisiae and completing the DNA sequence anabsis of the both and upstream downstream of Bud3p. Northem analysis Indicates that the remaining nine allele Our results suggest that minicrporation of nucleotides by Taq BUD3 transcript is cell cyde regulated, and examination of Bud3p is in polymerase is an efficient method of generating random sine mutations in a gne the progress. The associton of Bud3p with the neck filament proteins is currently. size of cLc42 (573 nt.). being tested by a directed two-hybrid screen. Tuesday. Cell Polarity (2072-2074) 357a

2072 2073 APICAL POLARITY OF A SUB-MEMBRANE INTERMEDIATE FILAMENT INDUCTION OF ALKALINE PHOSPHATASE BY CYTOSKELETON IN NON-BRUSH BORDER EPITHELIAL CELLS. ((M.L. BUTYRATE IN DIFFERENTIATING HUMAN Rodriguez, M. Brignoni and P.J.1. Salas)). Fundaci6n Campomar, Buenos Aires and ENDOMETRIAL CELLS ((H.Fleming, M.J. Begley, J.R. Dept. of Cell Biology and Anatomy, Univ. of Miami School of Medicine, Miami FL McDonagh, S.W. Wallace)) Department of Chemistry and 33136. Biocheniistry, Middlebusy College, Middlebury VT 05753

Although many pieces of evidence support the notion that the cytoskeleton plays Human endometrial cells (Ishikawa line) differentiate in culture in a role in epithelial polarization, no cytoskeletal component had been found to be response to a factor in fetal bovine serum (FBS). Butyrate inhibits cell specifically apical, except for some actin binding proteins. In this work, we report the proliferation and greatly enhances differentiation. Over a 48 hour apical distribubon of a 53 kDa cytokeratin. Furthermore, this cytokeratin co-punfied period, in response to FBS and 1mM butyrate, cells differentiate, with biotinylated apical plasma membrane proteins in high density complexes, polarize and detach from the dish resulting in the formation of hundreds obtained by sonication / rate centrifugation gradients. Differential biotinylation of the of dome structures in a previously undifferentiated monolayer of basolateral domain showed, using the same technique, that the 53 kDa protein is epithelial cells. At the same time changes in levels of a variety of mainly attached to the apical membrane, although a companion 58 kDa protein enzymes accompany these morphological changes. The enzyme alkaline attaches to both apical and basolateral membrane proteins. Immunoprecipitation phosphatase(ALP) has been studied for evidence of changing levels experiments indicated that a number of apical components are directly or indirectly accompanying cell differentiation. Low levels of ALP are found in linked to the 53 kDa protein, and metabolic labelling suggested that at least two undifferentiated cultures of endometrial cells. The inclusion of butyrate other proteins, besides the 53 kDa, do not biotinilate and are likely to be only in a culture of non-differentiated endometrial cells results in an induction cytoplasmic in these complexes. The 53 kDa protein was also present in human of ALP activity that is greatly enhanced as the cells differentiate and tissues and cell lines. These results indicate the existence of a terminal web-like become polarized. Cytochemical staining indicates that alkaline structure in, at least some, non-brush border cells, which attaches to the apical phosphatase levels are particularly high in the dome structures. Studies, domain and may play a role in apical polarization, specially during the acquisition of using inhibitors of ALP activity that distinguish among ALP polarity from non-polarized cellular stages. Supported by American Heart isoenzymes, have allowed us to demonstrate that a non-placental ALP is Association, Florida affiliate induced by butyrate in differentiating cells; distinguishing this effect from the previously reported induction of placental ALP in non- differentiating, proliferating cells by estoadiol.

2074 CADHERIN-MEDIATED CELL ADHESION INDUCED CHANGES IN CELLULAR PHENOTYPE. ((J. A. Mars, C. Auderson-Fllone, M. C. Jeong, I. R. NabI E. Rodripez-Boulm asd W. J. Nelson)). Department of Moleklar sad Cellular Physiolog, Stanford University, Stanford, CA 9430S-S426. Department of Anatomy and CeU BbIo , Corell University, New York, NY 10021 A primary function of cadheins is the regulation of cell adhesion. Here, we demonste that members of the cadhern superfamily differ i teir capacities to ioduce specialized epitdhal cell phnotype In stu, the retinal pigment epithelium (RPE) fonns cell-cell contacts within the monolayer, and at the apical membrane with the overlying nual ue; Nat, K+-ATPase and membrane-cytoskeleton distributions ar resutr d to the RPE apica membane domain, and desmnosomes are not formed on latral membranes. Thc RPE cell line RPE-J) expresses endogenous cadherin and forms a tight cell monolayer, but Nae. K+-ATPase is loalized to both apical and basal-lateral membrane domains. Expression of E-cadherin in RPE-J cells results in the restriction of Na+, K+-ATPase distribution to sites of cell-cell contact, and the reoa nizain and accumulation of the membrane-cytoskeleton to cell-cell contacts. The increased stability and accumulation of Na+, K+-ATPase in the basal-lateral plasma membrane correlates with a E-cadherin-induced redistribution and accumulation of fodin. E-cadhein induces the accumulation of the ankyrin-1/-2 isoforms which are notexpressed in the RPE-J cell or the retnal pigmentepithelium in situ. Significantly, E-calherin expression also induces the expression and assembly of desmosomes. These results demona tht pasicity in the structural organizato and functional phenotype of one epithelial cell-type can be directly regulated by the expession and distribution ofdifferent cadherin superfamily membes

Signal Transduction in Development (2075-2076)

2075 2076 INTRACELLULAR REDISTRIBUTION OF cAMP-DEPENDENT PROTEIN CHARACIERZATIONOFA PUTATIVE RECEPrORTYROSINE KINASE KINASE DURING LIMB CARTILAGE DIFFERENTIATION. ((Q. Zhang, D.W. FROM INTESlINAL EP1THEJLUMK ((M. D'Anico and D. Burgess)) Depart- Carr, K.M. Lerea, J.D. Scott, and S.A. Newman)) Dept. of Cell Biology and ment of Biological Sciences, University of Pitsburgh, Pittsbrh, PA 15260. Anatomy, New York Medical College, Valhalla, NY 10595, and Vollum Institute, Oregon Health Sciences University, Portland, OR 97201 A cDNA expssion libry pepaed from cells of the chicken intestinal Cartilage differenffation in chick limb bud precartilage mesenchyme is epithelium was screened with a polyclonal antibody to phosphotyrosi. Plaque accompanied by cAMP-dependent phosphorylation of a basic nuclear protein, puification yilded the partial sequence with a contanuous openreadingfame of p35 (Leonard & Newman, Dev. Biol. 120:92, 1987). We have now analyzed 1.7kB. Translation of this sequence revealed a molecule of 550 amino acids the distribution of the cAMP-dependent protein kinase (PKA), its substrate(s), (-63kDa) with nunewrous N-tesnnal N-linked glycosylation sites and a putative and putative A-Kinase Anchoring Proteins (AKAP) in the nuclear and rnsmmrane domaln. A search of sequence databanks yieled no matches to extranuclear fractions of precartilage and cartilage cells. The type II known tyrosine kinaas, although the clone showed limited similarity to the >- regulatory subunit (Rul) of PKA was abundant in precartilage nuclei, but chain of the insulin receptor. SDS lysates of cmb t E. coli in vivo labeled present in only trace amounts in cartilage nuclei. Correspondingly, p35, the with 32P.ot hate we electrophoresed on 10% SDS-PAGE gels and the major nuclear substrate of PKA, was phosphorylated in a cAMP-dependent fashion in precartilage nuclei, but not in cartilage nuclei. Lack of p35 resolvedproteins we tmnsferred onto mmilon. The resuldng blos contained phosphorylation in cartilage nuclei was due to the absence of PKA, since radioacdve bands which were sistant so KOH uetment. Phosphoamino acid exogenous PKA catalytic subunit caused phosphorylabon of this protein in analysis confirmed the presence of phosphotyosine in dese proteins. Western cartilage nuclei. A -150 kDa AKAP (nAKAP150) that bound specifically to the blots of m t cell exrctus with anti-phosphotyrosine yielded immunore- PKA Rll subunit was detected in precartilage nuclei but in trace amounts in active bands thatcouldbeefficindycompetedwith phosphotyrosine butnotwith cartilage nuclei. Nuclear fractionation studies indicated that nAKAP150 was phosphoserine or Nortern anlysis indicated that a 2.0kB with the nuclear whereas could be released primarily associated matrix, p35 mRNA was expsed in the pithe cells of the insine and in the pancra, by DNAse treatment. These observations indicate that co-localization of PKA although expresson was not found in the liver, brain, heut, gizrd, kidney, with p35 is required for p35 phosphorylation in the nuclei of precartilage cells. brast I expresson The developmentally-regulated nuclear AKAP may direct the localization of Rll muscleorlung. situ hybridizain studies have suwpported this in the precartilage nucleus and thus regulate cAMP-mediated nuclear events patter in the intestine and the pasceas. (Supported by NIH Grant DK 31643) during cartilage differentiation. (Supported by grants from NIH and AHA) 358a Signal Transduction in Development (2077-2082). Tuesday

2077 2078 ELEVATED INSULIN-LIKE GROWTH FACTOR-I RECEPTORS AND THE EXISTENCEIP3OF AND RYANODYNE RECEPTORS IN BOVINE PROTEOGLYCAN LEVELS IN THE AVIAN LOW SCORE NORMAL MUSCLE OOCYTES. ((C Yue, K. L White, W. A. Reed, T. D. Bunch)) ADVS Dept., D.C. J.D. WEAKNESS (S.G. Vellemanl,DcFarland2, Yeager', Biotech. CenterUtah State University, Logan, UT 84322 K.K. Gilkerson2, and J.E. Pesall2) 'Departments of Animal Science and Animal Genetics, University of Connecticut, Storrs, CT 06269 and 2Department of Animal and Range The existence of inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine Sciences, South Dakota State University, Brookings, SD receptor (RYR) was investigtedusing bovine oocytes by evaluating 1) injection 57007. of IP3R(1P3) and RYR (ryanodine (Ry), caffeine (Caf), cyclic ADP-ribose) agoniston on [Ca"i* release; 2)abblityofIP3RandRYRantagoniss (heparin(Hep; Low Score Normal (LSN) birds exhibit a loss of muscle ruthenium red and RYR)) to block the Ca2+- function as measured by an impaired ability to right IP3R), (RR; RYR), procaine (Pro; themselves when repeatedly placed in a supine position. The inducing ffctsofIP3,CafandRy;3)theedesensiifectofIP3 andRyon defect does not affect pectoral muscle myosin heavy chain I P3RandRYR;4)theabilityofRytopotendatetheeffectofCaf.InjectionoflP3 expression or myotube morphology throughout development (50-250 nM), (100-200KM), Caf (10-20mM), and cyclic ADP-ribose (0.5-4 (Muscle and Nerve, 16:881, 1993). In further consideration Ry induced Ca , while ofvehiclemedium(VM)hadnoeffect of the LSN defect, we have begun to investigate pM) relcasc injection extracellular matrix-cell signal transduction developmental Priorinjection of 1 mg/rnl Hep, but not RR (50PM), Pro (50 pM) orVMblocked pathways. Insulin-like growth factor-I (IGF-I) receptor [Ca2-]i release induced by 250 nM IP3. Prior injection of Hep(1 mg/ml) or VM binding was measured in pectoral muscle plasma membranes (1 %)dcidnoteffect[Ca2i.inducedby20mMCafor20 pMRy,butpmiorinjection isolated from 21 day embryonic and one week posthatch LEN of OM IM inhibited release induced by 20mM Caf. Prior and control birds. We have observed a 531 elevation in LEN 50 RRor50 Pro [Ca2J1] of RR orPro pM Ry IGF-I receptor concentration. However dissociation injection (200gM) (200 blocked 200 induced-[Ca2i] constants (Kds) did not vary significantly between LSN and release. After a 200 nMI 13-, or 200 pM Ry-induced[Ca2i] release, there was a control receptors,1 56xlO-8 M and1 73xlO-1, respectively. 10-15mnin refractory period during which, subsequent injections of the same We have also observed a significant elevation in LSN total agonistwereineffectivebutreciprocalinjectionsinduced[Ca2*] release. Injection and decorin levels at this glycosaminoglycan developmental of a subthreshold of the effect of low concentration stage. These data suggest that the LSN phenotype results concentration Ry potentlated Caf. RYR inbovineoocytesand from a modification in an extracellular-cell signal ThesedataindicateindependentlP3Rand coexist transduction pathway. stimulation with agonists of each induce [Ca2J. transients. Preliminay data indicate antagonists to these receptors reduce ferdlization rates.

2079 2080 ASSEMBLY OF A CONTRACTILE NETWORK IN THE CORTEX OF PKC AND PKM: A CELLULAR CHRONOMETER FOR REGULATING THE AMPHIBIAN EGGS IS REGULATED BY PROTEIN KINASE C. ((Martin REMODELING OF THE CONTRACTILE NETWORK OF THE AMPHBIAN EGG C. Yousef and David G. Capco)), Molecular and Cellular Biology/Zoology, ((J.H. Olson and D.G. Capco)), Molecular and Celhluar Biology/Zoology, Arizona State Arizona State University, Tempe, AZ 85287-1501 University, Tempe, AZ 85287-1501

The resumption ofmeiosis rapidly trigers the assembly of a contractile The conversion of an oocyteinto an egg qui nmeroustempolly programmed network in the cortex of amphibian oocytes. However, little is known structal and metabolic modificatos which act both at the corticl and the ineio r concerning the mechanisms regulating the assembly of this contractile cytoplasmic regions. One kinase, protein kiase C (PKC) servas to regule much of network. Recently, we have shown that drugs which activate protein kinase C this remodeling atempodly and spaally distinct fashion. As an immedate (PKC) can induce the full spectrum of alterations in the contractile network response to the appliation of progesterone, the natura tiger of meiotic maturaion, to we that active at the psma typically induced by progesterone, the natural trigger of meiotic maturaion Xesopsa1nvis oocytes demonste PKC becomes (Capco et al; J. Exp. Zool 264:395, 1992). We prpOsehut biedlg of membane. The oocye gins the ability to undero a conactile event detected the of a bisection of oocytes. At a prgestueto its reeptor actives phesphelpase C (PLC) whose mados fonnation contactie ing upon progestero-tated distnctly later the conbactle actvity radcay changes the axis of conftrce prdsct facilitste actvatie of PKC. Subsequendy, this kinase time, oriestston that of a ring into that of a cap upon bisection. The conical capthus phosphorylates cytoskeletal-associated proteins such as vinculin, resulting in from formed is comparable to the corical contactionseen upon fertilization of an amphibian recruitment and dispernement of proteins from the forming network. In egg. Our dat demonsaes tatthese distinct contrcte eves are rgulated by a support of this model we demonstrate that microinjection of PLC causes the single kinae, one form epgain the contactle ring, PKC, and therformregulting oocyte to acquire contractile ability as does microinjection of an active form the corticl cap, PKM. PKM is the cytosolic, active, coun_trpart of PKC produced by of PKC. Moreover, microinjection of antibodies to PLC, or inhibitors of cleavage of the membrae binding domain of PKC from its catlytic subunit We obliterates the ability of progesterone to induce contractility of the PKC, demonstrate the biochemica activity and prsence of PKCPIKM using phosphate cortex. Finally, injection of antibodies to some, but not all actin-binding incoporation and gel sft assays tding ito account the diffiag co-factor proteins the contractile ability, but the affects of these obliterates antibodies req s of thee relatd, yet distinctly diffrent kinses. Weshow that PKC is change as the contractile potential altes. The results suggst that the immediately activated upon application of progesterone to the oocyte, while PKM cytoplsmic signal transduction cascade initiated by progesterone acts to requirs 30 additional minutes before maximum activity is achieved. Since PKC and remodel the cortical cytoskeleton by phosphorylating actin binding proteins PKM are active in spataly distinct lcaons in the cell at temporally disinct times, this consequently altering their state of assembly within the contractile network kinase serves as a cellular chronometer to regulae theremodelling. Supported by NIH over time. Supported by NIH HD 27151. HD 27151.

2081 2682 CALCIUM DYNAMICS DURING STARFISH EARLY DEVELOPMENT. THE ECM AND GROWTH FACTORS COLLABORATE IN SEA URCHIN ((SA. Stricker)) Department of Biology, University of New Mexico, GASTRULATION AND IN GENE EXPRESSION VIA AN ECM RESPONSE ELEMENT. ((C.R. Tonkon, CA 8Sd, R.K. Rarnahaan, V. Govndwan. Albuqerque, NM 87131. J.M. George, and S. PatI) Departmentd Biology, Universtyd Houdon, Houson, TX 77204-5513. Oocytes of thestarfish Pirastr ochdaces weremicroinjected with Calcium plus B confocal Green dextran Rhodamine dextrn for dual-channd imai of The prooess d ga_tuton provIdes some d themoat kriporlrtand ng standIng calcium dynmics during oocyte muration, frtlization, a cleavage. Time- tion in ideep-m-rs biology. We preset evdne showIng thgrowth lpse images of dye-loaded specime were obtained every 15 sec for the first factors w h th rmt (ECM) psrt*te In s urehn few hoursofdevepmt and ratioed (CG dexIRB dex) to anlyze genesl a nd gen reguaton mwn in the a is arreed at the messnimbimuI stge and the biegeis481 the ral patten ofinclluar free calciumiom in dleaving -p-totcwpo inactvated when oclaogn orcths oorrgionint dlthe ECMred lsd No typically mbryos. prmie calcim spikes werc evident following addition previously shown tat the addllon of platet-derive! growth factor (PDGF) and of rnratoinducing hormone. However, conducted at 2 hr post- transtorming growth factora (TGF-a) rscue d m of ECM-dlrupted hormone trtment caused a global calcim wave that resulted in a proloned errmbryo and expression d LpSI gne. We report he that 1) A 125-bp (10-20 min) rise in the COG/RB ratio, and most zygotes displayed region in the promtr d the LpSI contains an ECM.reeponse cs elmdnt. The inesrtion of this elmed Int hot prom oe tha wer Independent multiple calcm spikes whose amplitudes were typically 0.14.5 that of the of the ECM causs the promote to require en Inta ECW for fertlizaon peak. Such repetitive calcium transients occred in predoymnany transcrIptir actvly. 2) Dbrupton of the interaction betwen were at the cortical cytoplasm ard obsrved timepoints corresponGdIn to Lytechsue PDGF and the ECM Inibn pgsutlon and Thes cytosis dung the first several cell cycles of both norma devlping studies suggest tha grwth fdaor algnng duIng Lythnus embryos ard cavg-rrested specimens that had boe ijected with colchicine. intera wih and requkes anhst ECM for acMy. a) Ard agn PDGF TGF-a antidb sthe respetve o Ihibit g adned In addion, risa the CG/RB ratio were sometmes evidelt aumd the tme of and and _p eL 1rthe im unoa elto w eshow th, td hmw nuclear envelope rakdown amd aapawe onst during first ceavage. Injecto lywe PDGF and TGF-a receptors are boated, prctd, on the of of but not de-N-sulated heparin, c1au abnrmal propagations of heprin, the snbrrt that rise to the god and sploulsa. 4) Pules studs show that the ferdlizat-xinduced calcium waves in a dose-dependent daion ad typically erribryos are ffnst to the growth factor antibody treatment smnetim from the abolisd post-fertilization calcium trients and dceavage. Based on correlatve 4-cel to the rneenchymn blkul dsges. Thee reult suggest that PDGF and the studies using caged IP3, hepari inte d with IP3-nded calcium releaue, TGF-a naik pa counction wkh the ECM are Iwolved in in the of calcium and/or early events of gastrulation and suggesting that release involved prouton thie free specffication8piculogenesia. morphognetic elevatiom that are observed in normally developing starfish embryos. Tuesday. Signal Transduction in Development (2083-2085) 359a

2083 2084 TORSO-LIKE, THE LOCALIZED ACTIVATOR OF THE TORSO RECEPTOR CELL DENSITY SENSING IN DICTYOSTELIUM. ((J.D. Bishop, D.F. TYROSINE KINASE. ((S. Savant-Bhonsale, and D. Monteli)) Dept of Biol. Lindsey, R.H. Gomer and P.J.M. van Haastert)) Dept. of Biochemistry and Chem., The Johns Hopkins University, 725 N. Wolfe SL, Baltimore, MD., 21205- Cell Biology and Howard Hughes Medical Institute, Rice University, 2185. (Spon. by S. Savant-Bhonsale) Houston, TX 77251 and Department of Biochemistry, University of Groningen, The Netherlands. Receptor tyrosine kInas (RTK) sgnaling controls growth and differentiation m a variety of eel types. Diferentiation of the r-segmented region at the The development of Dictyostelium discoideum is initiated when individual anterior and posterior poles of the Drosophib oentxyo reqtires the action of cells in a small area overgrow bacterial food source and starve. To seven matemally expessed genes including torso. The torso gene encodes of soil their cells a a RTK, which is expressed throughot the embryo btA activated specifcaly coordinate development, the monitor the extracellular level of protein, at the anterior and posterior poles. Genetic mosaic analysis has shown that CMF, secreted by starved cells. CMF is sequestered in vegetative cells and is the torso4ike (tsO gene is required during oogenesia in folicle cells each secreted upon starvation. Cells aggregate using relayed pulses of cAMP as the end of the oocyte. tsi is known to act prior to torso and i has been proposed chemoattractant. CMF regulates the signal transduction system which senses that tsl may encode the TORSO ligand or another activity contributing to the cAMP pulses, and cell aggregation is triggered when the density of starving activation of the TORSO RTK. cells in a given area is sufficient to cause extracellular CMF levels to rise above We have cboned the tsi gene by transposon tagging. The gene is expressed a threshold value. To examine the downstream effects of CMF, we probed as a 1.9 kb transcript in ovaries and embryos. In situ hybridizations in Northern blots of RNA from filterpad-developed CMF antisense and control ovaries revealed specific expression in follicle cells at the anterior and the cells with known developmentally regulated genes. For the cAMP pulse- posterior ends of the oocyte. Ubiquitous ectopic expression of cDNA genes and the absence of CMF during oogenesis, using a heat shock promoter, produced embryos with a induced D2, M3, phosphodiesterase, Ga2, phenotype similar to that resulting from constitutively active Torso alleles. prevents the expression from increasing 5 to 10 hours after starvation. On the These results suggest that ubiquitous tsi expression is sufficient to cause other hand, the pulse induced gene gp80 is expressed at a higher level in CMF uniform TORSO activation and that tsl normally controls the localized antisense cells than in wild type. For the cAMP pulse-repressed gene rasG, activation of the torso RTK. tsl encodes a novel protein with a putative N- and the possibly pulse-repressed phosphodiesterase inhibitor, message levels terminal signal sequence. We used TSL antbodies to stain early embryos. were the same or higher 5 to 10 hours after starvation in CMF antisense cells PreimTinary results show that TSL is present at both poles of the syncytial compared to control cells. We have previously shown that CMF regulates the bbstoderm embryo. These observations suggest that tsl could encode the cAMP-induced activations of calcium influx, adenylyl cyclase, and guanylyl TORSO tpand. We are currently testing this hypothesis. cyclase. Binding studies with whole cells indicate that CMF either binds to the cAMP receptor or that the CMF receptor is closely linked to the cAMP receptor. This suggests a mechanism whereby CMF regulates aggregation and gene expression by regulating cAMP signal transduction.

2085 IDENTIFICATION OF PROTEIN KINASES REGULATED DURING FERTnIZATION IN CHLAMYDOMONAS. ((V.Kurvari YZhang, Y. Luo and WSnell)) Cell Biol. & Neurosci, UT Southwestern, Dallas, T1X 75235. During ferdlization in Chlwydomonas, as gametes of opposite mating tpe adhere through flagellar gJycoproteins called agglutinins, they begin a complex series of signal transduction event culminating in the formation of a zygote. One of the early events in this pathway is the activation of a membrane-bound adenylyl cydase in the flagella. Previously we reported that the regulation of adenylylcyclaseiscoupledto agglutinin interactions bya staurosporine-sensitive acdvity, probably a protein kinase. Recently, using an in vitro phospoylation assay, we have discovered that flagellar adhesion leads to inhibition of phosphorylation of a 48 kD soluble flagellar protein. Results from biochemical studies have indicated that this inhibition is not due to regulation of a protein phosphatase, but to the inactivation of a protein kinase that may act upstream of adenylyl cyclase. Inhibitor studies and immunoblotting using anti- phosphotyrosine antibodies indicated that the 48 kD protein is tyrosine- phosphorylated in non-adhering flagella. Molecular doning and characterization by sequence analysisofa cDNAfor the 48 kD protein revealed about 20% identiy and 45% similarity at the amino acid level with inown protein kinases from other . Biochemical analysis of a bacterially expressed GST-fusion protein indicates that it is a substrate for a soluble flagelar protein kinase from Chlamydomoma Moreover, this protein kinase substrate itself is an active protein inase capabe of autopbophouylation s well as phosphoryladon ofmyelin basic protein. This protein and the molecule that phosphorylates it may be part of a novel signal transduction cascade in Cwanydmwnas. (Supported by NIH GM-2S661.) Neural Development H (2086-2087)

208 2087 AGREEMENT BEIWEEN IPR MODEL PREDICTIONS AND MAGNETIC GROWTH FACTOR REGULATION OF SURVIVAL AND DIF- FIELD INFLUENCE ON PC-12 CELL NEURITE PRODUCTION. ((CF. FERENTIATION IN THE SCHWANN CELL LINEAGE. ((R Mirsky, Blacikman, LP. Blanchard*, S.G. Benane, D.E. House)) US EPA, Research Ttiangle Park, NC 27711, *Bechtel R & D, San Francisco, CA 94119-3965. A Brennan, Z Dong, HJS Stewart, L Morgan, J Gavrilovic, *G Rougon, KR Jessen)) Department of Anatomy, UCL, Gower Street, London WClE 6BT, A recently developed ion parmetric resonance (IPR) model predicts magnetic UK. *CNRS UMR 9943, Universite d'Aix-Marseille II, 13288 MARSEILLE field interactions with biological systems based on a selective relation between the CEDEX 09, FRANCE. flux density of the static magnetic field (Bdc), the frequency and flux density (Bac) of the AC magnetic field, and the charge-to-mass ratio of ions of biological We have examined the ability of growth factors to influence Schwann cell devel- relevance (Bloelectromagnetics, 15:217,1994). To test this model, primed PC-12 opment and differentiation using in situ hybridisation, immunocytochemistry, 5 were Inside 5% cells, stimulated with ng/ml of nerve growth factor, placed a C02 northern and western blotting to monitor crucial changes in molecular phe- incubator containing a multiple-coll exposure system, where they were grown for 23 hours while exposed to specific 45-Hz Bac fields (determined by the IPR notype. Unlike Schwann cells, Schwann cell precursors (Jessen et aL Neuron, model), with a parallel Bdc of 366 mG and a perpendicular Bdc of less than 2 12, 509, 1994), cells that represent a distinct intermediate stage in the genera- mG. Cells exhibiting neurite outgrowth (NO) longer than the cell-body diameter tion of Schwann cells from neural crest cells, will undergo apoptotic cell death were scored as positive (FASEB J, 7:801, 1993). An earlier paper reported good within 24 hours of removal from axonal contact. A combination of IGF I or between model and NO inhibition for Bac between 0 and agreement predictions II and either FGF or 4 promotes short term survival in 468 mG rms as the cell response passed through maximal inhibition and returned 1,2 of these cells cul- to control level (Bioelectromagnetics, 15:239,1994). We now report continued ture. DNA synthesis is not induced under these culture conditions, even in consistency between the IPR model predictions and experimental data at higher the presence of forskolin. In contrast, this combination of factors and drugs AC magnetic field intensities (404-1109 mG rms). Specifically, in the extended causes proliferation in Schwann cells. Conditioned medium from DRG neurons range, the cells responded with distinctive, quasi-periodic cycles of inhibition promotes ionger term survival of Schwann cell precursors, and allows them to passing through a maximum, reuning to the control level, and then exhibiting a third maximum as Bac increases. An extension of the best mode fit obtained develop into S100 positive Schwann cells. Neuregulins have similar effects. In with the earlier low Bac data also fits these exmentl results well, predicting the Schwann cells cultured in defined medium, FGFs, PDGF BB and TGF#s sup- Bac values at which the cells demonstrate both maximal and minimal effects as press cAMP induced Pb mRNA and protein induction. In the case of TGFd, well as the relative magnitude of those maxima compared to the magnitude of the there is no requirement of DNA for suppression to occur (Morgan et first maximum observed in the 0-468 mG rms range. These results provide synthesis further support for the fundamental relationships embodied in the IPR model to aL Development, 120, 1399, 1994). TGFOs also induce higher levels of N-CAM predict biological responses at distinct AC and DC magnetic field combinations. and Li expression in cultured Schwann cells and suppress induction of 04 and CFB sppoed by EPA ad by DOE, IAG DEAID149CE34024; JPB sppword by galactocerebroside in reponse to intracellular cAMP eleation. Bechis R&D. i asact doss reflct EPA policy. 360a Neural DevelopmentII (2088-2093). Tuesday

2088 2089 DYMANICS OF NEURAL CREST MIGRATION FROM THE CHICK BRAIN MORPHOLOGY IN MURINE MUCOPOLYSACCHARIDOSIS HINDBRAIN REVEALED BY INTRAVITAL MICROSCOPY. ((E Birg- (MPS) Type VII ((N.J. Galvin, B. Levy, C. Vogler)) bauer, J. Sechrist, M. Bronner-Fraser and S. Fraser)) Division of Biology, California Department of Pathology, St. Louis University Institute of Technology, Pasadena, CA 91125 and Developmental Biology Center, Uni- Medical School, St. Louis, MO 63104. versity of California, Irvine, CA 92717. MPSs are inherited lysosomal storage diseases Neural crest cell migration in the hindbrain is segmental, with prominent streams of caused by deficiency of a lysosomal enzyme needed migrating cells adjacent to rhombomeres r2,r4 and r6, but not r3 or r5. Focal injections for degradation of glycosaminoglycans (GAGs). A of the lipophilic dye DiI clearly demonstrate that all rhombomeres, including r3 and r5, souse model of MPS VII shares features with human produce neural crest cells. We examined the dynamics of neural crest cell movement by MPS VII including severe -glucuronidase iontophoretically injecting DiI into small numbers of cells and repeatedly imaging their (,8-gluc) deficiency and cognitive defects. Central nervous progeny in living embryos over time using low light level epifluorescence microscopy. system (CNS) lysosomal storage occurs in both Labeled cells in the caudal ormid r3 move caudally within and along the dorsal neural murine and human MPS VII, but how it causes CNS tube surface toward the r3/4 border, and even into r4, from which they exit and migrate dysfunction is unknown. We described lysosomal toward the second branchial arch. Cells labeled in rostral r3 predominantly move rostrally to the r2/3 border before migrating toward thefirst branchial arch. Double storage and altered neuronal morphology with possible functional significance in MPS VII CNS. labeling shows that cells in ventrolateral r3 remain stable relative to the rhombomere Storage occured vessels, neurons, boundaries while those in the dorsomedial region shift caudally. DiI injections into r5 in meninges, ependyma, and glial cells. Storage was progressive show lateral movement followed by rostral and caudal migration along the forming otic vesicle into 2nd and 3rd arches. NF-M and HNK-1 antibody staining confirms this with age and varied in amount for different neuronal populations with high levels in pyramidal There appears to be a block by the thickened otic ectoderm adjacent to the pattern cells of hippocampal regions CA2, CA3, CA4 and in and similar but smaller blocks at r3 and elsewhere in the hindbrain are seen neural tube, cells. Histochemical staining of normal at various stages. These ectodermal wedgesseen in section appear to form a thin tunnel Purkinje mouse CNS indicated areas with high levels that may direct neural crest to openinp at r4 and mid r6. These results demonstrate of,0- gluc activity corresponded to areas of high that all rhombomeres initially give rise to neural crest cells, and that rostrocaudal neuronal storage in VII CNS. By EM, Purkinje contribute to the apparent segmental migration of neural crest cells. MPS movements cells with high levels of storage also showed condensed cytoplasm and alterations in dendritic ultrastructure. Morphometric analysis of MPS VII CNS showed decreased brain size and Purkirnje cell number.

2090 2091 A worm's eye view of unc-13 CONTRIBUTION OF NOTCH TO GLIAL FUNCTION IN ESTAB- Terese Rakow, Jeff Bender, Ichi Maruyama and Sydney Brenner LISHING BOUNDARIES IN THE NERVOUS SYSTEK DURING Dept. of Cell Biology, The Scripps Research Institute, La Jolla, CA METAMORPHOSIS OF DROSOPHILA. ((W.J. Costello)) 92037 Department of Biological Sciences/College of Osteopathic Medicine, Ohio University, Athens, OH Theunc-13 gene has been determined to affect the nervous 45701. system of C elegans. In unc-13 mutants, there is slow, irregular pharyngeal pumping, uncoordinated movement, and an insensitivity to Glial cells play a variety of important roles in cholinesterase inhibitors with an accumulation of acetylcholine. The nervous systems. During development, a major role for Unc-13 protein contains a diacylglycerol binding region (C1), consisting glia is forming pathways for neuronal migration and of cysteine repeats. Unc-13 also contains a calcium, phospholipid, and fiber tracts. Inherent in this role is establishing cytoskeletal element binding domain (C2). Electron micrograph boundaries in the nervous system. Notch (Ef), a neuro- reconstruction of the nervous system of unc-13 mutants reveals abnormal genic gene, appears to contribute to the function of gap junctions between major intemeurons. Also, there is abnormal the glia during neurogenesis. In late 3rd instar axonal morphology of the AS2 motor neuron. Thus unc-13 is thought to larvae and early pupae, there is a large accumulation be a novel component of a signal transduction pathway involved in of N product in two subsets of glia in the central nervous system development and function. Our current goal is toresolve nervous system. One subset forms a distinct boundary the expression pattern of unc-13. Due to the phenotype webelieve that it between the neuropil and cortex; the other subset is is expressed mainly in neurons, and possibly not in all neurons. We in the midline of the nervous system. By the middle have generated two fragments of the unc-13 protein to be used for of metamorphosis, the )I product in these glia subsets polyclonal antibody production. The antisera will be used for western has disappeared, coincedent with the establishment of analysis of unc-13 mutants and wild type nematodes and for the adult structure of the central nervous system. immunohistochemisrty. In concert with this, we are working to identify To test for the contribution of If product to the function of these glia subsets, the temperature- the 5 end of the mRNA which will be used to locate the region E M containing the unc-13 promoter on a cosmid containing genomic DNA sensitive allele of (1_) was used. When late surrounding the unc-13 gene. The promoter will be used to drive the 3rd stage larvae and early pupae are exposed to non- expression of a reporter gene in transgenic . permissive temperatures, disruptions occur in the formation of the central nervous system. The delinea- tion of boundaries by the glia subsets becomes less defined than in control organisms.

2092 2093

INSITU LOCALZAITIONOFTRAN(IT FOR VNTGRIN BETA-l, BETA- SEROTONIN DISTRIBUTION IN THE BRAIN OF THE 3 AND BETA-S IN THE DEVELOPING CHICK RETIN. ((D.B. Gevin, G.M. EMBRYONIC ZEBRAFISH. ((T.A. Corba, K. Garren and L.S. Ross)) Cai sd D.O. C3)) N Reseach Insitute, Universiy of Calforna at Neurobiology Program, Department of Biological Sciences, Ohio Sant Babaa, Sant Barbara, CA 93106. (Spon. by D.H. AndeoL) University, Athens, OH 45701.

ltegrn are a family of sarfc rcepto that have bees Aso We are investigatng the distrbution of serotonergic immunoreactve to mediate ad cell_xacear matix (Eas) iteromm for a widvariety cells in the zebrafish brain. Our results indicate that of cel type In prima retina cultres moderate imortant celar embryonic initdal activitie such aceurite, outgrowth h bees proposd dth serotonergic immunoreactive cells orginate in discrete clusters that are integri eff sim func WYO. In~the retina, in bilaterally symmetrical along the midline. The first immunoreactive cell fun .aeidence doesat exIt to pot d deveo bodies were labeled between 30 and 52 hours postfertilization. In 48 to patterns for wel 1t 54 hour embryos, two distinct clusters of sengic cells were seen in beta-, be-3 ad betS are a _exrsd in the developing chick retina dring the hypothalamus and ventral tegmentum. The hypothalamic cells were maod ftd wIth erntadon, uIrati and a al Our located near the midline, forming clusters of 10 to 15 dadrly labeled orthern show that the reltv abundance of ea oft cells. These hypothalamic cells extended processes that contacted the to be nzinm at embryonic day 6 (E), and decre e ventricular surface. The cells in the ventral tegmentum were also located is likely tht molcule such lmin viro ectin and collge play ctive role approximately to in developmntl events, and it of great ktine t to the cellular along the midline, forming larger clusters of 50 100 exprss of fteinthat have been show to madate with EC molecuit cells. Small diameter fibers were seen projecting laterally from these To ocaie the depatt for itegrin =RNA!&, in slu ventral tegmental cells and also to other areas of the tegmentum itself. In usinglabels RNA p a fi m chicken addition, serotonergic fibers were identified in the rostal telencephalon cDNA dooe for beta-, ho er studies showed in embryos at this stage. We are currently extending these findings by beta- i mmureiitpi b (RCt s), we founad detemining the precise timing of the initial immunoreactivity and the Beta-i cpreslos in wie and mat of the i i retinal distribution of immunoreactive cells in older embryos. precusn at EK At E12, bet localeudi to the developing RGC ay, a rio the layer (INL) Supported by NSF IBN-9222896. mueler'gsl ex By po al (P1), betl that is S tive" of cll day eqpei in prma re d to the RGCL Our resoks sw at that betl the developig retinainm extenive previuslyoberved. In iu for both bea3 and beta-S alao swes wide iprad expesion in Tuesday. Neural Development II (2094-2099) 361a

2094 2095 LINEAGE ANALYSIS OF OLFACTORY PLACODE A LONGITUDINAL ANALYSIS OF CELL LINEAGE IN THE OPTIC DEVELOPMENT IN THE EMBRYONIC ZEBRAFISH. ((L.K. Cole TECTUM OF THE EMBRYONIC ZEBRAFISH. ((M.M. Murray, L.G. and L.S. Ross)) Neurobiology Program, Department of Biological Craft, and L.S. Ross)) Neurobiology Program, Department of Biological Sciences, Ohio University, Athens, OH 45701. Sciences, Ohio University, Athens, OH 45701. The olfactory placode is unique among CNS structures in its ability to We are studying the roles of cell lineage and environmental interactions regenerate sensory neurons, which must then establish new synapses with in the development of the zebrafish optic tectum. Acetylcholinesterase the olfactory bulb of the telencephalon. Additionally, placode precursors (AChe) staining was used to determine when and where cells become give rise to cells which migrate to other brain regions. We are using post-mitotic. The optic tectum of the zebrafish differentiates in a lineage analysis to describe the normal pattern of olfactory placode rostrocaudal, lateromedial gradient. AChe staining was first observed at development in zebrafish embryos. Presumptive olfactory placode cells 30 hours of age, and only in the most rostrolateral regions. The extent of were labelled in blastula stage embryos by injection with a cocktail of staining gradually increased until it encompassed all but the most caudal fluorescent and biotinylated lineage tracers. Embryos were allowed to tectum at 48 hours. This age also marks the initiation of a marginal zone develop to various stages and then fixed for methacrylate embedding. of myelinated fibers that begins to form at the lateral aspect of the tectum. Serial sections were examined to characterize distribution patterns of By 60 hours, tectal cells migrated into this marginal zone, which clonally related placode cells. Progeny of successfully injected placodal continued to expand. At 1 week of age marginal zone cells formed the cells formed linear arrays, suggesting that early placodal cells distribute specific laminae characteristic of the mature optic tectum. In order to in an organized fashion. We are making time-lapse visual recordings of follow the dispersion of clonally related tectal cells, we injected cells labelled cells in living embryos, allowing us to describe directionality and with lineage tracer dyes at early stages of development. Tectal clones migration patterns of placodal progeny. resulting from blastomere injections disperse in either a radial or non- directional clumped pattern; there is also a bilateral symmetry to these Supported by NSF IBN-9222896. clonal dispersions. We also injected single midline cells of older embryos at the 5 somite stage. Clonal progeny of cells injected at this age form clones that are strikingly bilaterally symmetrical. These results suggest that tectal progenitor cells contribute progeny to both sides of the brain, even as late as 11-12 hours postfertilization. Supported by NSF IBN-9222896.

2096 2097 EXOCYTIC VESICLE TRAFFIC IN CULTURED HIPPOCAMPAL NEURONS. ((M. EVALUATION OF THE ROLE OF nWAVES" IN INTERSTITIAL BUDDING Martinic and G. Banker)) Department of Neuroscience, University of Virginia School AND GROWTH CONE REFORMATION IN CULTURED HIPPOCAMPAL of Medicine, MR4 Annex, Box 5148, Charlottesville, VA 22908. NEURONS. ((G. Ruthel and G. Banker)) Dept. of Neuroscience, University of Virginia School of Medicine, Charlottesville, VA 22903. Neuriteoutgrowth requiresthe delivery of newlysynthesized components along the exocytic pathway to the plasma membrane. We have used fluorescent derivatives We have previously described structures, referred to as 'waves', which travel down of ceramide which selectively partition to the Golgi apparatus and are incorporated axons neurons are a into Golgi-derived vesicles to examine exocytic vesicle traffic in hippocampal the of cultured hippocampal and associated with growth spurt neurons developing in culture. After 60 minutes at 370C, fluorescent puncta, when they reach the dp (Ruthel & Banker, Soc. Neuro. Abs. 1992). Waves are interpreted as presumptive exocytic vesicles, were distributed throughout both similar to growth cones in that they extend lamellipodia and filopodia and are axons and dendrites. Vesicles were distributed uniformly throughout the intensely labeled with rhodamine phalloidin and growth-cone specific antibodies. We microtubule-containing regions of neurites and were largely absent from actin-rich have noted two circumstane in which waves appear to become growth cones: when regions, such as the lamellipodia and filopodia of growth cones. Double-label studies a wave veers outward from the axon shaft to become the growth cone of a branch and with the endocytic markers sulforhodamine B and rhodamine-WGA confirmed that when a wave reaches the tip of an axon that has lost its growth cone. T'he current the ceramide-labeled vesicles were notthe product of endocytosis. Studies with the study was undertaken to address the question of whether waves are necessary for vesicle marker synaptic synaptophysin showed that ceramide-labeled and synaptic interstitial budding of branches and for growth cone reformation in cultured vesicles two In order to examine the comprise predominantly separate populations. hippocampal cells. We used time-lapse video microscopy to follow the development relationship between vesicle traffic and neurite outgrowth, vesicle density was of collateral branches. Collateral to at quantified in axons and dendrites at various stages of development. At day 1 in branches sometimes appeared arise random culture, axons are growing rapidly but dendrites are not. Despite this, the density sites along axons, but we also noted that whenever an axon looped to grow back along of ceramide-labeled vesicles in the two types of processes was similar. At day 7 in itself an interstitial branch formed from the looped region. Branching at a looped culture, when both axons and dendrites are elongating, the density of ceramide- region was coincident with the arrival of a wave in ondy 1 out of 16 cases, as labeled vesicles in dendrites was higher than in axons, but may reflect the larger compared to 7 out of 15 for non-looped regions. We also examined the formadon of diameter of dendrites. The data indicate that glycosphingolipid-containing vesicles new growth cones after transecting axons with a glass micropipet. Ihe formation of are delivered to both the axonal and somatodendritic domains of cultured a new growth cone at the tip of a trasected neurite was coincident with the arrival hippocampal neurons. This contrasts with the exocytic pathway in many polarized of a wave in 15 out of 39 cases. These results suggest that waves frequendy play a where epithelial cells, glycosphingolipid-containing vesicles are delivered to the role in the formadon of new growth cones during interstidal budding or following apical domain. Our results do not reveal an obvious correlation between the delivery axonal but are not necessary cones of transection, they for the formation of growth in exocytic vesicles to axons and dendrites and their rates of growth. Supported by these circumstances. NIH grants NS1 7112, T32HD07323, and IF32NS09342. Supported by NIH grants NS 7112 and HfDO7323.

2098 2099 GENERATION AND CHARACTERIZATION OF A MYC IMMORTAlIZED NEURAL CELLULAR DIFFERENTIATION OF PINEAL CELLS IN VITRO. CREST STEM CELL UNE.'M. Rao and D,J. Anderon'. Howard Hughes Medical (G.A. McNulty, S-Y. Tsai, P.T. Le, S. Silberman)) Dept. of Cell Biology, Institute, Divsion of Bilogy Caltech, Pasadena CA 91125. Neurobiology, Anatomy and Pathology, Loyola Univ. Stritch School of To study mouse neural crest development we have generated a rat IL monoclonal antibody to mouse low affinity NGF receptor(P75). We have Medicine, Maywood, 60153. utilized this antibody to show that p75 immunoreactive neural crest cells can give rise to neurons and glial cells in clonal culture. To obtain a large Cells of the pineal gland in the rat comprise a heterogenous population of identical precursor cells we have immortalized a population. Long term cultures of neonatal pinealocytes were pleuripotential precursor cell line using avian myc. The myc-1 cell line developed to further identify and characterize pineal cellular when on grown fibronectin is morphologically and antigenically similar to components 1-day-old Sprague-Dawley rat pineal glands were early migrating neural crest cells and does not neural and express glial and cultured + differentiation markers. Under conditions that generate neurons and glia in dispersed (DMEM 10% FBS) for 7-28 days. For primary crest cultures the myc-1 line undergoes differentiation into immunocytohemistry, the cells were fixed in 4% buffered postmitotic neurons and peripheral glia. Clonal culture of the immortalized paraformaldehyde and the reaction analyzed by either FrIC or HRP cells demonstrates that individual cells are bipotential and further when techniques. 7-day cultures contained S-antigen-positive pinealocytes cells are grown at colony density virtually all colonies contain both and a confluent monolayer of non-neuronal cells. The latter cells neuronal and non neuronal cells. Thus, the cell line generated from mouse included macrophages/microglia (OX-42+, OX-6+, Ac-LDL+), few crest is at least bipotential and identical to primary p75 immunoreactive few fibroblasts multipotential crest cells by a variety of criteria. astrCItes (S-100+, GFAP-), (pre-collagen 1+) and a We have begun experiments to analyze the ability of the cell line to population of unidentified cells. Additional cell types that appeared in differentiate into phenotypes other than neurons and glia as well as to 21-28 day cultures included striated myocytes, epithelioid cells and identify if neurons of both the sympathoadrenal as well as sensory lineage melanocytes.qHghly proliferating epithelial and pigment cells were are generated. We find that when myc-1 cells are co-cultured with the isolated from several long-term cultures. Co-loalization of keratin heart they generate neurons that are immunoreactive for tyrosine and S-antigen suggests that pinealocytes are the origin of the epithelial that the neurons have hydroxylase suggesting generated properties similar cells. Both to pinealocytes and the confluent non-neuronal cells sympathetic neurons. We have also begun to investigate the role of MASH expressed desmin. These findings demonstrate that the pineal is a (Mammalian achaete-scutehomologue) in specifying neural differentiation peripheral nervous system by overexpressing MASH in the cell line. useful developmental model for future studies of the effects of trophic factors and extracellular matnx on pineal cell differentiation. Supported in part by NSF (BNS-8801726) and the Bane Estate. 362a Neural DevelopmentII (2100-2105). Tuesday

2100 2101 STRUCTURAL CHANGE IN SYNAPSES IN LONG-TERM, ORGANOTYPIC SYNAPTICPORTERTIESNDURIIILDUING DEVLOPMENT. Ahnert-llitser, U. CULTURES OF HEPPOCAMPUS. ((K. Miyaguchi)) Laboratory of Neurobiology, Kutay, T.A. Rapoport*, and B. Wiedennua)) Department of Gastroenterology, NINDS, NIH, Bethesda, MD 20892. Benjamin FraklnNedic eat Center, Free UniversityBof ertin, inderturgdd 30, D-12200Berrin,nax-DotbrOkand CenterfCorleotecutr Medicine,n Robertt Berlin-Bush, termnvy. (Spon. by B. Wiedenam) Organotypic cultures of hippocampal slices from 6-8 day-old neonatal rata Rosste-Str1e, 15, B3i25 were maintained for periods of up to twelve weeks in vitro, by culturing them on in order to gain insight into the events thit take ptace when neuronat growth a porous membrane at interfacethe between air and culture medium. Typical cones are into we tested the hypothesis that The tre)modetted synapses. laminar organization and most of their thickness was maintained. covering neurotransitter-retated properties of presyntic terminats are atready glial layer was sufficiently thin that slam-freezing could be uaed to prepare slices present before synaptogenesis. The ontogeny of mrkers for the smatt syntic for freeze substitution followed by ultrastructural study. Comparison of CAl in vesictes and the fusion machinery were studied in parallel to the stimutated cultures of different ages showed that it retained normal ultrastructural morphology release ofredilotabeled SAA. for two weeks. After three weeks, concave-shaped spine synapaes, in which Primry cultures of hypothalmic, cerebellarnd corticat neurons, isolated cytoplasmic extensions from the postsynaptic density region embraced the froe mouse embryos on day 15 (Tixier-Vidat et at., J. Cell Sci. 105, brain presynaptic process, became prominant in CAl. After five weeks in vitro, some 935-47) as well as embryonic(1-218)), newborn and adult mouse whte were studied for the of protein spine synapses contained"spinules", finger-like projections of the postsynaptic expresion synaptophysin, S.V.2,SIAP25, rab and and membrane into the presynaptic terminal. Since this age, thepresynaptic terminal synaptotapin, 6p, synaptobrevin 1 by Western blotting the established mrker for synaptogenesis, gradually enlarged and the number of synapses having segmented post synaptic iummaohistochemistry. Whereas synaptophysin, was increasingly expressed during synaptogeresis, synaptobrevin densities incmased. After ten weeks in vitro, conspicuously enlarged presynaptic 11 proteinlevels were high at earlyeyryonic stage nd didnotincreae in terminals crowded with synaptic vesicles were often observed. Thesestructural perallel to synaptophysin. Parallel studieson GA5A secretion showed that changes are reminiscent of previously reported activity-dependent changes in regulated mino acid transmitter relese was simiar at all embryonic stges. synaptic morphology (Calverley and Jones. 1990. Brain Res. Rev. 15, 215-249). In smary, our data sugest that properties of mature synases are already, Taken together, these results suggest that long-term cultured hippocampal slices atleast in part, presentin immature neurons prior to synapse formtion. The could be an excellent preparation in which to examine synaptic plasticity and early developsent of these properties my also be crucial for a controlled development. synaptogenesis.

2102 2103 EFFECTS OF BLOCKJNG DYSTROPHIN SYNTHESISWITH ANTISENSE USE OFATRANSGENIC MOUSE UNE WHICH EXPRESSESAWNT-1 LACZ IN HUMAN FETAL NEURONS((F.Gemo, V.Sogos, M.G.Ennas, P.LOnali FUSION PROTEINTOANALYZE NEURAL CREST CELLS IN SPLOTCH MOUSE and IMussini)) School of Medicine, Caglari; C.N.R., Padova;iTALY (Spon.by EMBRYOS. ((G N. Serbedzuja and A. P. McMahon)). Dept. of Mol. and Cell. M.Presta). Blo. Harvardx University, Cambridge MA 01238. Dystrophinis the protein encoded by the Duchenne muscular dystrphy (DMD) gene. DMD gemn transcpts have beenidented andits products Mouse embryos homozygous for Splotch (Sp), a mutation in the Pax 3 gene, locaised Inthe developing and adut brain. Though, Its role Isstill unknown. show defects inthe neural tube and a reduction or absence of a number of The hYPohsss has boen advanced that Its deficency In brain might be neural crest derivatives, including the pigment cells, the sympathetic related to the cognItiveImpiafnent observedIn a high percenta of DMD ganglia, the spinal ganglia, the cardiac outflow tract and the enteric nervous patnts. In order to elucidat the PossIble damag due toiack of dystrophin system, with the effects increasing in severity along the rostro-caudal axis. In human Immatur neurons, webeated human fetal neuronal cultures with Previous experiments suggested that these deficiencies are due to a delayin dystrophin anlene ollgonucleotdes. Block of syntesIs was detected both neural crest cell emigration from the neural tube. Unfortunately, direct with Wesen blot andimmunoicytochemnatry after 4 days from tratment. At analysis this and other neural crest mutants has been difficult due the lack lIght micwcope, treated neurons showed an altered morphology, wtth of a noninvasive cell marker that labels both migrating neural crest cells enlargement of cell bodies and detachment of cells from the substrum. At and their derivatives. Recently, a stable transgenic mouse line was been E.M., neumns appeared filled with autophagosomes, whIch often were as generated In our lab, which expresses the Lac Z reporter genein the Wnt-1 large as the cell body. Ther content Included disrupted membranes, granules expression pattem, including in the dorsal portion of the neural tube, whic- and dersely- ed materal. Vacuoles were also prest in process which gives rise to the neural crest. Embryos from this line show B-gal activity apPeared abnornally enlarged. in emigrating, migrating and differentiating neural crest cells. Here, we Normal and trea coels w testd for their capability to respond to use this tmrngenicline to directly analyze the effect of the Sp mutation on stImulatIon of spocIfIc neurotraremlter rceptors. Results demonsrwted that neural crestcell migration and differentiation. Crosses between the Sp line block of dystrphin synt was corrlated wIh diminished calcium levels In and the Lac Z reporter line, have generated homozygous Sp embryos which response toglutamate. express the Wnt-I Lac Z fusion protein. By staining these embryos at (Supported by Telethon-rtaly to F.G.) different developmental stages we observed that neural crest cell emigration appears to begin at the appropriate time at each axla level. However, there is a severe reduction in the number of cell which emigrate in both the vagal and lumbar regions of the neural tube. In contrast to previous work in culture, we saw noevidence for any additional emigration from the neural tube beyond the normal period neural crest cell emigration.

2104 2105 TARGETED DISRUPTION OF THE GAP-43 GENE IN MICE ((Qixzbang THE ROLE OF CCAAT/ENHANCER BINDING PROTEIN IN Zhu and Jean-Pierre Julien)). Centre for Research in Nerosaences of McGill SCHWANN CEL. DIFFERENTIATION. ((Y Huan, JL Rutlowwsd, and University, The Montral General Hospital Research Institute. Monteal, GI Tennekoon))Depatinent of Pediatics and Neurology,The University Canada H3G 1A4 of Michigan, Ann Arbor, M 48109. CCAAT/enhancer binding prtein (CIEBP) is a tnsiptional factor that GAP-43 is a growth associated protein found almost exclusively in belong to lucine zipper (b2WP) family. Thbe of the neurons,typically in the grwth cone. GAP-43 is highly regulated at thelevel EP fily (CIEBPa, B. . a and a)tha dinmrize via a leucin rept of gee s. During development GAP-43 is first expressed when r CIBBPra is a 42d protein that is involved in the teminal role of in neuroblast start to extend ther processes Is peaks in the perinatal peiod then ntio of ad nic ti e. Weexamined the CEBPa Schwann cell diffeni ce the cells alipid-rich myelin. recedes High level expresson of GAP-43 resumes in the adult after axonal syntheiz membrane. During development of peripheral nervous system, the injury when conditions favour regeneraion. In vitro, overxpression of GAP- appearane of Cg P. and myelin P0 protein occurred in parallel and 43 protein by trandection in both the neuronal PC12 cell line and non- deceased simul eously after nerve injury. There was no change in the neuronal COS orNIH 313 cell lines cause extending long axon-like process mRNA levels ofadoinant neaive regulsar ofCEBP function, CHOP- in these cells Thus it seems like that GAP-43 might be emental for axonal 10. In cultured Schwann cells, CIEBPa tanswctivates Po gene expression to the as demonstrated by mobility shift, growth during development and regeneraion. Howeer the precis rols for by binding promoter and transient transfection assays. Furthermore, we and functional are still To Southwestern, GAP-43 in axonal regeneration recovery unimown. generated stable Schwann cell lines expressing sense and antisense address tbis question, we have dirted the GAP-43 gene in RI embryonc p42CIEBPe. The cels exprssing sense p42C/EBPa differentiated as stem celLs by homologous recombinaio The targeting vector contain a 1.1 evidened by increased expession of myelin Po, P2 prtes and the lipid kb TKMeo casseUe replacing a 1.2 kb HindII-BAgU fragment which encodes _tace.ebro- Moreover, in cells expresing sense p42CBBP dhere the firstexon of GAP-43, flanked by 8.1 kb at the 5' end and 4.1 kb at the 3' was a maied decrease in mitods mesd by BrdU mcsn xprton as end. Of 384 G418 reitantclones, 27 have the desired mutadon as determibed compared tocells antinse. These daa indicate that p42CPAmP eqxpssing cells. blot Heterozygous animals from two is involvedin growth arrest and differentiation of Schwann by Southern analysis. derived different (Supported by NIH. NS21700 and 29710.) GAP-43 tareed cell lines appear normal upon external obsvation. We are now in the pocess of analyzing offspring from tes betrozygous. These results will be presened the poser. arefive Tuesday. Neural DevelopmentII (2106-2111) 363a

2106 21nT EVIDENCE FOR AN ENDOGENOUS PROTEASE INHIBITOR IN SYNTHETIC NEUROSCIENCE: EXCITABILITY IN PROTEINOID DEVELOPING CHICK RETINA. ((B.W.Hanna, H.Peng, and J.B.Sheffield)) MICROSPHERES ((S.W.Fox, B.Yu, M.Li, and A.T. Philadelphia, PA Pappelis)) Coastal Research Development Institute Department of Biology, Temple Universtity, 19122. and Pharmacology Department, University of South Alabama, Mobile Al 36688 and Department of Plant We have shown that endogenous processes play a role in retinalneurogenesis Biology, Southern Illinois University, Carbondale in the chick. We have also shown that the embryonic vitreous contains a major gelatinolyticactivity of80kd, and a second of50kd. Th enzymes from both Synthetic neuroscience results from experimental tissues are inhibited by calcium depletion and by seine protease inhibitors. We findings: (a) thermal proteinoids are highly non- random, (b) the proteinoids self-organize in water now demonstr that protease inhibitor(s) are present in endogenous developing to bilayered polybiofunctional cells and networks, chick retina. As a test for retinal protease inhibitors (RPI), extracts of reina and and (c) the cells generate electrical signals in vitreous were incubated at 37C for two hours, and then subjected to response to stimuli such as light. Microspheres zymogaphy with gelatin as a substme. The retinal extract inhibited both of the from lecithin and a variety of aspartic acid-rich vitreous enzymes in a concentration dependent manner. The inhibitory activity proteinoids display signals that change with chang- remains in the supematant afte centifugation at 50,000 rpm for 15 minutes. ing pH, the pattern from each proteinoid being char- The inhibitor is stable at 37"C for up toseven days. The existence of this acteristic of that amino acid composition. The pro- teinoid cells have intrinsic lipid quality due to inhibitor is consistent with our finding that anti peptide antibodies to the 72kd hydrocarbon sidechains of amino acids (Lehninger gelatinae (a kind gift of Preston Alexander, Department of Ophthalmology, (1975). All types tend to form primitive gap junc- University of Oregon) identify a high molecular weight material in extracts of tions; those with a substantial proportion of tyro- retina on western blots. Similarly, zymograms of developing retina indicate a sine form dendritic networks. The magnitude of the high molecular weight protease activity. When retna cells are grown in vur, electrical signals in various proteinoids is related free of issue constraints, they releae a 72kd protease into the medium. This to tyrosine content, as well as to pH in individual assemblies. Other electrical properties have been same medium does not inhibit the vitreous proteolytic activity. We proposethat largely summarized in The Evolution of Information the retina in situ contains a balanced system consisting of proteas developing Processina Systems, K. Haefner (ed.) 1992. and inhibitor, and that the in vit conditions are not conducive to inhibitor fornation. Research aided by NASA, IBM, NSF, NFCR.

2168 216 THE CNIDARIAN ACHAETE-SCUTE HOMOLOGCNASH HAS BASIC FIBROBLAST GROWTH FACTOR STIMULATES PRONEURAL ACTIVITY IN DROSOPHILA. ((A. Grens andHR. THE LIMITED PROLIFERATION OF A MULTIPOTENT Developmental Biology Center, University of California, EMBRYONIC MURINE PRECURSOR CELL. ((C. Bjornson, S. Bode)) Weiss* and B.A. Reynolds)) NeuroSpheres, Calgary, Canada and Irvine CA 92717 *Neuroscience Research Group, University of Calgary, Calgary, Canada. We have isolated and characterized a complete cDNA of the Basic fibroblast growth factor (bFGF) has been shown to have a broad Cnidarian achaete-scute homolog (CnASH) from Hydra vulgaris. range of actions on embryonic CNS cells. These include: supporting The achaete-scute family of basic helix-loop-helix (bHLH) neuronal survival, enhancing neurite outgrowth, and stimulating the transcription factors is required for specfication of neural proliferation of astrocytes, unipotent (neuronal) and bipotent (neuronal/ in both vertebrate (mouse) and invertebrate astrocytic) precursors. Dissociated E14 striatal cells were plated precursors (25,000Yml) onto untrated tissue culture plastic substate in defined senm- only a single (Drosophila) systems. Hydra contains achaete-scute free medium with or without bFGF. In the bFGF-treated cultures dividing gene, in contrast to the multi-gene families observed in more cells were observed, after 3 days in vitro, which formed small clusters of complex organisms. There is an extremely high degree of amino cells (80 ± 10 Am in diameter) over the ensuing 6 days. These clusters acid conservation across the bHLH domain of CnASH when ceased to increase in size after 10 days in vitro No proliferation was compared to both vertebrate and invertebrate achaete-scute observed in the absence of bFGF. Cells within the clusters were undifferentiated as defined by nestin immunoreactivity and In vitro translated CnASH protein can form morphological proteins. criteria. Removal and plating of the clusters onto poly-L-ornithine-coated heterodimers with the ubiquitously expressed Drosophila bHLH coverslips, in bFGF-free medium containing 1% serum, induced cells protein daughterless (da) and these dimers bind to consensus within the cluster to migrate and adopt the morphology of differentiated achaete-scute DNA binding sites (E boxes) in a sequence-specific cells. Triple-label immunocytochemistry for MAP-2, GFAP, and 04 manner in gel retardation assays. Ectopic expression of CnASH in antigens revealed the presence of cells with the antigenic and morphological characteristics of neurons, astrocytes and wild-type late third instar Drosophila larvae and early pupae leads oligodendrocytes, respectively. These results indicate that bFGF can induce the limited proliferation of a to the formation of ectopic sensory organs. Expression of CnASH multipotent precursor cell which generates the three principal cell types of in Sc10-1 flies, which are achadte and scute double mutants, gives the mammalian CNS. The relationship between this multipotent bFGF- partial rescue, comparable to the degree of rescue obtained by responsive cell and a multipotent EGF-responsive stem cell, which we have ectopic expression of Drosophila achaete-scute genes. recently identified, is under investigation.

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MONITOFING THE NUMJ OF DOPAMINE NEURONS IN PRIMARY DYNAMICS OF GROWH CO R AND REGUATON BY TARGET TSSUE CULTURES OF IMDBRAN NEURONS. ((S.E. Podudo and R. Seelow)) ((GJQeEPeard DP9)D SpLd iScUi.d Jlno L606L7. Depeqme d Neuology, Tus Tech Unwrsty HIe Scbnc Cer, Lubbock, TX 79430. Ne w _d cons htaws cizmoplaows kernek d lponse

- Whbe prley ciur d neuron prepred from the mIdbraIai embryonIc lesion cinwaerdo ulowuIri fiesigon- haw adifresieed dcttssce ims1 olmorm cih iges. We on ft tlcs di Ism m Ste gae rate are qLute hom@geneow duIn the fIt thee days the nmber d dpa orphoaalog r-l d m -i lm -mle. Luvel crd dopmne neurnu low D.5-1.0%). Ev on d cltue condtr to by p A _ wmr Am W In diumbes on a scidrki in ft presence or paosidy Incem the number d dopamnhi neurons undermy. WhWi Sohesloo pail_4d dte ft deimlop meridy opp ,s' ('meeed miW lbe bu4 fo mola Xt opn nron don't InItM1y pIts or whihr the cells we down m m Compnie udyals di dds4M n w used lo psaswos cid sde regtgaitd durIn cure under ivedgitlo. To prepar the ctfturet the moRphonlogy.li u bsc dlew wd pe wI iN ymhfeb opello de nmbrdn from a d ebrync rt were dIcted and ajected to a h G ns m1 sW d Aip ic m um brW trypelnlitlon step hi medIun The s0ntened wae passed lg idhil remu ev d The mesiped od f qip erhi 5 t igsch and the ce uspenlon platd orto pder_*W coatd Ater one ep 0111l1 hour the mnedI (Dubeccos' hih gucoee wIth a mp d a des df Vs 2obeeridon pero,me de dV hug hibbkVse f me hietn 1% BSA, end 10% kbl calf erun) we chdgd to remow unpkted h o t c -n coaie crud1 ns& Gf onsi merdurgemert cels debris.lO the nes 2448 hourX the ctjue conested prmely byran icrmone In fVsm OradciVsd prioalonm hta# d vdsel le-rcaon Smbly, d neuron whIch utene long pocsses. We hem etabllehed mIcro gemwi cons ekdo deseemailedbvienn kwimes in moisbda-seirionW lmbb enzye samy to m_eeu tee hydrcom actity are the acvIty and h_munocytochurls to monor the number d dopwn*i a e-s-e in o rd du ob er p a phe wdm r. neuron AddIodnbFit s coibldonsdId not ltertheenzyme acvIty or numbr d neu poelvely wharbody to tyroine UopodM The pelodicly cl Vscyde m aI ch d ut ng L. Ruido hfidrcsYIe Changing the median to a b_ mewdum with pplemuto d ocmmrdlInm anli hc ressin t eIVosf ph cifts ydS& d W N2 or B27 did hew delnt egfet on the morphologyyd the cultuus. mg to ''d i d ThsmAied b isd seah cyds in pconsc not Hawr, nether meduin eltere the xnuber ddopqmln neron pree hI bc dsm gbve Vo pumi mu a hin m cius VarIous combntos of the above and edlln od grot cuIdIralhuo.Ih s be decwhase gon dw 7W in da Vlo eelVsdVspedmIite In b:tors are currudy under hwedtIg L Supported by NIH grt DA073B dwair dni uIbgd c _d o -eb Slice s mak used hin ftsk itWM a io i on hi f bla giteum Vso dmsus hIn W Au mo mop be midsed lo ft eut-Ies d a repnes (roPo ed byfU usR#swhbAA. 364a Neural DevelopmentII (2112-2117). Tuesday

2112 2113 UNINJURED ADULT MOUSE SCIATIC NERVE SUPPORTS OUTGROWTH BUT NOT QUANlTIATiVE ANALYSIS AND SIMULATION OF NEURONAL STABILIZATION OF ADULT MOUSE DORSAL ROOT GANGLION NEURITES. ((H.W. GROWTH CONE MIGRATION AND GUIDANCE. ((H.M.Buette and Luk and Z. Werb)) Laboratory of Radiobiology and Environmental Health and S. Gwydir)) Department of Chemical & Biochemical Engineering, Rutgers BiomedicalSaences Program, University of Califoria, San Francisco, CA. University, Piscataway, NJ08855. 94143-0750. pioneerng of neurl pathways hinges critically on the migration and guidance of the growth cone the embryo. While many of the factrsin Surgical transection of the adult mouse sciatic nerve is followed by a tlsi process have been elucidated much remains unclear about the macrophage-dependent process of structural remodeling known as Wallerian quanitative mechanisms by which the growth cone naviptes its degeneration, which is restrcted in the distal segment of the severed nerve and environmnent. As an aidto investigatingthese questions, we have been has been shown to be essential for subsequent nerve regeneration. To deter- developing matm_atcal models of growth conemigration and guidance mine what aspect of Wallerian degeneration is responsible for successful nerve based onexprimental observation of chick DRG andrat SCG neurons regeneration, we tested longitudinal cryosections of uninjured and 10-day post- growing on laminin and collagen in vitro. Tim lapse videomicroscopydata is a oil on a Macintosh sciatic nerves for their ability to support neurite outgrowth of a nearby obtained usingl00x immersion objective, digitized injury and with analysis routines custom adult dorsal root gangiion explant (DRG) in culture. Immunostaining with anti- ici, analyzed computer-assistedimuge designed to provideparamne of dynamic growth cone and filopodial body against GAP-43 revealed that DRG neurites were able to migrate on either morphology.The models developed from the data can be used tosimulate kind of cryosecton. Thus both uninjured and post-injury sciatic nerves are per- separate process involved growth cone migration, investigate missive substrates for adult DRG neurite outgrowth. However in prolonged cul- underlying molecular mechanisms, and examine different hypothese of ture, the DRG neuritesgrowing on uninjured nerve cryosections appeared to growth cone guidance.Thus far, we have been able todescribe the motion cone. degenerate into fragments while those on the distal segment of post-injury nerve of the growth cone and filopodial dynamics on themoving growth can be assembled into an integrated model of cryosections remained intact When cryosections of uninjuredsciatic nerve were These descriptions growth cone advance and, with assumptions, used to describe growth pre-incubated with macrophage-conditioned medium, they now support the appropriate coneresponse to directional cuessuch as guideposts orchemotactic and growth of stable neurites in prolonged culture. Taken together, these data sug- haptotacticgradients. Two types ofsimulation results can be obtained from gest that during Wallerian degeneration, macrophages infiltrating the injured the models: 1) quantitative predictions of growth cone success intrcing nerve may secrete diffusible factor(s) that transform the nerve environment into out specific pathways, and 2) computer grphics simulations showing one that supports stabilization of growing neurites. (Supported by OHER, US- visual realizations of the process. Both types of results will be presented. DOE contract # DE-AC03-76-SF010102) Supported by NSF BCS-9210540 and the WhitakerFoundation.

2114 2115 ASSOCIATION OF NONRECEPTOR TYROSINE KINASES AND CA++- NCAMTRIGGERING ACTIVATES P59FYN TYROSINE KINASE IN INDEPENDENT CELL ADHESION MOLECULES IN THE DEVELOPING NERVE GROWTH CONE-ENRICHED MEMBRANES. ((H.E. Beggs, OLFACTORY PATHWAY.((W. R Morse, J.G. Whitesides, C. R. Shanley, M. Schachner, and P. F. Maness)) Department of Biochemistry, A-S. LaMantia, & P.F. Maness)) Dept. Biochemistry, Univ. Of North Carolina University of North Carolina School of Medicine, Chapel Hill, NC School of Medicine, Chapel Hill, NC 27599 & Dept. Neurobiology, Duke 27599-7260. University School of Medicine, Durham, NC 27710. p59fYn is a nonreceptor tyrosine kinase of the src family that is an We have evaluated the relationship between the expression of the nonreceptor essential component of NCAM-directed neurite outgrowth. Analysis of tyrosine kinases pp60c-srcandp59fyn and several Ca++-independent cell cerebellar and sensory neurons from null mutant mice lacking the fyn adhesion molecules (CAMs) during the initial formation of the olfactory gene has revealed a dramatic impairment of neurite extension on pathway in the mouse. Immunofluorescent localization of pp6&c-src and NCAMl40-transfected fibroblasts. In contrast, src- and yes-minus p59fyn at embryonic days (E)I0.5 and 11.5 shows that both molecules are neurons showed no alteration in NCAM-dependent neurite outgrowth. expressed primarily along the nascent olfactory axons in the lateral cranial To test whether NCAM binding resulted in a specific change in fyn mesenchyme and processes in the ventrolateral aspect of the telencephalic kinase activity, growth cone-enriched membranes isolated from fetalrat vesicle. Localization of the sialylated form of NCAM, as well as LI and TAG- brain were incubated with antibodies directed against the extracellular 1 shows that the expression of these molecules coincides with that of the domain of NCAM p59fYn kinase activity increasedtransiently following nonreceptortyrosinekinases. Expression of other adhesion molecules including NCAM triggering by approximately two-fold as measured by in vitro laminin, fibronectin, and E-cadherin is not coincident with that of the knase assays using enolase as an exogenous substrate. Kinase activation nonreceptorkinases. These expression pattems are consistent with in vitro was accompanied by a similar increase in the autophosphorylation of neurite outgrowth studies that show an exclusive functional association of p59fYn. NCAM and p5QfYn did not appear to be physically associated, pp60c-src with LI and p59fyn with NCAM, but not with integrins, matrix because NCAM antibodies failed to immunoprecipitate any tyrosine adhesion molecules or cadherins. This suggests that redundant adhesive kinase activity either from growth cone membranes or cerebellar interactions via the Ca++-independent CAMs and the nonreceptor kinases may extracts. NCAM activation appeared to be specific to p59fYn, as NCAM constrain olfactory axon growth and guidance in vivo. We are currently binding did not result in a significant change in pp60C-srC tyrosine hypothesis by examining initial olfactory axon growth in evaluating this null kinase activity. These results indicate that NCAM triggers a positive lacking src genes. Src-minus mice show no mutant mice orhyn observable of kinase activity, leading to neurite anomalies in inital olfactory pathway formation at El 1.5. This supports the modulation p59fYn tyrosine redundant pathways hypothesis. outgrowth.

2116 2117 REGULATION OF T-CADHERIN EXPRESSION IN THE FLOOR ThE ROIEOF SOLUBLE CHONDROrr SULFATEPROSEOLYCANIN SENSORY PLATE BY COMMISSURAL AXON CONTACT. ((S. S. Kanekar and NEURIrE ouTRoMrwrt ((D. M. Snow)) Department of Cel Biology and B. Ranscht)) La Jolla Cancr Research Foundation, La Jolla, CA 92037. University ofMin , Minnapol MN 55455 (Spon. by F.O. Berglund.) Neuroanatomy, a The floor plate demarcates the ventral midline of the developing spinal Proteoglycans (PGs)ae complex macromolecules reported to have variety cord and is an intermediate rget for axons extending from the dorsally- of effects on neurons in vio and in vitro. The mechanism(s) by located commissural neurons. T-cadherin, a OPtI-linked, calcium- modulate neuronal behavior are unclear. To elucidate possible mechm of dependent cell adhesion molecule, 1i expressed the deve g chick and this study whedhr a inteion between PGs dongating neurites, exoained spial the formaton of the commissural trajectory. T- cord during solubl sulfate influenced neurite outgrowth is expressed in floor plate cells upon comsurl axon chIdroiMn proteoglycan (CSPO) cadhenn first chicken root contact As axons project contralaterally, T-cadherin is transiently diffently than substatum-bound CSPG. Dissociated doral expressed on the commissural axon senment in the floor plate and on the ganglion neurons were cultued on ther lminin (LN) o fib (EN). ventral midline cells, suggesting that T-cadherin is involved in bothseory neurite outgrowth-promotig glycoproteins. In hes cutue commisrall axon guidance.To determine whether floor plate expression CSPG was bound to FN or LN, or added to the clte modia T-cadherin is induced axonal contact, commissural neurons were of by e cment. In some cases, CS alone was added to dhe cuue prior to contact of the floor plate by in sis operations. eliminated axonal stuies T-cadherin was then examined in operaed embryos at later media. Using time lapse video mioscopy and image analysis, tee e,xpression ned: ra of neuuie developmentaf stages, when Tr-cadhein expression is dense in the floor 1) neuriteprodcon, 2) neurielgoutow, 3) plate of normal embryvos. Eimination of commissunl axon contact elongation, and 4)fSlopodl length. Theresults of these st cofim dt abolishes T-cadherin expression in floor plate cells, but not that of FPI, the effects of CSPG are concetration- and interaction-dependeLnt O LN, another floor plate marker. Tc in expression is not altered in the soluble CSPG had no effect on neurite or outgrowth, whik ventral spinal cord, including the motor neurons, or in the surwnding production somites. T-cadhenn expression in floor plate cells is specifically substratum-bound CSPG inhibited neurite production, reduced total wecit dependent on commissural aaxon contact, and not on any aspect of the length, and inhibited neunte outgrowth. In contrast, on EN, soluble CSPG surgical procdures, ince operated embryos with surviving commissual inhibited neurite on, decrasd neuie legth, and deceaed the re of axons in the floor plate retain T-cadherin in the ventral midline cels. In a manner. did induces neuite elongationmin concentration-dependent Soluble CSPO conclusion, this study demonstrates thsat commissura axcon contact of on either LN or EN. the floor plate and provides the first evidence for the not affect the length sensory growth cone Slopodia T-cadherin in a is to regulation of gene expression in the floor plate by signals from Given that substum-bound CSPG frm variety of source iniby commissural neurons. We thus propose that neurons can alter gene neunite outgrowth and to the rte of neurite ongaton, the re gt expression in their targets or surrounding tissue thus influencing the that POs may affect sensory neurons differently soluble for, than guidance cues in their environment and tiimatey the pattern of axon substamtm-bound, indiag diferent mechanisms of interation. This we* grwth- was supported by NIHgrants EY06331 wad HD19950, anda grantfrom the Afollowinglmerica ParalysisAs.s iadon Tuesday. Neural Development 11 (2118) 365a

2118 MUTATIONS THAT SUPPRESS GROWTH CONE REORIENTATIONINDUCEDBYECTOPICEXPRESSIONOF THE UNC-5 GUIDANCE RECEPTOR OF C. ELEGANS ((A. Colavita and J. Culotti)) Department of Medical Genetics, University of Toronto, Toronto M5S 1A8 Canada The UNC-5 guidance receptor, in response to the laminin-related UNC-6 path cue molecule, orients growing axons in a dorsal direction on the C elegans epidermis. When ectopoically expressed in a set of touch receptor neurons, which normally grow ventrally or longitudinally, UNC-5 is able to reorient their axons towards the dorsal side (Hamelin et al. Nature 34:327-330, 1993). This UNC- 5-induced reorientation is suppressed by mutations in unc-6, which is known from genetic evidence to act in the same pathway as unc- 5. We have screened for mutations that act like unc-6 mutations in their ability to suppress UNC-5-induced reorientation of the touch cell axons. Theoretically, these suppressor mutations may identify other axon guidance genes that act in the UNC-5/UNC-6 pathway. So far we have identified mutations in approximately six genes, including unc-6 (as expected), unc-40 (another axon guidance gene required for ventral and dorsal guidance, Hedgecock et al. Neuron 4:61-85, 1990), and unc-44 (a C elegwts ankyrin (A. Otsuka, personal communication). The other mutations are being further characterized genetically and phenotypically for axon guidance errors. Secretory Granules (2119-2122) 2119 2120 GRANULA FORMATION UNDER HOST CELL PROTEIN SECRETORY GRANULE CONTENT AND MEMBRANE PROTEINS SYNTHESIS SHUT-OFF. ((P. Samenfeld, 1. Loef, and H.-H. Gerdes)) AGGREGATE IN VITRO AT MILDLY ACIDIC PH: IMPLICATIONS FOR of D-69120 TARGETING OF PROTEINS TO THE REGULATED SECRETORY Institute for Neurobiology, University Heidelberg, PATHWAY. ((V. Colomer and M. J. Rindler)) Department of Cell Biology Heidelberg. (Spon. by W.B. Huttner) and Kaplan Cancer Center, NYU Medical Center, New York, NY 10016. Sorting of secretory proteins to dense core secretory granules is A major unresolved issue in the field of secretory granule biogenesis is to what thought to occur via the selective aggregation of these proteins in the extent the aggregation of granule content proteins under the ionic conditions pre- specific milieu of the trans Golgi network (TGN). Such aggregates are sent in the trans Golgi network (MN) and immature granules plays a role in the mostly mixtures of various secretory proteins. It remains to be sorting of regulated from constitutively secreted proteins. We have developed in established whether each secretory protein of such mixed aggregates vitro assays that reconstitute the precipitation out of solution of granule content is capable of being sorted to secretory granules if it were the only proteins from anterior pituitary, adrenal medulla, and acinar pancreas. Tbe ag- newly synthesized secretory protein present in the lumen of the TGN. greption occurs as the pH is titrated <6.5. All of the major polypeptides of the To address this issue, the intracellular transport of secretory proteins content form a precipitate that can be recovered in the pellet fraction after cen- was studied in the rat neuroendocrine cell line PC12, using an uifugation. By contrast, constitutively secreted proteins added to the assays re- main in the superatant. Among the individual content proteins tested, amylase, expression system in which host cell protein synthesis is blocked. For prolactin, and chromogranins can aggregate homotypically in the assay. The this purpose, a recombinant vaccinia virus was prepared which luminal domains of granule membrane proteins could also interact with content encoded human chromogranin B (CgB), a secretory protein which is proteins. A soluble form of the pancreatic membrane protein GP2 as well as packaged into secretory granules together with various peptide secretory forms of endocrine and neuroendocrine granule membrane proteins hormones and neuropeptides. The transport of hCgB was studied were found to co-precipitate with content proteins. The results provide strong under infection conditions in which host cell synthesis was found to be support for the hypothesis that spontaneous aggregation of proteins is completely blocked. Collectively our data show that hCgB alone has responsible for their sorting to secretory granules. They further support the idea all the structural information for being sorted to the regulated that co-precipitation of membrane proteins with content proteins plays a seminal secretory pathway. This in tum implies that the components involved role in the segregation of granule membrane proteins from those transported in granule formation (except for cargo proteins) can be recruited from directly to the cell surface. Such membrane-content protein interactions would pools that for several hours do not depend on ongoing protein also ensure the acquisition by the aggregating mass of content proteins of an appropriate membrane envelope during granule formation. Supported by grants synthesis. from the N.LH. and the American Heart Association, NYC affiliate.

2121 2122 PROLACTIN-ZINC INTERACTIONS IN SECRETORY GRANULES. ((TJ. THE NEUROENDOCRINE PROTEIN 7B2 FACILITATES THE Patel, Mary Y. Loenson, Jo-Wen Liu and Amese M. Walker)) Division of MATURATION OF PROHORMONE CONVERTASE PC2 of CA 92521. Biomedical Sciences, Univerity Califoia, Rierside, ((X Zbu and . Lindbaerg)) Departn of Biochesnisty and Molcular Previous studies have demstrated tpresence of Zn2+ in prolactin (PRL) Biology, Louiiana State Univety Medical Center, New Orleans, LA 70112 secretory granules and an effect of Zn on the conformation of PRL within (Spon. by x Zhu) tbecretoq granule (Endocrinoogy 126: 512). Here we report that PRL binds Z+ in solution, that PRL preparations contain divalent catins, some ofwhich The neuroendocrine protin 7B2 is a very wel conserved proten prese in are probably zinC and that the reverbl exraction of Zn from secretory prohormone-producing cells. Reoent studies have shown that in 27 kIDa granules changes the aissiblity of the C-terminus of PRL to specific vito ptelyti cleavage, Z+-PRLbinding in solution wa assessed using the 7B2 is a pote inhibitor of PC2, a subtllisin-related prohormone converta chrosnophqi- chelator, murexie. Free murexide bq a peak aheorbanc at 520 thougt to be involved in the pocg of a large number of p on nm and Zn '+-murexide at 460 nm. Addition of Zn+ to PRLsolutions (40 pM) Like 7B2, PC2 is also lcdively present in the replated secretory pahway of in the prsence of murexide (20 pM) prgpuced a third peak with an absorbance neurons and endocrine cells. Previu studies have shown that the biosynthetc max at 475 nm, indicative of a PRL-Zn+-muredde ccn,mel The size of the procssing of proPC2 is etremely slow in AtT-20 celsl In our study we 475 nm peak was dependent on the amount of added Znar up to the maxmum with tried, 150 pM Tlhe divalent cation content of purified NIDDK PRL was assesed transfected AtT-20 cels, which had previously been stably nsfted by matrix assisted time-of-flight mass analysi before and after treatment with PC2, with a rat 7B2-pCEP4 consuct. Hygromycin-restant clones were EDTA. EDTA treatment resulted in removal of a series of shoulder peaks of selected by RIA and Western blotting The biosynthetic pathway of PC2 was dightly greater mas than PRL from the main PRL peal Extaction of Zn2+ assessed in 7B2-expressing and control cels usng pulse-chased from isolated secretory granuks with EDTA (10 mM) and phenanthroline (3 fecotai OtiOur results show that: 1) transfected clones stably mM) alowed for specific C-terminal ceavage of PRL by glandular kallikrein (5 7B2 as as PC2; 2) 7B2 (27 but not the pg granule PRL + 100 ng enzyme in 10 mM Tris-HCI, 0.15 M Na%. 10 mM p- express well uroced WDa), mature mercaptoxhynol 0.25% triton X). Replacement of some of the Zn (addition fofm (21 kDa), could be e with PC2 in pulsed, but not of 5 mM Zn+'to EDTA -phenanthroine) inhibited one of the,hree spe;fic chased samps, at a molar ratio of 1.1:1; and 3) the PC2 mauration process in proteolytic ceavages This effect was not duplicated by either Ca-" or Mg'+. 7B2-expressi_ AtT-20 cells is faster than in cells without 7B2, with a T1/2 Prehminary expriments using a model suhate controlled for effects of (50 % conversion) of 1.7 versus 2.7 h. Our data strngly suggest that 7B2 is chelators and metal ions directly on enzyme activity. These results confirm that involved in the bioyhetic prcing of proPC2, perhp Zn does indeed affect the packing of PRL in secretory granuls and implies a preventing somewhat specific effect at the C-terminus of the mokalle. HD28726. prem acdvation ofthis enzyme. 366a Secretory Granules (2123-2128). Tuesday

2123 2124 CELLULAR DISTRIBUTIONS OF THE PROHORMONE PHOSPHORYLATION OF PEPTIDYLGLYCINE a-AMIDATING MONOOXYGENASE IN NEUROENDOCRINE CELLS. S. PROCESSING ENZYMES PC1 AND PC2. ((I. Lindberg, S. Ahn, and (H.-Y. Yun, L. Milgram, and B. A. Eipper)) Department of Neuroscience, The Johns Hopkins M.B. Breslin)) Department of Biochemistry and Molecular Biology, University School of Medicine, Baltimore, MD 21205. (Spon. by B. A. Eipper) Louisiana State University Medical Center, New Orleans, LA 70112. (Spon. by 1. Lindberg) Peptidylglycine a-amidating monooxygenase (PAM) catalyzes the COOH- terminal a-amidation of bioactive peptides. The highly conserved 86 amino acid COOH-terminal cytoplasmic domain (CD) of PAM-l (residues 891-976), a The prohormone convertases PCI (also known as sPC3) and PC2 type I integral membrane protein, contains several potential phosphorylation are known to mediate the proteolytic conversion of inactive sites. Purified rcombinant CD is phosphorylated by cAMP-dependent protein neuropeptide and hormone precursors to bioactive peptide products. kinaae (PKA) at Ser921 and by proin kinase C (PKC) at Ser932 and Ser937 in In this study we have used sucrose density centrifugation to vitro. Phosphorylation of PAM proteins stably expressed in AtT-20 determine the subcellular distributions of the various forms of PC1 corticotope tumor celis was studied using biosynthetic labeling with [32p]pO43- and analysis of immunoprecipitated PAM proteins. [32pJpO43- was efficiently and PC2 in three different cell types, AtT-20, beta TC3, and PC12 incorporated into integral membrane PAM (PAM-1 and PAM-2) but not into a cells. The former two cell lines naturally express PC enzymes, while soluble form of PAM (PAM-3) produced when the trmembrane domain is PC12 cell clones expressing PCs were obtained by stable eliminated by altemative splicing. Phosphoamino acid analysis of [32p]P043- transfection and verified by Westem blotting. Our data show labeled PAM-I revealed phosphoserine with lesser amounts of phosphothreonine. Truncaion of the COOH-terminal 40 amino acids (PAM- considerable cell-line specific variation in PC processing, with PC12 V936) abolished phosphorylation of PAM-1 in AtT-20 celis. Truncation of the cells exhibiting the most complete processing of both enzyme COOH-terminal 15, 19 or 23 (PAM-1/953) amino acids resulted in decreased precursors to 64- 66 kDa mature forms. While in all cell lines mature basal phosphorylation of PAM-1, as did deetion of residues (900-952) or (918-952), suggesting that more than one phosphorylation site exists in the forms of both enzymes were stored within particles having the same COOH-terminal domain of PAM. Truncation of the COOH-terminal 19 amino buoyant density as secretory granule markers, in some cell lines acids (PAM-1/957) led to increased phosphorylation at Ser937, suggesting that substantial amounts of mature PC1 and PC2 were also associated the COOH-terminal 19 amino acid region interferes with phosphorylation at with the Golgi marker. Interestingly, analysis of PC12 cells Ser937. Mutation of Ser937 to Ala (PAM-1/S937A) altered the distribution of stimulated to produce an endogenous peptide precursor, PAM proteins intemalized from the surface of AtT-20 cells. Since the COOH- not showed terminal domain contains signals goveming intracellular routing and proneurotensin, revealed that transfected PC1, but PC2, intemalization from the celi surface, phosphorylation of this domain may enzymatic activity against this precursor. regulate specific steps in the intacelular afficking of PAM.

2125 2126 LOCALIZATION OF A RAB3D-LIKE LOW Mr GTP-BINDING PROTEIN IN IDENTIFICATION OF NOVEL VESICLE-SPECIFIC ANTIGENS IN PC12 GASTRIC CHIEF CELLS. ((L.H. Tang, F.D. Gumkowski, D. Sengupta, E.M. CELLS. ((C.-J. Jeng and E. S. Schweitzer)) Dept. of Anatomy & Cell Konieczko, A.C. Lee and J.D. Jamieson)) Department of Cell Biology, Yale Biology, UCLA Medical School, Los Angeles, CA 90024. University School of Medicine, New Haven, CT 06510 We have isolated monoclonal antibodies that recognize two vesicle- Gastric chief cells represent a class of typical polarized exocrine cells. Regulated secretion from chief cells involves directed vesicle movement which culminates in specific proteins in PC12 cells. Both these antigens are co- the release of pepsinogen into the lumen of gastric glands. Regulated exocytosis precipitated by monoclonal antibodies against SV2. The first (7C8) is requires a group of proteins that confer membrane recognition, targeting and an integral membrane protein of MW 27 kD whose distribution on fusion. Rab proteins are postulated to act as regulators of membrane trafficking sub-cellular fractionation parallels that of SV2; i.e., it is a in exocytosis and endocytosis, including adipocytes which possess rab3D which component of both the large, dense-core vesicles and the small clear may be involved in exocytosis of the glucose transporter (Baldini et al., 1992, vesicles. lmmunofluorescence staining of PC12 cells shows a fine PNAS, USA, 89:5049). Previously, we reported a secretory granule membrane- punctate staining that is present in both the perinuclear cytoplasm associated rab3-like GTP-binding protein in pancreatic acinar cells. Using rab3D and in the neurite terminals, similar to SV2 and synaptophysin. polyclonal antibodies, we subsequently characterized a different rab3-like protein lmmunrohistochemical analysis indicates that the 7C8 antigen is in the pancreas (Konieczko et al., ASCB Meeting, 1994). This rab3D-like protein present in adrenal medullary cells but not in adrenal cortical cells, is now identified in chief cells and is predominately localized to pepsinogen and in gray matter in the cerebral cortex, but not white matter. It is granules. This secretory granule-associated rab3D-like protein behaved as an integral membrane protein since both 0.5 M NaCl and alkaline carbonate also present in endocrine, but not exocrine, pancreatic cells. These extraction failed to remove the protein from granule membranes. In addition, data suggest that the 7C8 antigen is specifically expressed in neural upon stimulation of gastric glands with 10 nM cholecystokinin, no significant and endocrine cells, where it is targeted to regulated secretory membrane dissociation of rab3D-like protein from the membrane to the cytosol vesicles. A second mAb (1 SC12) recognizes an integral membrane was observed. Immunocytochemistry using antibodies against rab3D and glycoprotein of MW -70 kD that is specific for the large, dense-core pepsinogen confirmed the chief cell localization of the rab3D-like protein. In vesicles of PC12 cells, as evidenced by the co-localization of 1 5C12 contrast, rab3D was not observed in acid secreting parietal cells as demonstrated binding with the norepinephrine-containing peak of density gradients, by immunostaining with a H/K ATPase antibody. Immunoelectron microscopy and its absence from the lower-density peak which contains the small, revealed an exclusive labeling of the rab3D-like protein on the cytoplasmic face clear vesicles. Immunofluorescence staining of PC12 cells with of the secretory granules. The results of the present study provide evidence that 1 5C12 shows a pattem similar to that for dopamine R-hydroxylase, chief cells possess a rab3D-like protein which is tightly associated with the consistent with its localization exclusively in the large, dense-core secretory granule membranes. Given its common localization in at least two vesicles. exocrine cells, the rab3D-like protein may play important role in the regulated exocytosis. (Supported by USPHS Grant DK 17389 to JDJ)

212T 2128 MELANOCYTES CULTURED FROM PALLID MICE ARE LESS MELANIZED THAN SIRUCIURE AND EXPRESSION OF THE GENE ENCODING C57 MELANOCYTES AND DEMONSTRATE DIFFERENCES IN PALUDIN (SAND PANTOPHYSIN, A UBIQUITOUS HOMOLOG OFTHE 4.2) LOCALUZATION. ((MA. Ringer, LH. Deocr, and C.M. Cohen)) Deparbmt of Bbomedial Resrch, St. Ebabehts Medicl Center, Boston, MA 02135 and NEUROENDOCRINE VESICLE PROTEIN SYNAPTOPHYSIN Deparmnt of Modicine and Anatonmy and Colular Biology, Tufts Univeity School ((N. Haass, U. Leimer, and R.E. Leube)) Division of Cell of Meddine, Boston, MA 02111. Biology, German Cancer Research Center, D-69120 Paldin (band 4.2) is a widely distributed protein orginaly isobed from the human Heidelberg, Federal Republic of Germany. (Spon. by W.W. ehocy in which It Is has a roleby the mintence of mmbrane sbbity. Franke.) However, a spedik fucon for peldin ha not been debtrmnd. Alhough peldin doee not have protein croee4inking ae lty,t is a member of the trangluhtamine gene farily. The peldn gene he been mappd to the pWd lcu on mous We have recently characterized an ubiquitously expressed chromoeome 2. PaEd Is one of 12 inIner ne taom In which eveal cDNA in rodents and human for a intrecelular organeles are abnoml aulng in pigmnt dilution, a prolonged coding pantophysin, bblee tme due to a pletelet derme granule defec, altered idney lys1ooral membrane protein closely homologous to synaptophysin secretion, end sveral other defect. We cultured melnocyte from the dorsl adn which is one of the major integral membrane proteins of of ped and C57 mice and found that altough both ped nd C57 meno exhlbd the expected morphlgil of mebnocyte, the peEd small electron-translucent, transmitter-containing vesicles mienocytes appered nmenized. Melnin assaya roveald the ped in neurons and of similar vesicles in neuroendocrine cells Isno had appox 20 fold le melanin than C57 al gh Leube. 1994. Differentiation they had at 2.5 tI the tyroeirnse actvity of C57 mebnoWte. Ebcron (R.E. 56:163-171). Comparison mcoecopy revealed that peldmelnoeomea appoar lee matue than mAn_oenes of the genes for synaptophysin and pantophysin (-24 kb) from C57 Many sucue which appeared to have reelted forn the also reveals a very similar exon-intron pattern. Using fusion of lyosom with melnoomwe wer present. No significant diferncee In acid ph actvitywer dobectd betwen psand C57 mobnocyte extra. specific reagents we show the ubiquitous expression of the We have detected any di ncs In the molcur weiht or intnsy of bends pantophysin gene at the nucleic acid and protein level and rcognized by palin anibodie In weatm blot analys of p d C57 melanocytes. In immunuexperimet pein antibodies recognize a emphasize the similarities with the synthesis of populaton of vesicles In pWd n ct. Peipheral sining of the velces cellubrevin. The possible role of pantophysin in the suggesed that pdin may be asscated with ot vkeal nemembrane. In C57 formation of vesicles is discussed. menocyt onlysubpopulat.ona of ca veulr sainig and the c specific appeared to be the le mebrnzed coe In the culto. Al ceb appeared to have some diffuse staing. Supported by NIH HL 37462. Tuesday. Secretory Granules (2129-2130) 367a

2129 2130 EXPRESSION OF gp300 IN MOUSE PANCREAS PARALLELS NOVELGRANULES FORMED BY RECOMBINANT FIBRONECTINS INAtT- ZYMOGEN GRANULE FORMATION ((R.C. De Lisle)) Dept. Anatomy 20 CELLS. ((A.M. Castle, J. E. Schwarzbauer, J.D. Castle)) Dept. of Cell and Cell Biology, Univ. Kansas Medical Center, Kansas City, KS 66160. Biology, Univ. of Virginia, Charlottesville, VA 22908 and Dept. of Molecular Biology, Princeton Univ., Princeton, NJ 08544' Sulfated macromolecules have been postulated to play a role in zymogen In the pituitary-derived AtT-20 cells, dimeric recombinant fibronectin granule biogenesis/maturation in the exocrine pancreas. The major sulfated polypeptides containing the N-terminal fibrir/collagen domain linked to the protein of the mouse pancreatic acinar cell, gp300, is a glycoprotein of C-terminal half of FN (RecFN l.,JC110) concentrate in novel granules prior -300 kDa which contains -40% of metabolically incorporated (3'S]sulfate in to secretion into the culture medium. Immunofluorescent localization shows the acinar cell. gp300 contains both N- and 0-linked oligosaccharides and that these granules are distinct from the endogenous ACTH-containing the sulfate is on 0-linked sugars. By immunolocalization and subcellular granules and localize to the cell body at sites distinct from lysosomes, fractionation, gp300 is a peripheral membrane protein on the lumenal face endosomes and ER (as marked by BiP) Unlike endogenous granules, their of the zymogen granule membrane. gp300 is not secreted in response to discharge is not stimulated by 8BrcAMP or phorbol esters, but they CCK8 stimulation of acini, so it is not a secretory product. These disappear with a half-time of - 3h after inhibiting protein synthesis. Turnover characteristics suggest that gp300 is a structural component of the granule is unaffected by chloroquine and does not appear to involve loss from the total pool of RecFN 10 (cells + medium) suggesting that the granules membrane and it may have a role in granule biogenesis. A prediction of l1,/C1 are secreted rather than degraded. Formation of these granules correlates should this proposed function is that gp300 expression parallel the with intracellular aggregation of dimeric RecFN l1,JC110 (as judged by appearance of zymogen granules during fetal development. By Westem sedimentation analysis); monomeric RecFN l1,/C110 does not aggregate blotting gp300 was first detectable on e15, the same time as amylase and it enters the endogenous ACTH-containing granules. Recombinant expression increases from protodifferentiated to differentiated levels and fibronectins containing only the C-terminal half of FN are found in the granules start appearing. Relative levels of gp300 paralleled changes in endogenous granules and do not form the novel granules showing a amylase from e14 through neonate and in adult. The developmental time requirement for the amino terminal domain of FN. Finally, ER export rates course of gp300 expression is consistent with its having a role in granule for the monomeric and dimeric RecFN 1,JCC110 are very similar suggesfing biogenesis. (Supported by the Pew Charitable Trusts and NIH grant no significant differences in folding. Together, these findings imply that DK46594.) aggregation is a prerequisite for forming the novel granules (as for vonWillebrand factor) but not for entry into ACTH-containing granules. Epithelia II (2131-2134)

2131 2132 IMMUNOCHEMICAL STUDIES OF THE Na+/H+ EXCHANGER STRUCTURE AND INHIBITORY ACTION OF PSORIATIC TISSUE-DERIVED ISOFORM NHEl IN THE MAMMALIAN URINARY EPITHELIUM ((D. CATHEPSIN L-SPECIFIC INHIBITOR (PSORIASTATIN). ((T. Takahashi1.2, T. Biemesderfer)) Department of Internal Medicine, Section of Nephrology, Hibinol .2, A. Takeds3 and P.F. Gofinckl1)) 1 MGH/Harvard Cutaneous Bioogy Yale University School of Medicine, New Haven CT 06510. Research Center, USA, 2Shiseido Science Research Laboratories, Japan, 3Depertment of Clinical Pathology, Showa University, Fujigaoka Hospital, Japan. The Na+/H+ exchanger isoform NHEI is expressed on the basolateral membrane of many epithelal cells where it participates in varied cellular Changes of proteinases and their inhibitors appear to be correlated with diseased functions including intracellular pH and cell volume regulation. Since the states. Here we report a 43 kDa cysteine proteinase inhibitor (psorastatin) which transitional epithehum of the mammalian urinary bladder is exposed to large was purified from psoriat epidermis. This inhibitor is distinct from cystatin, a well- fluctuations of uine osmolariy and pH immunochemical studies were known cysteine proteinase inhibitor, in terms of molecular weight and inhibitory performed to assess the level of expression and the subcellular localization action against varous cysteine proteinases. Psoriostatin specifically inhibits of NHEI in this epithellum. Immunoblotting studies comparing NHE1 in cathepein L with a KI value d 1.9 nM, bti has no effect on the lysosomal cysteine microsomes from rabbit bladder, stomach, ileum, colon and kidney showed proeinases cathepsin B and H. It also iacivates pant cysteine proteinases, such that the expression of this isoform in the bladder was higher than all other as papain, ficin and bromelain. Complex formation between psoriastatin and tissues except stomach. To immunolocalize NHEI in the bladder indirect cathepsin L was invesiated by Westem blot analysis. The 43 kDa band partially immunofluorescence microscopy was performed. Bladders were fixed either shifted to 35 kDa during incubation at pH 5.0, suggesting the release of a reaction filled or empty using periodate-lysine-paraformaldehyde. Immunostaining peptide in the process of complex fornation. Inhibitory activity was mairtained was performea on either semithin (0.5um) cryosections or on intact bladder throughout the incubation for up to 3 hours. lmmunohisto-chemical studies epithelia and examined with a Zeiss confocal microscoe. In the deeper non- revealed that psoritin first appears in the nuclei of cells above the basal layer polized cell layers staining was detected only alongt plasma menbrane. in the epidermis and subsequently in the lysosomes as the psoriatic condition No staining was seen in cells of the underlying connective tissue or smooth progresses. Amino acid sequence analysis revealed a close homology to muscle. However, the most intense staining was restricted to cells of the squamous cell carcinoma antigen which belongs to serine proteinase inhibitor superficial layer (umbrella cells) where labeling was observed in an family. However, psorastatin Wd not inhibit any seaine proteinases. mRNA was intracellular vesicular compartment Double-labeling with anti-NHEl and isolated from psoriatic tissue using shave biopsy. Complementary DNA fragments anti-Na,K ATPase antibodies demonstrated that the Na+/ exchanger and were obtained by PCR using several pnmers based on the amino acid sequence of Na pump colocalized only at the plasma membrane in both stretched and psorastatin, indicating activation of psoristatin gene in the involved epidermis. relaxed -bladders. These data demonstrate that the Na+/H+ exchanger isoform NHE1 is expressed at high levels in the urinary bladder epithelium on both the plasma membrane and within a vesicular compartment. The NHEl-vesicles may represent a novel intracellular organelle involved in the regulation of Na+lH+ exchange in this epithelium.

2133 2134 EXPRESSION OF EPITHELIAL STRUCTURES DURING THE EFFECT OF CANCER CHEMOPREVENTIVE DRUGS ON THE PROLIFERATION OF RAT COLONOCYTES IN ABERRANT CRYPT FOCI. ((GA Piazza, NS. Paranka, R. Panukcu, RW. CELLULAR MORPHOGENESIS IN VIVO. Ppeage ((PA and Wj. Bur, and DJ. Ahnen)) Cell Pathways, Inc. and Departnet of Vetean's Affairs Medical Center, Nelson)) Department of Molecular & Cellular Physiolog, Stanford Universaty Daever, CO 80220. (5pon. by CL. jones) School of Medcine, Stanford, CA 94305 The ainsnflannmaory drug, sulindacr and its suitone metabolie, FGN-1, have been shown to have chermopeventive activity for chemicallyyinduced colon cancer in rodent mrodels and are of crcal all work Thedevelopnent polarzed epithelia is in vtebrate Previoua being as cancer dnemopreventtve agents in hunam The azoxymethn (AOM)-nduced with embryonic Iddney organ cultures has demonstrated that interacon of colonic cancer model in rodents is being used to determine the cellular basis for the efficacy developing epitheial cells with the underlying basement membrane is necessary of these drus. One possible mechanism for the chemnoproectiv effect of these drus is for proper polarization of the epithelial cells Similar studies to determine the inhibition of colonic cell prdiferation. We have prviously shown that sulindac and FGN-1 do not sisnificantly inhibit poliferation in nonml or AOMtreaed fla colonic mucosa, nor in impo of the cadherin- mediated cell-cell contacts for epithlial devopmt AOMinduced tumors (ACR, 35:631, 1994). AOM induces numerous clonal neoplastic foci have proven equivocal however. In this study, we have charcterized the termed aberan crypt foci laCF) in the colonic mucmsa within wees of its administrmion. ACF expression and klcaization of cadherin-mediated cell-cell contacts, cell-substrate are thouglt to be the earliest neoplastic lsiom induced by AOM. Sulindac nd derivaties of the membrane skeleton, the tight unctionand markers of could prevent colon cancer by inhibWng the prolierdive roes of coonocyes within small ACF epihial polarity in the developing and adult mwuse kIdney. In the adult kidhey, so tha they do not to grow edo visible adenomas ad carcnomas. To test this hypodwesis. ACF in the colonic mucoas of AOMtreated rats were identified by methylene blue staining and exPresson levels of ankyrin, and fodrin very coordinately along the Na/K-ATPase, prolifermion was measured in the ACF and in the adjacent nomal appeanng colonic caypts by length of the nephron, suggesting that the Na/K-ATPase achieves its polaied the whole ctypt mitotc count echnique. ACF from AOmtreated rats were fund to be distribution in situ through selective retention in the basolaterl nmebrane. As hyperproldiersive when compand with noemal adacent cypts based on the number of mitons with the ion transport machinery, the components of the cell adhesion systems per cypt (ACF - 8.11 0.764. norma - 1.77 0.28; p<0.001) and the number ofdmitosis vary in their expression throughout the nephron. Ecadherin, -tenin, and per unit crypt knl h tACF - 0.076 0.005 nonxmal - 0.028 0.004; p<0.001). f- Ineresingy, the prolferaive cell zone within the ACF was not expanded rebtive to normal catenin are expresed ubiquitiously whereas p b ( tenin) is restriced to adjacent aty Sulindac and FGN-1 significandy (p<0.02) reduced the number of ACF aber endothelial celb and the cells of the thick limbs and distal epithelial 5, 10, and 15 weets of AOM nesanent tACF/colon ate 5 weeks treanent; control - 170.8 tubules. In the ascending thick limbs and distal tubules, plakoglobin is * 12, sulindac - 132.0 9.2. FGN-1 - 133.4 9.2)1 but drug treatment did not reduce coordinately expressed with cytokeratin K8, desmoplakin, and desmoglein, either the number of mitoss in ACF nor significardy affect crypt lnh. In addiion, neither suggesting that it is a desmosomal component in these cels. The role of sulinxac nor FGN-1 significantly afecued the ocation of the prolifeive cell zone within ACF. These daa, along with our previous observaions lad us to conclude tha mechanisms other plskoglbin in the endothela cels is unknown, but it may modulate the tightness than inhibition of colonic cell prolderamion acount for the cacer chemopreventire activity of of the endothelial cell-cell contacts and/or tight jnctons. The time course with sufindac and FGN-1. which these and other differences arise during Mg varies among the different proten and will allow us to better elucidate the roes these proteins and jnctional complexes play in the acquisition of epithelial polarityand function. 368a EpitheliaII (2135-2140). Tuesday

2135 2136 ROLE OF LUNG PHOSPHOLIPASES A2 (PLA,) IN DEGRADATION OF BLADDER EPITHELIUMI CANBE CULTURED FROMCYSTOSCOPIC BIOPSIES OF INTERNALIZED SURFACTANT PHOSPHOLIPID. ((A.B. Fisher and C. Dodia)) PATIENTS WITH INTERSTITIAL CYSTITIS. ((A.L. Trifillis, X. Cui, Institute for Environmental Medicine, University of Pennsylvania School of S. Jacobs, and J.W. Warren)) Departments of Pathology and Medicine, Philadelphia, PA 19104. Medicine, University of Maryland School of Medicine, Baltimore, MD 21201. Lung surfactant, the phospholipid-protein complex that regulates lung surface Interstitial cystitis is a chronic disease of unknown etiology is secreted and recycled from the alveolar space by the alveolar epithelial tension, characterized by bladder pain and urinary frequency and urgency. (type II) cells. Dipalmitoylphosphatidylcholine (DPPC), the major surfactant Bladder epithelium may be important in its pathogenesis: the phospholipid is intemalized by lung cells, degraded via PLA2 activity, and diagnostic criteria set forth by the NIDDK are visible epitheli- components reutilized. Lungs possess several PLA2 enzymes, including secretory al defects (Hunner's ulcers and epithelial ruptures); areas PLA2s (sPLA,), cytosolic PLA2 (cPLA,), and an acidic Ca:-independent PLA2 denuded of epithelium are commonly seen and defects in epitheli- (aPLA,). The relative roles of these enzymes in degradation of intemalized DPPC al permeability are characteristic. We report here the culture was investigated in isolated perfused rat lungs and type II cells in primary culture and characterization of epithelial cells from cystoscopic using p-bromophenacylbromide (BPB) and 1-hexadecyl-3-trifluorethylglycero-sn-2- bladder biopsies obtained from 7 female patients with intersti- phosphomethanol (MJ33) as inhibitors of sPLA2 and aPLA2, respectively. Withrat tial cystitis. Within 4-14 days cellular outgrowths appeared from explants incubated in cell medium. Monolayers reached lung and typeII cell homogenates, PLA2 activity at pH 8.5 was inhibited - 90% with confluence after 6 weeks. Cells of the monolayers were cyto- BPB and <5% with MJ33; at pH 4, values were < 10% and - 50%, respectively. keratin-positive and smooth muscle actin-negative, confirming was 2 h after endotracheal instillation of 3H-DPPC Degradation in rat lung studied their epithelial origin. They exhibited epithelial cell in mixed unilamellar liposomes. Degradation (control 34.7 ± 2.8 nmol PC/2 h) was ultrastructure including intermediate filaments and junctional decreased by 20% with BPB, 45% with MJ33 and 58% with both inhibitors. complexes. Vesicles bounded by a trilaminar plasma membrane and Degradation in isolated typeII cells (control 4.1 ± 0.05nmollmg cell protein/2 h) lateral interdigitations were also present. These cells have was decreased by 21% with BPB, 52% with MJ33 and 70% with both. The putative been passaged up to 6 times and have maintained their epithelial cPLA2 inhibitor, AACOCF3, did not affect 3H-DPPC degradation. These results cell characteristics. Epithelial cells may be targets for indicate that both secretory and Ca:-independent lysosomal-type PLA2s are involved initiating agents and inflammatory effects of interstitial in degradation of intemalized DPPC by lung cells under physiological conditions cystitis and should be useful for studies of the pathogenesis with the latter accounting for the greater fraction. of this disease. (Supported by NIH R01 DK 44818).

2137 2138 ALTERATIONS IN DIPEPTIDYLPHOSPHATASE IV AND ACID 3-DIMENSIONAL MAPPING OF EPITHELIAL INJURY AND REPAIR IN PHOSPHATASE ARE ACUTE SURROGATE MARKERS FOR DISTAL AIRWAYS ((C Plopper, L Van Wine, S NLshio, A Weir, N Tyler and NEPHROTOXICITY OF IMMUNOSUPPRESSIVE COMPOUNDS. ((J.W. M Voit)) School of Veterinary Medicine, University of California, Davis CA Woods', J. Barker*, S. Koprakt, F. Dumontt and I.I. Singer)) Depts. of 95616 Biochem. and Mol. Path. and tImmunology Research, Merck Research Epithelial cells lining the airways are for cyto- Laboratories, Merck and Co. Inc. Rahway, New Jersey, 07065 respiratory conducdng targets toxicity. Current approaches indicate dtat susceptibility within the epithelial population varies by cell type, airway level and between cells of thesame type in Nephrotoxicity is a major clinical issue in the use of the immunosuppressives, a local a lbe heterogeneit of the pulmonaryinjury/rsponse hampers sudies A FK506. Microscopic examination of both animal and m Cyclosporin (CsA) and alemp_ to define the cellular basis of susceptibility toinjury, the human renal tissue shows significant changes in the epithelial cells of the of protection and the prcess ofrepair. This is especially difficult when the target proximal tubule. Transisson electronmicroscopic examination of renal sections cell population is also the progenitor for repairas is the case with cytcrme P- from rats treated orally for 3 weeks with FK506 (10 mg/kg) or CsA (20 mg/kg) 450 activated cytotxicants, such asnapisthalene (NA), and the Clara (nonciliated demonstrated that: 1) tubule epithelial cells contained large, heterogeneous bronchiolar epithelial) cell. NA causes Clam cel injury which is dose dependen lysosomes, 2) the brush border of otherwise normal appearing cells was often site-specific and variable by degree of differentiation of individual target cells. either very disrupted or missing entirely, and 3) numerous necrotic cells Acute NA injury is characterized by swelling and exfoliation of injured cells within 48 houn. and reorganization of epithelium is complete 14 containing very few cytoplasmic components were present. These effects were Regeeration rarely observed in vehicle treated controls. To quantify these effects the days after injury. The purpose of this study was two fold: 1) to 3-dimensionally map the distribution of Clara cell injury produced by naphtalene in the mouse following procedures were developed; 1) lysosomes were stained using a and 2) to define the pattem of repopulation during repair using scanning laser histochemical technique to label a lysosomal hydrolase, acid phosphatase confocal microscopy of fluoescent nuclear dyes. In steady state, nuclei of (AP), 2) the brush border integrety was visualized using an immuno-perxidase bslnchiolar epithelium are in a regular pattem with clear boundaries between technique with an antibody specific for the brush border enzyme alveolar ducts and terminal bronchiolar epitheium. This pattem is disrupted by dipeptidylphosphatase IV (DPP), and 3) computer based analysis of digitized NA injury ina site and cel specific marner. Nuclear dyes indicate that there is an light microscopic images was used to quantify the pixel densities of these labels. uneven distribution of cytotoxicity: the majority of injured cels are in distal Using this analysis we demostrated that it was possible to detect significant bronchioles. After acute injury and exfoliation, surviving cells are clustered increases in AP and decreases in DPP compared to vehicle treated controls from around branch points of proximal airway genrations. Epithdial reorgaizaton in rats treated orally for 7 days with either CsA or FK506. Further we have found areas where there ar srvors involves neighboring interstitial cells. At 14 days, it possible to detect increases in AP labeling in animals which had been i.v. the regular nuclear palbem of epithelial cells is indistinguishable treated with FK506 for only 4 days. These relatively short term assays should from controls. Supported by NIH ES05707, ES0431 1, ES04699, ES06700 and facilitate the quantitation of nephrotoxicity in animals. ES07059.

2139 2140 MATERNAL BETA-1-ADRENERGIC BLOCKADE CAUSES IMPAIRED FETAL EXPRESSION OF TIA PROTEIN IN RAT EMBRYOS ((M. C. Williams, A. ALVEOLAR TYPE 11 EPITHELIAL CELL MATURATION IN RATS. ((D.M. Smith, Hinds, and A. Wetterwald)) Departments of Medicine and Anatomy, M. Bazalakova, and S.K. Sommers Smith)) Deparment of Biological Sciences, Boston University School of Medicine, Boston, MA 02118 and Institute Wellesley College, Wellesley, MA 02181. of Pathology, University of Bern, Switzerland.

We have previously reported that maternal treatment, via an implanted osmotic The Tla gene is expressed in high abundance in normal rat lung in pump, with the wide-range beta-adrenergic blocker propranolol on days 18-21 of alveolar type I cells (Rishi et al., in press) and in certain osteoblasts gestation in rats causes a significant decrease in fetal type II alveolar cell maturation (A. W.). PCR/northern analyses show that Tlac mRNA is marginally and fetal body weight. The present study was undertaken in order to determine if detectable in adult brain, kidney, and intestine. Abundant TIa mRNA similar treatment with specific beta-1 adrenergic blocking agents had similar effects. is found in gut and derivatives and in brain and neural derivatives Timed rats were implanted with osmotic pumps containing either pregnant S.D of day 10 embryonic rats and in day 12.5 lung bud. We used a Atenolol or Metoprolol, such that the pump delivered 0.21 mg/hr, or 0.47 mg/hr, monoclonal antibody (prepared by A. W.) and immunoperoxidase on day 18 of gestation, and the animals respectively. The pumps were implanted staining to determine where the mRNA is translated in embryos. On were killed at day 21. Other groups of animals were not handled prior to sacrifice at day 10 low concentrations of Tla protein are present in the neural and served as 'untouched" controls. We have previously reported data for day 21, tube. By day 13, the entire nervous system (brain, spinal cord, spinal fetuses from rats treated with saline-loaded pumps. At day 21, the fetuses were ganglia, optic cup, developing neurites) contains abundant Tla and lungs from two fetuses of each litter were studied by light and electron weighed, protein as do gut derivatives (esophagus, stomach, trachea, and microscopy. The results indicated that the fetal weights in both beta-blocked groups forming airways) and mesonephric tubules. In these organs and in were not different from saline-treated controls, nor from untouched controls. The the optic cup Tla protein is localized specifically to apical plasma fetal lungs in both beta-blocked groups, however, appeared less mature by light membranes; thus in the eye it resides at the interface between the than did controls of either kind. There were fewer, and less well- microscopy putative neural retina and pigment epithelium. By fetal day 16 developed alveoli, and much nore interstitial loose connective tissue. Eletron TIca mRNA is largely absent from neural structures but the protein is microscopy revealed alveolar epithelial cells with much more glycogen in their detectable. Although the level of expression to cytoplasm than controls, and apparently fewer lameliar inclusion bodies. The Tlct continues increase in the lung during development expression is decreased to finding of no difference In the fetal body weights, in contrast to the propranolol- very low levels in adult kidney, esophagus, and stomach. Expression treated group previously reported, suggests that the effect on the lung is specific to is also repressed in brain and other neural derivatives except the the blockade of beta-1-adrenergic receptors at a critical time in fetal lung choroid plexus and the eye where it is expressed at high levels in the development. Further studies are underway to quantify this effect. ciliary epithelium. The presence of Tla in transporting epithelia suggests that Tics protein may participate in regulation of ion/water fluxes. Supported by PPG HL 47049. Tuesday. Epithelia II (2141-2146) 369a

2141 2142 SAUNITY AND TEMPERATURE EFFECTS ON PROTEIN EXPRESSION, IN STRATIFIED SQUAMOUS EPITHELIA PRODUCE MUCI SITU ATPASE ACTIVMES, AND MORPHOMETRIC PROPERTIES OF MUCIN. ((T. Inatomi and I.K. Gipson)) Schepens Eye Research TELEOST GILL EPITHELIAL CELLS. ((D. Kukz and G.N. Somero)) Institute and Harvard Medical School, Boston, MA 02114. Department of Zoologa, Oregon State University, Corvallis, OR 97331-2914. The stratified squamous epithelia of the ocular surface and vagina of which are The gill epithelium of and eurythermai teleosts is directly exposed are mucus-coated nonkeratinizing epithelia, apices euryhline to abrasive from interactions to the ambient medium but has the ability to functionally compensate for sa- subject pressure epithelia-epithelia from eyelid or vaginal folds, respectively. Mucus coats on these linity (S) and temperature (T) effects and maintain enantiostasis. After 5 have been considered to be derived from the and cell isolation epithelia generally months acclimation of the fish (cdyb* mnirb subsequent specialized mucin-producing cells embedded either in the epithelia - T - on mo- we examined long-term effects of S (1.5 34 ppt.) and (7 26'C) or in adjacent tissues. In this study, we demonstrate that MUCI, lecular and cellular properties of gill epithelial cells. Using high-resolution which is the transmembrane epithelial mucin expressed by a large two-dimensional electrophoresis more than 300 cellular proteins were re- number of simple secretory epithelial tissues, is produced by solved and the pi's and M,s of cold and warm induced and S regulated pro- stratified corneal, conjunctival and vaginal epithelium. MUCI teins were calculated. In addition to changes in protein patterns we mRNA expression was detected in the RNA of stratified cultured demonstrate significant influences of both S and T on the specific activities of comeal epithelium by northern blotting. Using RT-PCR technique, the plasma membrane Na/K- and H-ATPases in living, permeabilized cells. MUCI expression was also observed in ex vivo conjunctival Compared to crude homogenates, the activities of these enzymes were con- epithelium. In situ hybridization study showed that all three sistently about fourfold higher in permeabilized cells, most likely due to the epithelia expressed MUCI mRNA. In vaginal epithelium, a higher mRNA was detected in the basal half of sidedness of vesides and to the loss of interactions during level of MUCI expression lipid-enzyme the cell and the extent of of MUCl mRNA tended homogenization. Significant differences in dependence on S but not T were layers, expression to decrease from basal to apical. These results suggest 1) MUC1 observed in the number, shape, and size of the metabolically most active chlo- mRNA is transcribed in the basal half of the and that of epithelium, ride cells. These results show that important molecular propertes gill epi- translation and glycosylation steps of mucin synthesis occur as the thelial cells are dependent on both S and T whereas only S has influence on epithelium differentiates to the,apical cell layers and 2) that stratfied the cellular level of organizaion. Supported by the German Academic Exchange as well as simple epithelia synthesize MUCI. (Supported by Service (DK) and NSF grnt IBN 92-06660 (GNS). EY03306 to I.K. Gipson.)

2143 21" POLYION-MEDIATEDTRANSFECIONOFCOMMA-IDMOUSEMAMMARYAND REGULATION OF EXPRESSION OF THE NA,K-ATPase IN MADIN DARBY PRIMARY BOVINE MAMMARY EPSTURLIAL CELLS BN VIMRO. (( D. A_utz, CANINE KIDNEY (MDCK) CELLS IN SERUM FREE MEDIUM. ((M. Taub, and C. N. Be_we P. A. Hiltner, and F. L. Schabacher)) Lab. Mol. & Dcv. BioL, Ohio Agric. C. Allen)) Department of Biochemistry, State Univ. of NY at Buffalo, Re antd Dcv. CeiSer, lbe Otio State Uniesity, Wooster, OH 44691. Buffalo, NY 14214. The role of and AMP in the Metods for non-gmli, nonviral tansfection of rwombninut DNA direcdy into targ Prostaglandin El (PGEI) cyclic regulating of the in MDCK cells has been studied. orns would facilitate hamangn theay and also al expreson of recombinsnt pmti activity Na,K-ATPase Previously, we that PGE1 and AMP increase the rate of in bovine mammasy celis in vwvo and fusaquest scfeioniuo mik. Polycabionnansftcon reported 8-bromocyclic ouabain-sensitive Rb+ vyi the Na,K-ATPase in MDCK cells. This syems we examned by adboladblled pa.sid uptake, nbdcuar distribution, ad umnan uptake effects was associated with an increase in the number of ouabain binding growth bomone (hGH) eNxp_sion for their ability to tansfect Comma-1D mwsc mamary sites, and an increase in the activity of the Na,K-ATPase in cell lysates (J. and pamay bovin cells in vito. isto whiole cells and mclearlocaizalon nummsy Uptake Cell. Physiol. 151:337,1992). In order to evaluate whether the increase wese much greater for poly-L-orzithine (ORN,) mediated Utsuftion compard to poly-L- in the number of ouabain binding sites could be explained by an increase in lysine or DEAE-dcxAE Absen of plasnud DNA in cytosoic frctiobs and ifs oalizaion the rate of biosynthesis of the Na,K-ATPase, 35S-labelling studies were in thl muclens suggeot thee ecndoomme of iitenalized DNA wa ot sale ling for conducted. Cell lysates were immunoprecipitated, separated on SDS gels expressin. Traistction with ORN gmvc moore efficiet cell tgeting and cear uptake and subjected to autoradiography. The results indicated an increase in the although hGH expreson wa than from DEAE-dxtn tumfcled cells. Mixtur of rate of biosynthesis of both the alpha and the beta subunits of the ORN, and DEAE-dcxtran iuread plasmid uptake and scklar localizao over cither Na,K-ATPase in PGEl and 8-bromocyclic AMP treated MDCK cells. In polycation alone in Comma-ID mousc mamnny cells. Molr ratios of 3:1 ORN.:DEAE- order to evaluate whether this increase in Na,K-ATPase biosynthesis could dextran and 10:1 polycaonDNA give optimal hGH expression (10-20 ugl modium) be explained by affects at the mRNA level, Northem analysis was perstngup to SO days post-tassfection. HigherpocDonDNArtio cauwd exsivecUl conducted. The results indicated an increase in the level of the mRNA for iy. Ihe addition of histamine deivatzed poly-1-glutarnic acid (GLU./His) to facile the beta subunit of the Na,K-ATPase in PGE, and 8-bromocyclic AMP polycationdiscionfom DNA in acidic endosomes iacred bGH expessio about five- treated cells, unlike alpha subunit mRNA. Similarly, nuclear runoff fold in Comma-ID and pimnay bovine mammary epithelial cells in vio, (ulOOng GEH/ml) transcription studies indicated an increase in the rate of beta subunit andreducedlcell iuy and death T sfectionof cell lines COS-1, MDGC, MDBK, 293, and mRNA transcription. A selective increase in Na,K-ATPase beta subunit Hep G2 shows each cell type to respond differely to aCsfection with DEAE-dcxm, the gene expression could presumably account for the increase in the level of polycaion mixture, or the ch bg-Ahf liGLU./His pobyon ltihmtonsystem. Polycation the Na,K-ATPase in PGE, and 84xomocyclic AMP treated MDCK cells, if for the of the and mixtmes pvde an eas, efficiest method for the liansfection of cutmed psnay or beta subunit levels are limiting assembly holoenzyme, its stabilization. NIH 9R01 DK4028607 to etblisepitelialcell lines with the capabilty for high exDemon for prolonged times in (Supported by M.T.) pfimasy cellswitht slkctionor isguM, as is neded for asfection of tart orpnm viwvo. Suppost by Natal. Daiy Prom. & Res. Board and Ohio Edison Pogmma finds

2145 2146 LACK OF RESPONSE OF SQUAMOUS CELL CARCINOMA LINES TO THIE WATER PERMEABILITY IN CACO-2 LAYERS GROWN ON PRODIFFERENTIATING ACTIONS OF 1,25 DIHYDROXY VITAMIN D AND TRANSPARENT FILTERS. M. Jouve, J. Bourguet, M. CALCIUM. ((A.V.Ratnam, M.J.Su,and D.D.Blkle)) Department of Medicine, Parisi, E. Caliot and S. Robine, Dept of Biology University of Callfornla,San Franclco,CA 94121. (Spon.by B.Halloran) CEA, Saclay and Institut Pasteur, Paris, France. Permeable have been used in the Normal Human Keratinocytes (NHK) differentiate from a highly proliferative supports widely basal cell to a terminally differentlated comilfed cell In culture In the study of cultured epithelial barriers. These presence of physiological levels of extracellular calcium. In studies require complete confluence and tight contrast,squamous carcinoma cells(SCC) differentiate less well than NHK junction's formation of the cultured cells. For under Identical condItIons. Our studies with Northem blot analysis elucidste water flow measurements, minimal unstirred layers that the differentiation process Is associated with a concomitant Increse In the expression of genes for Involucrln (I) and transglutaminase (TG) that in series are essential. One micron pores are Involved with cornited envelope formatIon. The mRNA for both I end TO transparent filters were used to support wer not detected In all the SCC lInes studIed vlz.,SCC4,12B2, 12F2,A431 epithelial cells from a CaCo-2 clone 1 cell line, snd HACAT,when they wer grown In MEMIKGM without serum. Additlon of allowing the visual monitoring of their growth and 5% FCS for 48 hours triggered the expression of these gnes which could restriction for the water then be maintained In offering insignificant subsequently KGM In the absence of serum. Serum measurement. The cells were seeded at 1 to was not required for InductIon of thes gones In NHK. In NHK,extracellular flow calcium (CaO)and 1,25 dlhydroxy vitamin D9 (1,25 D) stimulate the 5.105 cells/cm2. At confluence, the net water flow expression of I and TG In a dose dependent manner, while the SCC lines (Jw), transepithelial electrical resistance (TEER) faled to respond to thes factors regardle of whether thes cells had and potential difference (TEP) were measured (see been pr-exposed to serum. An Important factor that medIates 1,25 D Parisi et al., Pflugers Arch., 1993, 423, 1-6). stimulated gone expression Is the Vitamin D Receptor (VDR). However,VDR The results demonstrated that these when mRNA levels In all the SCC lines examIned were comparable to thos In NHK. cells, Furthermore, the VDR protein levels and afflnity In SCC lInes as assessed they have grown to confluence in 11 to 13 days, by Ilgand binding anaIs were comparable to those of NHK. Thes studies exhibited a TEP ranging from 0.2 to 1.4 mv and a Indlcate that serum, but not calcium or 1,25 D stimulate expression of I and TEER from 348 to 497 ohms/cm2. Jw, under a 5 cm TO genes In SCC lines, suggesting that a serum response element rether pressure difference, was than calcium or Vitamin D response element may specifically potentiats hydrostatic usually transcriptlon In the SCC lInes. Secondly, the mediators facilitating 1,25 D negligible ; it increased only moderately when the and celclum action on gen expresIon other then the VDR may be missIng serosal medium was made hyperosmolar with mannitol or defectIve In SCC lInes. Thus,SCC lines apper to leck some but not all (220 mOsm), in accordance thus with a low water the mediators by which NHK respond to the prodliferentating actions of permeability of the CaCo-2 cells plasma membranes. calcium and 1,25 D. Further experiments are Intended to elucIdate the defective pathway. 370a Epithelia II (2147). Tuesday

2147 OXIDATIVE STRESS INDUCES RELOCATION AND SYNTHESIS OF THE 27 KD HEAT SHOCK PROTEIN AND ISOFORMS IN NORMAL HUMAN KERATINOCYTES. ((J. W. Wongt, B. Sh2, M. MoCIaren2, R. R Nw5f2. and T. Shbamotol)) Depatmeisof 1 Eonknintal Toxioogy and 2Dermstology, School of Medicine, Unriversiy of Califrnia, Davis, CA 95616. Ahough classically induod after sress, heat shock or stress proteis are ubiquitouy expressed, suggest they have an essential celluhr function. HSP syneis ha been tudied hI many ce type and with various iiced stem which has ld us to examin H8P27 In oxidative-xtreseed keratinocytes. Cuured normal human derived from nonatal oreskin, treated wth hydrogen pex (H202)-eafnocytes,or tuyyrs de (t-BuOOH) at doses raging from5to 250 PM for 1 hour wer snWyzod for H8P27 xpr n. Indirecd m - oent stainin demon rated HSP27 is cded entirely In the cytoplasm Of control (unbtated) nd H202teabd ,ck white cl treated with t-BuOOH recate HSP- 27 to the nucleus. This idcafes that In otras of the general reactiviy of H202, the id-souble tIBuOOH Is more membra selective. We have pviously shown that he t-strs katMocytes express at ast four isoform, three bein pos transiional phosphorylated orms of the oroinal, wh isoeWctric pokingisng from 6 to 7. Uin SOS-PAGE or ver slab isoeectric koin, followed by Wesom bblt for HSP27, whob cell "ystes of treated and untreated cells were analyzed. Urdroeted ceiaexprsedtwo lsofornw of HSP27. Cells treated wth H202 atdomeas kwas 60sofr hr express new HSP27 bobms as well as an Cease in ttal HSP27 expressi. Cels treated at the highr doses (200 and 250 pM fr 1 hr) express four isolom which are more acidc than the two exprsd in untreated cell. For all treatmts, levels of the most basic boform derem whilb hose ol the rnwly brmred bofoms increase. Treatig keratinocytes with H202 or t-BuOOH did not affect the viabilky and pleration of the cell at c 100 pM. Comparon udies will be attempted with keraltocytes treated with t-BuOOH. Aithough tha putalt role of HSP27 i to protect cell. the dynamf cha s in H8P27 soen In keraiocytes after exposure to oxidaWe stress agges!t i may also play a role in survival and recovery of keratinocytes. Blood Vessels II (2148-2151).

2148 2149 A NOVEL NONMUSCLE FILAMIN LSOTYFE IN HUMAN OMENTUM SUPEROXIDE DISMUTASE EXPRESSION IN HUMAN OMENTUM MCROVASCULAR ENDOTHEUAL (HOME) CELLS. ((W. F. Paonl, N. MICROVASCULAR ENDOTHEBAL (HOME) AND MESOTHELIAL (MESO) Cblng-Welch2, Q. Wang1, Q. Su, R. P. Cambria2, H. Hectmn3, amdD. CELlS. ((N. Clug-Wlchl, W. F. Patton2, L Haste2, Q. Su, R. P. Cambria', an Sheprol)). 1Biology Da , Bosn Universty,Boso, Ma 02115, H. Hecizn3, D. Sher2)). 1Vascu ReseahL tY, 2VasculrReserhLabory, Massa Genr Hospital, and 3Surgery Ge l Hosbpitl, Bostn, MA. 02114, 2Biology Depa n Boston University, Dqstent, Havd Medicd Sdool, Bosto, Ma 02114 Bosto, Ma 0211 ad 3Surgy Deptmnt, Hard Mdical School, Bon, MA 02214. mbane-assocIated proedw, actn ad pefc b ptins play mIUbegXalinregni5UD5PEacelpn pe b ytybydUdgendoeil ceB Cer to surgical ala nrions the limitaton ofblood flow and oxygen ftb wnmusde nd swodi ddlivey to etime lanled vse trnbjecd artral occluson Prolongd nauacle ioypes har kn to be eqpessedInhom cells. Nonnucle fllamin even ca be deleterious to time normal vessd acitectUre. ( bP-280)s b stes for sef-oa , Itgal membanegl e admiofozyg after apeod oflch ia lea to a b ofrective andacti mRiItoat cAP-dm entdcadlu m oxygenradkals whicc damage_t cdli lnjuredby AIP depeon dring kdme tI_axGTP-tsite, tityand camn-actvatedcapein icemi le expresson oftie oxygn prhmt may be alterd by diffg cleavap dtes We found, by iamce mioopy, mony degees ofhypoxia, and reoygeno as we as expose to otlm cell tYpes. arey EC san meslmUc contan an abndanceofndonucle filmin wbile Indeed, cel Hln vasula graft tha ae equppedto catalyze Me HOME ceds express way lte. Inter, HOME cels contain a previousy dispropoatiotion ofoxygen dicals would, in put, Impaa e shot and long term unearaterxedfiamin iotype. MIefamin istype (isoeectric point, 7.2) was succesatulns ofsurgical arteial itvtIonM. Ime use ofHOME andMSO cells purfledbyac kbnatofprepave nodenatedngecpr cteiue, as aiy availe cdls for s odingascul pros o eely been denatringSDSectr _-B p eavge, advocatd but tie r ve nstvity of tiese cel to oxIdantury Is unknown, In vaedpbe HP Si peak frao wereaayzed by o1mated Ednan ths stdy, ftime ve amunts oft cellul atoxid e mes of HOME and dgadatond 11-16 amino ad _etcedaawasobtained.TLme purifieddn MESO cels we I iatLed Mm prmi MESO cel polypeptides (pI = 7.1- wa Identifed as a novd lanmlnlsotype th is 60.66% Identical with 70469; 75. l,- 20 kDa), found o be absm frm HOME cls by elcdMeawer dwpece.AcXbg fHamin homology to tlmepevioy c ized non1unc from human umbilca Identfi by ptedn s_eqen a lioforms of ma superoxide dismutase vein EC. FHiminffidsbu w ted by bndykinnnof InEC, chandin from a (Mn-SOD). Anayds oftime ges deamneddo HOMEad MESO cels conIned pericellulr disrbution to a diue cytimc di wItin two minetes 9.5ng and 2.2ng Cu-Zn SOD per ng oftot cellulrprotI rsewdvely. Only Flla o dlvrty nEC may pvidedifeentlal respondvenes to MESO cel conained Mn-SOD (47.3 n perg W ceulrprteln). ibs was cating aolda In vaiou vascula beds. coafinnedby gaphy s _emi . lbese resulta Indicate thatthe ty ofHOME an MESO cels to pcttielves againat oxida i Injury arequite differeIt. In tht MESO clSNs Mt HOME cel are equpped with tie antioxidt enzyme, Mn-SOD.

2150 2151 SUPPRESSION OF VASCULAR SMOOTH MUSCLE CELLr MErALLOPROTEINASE INHIBMON OF PROTEIN KINASE C l1 EXPRESSION PREVENTS PHORBOL ACnVFIBYBYM OVASCULARENDOTHFUALCELI ANDMESOTHsLAL ESTER-MEDIATED ENDOTHELIAL CELL RETRACTION. ((P.T. Vuong, A.B. CE8I. ((N a g-Wekh,R.HeRCmdCma))RaP. VVascarcPsaraclLsxmay, Malik, P.G. Nagpala, K. Mikocz, and H. Lum)) Department of Pharnacology, MussI uaettaGamma! Hosptl HardMedcal Siaol Boo, MA 02114. 'Department of Biochemistry, Rush Medical CollegeRush-Presbyterian-St. Luke's labial hypespleals(Ilt) lata pmutcaueofr-msmositw~vauombsmguyummmglo- Medical Center, Chicago, IL 60612. pasty. Vucub mmoa c (VSM) a lmtle _s lu fmi_oae atheextraul- hiarsuttuOM(Elt doedmatau hnm in the ahgasaOfIlL lime abiity OfVSM Activation of protein kinase C (PKC), a multigene family of serine/threonine cells ID aeCrammlgue. whbiwoudiitaticsval vehlgawiamaybercal kinase isoforms, is believed to be critical for endothelial cell retraction which is im ticdmedveosof DL P_esu by, hcmIsPre-in BeC as wl as* uomarem- responsible for increased vascular endotloial pemeability. However, the pinuifstmldgdmdot slceamcft iDdms VSM eltresma. Mmeuuof sa contribution of individual PKC isoforms to the regulation of endothelial barrier is inemml mIeoas"cehe.obnaiel OMECelIsummlmesaheMa (MMES)mcii amAb..- not known. In human dermal microvessel endotheUal celis (HMEC), we showed dmaounrceforaredinofyucdgatsmftbmeear5vocafadmamumoDat~sonaahie that one of the predominant PKC isofomns is PKCp. We investigated the tmtesiag bu dieectof mloproIelease activity byVSM cel ams umIlJ. timedeminshMedlgtkmcell to involvement of PKCPI isoform by stably transfecting HMEC with cDNA encoding iMme iWMklyoflHOWE mmIbMES callse leflundegeum tvity PKCPI in the antisense orientation via the retroviral vector PLNCX, which was by _mm aamus vei VSM celaft w s estigad, leekffeofHOWEmed madum (11D.M dWMES l-erhamdmedl.a (MESO-CMi) on ftimewxoaicti controlled by a consvttive promoter. Immunocytocherical analysis indicated .metp__ seby VSM ce a zby SDS-alula smi zyogrmphy. that the antsen transfection inhibited -50% of the PKCPI expression. We also Pri toeztare VSMcel weinma forr24 bows in HOME-CM or MESO.OC. determined the ab l eledical impedance (TEl), a measure of cell Zgaphy of VSM odl exzus= sddM r7da2Ok7Ogeboiydc =ye(tape IV shape dynamics in real tme as an index of endotheial cel retraction in confluent pr_ )lg_ a_ amOr omofo r60600ld_m (wdvwptypeIV_on.se). monotayers of control (non-transfected) and HLIEC transfcted with the PKCP1 imaeat itydoftdaasaaw eea.phlyhRdadby 1,10 _ihemelI antsense vedor. Treabtent of cois with 1 pM phorbol 12-myristate 13-acetate cma&amkmg dikamtigeseuae aImatca O(VSM alls with hOME-0ator (PMA) decreased the TEI to 30% below baseline in the control cells, indicative of ME-SO.Oa faridin thasp asiefhSs-ullquutainuealieqydwpetiumcoftwo retraction of endothellal cells wth widening of intercllularjunctions. The aladiue inlimsofeadMr51,000 mdMr23,000. Pode duatasgedadoti of inhibition of PKCP1 expression prevented the PMA-induced decrease in TEI. WON=mila mirllutnmwasaisomsedisiportby vbkmeinhitm nsaeetadby These results that is an isoform HOIEmi ella Remezyo d d maboIhHiOMEWdSO suggest PKCP1 important regulaUng endothelial mil umsodmMr 140,00,125,000, md40_000ai insm in dr rcasmdioaad cell retraction and may contribute to endothelal permeability folowing activaton amodkm. ianmserm s_&at biI eubod anct. ofhbtmby VSM cell wel of PKC. (Supported by HL45638, HL27016, and American Heart Associstion of as HO mi MESO absy be _pe4mi coehrladgedmeIDVSM cel chicago). uigeak mdimaoaseesaacof slhypuplin Tuesday. Blood Vessels II (2152-2157) 371a

2152 2153 NEOVASCULARIZATION OF THE NAIGILIAN CORNEA IS PRZEVNTED BY AN HOMOCYSTEINE METABOLISM IN VASCULAR CELLS AND TISSUE: INHIBITOR THAT IS DEFICIENT IN MICE MUTANTS AT Two DIFTHRENT EVIDENCE FOR AN INACTIVE TRANSSULFURATION PATHWAY. LOCI. ((P. Gillis, O.V. Volpert and N. Bouck)) Departmnt of ((D.W. Jacobsen, S.R. Savon, K. Robinson, R.W. Stewart and P.E. DiCorleto)) Microbiology-Imunology and R. H. Lurie Cancer Center, Departments of Cell Biology, Cardiology, and Thoracic and Cardiovascular Northwestern University School of Medicine, Chicago, IL 60611. Surgery, The Cleveland Clinic Foundation, Cleveland, OH 44195 Recent epidemiological evidence strongly suggest that hyperhomocysteinemia Blood vessels are normally excluded from the corneas of is an independent risk factor for the development of cardiovascular diseases. This to be due to the healthy adult mammals. is thought Homocysteine (Hcy), a byproduct of methionine metabolism, can be presence of inhibitor(*) of neovancularization. An activity remethylated to methionine, or converted to cysteine by entry into the has been extracted with PBS from souse and bovine corneas that tranasulfuration pathway (TsP). Remethylation of Hcy is catalyzed by B 12- blocks capillary endothelial cell migration in a Boyden chamber dependent methionine synthase while the TsP is initiated by B6-dependent towards angiogenic factor bFGF (10 ng/ml). This activity was cystathionine b-synthase (CBS). Each pathway consumes ca. 50% of intra- present in the conditioned mdia of cultured stromal cellular Hcy if both enzymes are expressed (Finkelstein, 1990). Although recent fibroblasts and extractable from isolated stromal tissue. It studies suggest that Hcy may exert its atherogenic effect by disrupting inhibited endothelial cell migration in a dose dependent manner endothelial function, little is known about the metabolism of Hcy in vascular with half maximal activity at 2 ug/ml total protein. Similar cells and tissues. To obtain evidence for TsP activity, we have measured CBS in extracts of the vascularized corneas that occur naturally in extracts from cultured human aorfic endothelial cells (HAEC) and smooth muscle hairless (hr/hr) and in nude (nu/nu) mice contained activity cells (HASMC), umbilical vein endothelial cells (HUVEC), intemal mammary capable of inducing endothelial cell migration. Full-thickness arteries (IMA) and rat liver (positive control; specific activity = 184 nmolUhr/mg protein) using a sensitive radioisotopic assay (Mudd et al., 1965). Low-passage of nu/nu and hr/hr mouse corneas induced fragmnts HAEC from 3 different isolates and HUVEC from 7-9 pooled cords had little or neovascularization in the rat cornea corneas (4/4 positive). no detectable CBS activity (< 1 nmolVhr/mg; range = 0 to 0.75 nmolUhr/mg). In Mixing experimnts demonstrated that the inhibitory activity contrast low-passage HASMC from 4 different isolates had CBS activities extracted from wild-type corneas could inhibit stimulatory ranging from 3.02 to 6.21 nmol/hr/mg. However, extracts of human IMA had activity extractable from vascular corneas. Inhibitory little or no detectable CBS activity suggesting that expression of CBS may be activity was heat- and trypsin-resistant yet >3 kDa in size, induced in cultured HASMC. The apparent lack of TsP activity in cultured suggesting it is due to a novel inhibitor. Identification of endothelial cells and IMA suggests that vascular endothelial cells may be cornea-derived angiogenesis inhibitor may reveal the mchani particularly sensitive to the increased levels of circulating Hcy found in 30 to underlying corneal avascularity and provide a therapeutic tool 40% of patients with cardiovascular diseases. to combat neovascularization associated with corneal disorders.

2154 2155 ATHEROSCLERarIC LESION-DERIVED MACROPHAGES HAVE REGULATION OF ENDOTHELIAL CELL VON WILLEBRAND DIMINISHED CAPACITY TO ADHERE TO ENDOTHELIAL CELLS. FACTOR BY VASCULAR ENDOTHELIAL CELL GROWTH FACTOR. ((Shankar, R., Byrne, C. E. and J. L. Gray.)) Dept. Surgery, Lyola ((S. Tannenbaum, S.Rosenfeld, E. Chao, and W. S. Arnold*.)) Hematology University Medical Center, Maywood, 1L40153. Section, Clinical Center and Surgical Metabolism Section*, NCI, NIH, Monocyte/macrophages are an integral cellar component of all Bethesda, MD 20892 (Spon. by H.K. Kleinman) stages of atherosclerotic lesions. Previous electron micrscopic studies sugested that macrophage foam cells reentered circulation from early von Willebrand factor (vWf) is a multimeric adhesive glycoprotein which lesions, thus forming a ipid clearance" system. We hypothesize that is synthesized and stored in endothelial cells and megakaryocytes. Tumor this clearance system fails as the lesion progresses because the lipid vasculature contains increased amounts of vWf. Vascular endothelial cell laden macrophaghes have a diminished capacity to adhere to endothelial growth factor (VEGF), an endothelial cell-specific growth factor produced cells and thus fail to exit the lesion. Here we have compared by tumors and important in angiogenesis, was studied for its ability to thioglycollate- elicited rabbit peritoneal macrophages (PM) and regulate the synthesis and storage of vWf. Human umbilical vein atheroslerotic lesion- derived macrophages (AthM0) in terms of their endothelial cells treated with recombinant VEGF, 0.3ng to 300ng/ml, had a ability to adhere to unstimulated and IL.1 stimulated human umbilical dose-dependent increase in cellular vWf to a maximum of 450% compared vein endothelial cells (HUVEC) usin 51Cr labeled macrophages. AthM0 with untreated cells. Basic fibroblast growth factor which alone was withou were isolated by enzyme digestion and density gradient separation fiom effect on cellular vWf, was synergistic with VEGF. VEGFs' ability to atherosclerotic lesions of 2% cholesterol diet fed rabbits. Our results increase cellular vWf was noted by 8h and increased steadily over time: indicated that 2.7 fold more PM compared to AthM0 adhered to VEGF IOOng/ml, % vWf compared with untreated cells; 8h 130%, 24h unstimulated HUVEC. When HUVEC were stimulated with IL-1 150%, 48h 190%, 72h 300%. Although VEGF caused a modest release of (10ng/ml) for 6 h to upregulate endothelial adhesion molecules, there vWf into supematant acutely, there were no major differences in the vWf in was approximately a 3 fold increase in PM adhesion. AthM0 adhesion on cell supematant over time. VEGF primarily increased the extremely high the other hand, did not change from that of the unstimulated controls. molecular weight multimers of vWf found in storage sites and 125I streptavidin binding asay showed that compared to PM, AthM0 subendothelial matrix. Since cells were plated at high density, cell numbers had significantly reduced expression of both CD11b and CD18 while and 3H-thymidine incorporation were the same in treated or untreated cells. maintaining the same levels of MHCII expression. These results suggest Thus the mitogenic effect of VEGF was separable from its effect on vWf. that AthM0 have impaired adhesion to endothelial celis possibly due to Although vWf mRNA has never been reported to be upregulated by any reduced expression of CD11/CD18 integins agent, cells treated with VEGF contained increased vWf mRNA. Regulation of vWf by VEGF may be important to tumor neovasularization.

2156 2157 SHEAR STRESS INCREASES AGONIST INDUCED RELEASE OF vWF CALCIUM SENSITVITY OF VASCULAR SMOOTH MUSCLE CELLS IS NOT FROM HUMAN ENDOTHELIAL CELLS. ((M. U. Nollert and B. Mathew)) ALTERED BY HYPERGLYCEMIA BUT IS AGONIST DEPENDENT. ((M.J. Cipola and C.T. Harker)) Dept. of Surgery, Div. of Vascular Surgery, OHSU, Portland, OR 97201. School of Chemical Engineeri and Materials Science, University of Oclahoma, Norman, OK 73019 Elevated levels of gLcose in the culture media of vascular smooth rrmiscle ceNs (VSMC) has been shown to after activity of two cellular enzymes, protein kinase C The endothela cell layer lining the vasculature responds to changes in fluid (PKC) and (Na,K)-ATPase. PKC activity is enhanced in high glucose by increasing mechanical shear stress by generating itacellular second messengers leading flux through the sorbiol pathway, causing increased de novo synthesis of 1,2-diacyl- to alteons in protein synthis and release. We have emined the response snglycerol (DAG), a precursor to PKC activation. Previous work from this lab has shown that elevatg gicose in the superfusate of iolated rat mesentenc artenes Of cells to arterl and venous levels of shear ess in a parallel signfcandy enhanced contractions to both lndolactam-V (IND, a specific PKC plate flow chamber. Human ealotdeial cells were obined from the umbilica activator) and potassium chlode (KCI) depolarIzafton, thus corinng the resuits vei and were cuwtred on gsssi;des usig sandard metxods. respone found in cell cuiture. The presert study was aimed to deternune whether this of the cells to the initiaon of arterial (25 dynes/cm2) and venous (4dyMecm2) augmernted response is due to aktered Ca- sensi of the VSMC contractile shear stres was by the release of von Willebid's epparatus. Mesenteric arteries from male Wstar rats (inner dia.<2SOpm; n-13) were Factor (vWF) using a sensitive EUISA. The initiation of flow, by itself, causes dissected in a zero Ca- PSS soluion, mounted on a specaized arteriograph and lumen diameter measured. Ca- was only a small increse in the relase of vWF. However, the agonis (either (Intralurinal P-SommHg) contiously cumulatively added back to the solufion and a Ca" dose-response curve (0.01-3.0mM) hiSamin (107M) of thrmbin (0.1 U/ml)) induced rekase of vWF was was generated in the presence of either IND (3pM) or KCI (60mM) in 11 mM glucose inreased in the presene of fluid flow. Cells exposed to and repeated in 44mM glucose after a 2h inubation. Alhough the amount of Ca- hismie in the presnce of flow rekased more than twice as much vWF as necessary to corract the vessels 50% of maximum (EC,) was significanty less in cells exposed to agonist is static conditions. Similar results were obsved for vessels contracted with IND vs. KCI (p0.01, both glucose concenitrations), elevating cells exposed to thrmbin and shear stres. These results are importat in gLcose trom 11mM to 44mM did not after the Ca- senst of vesseis contracted with either IND or KCI: for IND-0.18±0.05 in 11 mM and 0.21±0.05 in 44mM understai the biolog of i cells in an enviren that includes EC,w(mM) fluid flow. gluoose (p>0.05); ECD(mM) for KCI-059±0.09 in 11mM and 0.60± 0.10 In 44mM glucose (p>0.05). These results show: 1) Ca" sensitivity of VSMC can be modulated by coimpbx signaltanduction cascades asoated withcoritractile agonist stimiaton

not due to icreased Ca- sensivty of VSMC, but possbly to alterations In menrbane purmp activity could lead lo increased depoariation and/or aitered [Cal. 372a Blood Vessels II (2158-2163). Tuesday

2158 2159 N-ACETYLCYSTEINE (NAC) ALTERS GLUTATHIONE LEVELS PHENOTYPIC CHARACTERIZATION OF AORTIC SMOOTH MUSCLE CELLS CLONED AND REDOX STATE IN HUMAN UMBILICAL VEIN FROM ADULT RATS. ((M.L. Bochaton-Piallat, P. Ropraz, F. Gabbiani, and G. ENDOTHELIAL CELLS (HUVEC): EFFECT ON NF-icB Gabbiani)) Department of Pathology, University of Geneva, Switzerland. BINDING ACTIVITY. ((E. J. Poptic, Y-C. Chai, R. Faruqi, G. M. Arterial smooth muscle cells (SMC) assume different phenotypic features in Chisolm and P. E. DiCorleto)) Dept. of Cell Biology, Cleveland vitro according to their situation in vivo at the moment of collection: SMC Clinic Research Institute, Cleveland, OH 44195 cultured from the aduit aorta have a "hills and valleys" growth pattem and a serum dependent growth whereas those cultured from the intimal thickening The transcription factor NF-cB regulates the expression of several after endothelial denudation have an epithelioid growth pattem and a serum cytokine-inducible genes. Its activation appears to be a function of independent growth (Walker et al., Proc Nat Acad Sci USA 83:7311, 1986). the redox state of the cell which is maintained principally by This situation suggests that SMC are heterogeneous in their response to intracellular glutathione. We investigated the effect of NAC, a microenvironmental factors. In order to characterize SMC heterogeneity, we water-soluble antioxidant, on reduced (GSH) and oxidized (GSSG) have cloned SMC from adult rats. Fourteen clones were studied between glutathione levels in IL-1-stimulated HUVEC. NAC (20 mM) the fifth and fifteenth passage for their morphology, cytoskeletal features caused a 4-fold increase in GSH and a 6-fold decrease in GSSG. (i.e. expression of a-smooth muscle actin, desmin and smooth muscle These NAC-induced changes yielded molar ratios of intracellular myosin), replicative and migratory activities. Generally, clones were more GSH to GSSG that ranged from 40 to >400. Using electrophoretic dfferentiated than their parental population as far as their cytoskeletal mobility shift assays, we have examined in vitro the effect of simi- equipment is concemed. However, clones were heterogeneous in their lar ratios of GSH to GSSG on NF-icB binding to oligomers corre- morphology, replicative and migratory activities. They could be distributed in sponding to the NF-icB consensus DNA binding sequences in the E- 4 classes: (1) spindle cells with a "hills and valleys" growth pattem and a low selectin and VCAM-1 promoters. Nuclear extracts from IL-1 migratory activity; (2) epithelioid cells growing in monolayer with high treated HUVEC were used as the source of NF-icB. Maximum replicative and migratory activities; (3) thin-elongated cells growing in binding occurred at a GSHIGSSG ratio of 200 while minimal bind- multilayers with an extremely high proliferation rate and a high migratory ing was observed when the ratio was <20 or >400. Taken together, activity; and (4) cells becoming senescent before the tenth passage. Our these data suggest that changes in the ratio of GSH to GSSG in results suggest that a small proportion of SMC present in vivo in aduit rat HUVEC may result in altered binding of NF-KcB to DNA and thus aorta can acquire in culture features similar to those of SMC cuitured from In the same some cells can evolve to a may play a regulatory role in cytokine-induced E-selectin and intimal thickening. condition, senescent phenotype. (Supported by the Swiss National Science Foundation. VCAM-1 expression. (HL34727). Grant Nr 31.30796.91).

2160 2161 A COMPARISON OF UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC) AND COACT-DEPENDNTENDOTHELIALCLASS N HLAGENEACTIVAT1ON DERMAL MICROVASCULAR ENDOTHELIAL CELLS(HMVEC) WITH RESPECT INDUCED BY NATURAL KILLER CELLS IS MEDIATED BY IFN- TO ADHESION MOLECULE EXPRESSION, TISSUE FACTOR ACTIVITY, AND DEPENDENT AND INDEPENDENT MECHANISMS. ((CA Wtson, NEUTROPHIL KILLING. ((P.1 Lelkes-, M. Sh511nan., D.M. Wankowskid, S. Zhang-, P. Petzelauer, J. Zhou, R. Pardi, and J.R. Bender)) Yale University J. Varani, M. Dare+, J.Shen and S. Karrniol 'Dept Medicine, Lab Cell Biology, School of Medne, New Haven, CT 08536 (Spon. by J.R. Bender) Univ Wi8c Med School, MiMwpukee, WI 53201, + Dept Pathol, Univ Mich Med School, Ann Arbor Ml 48109, Clonetics Corp, San Diego Ca 92123. Natural killer lymphocytes adhere avidly ID alogenric endothellal cells (ECs)4 nd Iuce their menbrane of MHC clas In Three different preparations of early passage (<8) aduit microvascular endothelial expression 11 antgens vitro. Endoteal class 11 expression augmentB EC-driven CD4+ T cell cells from breast reduction exhibited a histiotypic cobble stone morphology, took up fluorescence-tagged acetylated LDL and stained uniformly positive for extabllehed proiferation hI vito, and may amplify T col recruitment and clonal endothelial cell markers, such as von Willebrand factor and PECAM-1. During expansion in vho. Utlking an ex viva lymphocyeskin organ cocuiture prolonged cuiture beyond passage 10, the cells underwent morphological changes model, NK cells oould be found EnIng and indcing cass 11 HLA on and lost some (e.g. PECAM-1, vWF), but not all of their characteristic markers (the mirovessel endothelium. Because IFN-y i te only known inducer of EC cells remained Dil-acetylated positive). In comparison to HUVEC, HMVEC formed class 11 HiA, EC responses to IFN-y and abogenec NK cels were more speedily (4 vs. 18 hours) capilary-like strutures on a reconstituted membrane compared In a variety of assays. Utilizing both neutralizing anti-IFN-y assembly (MATRIGEL), and responded differentiy to cytokine activation (eg, in and anti-IFN-y receptor (IFN-y R) antibodies, a spectrum of IFN-y - terms of induction and expression of a) cel adhesion moecules, VCAM-1, ICAM-1 dependence was observed for NK-medated EC HLA-DR induction, from tissue In HMVEC a and PECAM-1, and b) factor acvy. addition, exhbited unique negligble to moderate. Transwel experiments displayed that direct NK- pattem in the expression of both adhesion molecules and of tissue factor in EC contact Is required and antibody lnhbWlon studies indiated that the response to mechanical cycli which is distinct from responses P2 actvabon by strain, integrIn- ICAM-1 pathway(s) Is critcal In the generation of these of HUVEC. HUVEC and HUMVEC underwent neutrophil killing as fjrher, responses. The use of HLA-DRa promoter constucts in transient measured by Cr reease and failure to replate. HUVEC lost sensitivity as a function of passage while HMVEC did not. This killing could be blocked by DMTU, transfection assays demonred that the highly conserved X and S catalase and deroxamine, but not by SOD, implying hydroxyl radical mediaton. transcripion boxes are required in both IFN-y and NK-medlated gene In a model of spontaneous endothelial cell injury, as a function of passage number, activation. Taken together, these results indiate that human ailogeneic HUVEC demonstrate a progressive increase in cell death, while HMVEC did not. NK nphocy can induce EC dms 11 HLA gene actvation In an adhesion- This age-related injury was mediated by protein kdnase C and not by the agens that dependent, IFNy-ldspendent fashion, and suggest that, In concert with protect the cells againt oxidant injury. any IFN-y-dependent oomponent, this oould represent an efficient mode of endothelial activation and immune aiation viha.

2162 2163 SPATIAL-TEMPORAL EXPRESSION OF THE SMOOTH MUSCLE LOCALIZATION OF HUMAN VLDL RECEPTOR umRNA IN HUMAN CELL PHENOTYPE IN CHICK AORTA. (( J.M. Benson and Z. BLOOD VESSELS BY IN SITU HYBRIDIZATION. {(H.A.B. JIN N.E. Yablonka-Reuveni )) Department of Biological Structure, School of MULTHAUPT1, C.P. ARENAS-ELLIOTT1, H. , GAFVELS3, K. R. McCRAE2, M.J. WARHOL1) ) 1PENNSYLVANIA Medicine, University of Washington, Seattle, Washington 98195. HOSPITAL AND 2TEMPLE UNIVERSITY SCHOOL OF MEDICINE, PHILADELPHIA, PA, AND 3KAROLINSKA INSTITUTE, HUDINGE, We are analyzing smooth muscle ( SM ) cell phenotypes in developing and SWEDEN. adult chicken aortas. In the adult thoracic aorta there are two morphologically distinct cell populations organized into alternating The hVLDL receptor (VLDLR) is a member of the lamellae. One population ( SM+ ) exhibits a typical smooth muscle family of lipoprotein receptors which include the LDL receptor (LDLR), LDL receptor related protein/a;MR phenotype ( expressing cytoplasmic proteins such as SM or actin, SM (LRP) and GP 330. Little is known about the physio- myosin, SM calponin, myosin light chain kinase and desmin, and colocating logy or cellular distribution of the VLDL receptor. with extracellular basement membrane proteins ). The second population In order to define the distribution of this receptor ( SM- ) lacks these SM characteristics. On embryo day 7, SM+ cells are in normal and diseased human vascular tissue, we found in a multilayered continuum directly under the endothelial cells and performed fluorescence in situ hybridization on surgical specimens of normal and atherosclerotic are the only cell type present in the media of the aorta and the aortic arches. blood vessels. Endothelium within both small and By embryo day 16 the thoracic aorta displays the lamellar ( SM+ ) and large caliber vessels contained VLDLR mRNA. VLDLR interlamellar ( SM- ) morphology found in the adult, with a wide band of aRNA was also expressed within the vascular smooth SM- cells in the sub-intimal portion of the media. The abdominal aorta, muscle in both normal vessels and atherosclerotic from embryo to adult, does not develop this alternating lamellar plaques; however, it was not detected in fibroblasts morphology. In addition to these spatial changes in SM organization, there or other connective tissue cells. Although the physiologic function of the VLDLR is still uncertain, are sequential, temporal changes in protein expression during embryonic these findings suggest a role for this receptor in development. For example, fibronectin, collagen IV and SM actin appear peripheral transport of apoE-containing lipoproteins. first, followed by desmin and laminin at embryo day 9. Later, on embryo Furthermore, expression of this receptor by vascular day 11, calponin is ex.pressed. These spatal and temporal changes indicate cells involved in the formation of atherosclerotic the dynamic naure of the developing aorta. ( Supported by NIH and AHA ). plaques suggest a potential role for the VLDLR in facilitating this process. Tuesday. Blood Vessels II (2164-2165) 373a

2164 2165 NITRIC OXIDE PRODUCTION IN, LYMPHATIC 2NDOTHETIAT EXPRESSION OF INDUCIBLE NTRC OXIDE SYNTHASE (iNOS) IS CELeS IF VITRO. ( (L.V. Leak, J. L. Cadet , C. Grif- REQUIRED FOR PRODUCTION OF ANGIOGENIC ACTIVITY BY fin')), Department of Anatomy, Howard University, Washington, DC 20059, Molecular Neuropsychiatry MURINE MACROPHAGES AND IC-21 CELLS. ((S.J. Leibovich and J. Section,NIH, NIDA, Baltimore, MD, 21224. Golczewski)) Department ofAnatomy, Cell Biology & Injury Sciences, The New Jersey Medical School, UMDNJ, Newark, NJ 07103. To examine a possible involvement of nitric oxide (NO) as a lymphatic endothelial-derived relaxing We have shown previously (PNAS, 91,1994,4190-4194) that production of factor we have isolated and cultured lymphatic endothelial cells (LEC) from ovine mesenteric angiogenic activity by endotoxin (LPS)-activated human blood monocytes lymphatic vessels. The production of NO by LEC was requires an L-arginine-dependent iNOS pathway. In this study, IC-21 cells and quantitated by the colorimetric Griess reaction for macrophages from thioglycolate-induced Balb-c mouse peritoneal cavities were NO2 in culture medium conditioned by confluent activated with and/or LPS Production monolayers of LEC for 24 to 48 hr. Basal levels of interferon-y (100u/ml) (lOng-1 g/ml). of NO6production by confluent LEC o?golayers was 60 uM/ angiogenic activity was then tested, using the in vivo rat corneal bio-assay and 10 cells/24 hr, and 70 uN/10 cells/48 hr. To the in vito assay ofbovine aortic endotheial cell chemotaxis across gelatin- determine if the production of No could be induced coated 8g polycarbonate filters in a multi-well chemotaxis chamber. Production with various mediators, confluent LEC sonolayer of angiogenic activity required the presence ofL-arginine in the culture media; cultures were treated with histamine, LPS and vari- ous cytokines. The results to date show a dose de- D-arginine did not substitute for L-arginine. The NOS inhibitors Ng-mono- pendent response with h itamine producing the great- methyl-L-arginine and Ng-nitro-L-arginine methyl ester inhibited NO est response (120 /10 cells/48h at 1mM) compared production by the cells, and also inhibited production ofangiogenic activity. with LPS (98 uN/10 cell/48 h at 100 nM). The induc- While the production ofthe angiogenic cytokine tumor necrosis factor-a ible N02 production was significantly reduced with the NO synthase inhibitor N-nitroarginine methyl (TNFct) by these cells, as determined by ELISA, was not significantly decreased ester at 250 uM concentration. These results suggest by the NOS inhibitors, TNFx bioactivity, as determined in the L929 cell cyto- a possible involvement of NO in the vasodilation of toxicity assay, was markedly decreased. Our results suggest that inhibition of lymphatic'vessels in regulating the lymphatic vascu- NO production disrupts the balance ofproduction ofTNFa and TNFa soluble lar tone for lymph propulsion through lymph vessels and lymph nodes. binding proteins (TNFa-BPs), resulting in a net decrease in bio-active TNFac in the conditioned media. (Supported in part by NIH grant RO1-GM29135). Host-Parasite Interactions (2166-2169)

2166 2167 ACTIVE IVASION OF HOST CELLS BY TOXOPLASMA GONDII ACIIN-BINDING PROTEINS OF THE MALARIA MEROZOITE IS MEDIATED BY A PARASITE ACTIN-BASED MECIANISM. ((. Tardileux and G. E. Ward)) Laboratory of Malaria Research, NIAID, NI, ((L D. Sibleyl J. Dobrowolskil, J. H. Morisaid2, and J. E. Heuser2)) Bethesda, MD 20892 Deprmet of Moleua Micobiology1 and Cell Biology2, Washington Univerity School of Medicine, St Louis, MO 63110. Pretreatment of malaria merozoites with cytochalasin B (ICO5- 500 ng/ml), cytochalasin D (IC5o- lOnglml) orla li A (ICso - 50ng/ml) inhibits

their to invade et e, suggesting a role for parasite actin in Toxoplasmais uniqwely able to actively invade vtually all ypes of ability invasion. We are nuleated celsfrom wari-bkooded We have used high using G- and F-actin affinity chromatography (Miller and Alberta, PNAS X, 4808-4812) to merozoite actin-binding proteins resolution video microscopy to densta that active invasion occurs identify in 15-30 sec, does not induce nmbrane ruffling or redisribution of which may play a role in regulating actin function during invasion. The columns are either monomeric or host-cell cyskel if _ andresultS in frmation of a prepaed using polymerized (phalloidin- stabiliz) actin purified from rabbit skeletal muscle. Extracts ofPlaswodiwn ly con vacuole sundingthe parasite. In contast, knowlesi merozoites are passed over the columns in low salt buffers containing phagocytosis ofToxoplasm requies 24 mn, occurs by membrane nrling, involves extenive reognization of host-cell microfilaments, Ca?* or EGTA, and bound prtins are eluted with high salt, Mg2+ and ATP. highly enriched, and partially overlapping sets of proteins are and lads to formaton ofaspacious phagome. Phagocytosed Reproducible, paasies we often able to escape into a secondary vacuole by a eluted from the G- and the F- columns; these proteins are not bound to control (bovine serum albumin) columns. One of the proteins which binds to F-actin process similar to invasion atthe plasma ane. Pagme escape was detennined by tryptic peptide microsequencing to be Pkzlmodwn heat may be an inmtat nmchaism forinclr survival as only vwacuoles ocupied by actively invading parasies are able to avoid shock protein 70 (hsp7o). This identficaton was confinned by Western blotting with anti-hsp70 antibodies. Hsp7O is thought to promote F-actin pagocytic/endocytic fusion. Toxoplsma cells lack lo capping in other cells (Eddy et al, J. Biol. Clem. 2X 23267-23274; Haus et al, organeles but display dng motility on solid substates that is EMBO J. , and it may a similar role in malaria merozoites. characterized by clocis helicl rotation along the long axis of the 3763-3771), play body. Gliding motilityandceimvasion occurred at similar speeds of 1- A second protein of interst is a 75 kDa protein (p75) which binds to both G- and F-actin. sequence no 2 microns/sec andweze both specifically blocxked by cyqhlasins Preliminarsy analysis of p75 shows significant Invaion sudies using a combination of host-cell or Toxoplasma cell homology to any other proteins in the database. This protein is phosphorylated, and its intacton with actin is calcium dependent nmutants, that ae resistant to the actions ofcytochalasins, indicate that motility and invasion ar dependent on an actin miurofilament-based Antibodies against hsp70 and p75 will be used for immunolocalization and biochemical studies, in an attempt to determine the function of these proteins. motor in the paasite. Such a model is supponed by a newly identified system of micfiln lying beneath the parasit plasa membane. Actin affinity chromatography may be a useful tool for identifying merozoite actin-binding proteins which play arole in invasion.

2168 2169 REGULATION OF ACTIN mRNA LEVELS: CHANGES IN CELL SHAPE AND INTRACELULAR LOCALIZATION AND INHIBITION OF REORGANIZATION OF ACTIN IN Entamoeba histolytica. ((R. Manning- TRYPANOSOMA CRUZI CYSTEINE PROTEASE Cela, and 1. Meza)) Departamento de Biologfa Celular, Centro de (U.C. Engel, and J.H. McKerrow)) Dept. of Pathology, UCSF, San Investigaci6n y de Estudios Avanzados del I.P.N. Mdxico D.F. Mexico. Francisco, CA 94143-0506. Entamoeba histolytica is a pathogenic ameba that causes amebiasis in cruzi is a protozoan parasite that multiples humans. The toxic effects of E. histolytica require direct contact of Trypanosoma in mammalian A trophozoites with target cells which in turn relies on amoebic motility and intracellularly cells. cysteine protease (CP) of an organized actin cytoskeleton. Actin gene expression during growth, this appears to be essential to this process. Specific changes in cell shape and reorganization of the actin cytoskeleton were Inhibitors of CP such as (Z)-Phe-Ala-fluoromethyl ketone (FMK) studied in trophozoites of Entamoeba histolytica. Actin synthesis arrest intracellular replication without significantly affecting the drastically changed during growth showing that the expression of the host cell. Selective in viwo inhibition of the CP of the parasite actin gene is regulated at the level of actin mRNA production in cells that can be achieved using synthetic CP inhibitors. We have localized show changes in movement and shape. Furthermore, rounded suspended the CP on live and fixed cells of the different stages of the cells showed a decrease of 56.6% and 50% of actin mRNA levels with organism using a biotinylated peptide-FMK and hyperimmune respect to attaching or reattaching cells, further supporting a control of serum against the recombinant enzyme. Confocal microscopy actin gene expression in response to cell morphology. The treatment of derived images of epimastigotes show that the CP is stored in trophozoites with forskolin t0 pM), phorbol myristate acetate tOpM), vesides localized preferentially to the posterior end of the dibutyryl cAMP 0 mM) and cytochalasin D 110pM), all of them agents that induce the reorganization of actin microfilaments, produces the organism in opposition to the Golgi apparatus. In intracellular upregulation of actin mRNA levels in trophozoites. These experiments amastigotes, the CP is contained in vesides dispersed suggest that the transcriptional control of actin gene expression is linked throughout the cell, and appears also to be present in patches on to the growth, changes in cell shape and activity and reorganization of the surface of the organism. The data indicates that targeting actin filaments of trophozoites of E. histolytica, as has been shown for and intracellular trafficking of the CP in T. cruzi is stage specific. higher eukaryotic systems. 374a Host-Parasite Interactions (2170-2175). Tuesday

2170 2171 MOBILIZATION OF Ca2+ FROM INTRACELLULAR STORES IN HOST GIARDIA LAMBLIA: IMPLICATIONS OF A HIGHLY DEVELOPED CELLS BY A PROTEOLYIICALLY-GENERATED Trypanosoma cruzi FACTOR. ENDOMKEBRANE SYSTEM PRESENT DURING GROWTH, ENCYSTATION, AND ((B. Burleigh, A. Rodriguez, M. Rioult, N. Andrews) Department of Cell EXCYSTATION ((J.M. McCaffery* and F.D. Gillim)) *Division of Biology, Yale University School of Medicine, New Haven, CT 06510). Cellular and Molecular Medicine, 'Departaent of Pathology, University of California San Diego, La Jolla, CA 92093 The invasive form of the intracellular protozoan parasite, Trypanosoma cruzi, induces repetitive increases in the cytosolic free Ca2+ concentration ([Ca2+]i) in G. lamblia, an early , is reported not to have Golgi-sediated sorting and protein transport functions. host cells (Tardieux et al. 1994. J. Exp. Med. 179:1017). The Ca2+-signaling Recently, we characterized the endoissbrane system in G. activity in trypanosomes is soluble, does not bind to ConcanavalinA and is laiblla throughout the life cycle. We found endoiembrane inhibited a subset of serine and cysteine inhibitors. A 120 strongly by protease organelles and structures comparable to those of higher cells, kDa alkaline peptidase (Santana et al. 1992. BBRC 187:1466) was found to be including abundant rough ER, transitional elements, tubular- the major hydrolytic activity associated with the ConA unbound fraction of vesicular elements, and Golgi-like smooth perinuclear membrane and is sensitive to the same subset of inhibitors that effects trypomastigotes cisternae, as well as the lysosome-like peripheral vacuoles the Differential of extracts and intact Ca2+-signaling activity. sensitivity described earlier. We also observed numerous 50-80 nm to inhibitors that a parasites protease suggests proteolytic processing step may vesicles, many of which were coated, that were similar to the occur in a trypomastigote intracellular compartment, generating a short-lived small transport vesicles thought to effect protein transport soluble factor with [Ca2+li agonist activity. Parasite-induced Ca2+ transients between ER and Golgi compartments in higher cells. in the host cell are caused by Ca2+ release from intracellular stores, as they can Significantly, these elements were observed throughout the be blocked by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+- life cycle. In addition, by means of imauno EM, we traced the ATPase, while not being affected by the absence of Ca2+ in the extracellular transport of TSA 417, a variable surface protein that marks constitutive transport to the plasmalemma, and 8C5, a medium or Ca2+ channel blockers. Inositol are major by phosphates produced cyst wall epitope that marks a regulated secretory pathway. the first minutes after addition of the active continuously during extract, These results strongly support the existence of well-developed indicating that IP3 probably mediates the release of Ca2+ from the ER stores. constitutive and regulated secretory pathways, suggesting that No inositol phosphates are released in the presence of protease inhibitors, or G. lamblia has a complex endomembrane system for protein by extracts of the non-infective stage of T. cruzi. These findings suggest that the sorting and transport. proteolytically-generated trypomastigote [Ca2+1i-agonist binds to a host cell receptor coupled to phospholipase C and inositol phospholid hydrolysis.

2172 2173 IDENTIFICATION OF ELEMENTS OF THEENDOPLASMIC REIICULUM- SPHINGOMYELIN TRAFFICKING IN TOXOPLASMA-INFECTED HOST GOLGI COMPLEX IN EntsmoI bisWlyj (A. Mazmzco, M. Benchimol, and CELLS (( K. Kim, K. Haldar, J.C. Boothroyd)) Dept. of Micro. & W. de Souza). Instituto de BiofSsica - UFRJ, Rio de Janeiro, 21941, Brasil mnd Immunology, Stanford University School of Medicine, Stanford, CA Centro de Bioci6ncias e Biotecnologia, IJENF, Campos, RJ, Brasil. 94304 (Spon. by T.V.McDonald.) Toxoplasma gondii is an intracellular which Entamnoba histolvtica is one of the most important and common protoon obligate parasite and replicates within a parasitophorous vacuole. which infects the human intestinal tract Previous studies have The vacuolar morphological membrane is derived from the host plasma membrane but does not shown that E. as well as amoeba have a histoLtica odthr species, very primitive contain any known host proteins. In addition, the parasitophorous cytoplasm lacking organelles such as mitochondria, peroxisomes, Golgi complex vacuole does not acidify and is resistant to fusion with host cell and endoplasmic reticulum (ER). We decided to use fluorescent dyes which trafficking compartments including endosomes and lysosomes. have been shown to concentrate into the endoplasmic reticulum - Golgi complex Despite this apparant lack of interaction with host organelles, the system and enzyme cytochemistry to detect the presence of these organelles in vacuole is actively modified and grows dramatically during the E. histoytica and E. moshkovgski. Incubation of the cells in the presence of intracellular development of the parasite. As part of an effort to DiOC6, which binds to elements of the ER, revealed by confocal laser scanning understand the interaction of the parasite with its host, we have microscopy (CLSM) the presence of fluorescent labeling in the nuclea examined the transport of lipids within parasite-infected host cells membrane and a network of elements distuibuted thoughout the cytoplasm. using the fluorescent lipid probes. Infected cells were loaded with Transmission electron microscopy revealed the presence of a lage number of Bodipy-sphingomyelin at 0 degrees. After warming to 37 degrees, vacuoles labeled with Zinc iodide-osmium tetroxide and showing reaction infected cells internalized sphingomyelin with initial product indicative of the pfesence of glucose-6-phosphatase activity. in a few accumulation of label in the perinuclear area. With further incubation at 37 degrees, label was delivered to the cells images of the ER were seen. Incubation of the cells in the presence typical parasitophorous vacuole. Vacuolar labeling was temperature of C6-NBD ceramide and observation by CLSM revealed the presece of dependent; incubations at 0, 15 and 20 degrees resulted in label labeled vacuoles. Reaction product indicafive of thiamino-pirophosphe remaining at the plasma membrane. The time course of labeling activity was observed in some cytoplasnic vacuoles. These observations show was consistent with delivery of sphingomyelin to the vacuole after that elements of the ER and Golgi complex we present in E. histdytc it had reached the pericentriolar sorting endosomes. Further although do not show the typical aruay found in other oukwyotic cells. experiments, using characterized host cell markers, are being Support by CNPq and FINEP done to to determine if delivery of sphingomyelin to the parasitophorous vacuole occurs via the plasma membrane recycling pathway or via another mechanism.

2174 2175 LEISHMANIA-VECTOR INTERACTION: COMPETENCE OF THE STRUCTURES OF THE 0-OLYCANS OF THE GLYCOCALYX OF sC aTOS( M NW da J.P. M A. PHEBLOTOMINE SAND FLIES FOR DIFFERENT SPECIES OF ((K H-. Kh8o,S. )F. Calmd. K L, ) hspe Len 0Cdw o 8 _ RF Cdnmm ReseP d, PaAlo. LEISHMANIA IS CONTROLLED BY STRUCTURAL POLYMORPHISMS IN Codeep. THE SURFACE LIPOPHOSPHOGLYCAN.(('P. F. P. Pimenta, 2E. M. B. C_e1on_. bv a t eom ke ma t1 m eoved by a I n- 94vos*x 01.w Saraiva, 'G. Modi and 'D. L. Sacks)) Laboratory of Parasitic Diseases' and me -sge by anw "i In to of Ib da Ieweamid n cmfplemmt by fm idnh m pa el. Pusled Atoo*x b prnbdsty a B ad AtoOg_ retre by ofMalaria Institutes Laboratory Research2, NIAID-National ofHealth, Bethesda, re6mwSM mid d wda sd by q kbon* cdi e- spe n ey (FAa-M ), MD 20892. (Spon. by A. Sher). k wmiatyidR g Pa u a m midN NM1bR.hssgem &§u vew efim- belee. Therem m ,mwac *ey 1I or 2 epedt uat. - m Tha lt %x=obnos *buootbe de X _1_ N- Leshimania are tismitted to their msmnalian hosts via the bites No parasites N reeme h mpe usL Ther rdnd m m uwq um bc1me1 of an infected female sand fly. There is considerable evidence to suggest that some parasite species can only be trnsmitted by certain pheboomine species and not others. Comparison of a large number of vector/paraiste pairs revealed that species-specific differences in vectorial competence were in every case directly corelated with the ability of promastigotes to attah to the sand fly midgut, the variable outcomes of which were controted by stnuctural polymorphisms in the surface lipophosphoglycan (LPG) of the parasite. lbe LPG is not only involved but required for midgut binding. The ability of Phleboomus ppapasi to transmit only L. major sp. could be attributed to the unique, highly substituted nature of its LPG that provides for multiple terminally exposed B-linked galactose residues for bindig. While the relatively rA-n unsbstituted LPGs of other leishmanial species were unable to mediate promastigote attachment to P. papatasi, they could mediate binding to midguts of P argendpes. Our data suggest tha Pargenipes midguts possess a receptor, lacking in P.papatasi, for a relatively conserved oligosaccharide on parasite LPG. The data suggest that phlebotomine vectors differ with respect to the parasite recognition sites which they express, and that midgut adhesion is a The moek _ hI pot byX IdIM Reedi Coun, Welsme TnK NH sufficiently critical component of vectorial competence as to provide the woAs2301a. evolutionay drive for LPG structural poymorphisms. Tuesday. Host-Parasite Interactions (2176-2181) 375a

2176 211T INVASION OF EPITHELIAL CELLS BY SALMONELLA THE INTRACEALULAR FATE OF SALMONELLA TYPHIM/JRIUM ((J. A. Theriot, R. Roy, and J. E. Galin-)) TYPHIMURIUM. ((MIathman, D. Russeilt, and S. Falkow*)) Whitehead Institute for Biomedical Research, Cambridge, MA *Dept of Microbiology., Stanford Univ. Sch. of Med., Stanford, CA 02142, and *Department of Microbiology, State University of 94305 and tDep of Molcular Micobiology., Washington Univ. Sch. New York, Stony Brook, NY 1 1794. of Med., St. Louis, MO 63110.

Salmonella typhimudum, a common cause of food poisoning, Sabuonella cies and replcate within membrane bound can induce actin cytoskeletal rearrangements in macrophages vacuoes in a variety ofmanmalian cel types. We analyzed the and epithelal cells by signaling through the host cell plasma fate ofthe Sapbnoneila him (STP) in membrane. When wild-type S. typhimurum come close to a bone marw-dived murinemacphaes. To deterne the position Henle-407 intestinal epithelial cell, the oell throws up large actin- of the STPin the host cdl's adocytic pathway, immunoelectron miro- rich membrane ruffles caled asplashes that engulf the . scopy was used to detect the presence or absene ofphysiological figands indicative of a maturing incwlding the cation- or invade invA- mutant bacteria do not induce splashing cells. inpendent mannose--phosphate (CI-M6PR), the late endo- However, invA- bacteria do invade Henle-407 calls in the sonI lysosomal ma-ker LAMP-1, and a 1k10kD Ianmemrane proein presence of extracellular EGF (epidermal growth factor). We assoiased with thepo -ATPase responsible forvacuoaraciica- have used videomicroscopy to demonstrate that EGF-rescued ion. Our findings ind e that thae may be two sof early invasion by invA- bacteria involves the formation of apparently p onethat pmeeds along the host cell's lysosomal degrada- normal splashes. EGF alone does not cause extensive ruffling in tive pathway and a second consin ofspaciou vacuoles contining what appear to be unscated baera that persist in the cell. This second these cells. Splash events are often dustered; that is, if a splash p ultion from late somal es but appar- on one very acquir is induced particular cell, it is ikely that another endy inhibits lysosonal content mixing. Fnally, we stuid delivery of splash will occur within a few minutes elsewhere on the same endocytc makers to the mature STPs. These phagosomes cell, or nearby on a neighboring cell. We have preliminary were found to be amoesibie to fluid-phase makes, ligands endocytosed evidence that leukotriene D4, which is made in response to EGF viar o -mediated chni, and no-specifically labekd con- receptor activation in these cells, may sensitize them and stituents ofthe m opha p l These findings indicae that, promote the clustering phenomenon. although the mature SM Lies within the norml endocytic flow of the host cdL the matration prcess of this ona rm isnmdulated.

2176 2179 REACTIVE OXYGEN INTERMEDIATE-MEDIATED KILLING OF DIFFRENTIAL CYTOKINE GENE EXPRESSION IN HUMAN INTRACELLULAR EHRLICHIA BY MONOCYTES TREATED WITH MONOCYTIC CELL IUNE THP-1 INFECT WITH ERLICHI LIPOPOLYSACCHARIDE. ((U.A.K. Ran' and Y. Rikihisa'-')) Molec:ur, CHAFFEENSS AND EHRICIAU SENNMFJU. ((E. Lee', and Y. Cellular and Developmental Rlkihisa'2)) Moiecular, Celular, and Developmental Biology Progam1 and Biology Pro' and Demewnt of Veterinary Pathobiology2, The Ohio State Department of Vetrinary Pathobiology2, The Ohio State University, University, Columbus, OH 43210. Columbus, OH 43210.

itacear which Ehrlichia spp. are gram-negatve obligate bacteria prinmariy Ehichia spp. are gram-negative obligate intracellular bacteria which infect infect cells ofthe monocye-macrophage or graislocyte lineage. E. is the daffee cells of the monocyte/maarphage lineage. E.chaffeis is dte etiologic etiologic aget of human ehrlictiosis and E. risuil is the etiologic agaet of Potomac agent of h _man ehrchios's in the United States and Europe and E.sennesu horse fever. The purpose of the present study was to aceriz tmecmnim human Senneu ois in Far East involved inehrichial growth inhibition by lipopolysacchwride(LPS)-acted huan is the etiologic agent of Asia. Cytokis released by m play a val roe in the host immu and mne monocytes. E. coi LPS and LPS derived from Panoea agglomerwa response to inaelhular bacterial infctidn. The expression of mRNA of which is found as a major co i in whe flour and tus considered fer are in ekiuki, eukin-6, tumor necrosis and tranfo used in this study. When phorbol myrisdc acetate(PMA)teated human monocyte factor-a, human cell line in reonse cell line THP-1 cells were ifected wih E. chaffem or muse peril growth factor-B in monocytic keukem THP-1 were e machages were infected with E. risdci and sub ly ed in vttro with E. to either dehchal infecon or E.coli popolyaccharide by mRNA ex ion all or P. agglomerans LPS, they acquired the dos-depedent capacity to adicate Notrm blot analysis. Ecoll induced of four intcellular E. diaffeensi or E-risticii, respctively. Thiemaxinum in vitro cytokines examined. E.Kchqgff is induced the tans on of IL-lB and antehrtichalactvity was atained with 40 ng/ml of P. agglomes or E. LPS. TGF-B mRNA in simil amounts and rats to those produced by LPS. The involvemet of reactve oxygen inteediates (ROI) in the aniehrtichin l Lower level of TNF-a mRNA was induced by E.cdaffe.s than that by mechanis induced by LPS was exmined by using vaous scavengers specific to LPS. IL-6 mRNA was not inuced respons to E.chffeensls. In each ROI. The ichial civity induced by E. cobl or P. agglo.ranLPS was contst, E.senetu did not induce TGF-B, TNF-a, and IL-6 mRNA completely bloked by scavegers of ROI. 11e result indicates an ielial expession. ILL18 mRNA expression in response to E.senneu was aciqv of ROI induced by LPS tatun. Using marie odel of Potomac hors considerably kss than that induced by cither LPS or E.cwffeensu. The fever, oral administration of 20 ng/ml of P. agglonea or E. oiw LPS in water resls suggest that the expression of cytokine genes in monocytes in compleely mice from clinical and death prevented developing sigs upon challeng response to E.coll LPS, E.ch dsis, and E.senne3 is diffe y a of P. with E. risdaii. In vivw findi sWport thrpeutic potel aggomeas at the LPS for ehrlichiosis. regulated iptional

21U 2181 CHARACTERISICS OF A SYMBIONT-PRODUCED PROrEIN IN TRANSMISSION OF BACTERIOIDS TO OOCYTES OF EUSCELIDIUS xD AMOEBAE. ((.W. Pak and K.W. Jeon)) Dqetment of Zoobgy, VARIEGATUS ((W.W.K. Cheung)) Biology Department, University of lbnnessee, Knoxville, TN 37996. Chinese University of Hong Kong, Hong Kong.

Endosymbwtic Xbateria in the xD strain ofAmoeba proteus produce a The leafhopper Euscelidius variega tus harbours amount of a uniqu 29-kDa prten (Sx29), much of which is tans- symbiotic bacterioids a, t, and sometimes pathogen ported into the amoeba cytoplam. Vk obtained the complete nucleodide BEV inside its . Bacterioids a and t have sequece of the gene encoding Sx29 and its deduced amino-acid s- vegetative and migratory forms. BEV is a rod-shaped quene. The protin contains 257 amino-acid reidues, with a calculated bacterium measuring 0.5 pm x 4 pm. The transmission agreement with 29-kDa siz est d by SDS- pathway of these microorganisms from the bacteriomes no signl peptidle, sequence and no nodceable to the oocytes of a young adult is not fully under- domains. A cloned Ws29 gene exprssd in tasoed E. stood. This study aims to trace their migration coil with or without IPTO, indicating that the gene is contralled by its routes at the ultrastructural level. own promoter. The Sx29 proten poduced by tra Ewcoi is also Ovaries of two days old female E. variegatus were tran across bacterial mebranes into the culture medium. The dissected out and fixed in 2.5% glutaraldehyde and anino-acid sequece of Sx29 isolated fm post-fixed in 1 % osmium tetroxide (both in sodium coi and the cuture medium were identicl, indicatin that thee is nO cacodylate buffer). Tissues were blocked in Spurr dea of NH2terminal peptide during transport. It aea that the resin. Ultrathin sections were cut, routinely stained protin is rnsord wihout involving a squence. Wt identified for electron microscopic observations. A JOEL JEM- a nscrption-initiaion point by a pi xe rassay. The 1200EXII was used. region was found to be located 150-210 bp from the Migratory a and t bacterioids were transferred to codon, as confirmed by a series of deletion experiments. The core pro- oocytes via bacteriocytes, and commensal BEV migrated moter region did not contain any squen similar to the consensus independently at the same time. This took place on reconition hxanmrs. The pres role of Sx29 is not yet known but the the second day after the young adult had emerged, and proin is thought to play an important role in t host-symbiont intrac- had to be completed within 24 hours. These bacte- was suported by a gant from the National Science rioids had to overcome the host cells' defence mecha- Foundation.] nisms, and penetrate through the follicular epitheli- um (at the wedge cells) in order to infect a develop- ing oocyte. If they failed to reach the oocytes in the specified period they would be destroyed by the host's macrophages and lysosomes.