1 T' CONICYT.d4h COMISIÓN NACIONAL DE INVESTIGACIÓN CIENTÍRCA Y TECNOLÓGICA 4 GOBIERNO DE CHILE COMISION NACIONAL DE INVESTIGAC1ON CIENCIA Y TECNOLOGIA VERSION OFICIAL FECHA: 01/04/2010 PROYECTO REGULAR N INVESTIGADOR RESPONSABLE: KATHLEEN ELIZABETH WHITLOCK LEANING FONDO NACIONAL DE DESARROLLO CIENTIFICO Y TECNOLOGICO (FONDECYT) Bernarda Mrín 551, Prvidencia - casilla 297-y, Santiag 21 Telefn: FAX O
2 INFORME FINAL PROYECTO FONDECYT REGULAR AÑO ETAPA: 2009 DURACIÓN: 3 añs N PROYECTO: TÍTULO PROYECTO: NEURAL DIFFERENTIATION AND PLASTICITY IN THE OLFACTORY SENSORY SYSTEM OF THE ZEBRAFISH BIOLOGIA CELULAR DISCIPLINA PRINCIPAL: GRUPO DE ESTUDIO: BIOLOGIA 2 INVESTIGADOR(A) RESPONSABLE: KATHLEEN ELIZABETH WHITLOCK LEANING DIRECCIÓN: COMUNA: Playa Ancha CIUDAD: Valparais REGIÓN: V REGION FONO: INFORME OBJETIVOS Cumplimient de dm11idzina N 2 Objetivs planteads en el Pryect. Recuerde que ls bjetivs de] pryect n se refieren a listar actividades lei.z nhiptivcs drrnl12t1ns ls 2 OBJETIVOS CUMPLIMIENTO FUNDAMENTO Aa) T determine the phentypes that result frm gene knck-dwn f the patterning genes imprtant t setting aside the precursrs f the lfactry placde and cranial neural crest. This includes describing the phentype f the existing dg-eared (eyal) mutant. TOTAL Ab) T visualize the effects n cells mvements that may result frm the knck-dwn/lss f gene functin. Wc will visualize the celis mvement using GFP unes and marking celis f interest with fluresceinated caged dextrans PARCIAL Wc characterized the phentype f the dg-eared (eyal) mutant and realized that it is nt useful fr ur gals j.c. it reduces but des nt eliminate the lfactry placde. T date we have fund n bvius defects in the lfactry rgans f the dysseus (cxcr4b: Knaut et. al., 2003) and medusa (cxcr7: Valentin ci al., 2007)) mutants. Thus we have cncentrated n the aspects f ur prject invlving the frmatin f the placde thrugh ccli migratin. we used the sx lo::gfp une t visualize cranial neural crest cells (CNCCs) and labeled the placde dmain with Dii (see previus reprt). but were never able t btain clean labeling with Dii. Therefre we successfully generated a six4. 1: :mcherry une that expresses mcherry in the lfactry placdes. This line is extremely useful fr the ther experiments in this bjective (see beiw) Using sxlø::gfp and six4.1::mcherry reprter lines we are currently visual izing the mvements f CNCCs and OPCs in living embrys. Because t date nne f the chemkines mutants shw an bvius lfactry placde defect we have nt made the crsses t intrduce the reprter Unes int the chemkine mutant backgrunds.
3 3 A.c) T determine whether any f the effects TOTAL Using sxiø::gfp reprter une t visuatize the bserved in the gene knck-dwnliss are mvements f CNCCs in living embrys. Our phencpied by expsure f the early develping mst in depth anaiysis is with zebrafish embry t man-made chemicais knwn t be embrys expsed t aichi and pisc. Zebrafish present in the envirnment specificaily embrys incubated with Ethanl r Pisc shwed anti-fungai agents reduced expressin f chemkine cxcii 2a and its receptr cxcr4b. Our results suggest that alchl affects OP prgenitr rganizatin, CNC migratin, and enhances celi death. The studies we carried ut n the effects f anti-fungais were nt vaiid because the dsage necessary t bserve an effect in neurai crest migratin was utside the wrking cncentratin usuaily detected in the envirnment. We have ais anaiyzed a class f rganphsphate pesticides used in the aquacuiture industry t cmbat sea tice L determine whether they affect develpment f vertebrate embrys. These substances (Ivermectin) have a prnunced effect n the neurmuscuiar system resulting in permanent paralysis f the embrys, a phentype nt f interest t ur studies. 4 Objective B.a: T deveip ur methds fr PARCIAL Using ur new system fr analysis f zebrafish assaying behavir in the develping and adult behavir (Viewpint Life Sciences) we have zebrafish. Specificaiiy t determine the drs t tested the ability f the sftware t track multiple which a zebrafish will imprint, thus giving us fish in a behavir trial. Our initial experiments behavir assays fr dr t which we can then shw that we can track dr preferences in grups (thereticaliy) assign a genmic respnse. f fish vertime. Our preiiminary data demnstrate a strng crreiatin between lfactry sensry behavirs and activatin f the immediate early gene c-fs in the lfactry epitheiium and hypthalamus. We are cntinuing these studies. 5 Objective B.b: Our secnd gal f sectin "B" is PARCLkL Of the apprximately 160 ifactry receptrs we t take advantage f the recent reprt f the identified 34 that fit the specified criteria fr tx2 sequences f the ifactry receptrs in the binding sites. Currently we are cncentrating n zebrafish and use this genmic infnnatin t fur ifactry receptrs that we have cnfirmed determine whether the ifactry receptr are expressed in the ifactry epithelium using in expressin is affected by the envirnment. situ hybridizatin. Base n new dala that Because f the way we have designed these zebrafish are strngiy attracted t teststerne we experiments (L islate RNA frm animals after have identified a small subset f ifactry recrding their behavirai respnse) we wili be receptrs that have ARE binding sites in the abie t crreiate these genmic data with a upstream regin suggesting that their expressin functinally reievant event. may be reguiated by teststerne.we have shwn that the expressin f immediate early gene (IEG) c-fs in the lfactry epithelium is mdulated by teststerne, GnRI-I, natural drs f mate and female fish, and amin acids.we are cntinuing analysis by QT-RT-PCR. Otr(s) aspect(s) que Ud. cnsidere imprtante(s) en la evaluación del cumplimient de bjetivs planteads en la prpuesta riginal en las mdificacines autrizadas pr ls Cnsejs.
5 Kathleen Whitlck IRESULTADOS OBTENIDOS Objective A.a: T determine the phentypes thaf result frm gene knck-dwn f fhe patterning genes imprtanf f setting aside the precursrs f the lfactry placde and cranial neural crest. This includes describing the phentype f the existing dg-eared (eya 1) mutant. We characterized the phentype f the dg-eared (eyal) mutant and realized that it is nt useful fr ur gals i.e. it reduces but des nt eliminate the lfactry placde. T date we have fund n bvius defects in the lfactry rgans f the Odysseus (cxcr4b: Knaut et. al., 2003) and medusa (cxcr7: Valentin et. al., 2007)) mutants. Thus we have cncentrated n the aspects f ur prject invlving the frmatin f the placde thrugh celi migratin. In rder t understand the cellular mvements and interactins that take place during lfactry placde develpment, we used the sx10::gfp line t visualize cranial neural crest cells (CNCCs) and labeled the placde dmain with Oil (see previus reprt). but were never able t btain clean labeling with Dii. Thus we wrked t generate a transgenic line t visualize lfactry precursrs celis (OPCs). Previus wrk in ur lab shwed that the six4. 1 gene is expressed in OPCs in a dmain partially verlapping with dlx3 (Harden & Whitlck, unpublished). Because f the mre restricted expressin pattern f six4.1 we fcused n this gene t generate a transgenic reprter line expressing mcherry in the O POs. Figure 1: A) Frntal view f 36 hpf six4.1::mcherry embry with mcherry expressin (red) in the develping lfactry rgans. 8) CelIs expressing sxlü::gfp in the lfactry rgan f the5 day pst-fertilizatin zebrafish. The labeling f the cilia (red) des nt c- lcalize with the prcesses f these cells suggestirig they are nt ciliated lfactry neurns and may be glial cells. Initial attempts t generate this line using 4, 6 kb upstream as well as BAC cristructs were unsuccessful. Thus we identified cnserved sequences, upstream and dwnstream f six4. 1 gene, by philgenetic ftprinting and clned in a transpsable expressin vectr. The six4.1::mcherry embrys express mcherry in the lfactry placdes (Fig. la) as well as in restricted ppulatins f cells in the ear and lateral Une. We are using this line, in cmbinatin with the sx10::gfp line, t analyze the celi mvements f OPCs and CNCCs during early smitgenesis. In ur preliminary analysis f CNCC differentiatin we describe a restricted ppulatin f sxl0::gfp expressing cells in the lfactry rgan at 5dpf (Fig. IB). This maintained expressin suggests they may be gua cells. In cntrast t ther cranial nerves where the gua are crest derived, the gima f the lfactry sensry system are thught t arise frm the lfactry placde. Thus the pssibility that these cells crrespnd t glial cells and arise frm CNCCs wuld make the lfactry sensry system mre similar t ther cranial nerves. This wrk was carried ut by pst-dc Luisa Pereir. Related øublicatins: Paul, T. A., Rvnak, J., Quackenbush, S. L., Whitlck, K. E,, Zhan, H., Gng, Z., Spitsbergen, J., Paul R. Bwser, P. R., and Casey, J. W. (2010) Transgenic expressin f Walleye Dermal Sarcma accessry genes in zebra fish des rit result in fissue prliferatin. Marine Bitechnlgy, in press. Objective A.b: Te visualize fha effects n ce/ls mvements that may result frm the knck-dwn4ss f gene functin. We will visualize the cells mvement using GFP lines and marking ce/ls f inferest wifh fluresceinated cagad dextrans.
6 Kathieen Whitlck Using sxlo::gfp and six4.1::mcherry reprter unes we are currently visualizing the mvements f CNCCs and OPCs in living embrys. Because t date nne f the chemkines mutants shw an bvius lfactry placde defect we have nt made the crsses t intrduce the reprter unes int the chemkine mutant backgrunds. Figure 2. Frntal view f zebrafish embry expressing sxlo::gfp and a rhdamine marked actin t visualize celi structures. The reslutin f the actin labeling was t pr t c-lcalize the red flurescence with filpdia f the migrating crest cells. This pht wn an award in the cmpetitin at Ceil Bilgy Meeting in Pucn. Wrk f graduate student Katica Bric. Objective A.c: T determine whether any f the effects bseried in the gene knck-dwn4ss are phencpied by expsure f the early develping embry f man-made chemicais knwn f be presenf in the envirnmenf specifically anfi-fungal agents. We have expanded ur analysis f alchl effects n deveipment thrugh analysis f ceil death, migratin and quantitative analysis n the alchl cncentratin in the embry. We have shwn in agreement with ther published wrk (Tanguay and Reimers, 2008), that alchl increases the level f cell death abye reprted backgrund (Sanders and Whitlck, 2003). In additin we have analyzed the mrphlgical abnrmalities as well as effects n celi migratin as assayed thrugh time lapse in living embrys (Fig. 2, 3) and gene expressin f receptr ligand cmplex. This wrk is being prepared as a manuscript. The studies we carried ut n the effects f anti-fungals were nt valid because the dsage necessary t bserve an effect in neural crest migratin was utside the wrking cncentratin usually detected in the envirnment. We have als anatyzed a class f rgariphsphate pesticides used in the aquaculture industry t cmbat sea tice t determine whether they affect develpment f vertebrate embrys. These substances (Ivermectin) have a prnunced effect n the neurmuscular system resulting in permanent paralysis f the embrys Figure 3: Still images cllected frm time lapse z-stacks (spinning disc) f living sxlo-gfp embrys. Drsal view f sxlü-gfp embrys at 6-14 smites A) Celis initiate migratin anterir at 6-8 smites with anterir brder mving alng neural tube (arrwheads). B) As develpment prceeds the cranial rieural crest cells becme travel further anterir mving alng edge f neural tube (14 smites, arrwheads). C) Cntrl embry at 10 smites..d) Embry treated with 200mM ethanl shws accumulated celis that mve anterirly as a dispersed grup. Frm manuscript in preparatin: Katica Bric & K.E. Whitlck. We are studying the effects f envirnmentally induced disruptins iri the nervus system develpment using zebrafish (Dani ren) as a mdel. Alchl is an envirnmental teratgen that, when cnsurned c
7 Kathleen Whitlck during pregnancy, causes cranifacial malfrmatins as well as a spectrum f neurlgical and behaviral disrders, knwn as Fetal Alchl Syndrme (FAS). Cranifacial structures arise frm cranial neural crest(cnc) and is well accepted that these cells are affected by alchl; nevertheless hw alchl exerts its effects n them is nt well understd. Pre-migratry CNCs share a cmmn brder with the celis f the lfactry placde (OP) fleld, therefre we prpsed that alchl may affect the differentiatin f these prgenitrs as well. In additin we prpsed that OP and CNC prgenitr migratin is disrupted by expsure t alchl. Zebraflsh embrys incubated with Ethanl r Pisc and analyzed fr expressin f chemkine cxcll2a and its receptr cxcr4b, genes esseritial in OP develpment shwed reduced expressin in animals expsed t Ethanl/Pisc. We are adding additinal data at this time by analyzing the chemkine cxcll2b and its receptr cxcr4a, which are expressed in CNC. Using sxlo::gfp embrys we fllwed CNC migratin in living embrys (Figs. 2, 3). E analyzed mrphlgy, mrtality, structural malfrmatins, and cell death in cntrl and treated embrys. lmprtantly we measured the cncentratin f ethanl in the tissue at the end f the incubatin perid. Our results suggest that alchl affects OP prgenitr rganizatin, CNC migratin, and enhances celi death. Als, alchl prduces cranifacial abnrmalities very similar t the malfrmatins seen in FAS patients. Recently we have started examining the structure f primary cilia f CNCs. We are interested in this ceil structure because it has been shwn that in certain syndrmes shwing cranifacial dysmrphlgy similar t FAS patients have altered cilia. Furthermre recent data suggest that snic hedgehg signaling is transduced thrugh cilia, thus explaining the link between shh pathway mutant phentypes and cilia malfrmatins. Cnsequently, we hypthesize that alchl may als disrupt CNC's primary cilia mrphlgy. Finally, we think this research will allw us t develp an EcDev" system mdel fr assessing hw teratgens affect develpment f the early embry. Objective B.a: T develp ur methds fr assaying behavir in the develping and adult zebra fish. Specifically t determine the drs f which a zebra fish will imprint, thus giving us behavir assays fr dr f which we can then (theretically) assign a genmic respnse. While waiting fr ur behavir system t arrive and be installed we cllabrated with the Darwin Initiative (based in England, Chilean representatin: University de Ls Lags) n a prject n lfactry learning in puye (Galaxias maculatus), a native Chilean fish. We have shwn using puye ppulatins, allpatric and simpatric with brwn trut, that native flsh (puye) grwing up in rivers where trut and salmn are dcumented shw an avidance respnse t the scent f these predatrs. Puye frm streams lacking predatrs shw n fear respnse t the scent f predatrs. Thus we have shwn that the puye appear t learn the drs f predatrs (invasive salmnids). This wrk was carried ut by Paul Hwes with the help f Jessica Stephensn (bth British students). Jessica"s prject was t test whether puye respnded t alarm phermne, she wrked as a vlunteer carrying ut this wrk and surprisingly the puye shw n respnse t alarm phermne, suggesting, as reprted in the literature that there is a strng learned cmpnent in this respnse (Stephensn et al submitted). Jessica returned t d her Masters Thesis with me and used zebrafish t shw that lfactry cues mdulate visual sensitivity in zebraflsh (tested as individuals). Using ur new system fr analysis f zebrafish behavir (Viewpint Life Sciences) we have tested the ability f the sftware t track multiple fish in a behavir trial. This mdule f the sftware is new, develped in part using data we prvided Viewpint Life Sciences. Our initial experiments shw that we can track dr preferences in grups f fish vertime (Fig. 4). With these type f data we are starting t develp crrelatins between behavirs and changes in gene expressin within the lfactry sensry system. We knw that the dr f male flsh induces expressin f c-fs in the lfactry epithelium and hypthalamus f develping zebraflsh (see belw Maturana Masters thesis). Thus there is a strng crrelatin between lfactry sensry behavirs (Fig. 4) and activatin f the immediate early gene c-fs in the lfactry epithelium and hypthalamus (Fig. 6). Related øublicatins/theses: Hwes, P.N Predatr recgnitin f invasive salmnids by native galaxiids. MSc thesis. Swansea Universily, loop.) Whitlck c-tutr Sfephensn, J. Yung, K, and Whitlck, KE (2010)" Testing fr alarm cue respnse in a circumplar galaxlld, Galaxias macula tus" Fisheries Management and Eclgy in revisin. Sfephensn, J. (2010)Olfactry cue mdulatin f visual sensitivify in the zebra fish Dani ren. MSc Thesis, Bristl University in revisin.
8 Kathleen Whitlck Whit/ck, K.E. (2010). Olfactry Behavir in Zebra fish: Making Scents f a Changing Wrld. IN: Zebra fish mdeis in Neurbehaviral Research, Humana Press, in press. Figure 4. Upper graph depicts mvement f fish acrss three defined areas in presence f cntrl water. The fish shw n preference fr any given regin (Arealyellw; Area2-green, Area3-red). In cntrast the lwer graph shws a clear preference fr Area 3 (yellw) after the dr f male fish was added (arrw). Area 3 is the regin where the drant was intrduced t the testing tank. In these trials fish were tested fr 90 secnds. Adult, wild type zebrafish f the strain NWT and Crnell were used. This wrk is being carried ut by Alejandra Pnce, a dctrate student in (he lab. Objective B.b: Our secnd gal f sectin B" is f take advanfage f fha recent reprt f the sequences f the /fact,y recepfrs in the zebra flsh and use fhis genmic infrmatin f determine whether the lfacfry receptr expressin is affected by the envirnment. Becaus f fha way we have designad fhese experiments (f s/ate RNA frm animais after recrding their behaviral respnse) we wi// be ab/e f crre/ate these genmic dafa with a funtinaily re/e vant evenf. With (he purpse f identifying specific dwnstream targets f tx2. (transcriptin factr: see ur wrk in Harden et al 2006), we usad a biinfrmatics apprach t lk fr tx2 binding sites in regins upstream f the lfactry receptr genes. Of the apprximately 160 lfactry receptrs we identified 34 (Fig. 5A) that fit the specified criteria fr tx2 binding sites. Currently we are cncentrating n fur lfactry receptrs (Fig. 5B) that we haya cnfirmed are expressed in the lfactry epithelium using in situ hybridizatin. Fliwing up n the findings that zebrafish are strngly attracted t teststerne (used as an dr, Alejandra Pnce, dctrate student unpublished data) we have identified a small subset f lfactry receptrs that have ARE binding sites in the upstream regin suggesting that their expressin may be regulated by teststerne. This wrk is being carried ut by Cristian Calfun, undergraduate thesis student and a new technician Pabl Castill. Figure 5. A) Olfactry receptrs cntaining fx2 binding sites in upstream regins. Of these 34 genes we have chsen fur (B) t use as diagnstics fr QT-RT-PCR after cnfirmirig the expressin in the develping lfactry epithelium by in situ hybridizatin (see previus reprt). A B 0R102-1, 0R102-3, 0R R OP , 0R103-2, 0R R106-8, 0R OR11I-8, ORII1-il: 0R TAT'rGTAMCGTTTACTAATGCAGGGTTT'TA R115-1, 0R115-5, 0R R OR R R121-1, 0R125-1, 0R125-2, 0R R126-5, 0R128-1, 0R128-6, ACCACA CAGGTGCATGCCTTTACCAATTTCT R128-10, 0R132-1, 0R R133-1, OR13-5, 0R133-9, 0R R R R137-3, 0R GCCCCT TGCAGATG(3ACGCATCAGATGTGGG OP GGGTTA rgactgtcattt3tatgtgacmatg c
9 Kathieen Whitlck We have shwn that the expressin f immediate early gene (IEG) c-fs in the lfactry epithelium is mdulated by a variety f drs including frms f prgesterne and prstaglandin (MacKenzie and Whitlck unpublished), and nw teststerne, GnRH, natural drs f male and female fish (Fig. 6), and amnin acids (Maturana and Whitlck, unpublished). Maturana, C (2010) Efect del mediambienfe sbre la expresin de c-fs en pez cebra (Dani ren). Universidad de Valparaís, Magíster en Ciencias Bilógicas Mención Neurciencia Figure 6. Embrys expsed te water cntaining adult male fish dr increases c-fs expressin in the lfactry epithelium and hypthalamus. Ventral View with anterir tward the tp. (A) Head f 48h embry shwing celis in lfactry rgans (B) higher magnificatin view f hypthalamic c-fs expressin (C) high magnificatin view f ene lfactry rgan shwn in (A). (D) Head f 48h embry shwing celis in lfactry rgans f embrys expsed t water with drants f adult male fish. (E) Hypthalamic regin shwing increase in c-fs expressin (see cntrl B). (E) High magnificatin view f ne lfactry rgan shwn in (D). One bserves iricreased expressin f c-fs in the lfactry epithelium (arrwhead) and hypthalamus (arrws) when the juveniles are expsed t water cntaining adult male fish. Scale Bar: A, D=35pm;B, E=25pm;C,F=2Opm. References Harden MV, Newtn LA, Llyd RC, and Whitlck KE. (2006). Olfactry imprinting is crrelated with changes in gene expressin in the lfactry epithelia f the zebrafish. J Neurbil., Nv;66(13): Knaut H, Werz C, Geisler R, Nüsslein-Vlhard C; Tübingen 2000 Screen Cnsrtium. (2003). A zebrafish hmlgue f the chemkine receptr Cxcr4 is a germ-cell guidance receptr. Nature. Jan 1 6;421 (6920): Miyasaka N, Knaut H, and Yshihara Y. (2007). Cxcl12/Cxcr4 chemkine signaling is required fr placde assembly and serisry axn pathfinding in the zebrafish lfactry system. Develpment., Jul;1 34(1 3): Sanders LH, and Whitlck KE. (2003). Phentype f the zebrafish masterblind (mbl) mutant is dependent n genetic backgrund. Dey Dyn., Jun;227(2): Tanguay RL, and Reimers MJ. (2008). Analysis f ethanl develpmental txicity in zebrafish. Methds Ml Bici. 447: Valentin G, Haas P, and Gilmur D. (2007). The chemkine SDFIa crdinates tissue migratin thrugh the spatially restricted activatin f Cxcr7 and Cxcr4b. Curr Bil. Jun 19;17(12): Whitlck KE. (2006). The sense f scents: lfactry behavirs in the zebrafish. Zebrafish. 3(2):
10 OTHER ACHIEVEMENTS OF THE PROJECT: - Research visit(s) t ther institutin(s). - Outreach activities related t the prject's main tpic. - Any ther cntributin, nt addressed elsewhere, that yu cnsider imprtant. - Frmación de recurss humans exceptuand tesistas ya infrmads. - Actividades de difusión yi extensión en la temática del pryect. - Cualquier tr lgr n cntemplad en ls ítem anterires y que Ud. quiera destacar. The maximum Iength fr this sectin 1$ 1 page. (Anal r Verdana, fnt size 10). Frmación de recurss humans excetuand tesistas va infrmads. 1, like ther research scientists, participate in the frmatin f "capital human" thugh the training f peple in my lab including undergraduate students (1), graduate students (4), and technicians (3). 1 have trained ali f my technicians in the techniques f fish care, micrscpy and imaging, and develpment f early embrys. Theses cmpleted: Hwes, P.N Predatr recgnitin f invasive salmnids by native galaxiids. MSc thesis. Swansea University, 100 p.) Whitlck c-tutr Stephensn, J. (2010)Olfactry cue mdulatiri f visual sensitivity in the zebrafish Dani ren. MSc Thesis, Bristl University in revisin. Whitlck c-tutr Maturana, C (2010)Efect del mediambiente sbre la expresin de c-fs en pez cebra (Dani reni). Universidad de Valparaís, Magíster en Ciencias Bilógicas Mención Neurciencia. Whitlck tutr Actividades de difusión v/ extensión en la temática del prvect. KE Whitlck, Directr de Prgrama Ciencia al Tir. Fr this utreach prgram we develp science wrkshps that are taught nce weekly at the lcal schl Escuela Republica Arabe Siria in Valparais, ne f the wrst public schls in Chile as judged by results n the SIMCE (natinal evaluatin) test. This prject was funded by: Pryección al Medi Extern (Outreach). Iniciativa Cientifica Mileni (Millenium Science Initative). Talleres Cientificas (Science Wrkshps). Rle: Principal Investigatr/Directr - Any ther cntributin, nt addressed elsewhere, that vu cnsider imrtant. 1. 3rd Strategic Cnference f Zebrafish lnvestigatrs, This meeting is a cmpetetive meeting where ne must submit a Curriculum. 1 was accepted and attended this meeting January 2009, Asilmar, Califrnia, USA. 2. Crdinadr: Simpsi:"Efects del medi ambiente sbre el desarrll"xx Reunin Anual Sciedad Chilena de Reprduccin y desarrll, La Serena, Chile Presentacin: Efects de mediambiente en el desarrll del embrines de pez cebra: lres, alchl, y pesticidas K. E. Whitlck 1, K Bnic, M. V. Harden, M. G. McKenzie and C. Maturana Septiembre 2009, La Serena Chile 3. Organizer and Instructr, Internatinal Curse in Develpmental Bilgy, CIMARQ, Universidad Andres Bell, Quintay Chile January 2010
11 PR03ECT SUMMARY: Usirig nri-specialist language, prvide a precise and brief descriptin f the prject gals, bjectives and results btained. This summary may be published in CONICYT's web page. The maximum Iength fr this sectin is 1 page. (Anal r Verdana, fnt size 10). The develping peripheral nervus system s initially rganized thrugh ceil migratin with the neural precursrs aggregating t frm the placdes frm which the sensry neurns will differentiate. Fr example we have demnstrated that the lfactry placde f the zebraflsh arises frm a fleld f cells in (he anterlateral edge f (he neural plate and des s in the absence f celi divisin (Whitlck & Westerfield 2000; Whitlck, 2004a, b), and that there appears t be little mixing f the lfactry placde precursrs with the adjacent migrating cranial neural crest (Harden and Whitlck, unpublished). The lfactry system is unique amng the sensry systems in that it generates neurns thrughut the life f the animal and these new neurns are generated frm a basal celi ppulatin within the lfactry epithelium. The generatin f a differen(iated lfactry sensry neurns (OSNs) is mre cmplex than many ther sensry systems because each sensry neurns will express ne f 160 (fish) t 1,000 (muse) lfactry receptrs. The mechanisms cntrlling (he differentiatin f OSNs are nt well understd. We have shwn that in additinal t internal, pre-prgrammed mechanisms the envirnment can als affect the differentiatin f neurnal precursrs within (he develping lfactry sensry system (Harden et al 2006). Strikingly we discvered that a transcriptin factr, tx2, is up-regulated when the zebrafish are raised in a specific dr envirnmen( but nt when raised in dr envirnments we previusly described (amin acids: Vitebsky et al 2005). These finding cupled with ur recent analysis indicates that (his highly cnserved transcriptin factr plays a rle in cn(rlling neurgenesis in bth the central and peripheral nervus system. We have identified genes cding fr lfactry receptrs that are ptential targets fr regulatin by tx2 (ranscriptin factr. Furthermre we have shwn that drs elici( specific respnses in zebrafish, fr example scial cues such as drs f ther fish are attrac(ive, and that expsure t (hese drs triggers changes in immediate early gene expressin (c-fs) in the nervus system. Thus we are piecing tgether the pa(hways by which envirnment signals, in (his case drants, elicit changes in gene expressin that allw greater plas(ici(y in gene expressin during develpment f the animal. Cupled with ur analysis f the envirnment affects n gene regulatin in the lfactry epithelium we have als analyzed (he celi mvements that cntribute t the frmatin f the lfactry sensry system and surrunding facial structures. Thrugh this analysis we have shwn that alchl affec(s the migratin f (he cranial neural crest cells and lfactry placde precursrs by reducing expressin f specific genes and causing celi death. Thus we have speciflc tls t explre (he cell defects caused by ther teratgenic agents, in particular thse tha( cause defrmities f the cranial facial structures and underlying nervus system. References Whitlck KE, Westerfield M. The lfactry placdes f the zebrafish frm by cnvergence f cellular fields at (he edge f (he neural plate. Develpment Sep;1 27(17): Whitlck KE, Wlf CD, Byce ML. (2003) Gnadtrpin-releasing hrmne (GnRH) cells arise frm cranial neural crest and adenhypphyseal regins f the neural plate in (he zebrafish, Dani ren. Dey Bil. May 1 ;257(1): Whi(lck, KE (2004a) Making Scents: Develpmen( and Functin f the Olfactry Sensry System. In: Fish Develpmental Bilgy and Genetics, Mlecular Aspects in Fish and Marine Bilgy. Zhiyuan Gng & Vladimir Krzh, Wrld Scientific, Singapre Whi(lck KE. (2004b). A new mdel fr lfac(ry placde develpment. Brain Behav Evl. 64(3): Review. Vi(ebsky A, Reyes R, Sandersn MJ, Michel WC, Whi(lck KE. (2005). Islatin and characterizatin f (he laure lfactry behaviral mutant in (he zebrafish, Dani ren. Dey Dyn. Sep;234(1 ): Harden MV, Newtn LA, Llyd RC, Whitlck KE. (2006) Olfactry imprinting is crrela(ed with changes in gene expressin in the lfactry epi(helia f the zebrafish. J Neurbil. Nv;66(13): Whitlck, K.E. (2008). Develping a sense f scen(s: Plasticity in lfac(ry placde frmatin. Brain Res Buli. Mar 1 8;75(2-4):340-7.
12 PRODUCTOS ARTÍCULOS Para trabajs en Prensa/ Aceptads/Enviads adjunte cpia de carta de aceptación de recepción. N : 1 Autr (a)(es/as): Kathleen Whitlck, Hannah Kim, Carla Maturana, Jaquin Letelier Nmbre Cmplet de la Revista: Mechanisms f Develpment Títul (Idima riginal): Disrupted patterning f the hypthalamus results in Kallmann Syndrme like defects in the zebrafish Indexación: ISI ISSN: Añ: 2009 Vl.: 126 N : Suppl Páginas: S124 Estad de la publicación a la fecha: Publicada Otras Fuentes de financiamient, si las hay: NIH/NICH DRO 1HD Envía dcument en papel: Archiv Asciad al artícul: n Whitlck_ISDB_Published_abstract.pdf N : 2 Autr (a)(es/as): Katica Bric, Kathleen Whitlck Nmbre Cmplet de la Revista: Mechanisms f Develpment Títul (Idima riginal): Effects f teratgenic agents n zebrafish (Dani ren) nervus system develpment Indexación: ISI ISSN: Añ: 2009 Vl.: 126 N : Suppl Páginas: S131 Estad de la publicación a la fecha: Publicada Otras Fuentes de financiamient, si las hay: 11CM P ` Envía dcument en papel: Archiv Asciad al artícul: n Bric_Published_Abstract.pdf N : 3 Autr (a)(es/as): Jaquín Letelier, Kathleen Whitlck Nmbre Cmplet de la Revista: Mechanisms f Develpment Títul (Idima riginal) : Effect f the disruptin f prkineticm2 system in the develpment f zebrafish