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[(community,)-361(since)-355.2(the)-357.8(regionalized)-358.8(functions)-357.4(of)-357.3(many)-359(genes)]TJ T* [(could)-484.2(be)-484.9(readily)-482.5(explored)-487.5(during)-484.3(larval)-481.7(and)-480.1(adult)-488.4(stages.)]TJ T* [(While)-477.3(it)-478.2(is)-479.1(not)-474.5(clear)-477.1(whether)-483.6(shRNAs)-479.5(would)-480.2(be)-472.2(properly)]TJ 0 -1.2266 TD [(processed)-578.6(during)-572.8(blastula)-570.1(stages)-576(due)-567(to)-572.3(limiting)-576.4(shRNA)]TJ 0 -1.2203 TD [(processing)-302.5(activity,)]TJ 6.7246 0 0 5.9768 137.9905 461.1967 Tm (26)Tj 8.9663 0 0 8.9663 147.3448 457.2282 Tm [(RNAi)-293.7(may)-298.7(be)-295.2(able)-291.9(to)-300.4(substitute)-299.1(for)-294.8(an-)]TJ -9.516 -1.2203 TD [(tisense)-288(MOs)-281.4(later.)-285.2(If)-287.1(targeting)-288.7(efciency)-285.1(is)-283.1(high)-286.6(enough)-287.1(\(per-)]TJ 0 -1.2266 TD [(haps)-239.5(50%\),)-244.3(it)-237.9(would)-246.2(be)-238.3(worthwhile,)-245.2(and)-239.8(feasible)-246.5(to)-237.2(produce)-248.6(a)]TJ 0 -1.2203 TD [(library)-383.5(of)-388.9(shRNA)-383.4(constructs)-389.7(targeting)-389.9(all)-379.6(or)-389.2(most)-381.5(zebrash)]TJ T* (genes.)Tj 1.1128 -1.2266 TD [(An)-308.3(elegant)-308.3(study)-307.7(from)-305(Dong)]TJ /F6 1 Tf 13.0884 0 TD [(et)-311(al.)]TJ /F5 1 Tf 6.7246 0 0 5.9768 207.0425 406.4314 Tm (27)Tj 8.9663 0 0 8.9663 216.5669 402.4062 Tm [(showed)-309.2(that)-307.3(shRNAs)]TJ -17.2363 -1.2203 TD [(injected)-400.1(into)-402.2(zebrash)-397.4(embryos)-403.1(or)-395.5(expressed)-401.6(from)-399.8(the)-402.1(zeb-)]TJ T* [(rash)-362(miR30)-364.8(backbone)-365.9(could)-364(mediate)-364.3(RNAi.)-366.1(Assays)-363.4(in)-367.3(this)]TJ T* [(study)-295.1(targeted)-299.2(an)-293.8(EGFP)-298.4()55(sensor)50.1()-292.7(o)0(r)-294.3(integrated)-299.8(transgene,)-300.4(or)]TJ 0 -1.2266 TD [(the)-300.9(endogenous)]TJ /F6 1 Tf 7.2903 0 TD (chordin)Tj /F5 1 Tf 3.3448 0 TD (and)Tj /F6 1 Tf 1.9727 0 TD (gata1)Tj /F5 1 Tf 2.4976 0 TD [(genes.)-296.4(Although)-298.9(results)-305.8(in)]TJ -15.1054 -1.2203 TD [(this)-723.2(study)-718.7(were)-716.6(promising,)-723.3(RNA)-720.1(knockdown)-720.6(was)-716.9(not)]TJ T* [(quantied,)-325(rescue)-324.9(of)-319.4(phenotypes)-328.1(with)-325.2(normal)-323.6(RNA)-321.8(was)-318.6(not)]TJ 0 -1.2266 TD [(performed,)-508.8(and)-505.4(tissue-specic)-515.5(LOF)-503.6(was)-508.3(not)-506.1(analyzed.)-502.6(To)]TJ 0 -1.2203 TD [(address)-527.2(these)-523.4(issues,)-523.8(we)-520(present)-525.5(a)-524.3(brief)-521.2(report)-524.4(of)-521.7(results)]TJ T* [(obtained)-668.3(using)-671.6(shRNAs,)-672.1(expressed)-673.4(in)-664.5(an)-666.8(F0)-667.8(transgenic)]TJ 0 -1.2266 TD [(assay,)-628.6(targeting)-623.8(the)-629.7(genes)]TJ /F6 1 Tf 12.9304 0 TD (wnt5b)Tj /F5 1 Tf 3.1804 0 TD (and)Tj /F6 1 Tf 2.3015 0 TD (zDisc1)Tj /F5 1 Tf 2.7694 0 TD [(,)-622.6(each)-628(with)]TJ -21.1818 -1.2203 TD [(previously)-266.2(described)-268(morphant)-268.4(and)-258.8(mutant)-268.3(phenotypes.)]TJ 6.7246 0 0 5.9768 284.3149 285.8456 Tm (2831)Tj 8.9663 0 0 8.9663 62.022 270.9353 Tm [(Our)-352.9(results)-356.3(add)-353.3(further)-354.7(support)-357.1(that)-351.6(shRNA-mediated)-359.1(LOF)]TJ 0 -1.2266 TD [(is)-415.8(an)-420.2(efcient)-420.9(mechanism)-422.8(to)-414.2(inhibit)-420.6(gene)-424.9(expression)-421(in)-417.9(the)]TJ 0 -1.2203 TD (zebrash.)Tj /F4 1 Tf 27.9978 41.5605 TD [(Materials)-339(and)-331.9(Methods)]TJ /F10 1 Tf 0 -1.8336 TD [(shRNA)-336.3(design)-336.6(and)-329.4(purication)]TJ /F5 1 Tf 1.1128 -1.8336 TD [(Hairpins)-708.8(for)]TJ /F6 1 Tf 6.5063 0 TD (zDisc1)Tj /F5 1 Tf 3.4776 0 TD (and)Tj /F6 1 Tf 2.3774 0 TD (wnt5b)Tj /F5 1 Tf 3.25 0 TD [(were)-704(designed)-707.6(using)]TJ -16.7241 -1.2266 TD [(Invitrogen)-900.3(Block-IT)-897.5(RNAi)-894.4(Designer)-893.4(software.)-894.9(Hairpin)]TJ 0 -1.2203 TD [(oligonucleotide)-577.9(pairs)-575.5(were)-571.2(puried)-569.3(by)-571.6(UREA-PAGE,)-574.3(an-)]TJ T* [(nealed)-249.5(by)-242.8(heating)-248.6(to)-243.5(95)]TJ /F9 1 Tf 10.1103 0 TD ()Tj /F5 1 Tf .3983 0 TD [(C)-240.4(and)-246.2(slow)-249.5(cooling)-241(to)-249.8(10)]TJ /F9 1 Tf 10.7489 0 TD ()Tj /F5 1 Tf .392 0 TD [(C.)-243.4(We)-245.5(have)]TJ -21.6497 -1.2266 TD [(previously)-380(detailed)-379.6(these)-378(methods)-375.1(on)-372.9(ZFIN.)]TJ 6.7246 0 0 5.9768 491.244 548.9006 Tm (40)Tj 8.9663 0 0 8.9663 501.3353 544.8755 Tm [(At)-374.9(least)-371.5(four)]TJ -20.9984 -1.2203 TD [(hairpins)-389.8(per)-381.3(gene)-380.7(were)-387.8(tested.)-381(The)-385.7(guide)-378.9(strand)-387.8(siRNA)-383.5(se-)]TJ T* [(quence)-381.3(was)-375.5(designed)-378.9(to)-376.3(be)-371(a)-372.6(complete)-384.2(match)-373.3(to)-376.3(the)-376.8(target,)]TJ 0 -1.2266 TD [(which)-188.3(is)-181.9(anticipated)-184.7(to)-180.3(lead)-183(to)-186.6(cleavage)-183.8(of)-180.3(the)-180.8(target)-188.8(RNA)-182.7(after)]TJ 0 -1.2203 TD [(siRNA)-453.1(binding.)]TJ 6.7246 0 0 5.9768 376.4976 505.0203 Tm (19)Tj 8.9663 0 0 8.9663 387.2692 501.0518 Tm [(When)-454.6(annealed,)-451.3(each)-451(hairpin)-451.7(contains)-456.3(a)]TJ -8.2767 -1.2203 TD [(BbsI)-282.7(compatible)-284.2(site,)-280(for)-282.2(insertion)-284.6(into)-282(the)-288.3(miR30)-276.2(backbone.)]TJ /F10 1 Tf 0 -1.9475 TD [(Short)-334.9(hairpin)-336.2(sequences)-334.9(used)]TJ /F6 1 Tf 0 -1.8842 TD (wnt5b)Tj /F5 1 Tf 2.8896 0 TD (ORF268for:)Tj -2.8896 -1.2266 TD (5)Tj /F9 1 Tf .4995 0 TD ()Tj /F5 1 Tf .2466 0 TD [(GGCT)12(AGCCA)10.8(ACTCG)15.5(TGGTT)12.5(TGGTC)15.3(ATTACT)13.3(GGT)12.1(GCAC)]TJ -.7461 -1.2203 TD (ATGATGGAGTAATGACCACCACGAGTTGGCC3)Tj /F9 1 Tf 22.8636 0 TD ()Tj /F6 1 Tf -22.8636 -1.2203 TD (wnt5b)Tj /F5 1 Tf 2.8896 0 TD (ORF268rev:)Tj -2.8896 -1.2267 TD (5)Tj /F9 1 Tf .4995 0 TD ()Tj /F5 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0 TD (UTR1800for:)Tj -3.6357 -1.2203 TD (5)Tj /F9 1 Tf .5058 0 TD ()Tj /F5 1 Tf .2529 0 TD [(GG)-10.2(CT)-9.5(AGC)-13(AGG)-11.6(GT)-8.1(CT)-9.5(TTC)-12.5(TTG)-11.1(TA)-10.7(TA)-10.7(GAC)-13(GCT)-8.2(CT)-9.5(TA)-10.7(AC)-7.9(T)]TJ -.7588 -1.2266 TD .018 Tc [(G)-6(G).3(TGCATGATGGAGT)-4(TAAGAGCGT)-4(C)-1.1(TATACGAAAGA)]TJ 0 -1.2203 TD 0 Tc (CCCC3)Tj /F9 1 Tf 3.3385 0 TD ()Tj /F6 1 Tf -3.3385 -1.2203 TD (wnt5b)Tj /F5 1 Tf 2.8896 0 TD (3)Tj /F9 1 Tf .4995 0 TD ()Tj /F5 1 Tf .2466 0 TD (UTR1800rev:)Tj -3.6357 -1.2267 TD (5)Tj /F9 1 Tf .5058 0 TD ()Tj /F5 1 Tf .2529 0 TD [(GGC)-10.3(TG)-8.1(GGG)-8.9(TCTT)-9.2(TC)-9.5(GTA)-9.5(TAG)-9.5(AC)-7.9(GCT)-8.2(CTTA)-13.9(AC)-7.9(TCC)-9.7(ATC)]TJ -.7588 -1.2203 TD [(ATGCACCA)12.5(GTTAAGA)11.3(GCGTCTAT)10.7(ACAAG)9.5(AAAGACCCT)]TJ T* (GCT3)Tj /F9 1 Tf 2.5734 0 TD ()Tj ET 62.022 218.608 486.198 .51024 re f BT /F7 1 Tf 8.9663 0 0 8.9663 62.022 206.7023 Tm [(FIG.)-273.6(2.)]TJ /F5 1 Tf 4.034 0 TD [(Inhibition)-277.1(of)-268.8(endogenous)]TJ /F6 1 Tf 11.318 0 TD (wnt5b)Tj /F5 1 Tf 2.8263 0 TD [(expression.)-278.5(The)-271.8(3)]TJ /F9 1 Tf 7.5938 0 TD ()Tj /F5 1 Tf .2466 0 TD [(UTR1956)-278.6(shRNA)-269.6(targeting)]TJ /F6 1 Tf 12.0641 0 TD (wnt5b)Tj /F5 1 Tf 2.8263 0 TD [(was)-268(expressed)-281.4(from)-267.1(an)-274.8(intronic)]TJ -40.9092 -1.1128 TD [(miR30)-390(backbone,)-400.4(under)-394(the)]TJ /F6 1 Tf 12.8039 0 TD (b-actin)Tj /F5 1 Tf 3.1615 0 TD [(promoter,)-401.2(using)-393.4(the)-395.8(vector)-389.6(pTolDest-bactin-actin/intron-miR30-CFP-pA)-411(\(see)-399.9(Materials)]TJ -15.9654 -1.1128 TD [(and)-239.8(Methods\).)-240.7(Transient)-242.8(transgenesis)-246.8(was)-236.4(induced)-241.1(using)-241.6(Tol2)-236.4(transposase.)]TJ /F7 1 Tf 32.7969 0 TD (\(Aa\))Tj /F5 1 Tf 2.1498 0 TD [(Vector)-246.2(schematic.)]TJ /F7 1 Tf 7.9163 0 TD (\(Ab\))Tj /F5 1 Tf 2.2573 0 TD [(Relative)-239.4(expression)-244(of)]TJ /F6 1 Tf -45.1203 -1.1065 TD (wnt5b)Tj /F5 1 Tf 3.0224 0 TD 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[(EcoRI)-277(and)-277.8(tr)11.5(anscr)14.8(ibi)13.8(ng)-286(wit)13.3(h)-287.9(T)0(7)-280.7(pol)11.3(ymera)16.9(se.)]TJ /F10 1 Tf 0 -2.2762 TD (Microscopy)Tj /F5 1 Tf 1.1128 -1.8336 TD [(Methods)-268.9(for)-263.2(brighteld)-266.6(and)-265.1(uorescence)-270(microscopy)-265.9(have)]TJ -1.1128 -1.2203 TD [(been)-614.6(described)-615.7(previously.)]TJ 6.7246 0 0 5.9768 171.2125 616.1951 Tm (38)Tj 8.9663 0 0 8.9663 183.4582 612.2266 Tm [(Confocal)-612.6(imaging)-610.7(was)-615.8(per-)]TJ -13.7966 -1.2266 TD [(formed)-333.2(on)-328.6(a)-322(Zeiss)-332.8(LSM710,)-324.2(after)-329.8(xation)-331(in)-323.1(2%)-329.9(TCA)-327(\(acety-)]TJ 0 -1.2203 TD (lated)Tj /F12 1 Tf 2.4596 0 TD (a)Tj /F5 1 Tf .4995 0 TD [(-tubulin\))-284.6(or)-281.7(4%)-279.3(PFA)-279.1(\(phalloidin\).)]TJ /F10 1 Tf -2.9591 -2.2193 TD (Immunohistochemistry)Tj /F5 1 Tf 1.1128 -1.8336 TD [(Whole-mount)-204.3(immunostaining)-202.8(used)-201.6(mouse)-198.9(anti-acetylated)]TJ /F12 1 Tf -1.1128 -1.2266 TD (a)Tj /F5 1 Tf .4995 0 TD [(-tubulin)-378.9(\(Sigma,)-380(1:1000\).)-370.6(Goat)-374.6(anti-mouse)-383.3(Alexa)-373.2(Fluor)-379.5(488)]TJ -.4995 -1.2203 TD [(\()-89(Molecular)-268.6(Probes,)-272.6(1:500\))-267(was)-274.3(used)-271.1(as)-266.4(a)-271.4(secondary)-267.2(antibody.)]TJ T* [(Staining)-415.1(by)-413.5(phalloidin)-413.2(Texas)-410.6(Red)-411.8(was)-413.4(performed)-417.4(as)-405.5(previ-)]TJ 0 -1.2267 TD [(ously)-285.3(described.)]TJ 6.7246 0 0 5.9768 123.7039 514.0912 Tm (31)Tj /F10 1 Tf 8.9663 0 0 8.9663 59.7543 488.1826 Tm [(Brain)-334.6(ventricle)-340.7(injection)-334.1(and)-329.4(imaging)]TJ /F5 1 Tf 1.1128 -1.8336 TD [(Brain)-244.1(ventricles)-246.7(were)-242.4(injected)-248.4(with)-243(Texas)-246.2(Red)-241.1(dextran,)-246(and)]TJ -1.1128 -1.2266 TD [(superimposed)-602.1(brighteld)-595.4(and)-593.9(uorescence)-598.8(imaging)-591.8(were)]TJ 0 -1.2203 TD [(performed)-284.6(as)-285.4(described)-280.6(previously.)]TJ 6.7246 0 0 5.9768 199.1621 453.77 Tm (33)Tj /F10 1 Tf 8.9663 0 0 8.9663 59.7543 427.918 Tm [(RNA)-338.6(rescue)-330.5(assays)]TJ /F5 1 Tf 1.1128 -1.8336 TD [(Capped)-232.9(RNA)-226.9(was)-230.1(synthesized)-228.8(using)-229(the)-231.4(Message)-232.8(Machine)]TJ -1.1128 -1.2266 TD [(kit)-204.4(\(Ambion\),)-206.6(and)-201.9(injected)-210.4(at)-202.3(one-cell-stage.)]TJ /F6 1 Tf 18.9561 0 TD (hDisc1)Tj /F5 1 Tf 2.8327 0 TD (,)Tj /F6 1 Tf .4489 0 TD (wnt5b)Tj /F5 1 Tf 2.7568 0 TD (and)Tj /F6 1 Tf -24.9945 -1.2203 TD (mcherry)Tj /F5 1 Tf 3.5535 0 TD [(mRNAs)-219.5(were)-217.1(transcribed)-224.9(from)-216.5(pCS2-hDisc1,)]TJ 6.7246 0 0 5.9768 266.4566 393.5054 Tm (31)Tj 8.9663 0 0 8.9663 275.1873 389.5369 Tm (pCS2-)Tj -24.0271 -1.2203 TD [(wnt5b)-768.2(\(very)-765(kindly)-762.1(provided)-767.4(by)-761.3(Dr.)-763.8(Diane)-766.5(Slusarski\))]TJ 0 -1.2267 TD [(and)-480.1(pCS2-mcherry)-473.8(\(kindly)-478(provided)-476.6(by)-476.7(Dr.)-472.9(Frank)-474(Gertler)]TJ 0 -1.2203 TD [([Massachusetts)-514.2(Institute)-518.8(of)-502.8(Technology]\),)-517.6(respectively.)-511.1(For)]TJ T* [(rescues,)-228(200)-162.9(pg)-220.7(\()]TJ /F6 1 Tf 7.0121 0 TD (zDisc1)Tj /F5 1 Tf 2.7694 0 TD [(\))-221.8(and)-220.9(2)-163.9(p)0(g)-220.8(\()]TJ /F6 1 Tf 4.7928 0 TD (wnt5b)Tj /F5 1 Tf 2.7758 0 TD [(rescue\))-217.9(were)-223.4(injected,)]TJ -17.3501 -1.2266 TD [(with)-293.5(an)-293.7(equivalent)-288.6(amount)-295.9(of)]TJ /F6 1 Tf 13.4046 0 TD (mcherry)Tj /F5 1 Tf 3.623 0 TD [(RNA)-290.1(injected.)-295.5(Results)]TJ -17.0276 -1.2203 TD [(obtained)-409(with)-407.4(or)-401.8(without)-405.6(injection)-406.9(of)]TJ /F6 1 Tf 17.2426 0 TD (mcherry)Tj /F5 1 Tf 3.7305 0 TD [(mRNA)-407.8(were)]TJ -20.9731 -1.2203 TD (indistinguishable.)Tj /F10 1 Tf 0 -2.447 TD [(Morpholino)-340.4(oligonucleotide)-334.2(injections)]TJ /F5 1 Tf 1.1128 -1.8336 TD [(Antisense)-248.5(morpholino)-243.1(oligonucleotides)-244.1(\()-82.7(MOs;)-240(Gene)-242.2(Tools,)]TJ -1.1128 -1.2203 TD [(LLC,)-496(Philomath,)-499(OR\))-494.6(were)-495.3(as)-494(follows:)-493.2(z)]TJ /F6 1 Tf 18.7285 0 TD (Disc1)Tj /F5 1 Tf 2.3332 0 TD [(MO,)-491.6(5)]TJ /F9 1 Tf 2.9654 0 TD ()Tj /F5 1 Tf .2466 0 TD (-TCG)Tj -24.2737 -1.2203 TD (CAGTTTTGTCTTACCTGTCCTC-3)Tj /F9 1 Tf 15.6429 0 TD ()Tj /F5 1 Tf .2529 0 TD (;)Tj /F6 1 Tf .6007 0 TD (wnt5b)Tj /F5 1 Tf 2.5545 0 TD [(MO,)-346.2(5)]TJ /F9 1 Tf 2.82 0 TD ()Tj /F5 1 Tf .2466 0 TD (TGTTTAT)Tj -22.1175 -1.2266 TD [(TTCCTCACCATTCTTCC)-246.5(G-)-239.5(3)]TJ /F9 1 Tf 13.4994 0 TD ()Tj /F5 1 Tf .2466 0 TD [(;)-243.2(M)0(O)-235.8(\()0(1)-170.7(nl\))-241.3(was)-236.4(injected)-242(into)-244.1(a)]TJ -13.746 -1.2203 TD [(single)-338.6(cell)-328.7(of)-332(a)-334.6(12)-327.3(cell)-335(embryo,)-328.8(using)-336.5(3.5)-166.3(ng)-330.3(for)-332.7(both)-333.5(z)]TJ /F6 1 Tf 24.3369 0 TD (Disc1)Tj /F5 1 Tf -24.3369 -1.2203 TD [(MO)-267.4(and)]TJ /F6 1 Tf 3.9202 0 TD (wnt5b)Tj /F5 1 Tf 2.82 0 TD (MO.)Tj 6.7246 0 0 5.9768 137.8204 223.6535 Tm (16,31)Tj 8.9663 0 0 8.9663 155.3385 219.685 Tm [(For)-260.8(both)]TJ /F6 1 Tf 3.9898 0 TD (wnt5b)Tj /F5 1 Tf 2.8137 0 TD (and)Tj /F6 1 Tf 1.9411 0 TD (zDisc1,)Tj /F5 1 Tf 3.2816 0 TD (a)Tj /F6 1 Tf .7651 0 TD (p53)Tj /F5 1 Tf 1.4985 0 TD (MO)Tj -24.9502 -1.2266 TD (\(5)Tj /F9 1 Tf .8283 0 TD ()Tj /F5 1 Tf .2466 0 TD (GCGCCATTGCTTTGCAAGAATTG-3)Tj /F9 1 Tf 17.0086 0 TD ()Tj /F5 1 Tf .2466 0 TD [(\))-449.4(was)-445.1(used)-448.2(at)-448.9(1.5-)]TJ -18.3301 -1.2203 TD [(fold)-300.6(greater)-302.8(than)-297.6(the)-300.9(mass)-296.9(of)-300.4(experimental)-302.8(MO)-299(to)-294.1(reduce)-301.3(off)]TJ T* [(target)-283.7(effects.)]TJ 6.7246 0 0 5.9768 112.1385 190.7716 Tm (16)Tj /F10 1 Tf 8.9663 0 0 8.9663 59.7543 164.863 Tm [(Fish)-335.3(embryos)]TJ /F5 1 Tf 1.1128 -1.8336 TD [(Embryos)-526.3(were)-526.9(obtained)-529.2(from)-526.3(natural)-527(spawnings)-532(of)-521.7(an)]TJ /F6 1 Tf -1.1128 -1.2203 TD (AB)Tj /F5 1 Tf 1.6376 0 TD [(strain.)-309(Developmental)-317.5(stages)-316.7(are)-309.9(reported)-312.3(as)-310.6(hours)-313(post-)]TJ -1.6376 -1.2203 TD [(fertilization)-324.8(\(hpf)-87.9(\))-322.9(a)0(t)-316.1(2)0(8)]TJ /F9 1 Tf 10.0598 0 TD ()Tj /F5 1 Tf .3983 0 TD [(C.)-319.2(Disc1fh291)-323.6(mutants)-320.2(were)-324.6(isolated)]TJ -10.4581 -1.2266 TD [(from)-286(a)-277.7(library)-282.4(of)-281.4(8600)-276.3(ENU-mutagenized)-289.3(F1)-275.8(sh.)]TJ 6.7246 0 0 5.9768 252.907 119.5653 Tm (31,44)Tj /F4 1 Tf 8.9663 0 0 8.9663 59.7543 95.6409 Tm (Results)Tj /F5 1 Tf 1.1128 -1.8336 TD -.0141 Tc [(We)-368.1(us)-14.1(e)10(d)-364.9(two)-371(g).5(enes)-363.2(as)-369.5(t)4.4(e)-1.4(s)-14.1(t)9.4(s)-363.2(o)-14.1(f)-352.5(s)-14.1(h)12.8(R)-14.1(N)13.3(A)-369.6(efcacy:)]TJ /F6 1 Tf 21.0616 0 TD (wnt5b)Tj /F5 1 Tf 2.8453 0 TD -.0144 Tc (and)Tj /F6 1 Tf -25.0198 -1.2203 TD -.0135 Tc [(zD)-13.5(i)8.5(s)4.8(c1.)]TJ /F5 1 Tf 3.3385 0 TD 0 Tc [(F)14.6(o)0(r)-389.2(e)12.8(a)13.1(c)13.4(h)-401.7(g)14.6(e)12.8(n)15.6(e)12.7(,)-401.3(LO)25.6(F)-402.8(p)17.6(he)22(n)15.6(o)16.2(t)12.2(y)14.6(pe)24(s)-399.7(h)0(a)28.7(v)0(e)-392.7(b)14.6(e)12.7(e)12.8(n)-401.7(r)18.3(e)12.8(p)0(o)21.2(r)18.3(t)12.2(e)12.7(d)16(,)]TJ -3.3385 -1.2267 TD [(wi)26.5(t)12.2(h)-306.9(a)-309.3(s)0(i)26.8(m)15.1(il)24.6(a)13.1(r)-304.2(ph)20.5(e)19.1(n)0(o)25.5(t)12.2(y)14.5(p)0(e)-298.4(i)15.4(s)-311.1(o)16.2(b)14.6(s)0(e)24.1(r)0(v)30.2(e)12.8(d)-306.5(i)0(n)-297.8(b)14.6(ot)28.4(h)-313.2(g)14.6(e)12.8(n)15.6(e)12.7(t)12.2(i)15.4(c)-309.1(m)15.1(u)0(t)23.5(a)13.1(n)15.6(t)12.2(s)]TJ 28.0042 74.5598 TD [(a)13.1(n)15.6(d)-445.6(i)15.4(n)-439.7(m)15.1(or)21.8(p)17.6(h)0(a)22.4(n)15.6(t)12.2(s)-443.9(\()12.2(i)15.4(.)16.1(e)12.7(.)16.1(,)-445.5(a)13.1(f)12.2(t)18.5(e)12.7(r)-443.3(a)13.1(n)15.6(t)12.2(is)26.8(e)12.7(n)0(s)26.9(e)-442.5(m)15.1(o)0(r)21.8(p)17.6(ho)25.5(l)15.4(i)0(n)24.7(o)16.2(-)12.2(m)15.1(od)25.8(i)25.1(e)12.8(d)]TJ 0 -1.2203 TD [(o)16.2(l)15.4(ig)23.7(o)16.2(n)0(u)26.9(c)13.4(le)28.2(ot)22.1(i)15.4(d)16(e)-189.6(i)15.4(nj)23.3(e)12.8(c)13.4(t)12.2(i)15.4(o)16.2(n)0(\))21.4(.)-186.3(T)16(h)15.6(e)12.8(s)0(e)-178.3(p)0(h)26.9(e)12.7(n)15.6(o)0(t)22(y)14.6(p)17.6(e)12.8(s)-191(p)17.6(ro)21.8(v)18.2(i)0(d)25.1(e)-189.6(a)-189.2(b)14.6(a)13.1(s)17.7(i)15.4(s)-191(f)12.2(o)16.2(r)]TJ 0 -1.2266 TD [(c)13.4(o)16.2(m)15.1(p)0(a)24.4(r)0(i)27.4(s)0(o)27.5(n)-534.5(wi)26.5(t)12.2(h)-534.5(a)13.1(n)-534.5(sh)26.9(R)14.7(N)12.7(A-)23.4(m)15.1(e)12.7(d)16(i)15.4(a)13.1(t)12.2(e)12.7(d)-534.1(ph)26.9(e)12.7(n)0(o)25.5(t)12.2(y)14.6(p)0(e)24(.)-534(I)16.9(n)-534.5(t)12.2(h)0(e)28.3(s)0(e)]TJ 0 -1.2203 TD [(s)17.7(t)12.2(ud)27.3(ie)28.2(s,)-592.3(sh)26.9(RN)27.4(As)-597.1(w)17.3(e)12.8(re)-588.6(e)12.7(x)0(p)21.8(r)18.3(e)12.7(s)0(s)22.7(e)19.1(d)-610(f)18.5(ro)21.8(m)-604.6(a)-600.2(z)13.1(e)12.7(b)14.5(r)0(a)25.1()16(sh)-592.7(m)15.1(i)15.4(R)14.7(3)13.1(0)]TJ T* [(b)14.5(a)13.1(c)13.4(k)14.5(b)14.6(o)16.2(ne)22(,)-508.7(w)0(i)26.5(t)12.2(h)-509.2(t)12.2(h)15.6(e)-512.1(c)13.4(a)13.1(ss)29(e)12.7(t)12.2(t)12.2(e)-512.1(d)16(r)0(i)27.4(v)0(e)24.7(n)-515.5(b)14.6(y)-510.3(a)-511.7(g)14.6(e)12.8(n)15.6(e)12.7(r)0(a)25.1(l)-509.4(o)16.2(r)-512.9(t)12.2(i)15.4(ss)22.7(u)17.6(e)12.7(-)]TJ 0 -1.2266 TD [(s)17.7(p)0(e)24(c)13.4(i)15.4()0(c)-173(p)17.6(ro)28.1(mo)25(t)12.2(e)12.8(r)18.3(,)-186.3(a)13.1(n)15.6(d)-180(t)12.2(e)12.7(s)0(t)23.5(e)12.7(d)-180(i)15.4(n)-180.4(a)-182.9(t)12.2(r)0(a)25.1(n)15.6(si)26.8(e)12.7(n)15.6(t)-183.8(\()12.2(F)14.5(0)13.1(\))-183.8(t)12.2(r)18.3(a)13.1(ns)26.9(g)14.6(e)12.7(ni)24.7(c)-182.7(a)13.1(s)17.7(s)0(a)24.5(y)]TJ 0 -1.2203 TD [(\()12.2(F)14.6(i)15.4(g)14.6(.)-192.6(1)13.1(A)-191.1(a)13.1(n)15.6(d)-192.7(B)16(\))12.2(.)-192.6(T)16(he)-180.3(a)13.1(s)0(s)29(a)13.1(y)-194.1(w)0(a)30.5(s)-197.3(e)19.1(m)0(p)26.4(l)0(o)25.3(y)14.6(e)12.8(d)-192.7(t)12.2(o)-192.5(r)18.3(a)13.1(pi)26.7(dl)25.1(y)-187.8(t)12.2(e)12.7(s)0(t)-178.8(s)0(h)26.9(R)0(N)27.4(A)]TJ T* -.0137 Tc [(efcacies)-13.7(,)-182.3(and)-200.1(h)1.9(as)-198.4(t)4.8(h)-13.7(e)-187.7(a)-.6(dv)-13.7(a)17.7(n)-13.7(t)7.7(a)-.6(ge)-197(t)4.8(h)-13.7(a)8.7(t)-197.5(ef)4.8(fects)-198.4(c)-.3(an)-194.1(be)-197(exami)-13.7(n)11(ed)-193.7(i)-13.7(n)]TJ 0 -1.2266 TD 0 Tc [(t)12.2(h)15.6(e)-297.1(F)14.6(0)-296.7(\()12.2(F)14.6(i)0(g)23.7(.)-293.8(1)13.1(C)12.5(\))12.2(.)-293.8(Th)25.2(i)15.4(s)-298.5(t)12.2(y)14.5(pe)-285.8(o)16.2(f)-304(a)19.5(n)0(a)22.4(l)15.4(y)14.6(s)0(i)26.8(s)-298.5(h)15.6(a)13.1(s)-298.5(t)12.2(h)15.6(e)-297.1(di)25.1(sa)24.5(d)16(v)0(a)31.4(n)0(t)21.4(a)13.1(g)14.5(e)-297.1(o)16.2(f)]TJ 0 -1.2203 TD [(m)15.1(o)16.2(sa)24.5(i)15.4(c)13.4(i)15.4(s)0(m)26.4(,)-287.4(w)17.3(h)0(e)28.3(r)0(e)-272.5(f)12.2(e)12.7(w)17.3(e)12.7(r)-285.2(t)12.2(h)15.6(a)13.1(n)-287.9(1)13.1(0)19.5(0)13.1(%)-286.1(o)16.2(f)-285(c)13.4(e)12.7(l)15.4(l)0(s)-270.4(b)0(e)27.3(c)13.4(om)25(e)-284.4(t)12.2(ra)31.4(ns)26.9(ge)27.3(ni)24.7(c)]TJ T* [(a)13.1(n)15.6(d)-407.7(e)12.7(x)16.8(p)0(r)29.6(e)12.7(ss)-394.6(t)12.2(h)15.6(e)-404.6(s)0(h)26.9(R)0(N)21.1(A)17.6(,)-407.6(r)0(e)31(d)0(u)27.3(c)13.4(in)24.7(g)-402.8(t)12.2(he)-395.3(m)15.1(a)13.1(g)14.5(n)15.6(it)27.6(ud)27.3(e)-410.9(o)16.2(f)-405.1(s)0(h)26.9(R)14.7(N)12.7(A)]TJ T* [(e)12.7(f)12.2(f)18.5(e)12.7(c)13.4(t)12.2(s.)-282.4(In)-283.7(or)21.8(d)16(e)12.7(r)-297.9(t)12.2(o)-293.6(o)0(v)28.1(e)12.7(rc)25.3(om)25(e)-297.1(t)12.2(h)15.6(i)15.4(s)-298.5(d)0(i)25.1(s)17.7(a)13.1(d)0(v)27.9(a)13.1(nt)27.8(a)13.1(g)14.6(e)12.7(,)-300.1(w)17.3(e)-303.4(c)13.4(h)15.6(o)16.2(s)0(e)-285.7(e)12.7(m)15.1(-)]TJ 0 -1.2266 TD [(b)14.5(r)0(y)26.5(o)16.2(s)-393.3(e)12.7(xp)28.1(re)24.7(ss)29(in)24.7(g)-390.1(C)12.5(F)14.6(P)-393.4(e)12.7(x)0(t)22.7(e)19.1(ns)26.9(iv)27.3(e)12.7(l)0(y)23.7(,)-388.6(a)13.1(n)0(d)-379.4(s)0(c)24.7(o)16.2(re)24.7(d)-395(t)12.2(h)15.6(e)12.7(se)-380.6(f)12.2(o)16.2(r)-392.7(p)0(h)26.9(e)12.7(-)]TJ 0 -1.2203 TD [(n)15.6(o)0(t)28.4(y)0(p)25.8(e)12.7(s.)-472.1(E)16(a)13.1(c)13.4(h)-490.2(s)17.7(h)0(R)24(N)12.7(A)-481.9(e)12.8(xp)28.1(re)24.7(ss)29(io)25.3(n)-490.2(c)19.7(on)25.5(st)23.5(ru)29.6(c)13.4(t)-487.3(wa)30.5(s)-488.2(a)13.1(ss)29(a)13.1(y)14.6(e)12.7(d)-483.5(in)]TJ T* -.0143 Tc [(at)-375.2(leas)-14.3(t)-363.8(t)-2.1(wo)-377.5(in)-14.3(d)10.9(e)-1.6(p)-14.3(e)9.7(nden)-14.3(t)-365.9(e)-1.6(xp)-14.3(e)9.7(r)-14.3(i)13.1(m).8(ents)-14.3(,)-366.3(w)3(ith)-378.1(t)-2.1(otal)-378.2(nu)-14.3(m)12.1(b).3(er)-14.3(s)-364.1(o)1.9(f)]TJ 0 -1.2266 TD 0 Tc [(e)12.7(m)15.1(b)14.6(r)0(y)26.5(o)16.2(s)-279.5(e)12.7(x)16.8(a)13.1(m)15.1(i)15.4(ne)22(d)-274.9(i)15.4(nd)25.2(i)15.4(c)13.4(a)13.1(t)12.2(e)12.7(d)-274.9(i)15.4(n)-275.3(t)12.2(h)0(e)-262.5(r)0(e)24.7(l)15.4(e)12.7(v)0(a)25(n)15.6(t)-278.7(s)0(e)30.4(c)13.4(t)12.2(i)0(o)25.3(n)-275.3(b)14.6(e)12.8(lo)25.3(w.)]TJ /F10 1 Tf 0 -3.054 TD [(shRNAs)-335.8(inhibit)]TJ /F2 1 Tf 6.9109 0 TD (wnt5b)Tj /F10 1 Tf 2.9907 0 TD [(expression)-335.9(effectively)]TJ -9.9017 -1.2203 TD [(and)-329.4(specically)]TJ /F5 1 Tf 1.1128 -1.8336 TD [(The)-581.7(rst)-580.9(gene)-583(targeted)-583.8(was)]TJ /F6 1 Tf 13.7144 0 TD (wnt5b)Tj /F5 1 Tf 2.5545 0 TD [(,)-578.3(considered)-585.9(a)-581.2(non-)]TJ -17.3817 -1.2203 TD [(canonical)-621.2(wnt,)-621.7(which)-624.5(is)-618.2(expressed)-622.9(in)-620.2(the)-623.4(mesendoderm)]TJ 0 -1.2266 TD [(and)-328.4(tail)-329.5(bud.)-327.8(The)-328.8(LOF)-332.9(phenotype)-333.1(for)]TJ /F6 1 Tf 16.8822 0 TD (wnt5b)Tj /F5 1 Tf 2.8769 0 TD 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[(the)-218.7(Tol2)-223.8(method,)]TJ 6.7246 0 0 5.9768 376.8944 357.1653 Tm (32)Tj 8.9663 0 0 8.9663 385.6251 353.1967 Tm [(and)-220.9(examined)-223.9(at)-221.3(24)-163.4(hpf)-226.5(for)-218.9(a)-220.8(bent)-229.5(tail.)-218.6(One)]TJ -8.3399 -1.2266 TD [(hairpin,)-214.3(mapping)-219.8(to)-211.9(3)]TJ /F9 1 Tf 9.4148 0 TD ()Tj /F5 1 Tf .2466 0 TD [(UTR)-217.3(in)-215.6(position)-215.1(1956)-219.3(gave)-213.2(a)-214.5(phenotype)]TJ -9.6614 -1.2203 TD [(and)-688.8(this)-691.6(was)-691.7(used)-694.8(in)-689.8(subsequent)-700.7(assays.)-686.9(ORF269)-693.1(and)]TJ T* (3)Tj /F9 1 Tf .4995 0 TD ()Tj /F5 1 Tf .2466 0 TD [(UTR1299)-335.5(hairpins)-332.9(also)-335.9(gave)-327(weaker)-334.9(phenotypes.)-337.3(F0)-332.7(trans-)]TJ -.7461 -1.2266 TD [(genic)-535.3(embryos)-529.6(were)-533.2(sorted)-539.3(for)-528.7(high)-533.2(CFP)-537.1(expression)-534.8(and)]TJ 0 -1.2203 TD [(harvested)-306.1(at)-309.8(24)-163.4(hpf)-308.7(for)-307.4(qPCR)-307.4(analysis,)-310.3(which)-308.4(showed)-309.2(an)-306.4(av-)]TJ T* [(erage)-270(of)-268.8(71%)-266.2(\()]TJ /F6 1 Tf 6.1965 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(5)-271.4(independent)-274.1(experiments,)-272.3(82)-264.6(total)-272.3(control)]TJ -7.6318 -1.2266 TD [(and)-467.5(78)-473.2(total)-474.7(experimental)-473.5(embryos)-472.7(examined\))-477.3(decrease)-471.4(in)]TJ /F6 1 Tf 0 -1.2203 TD (wnt5b)Tj /F5 1 Tf 2.8453 0 TD [(RNA)-302.8(after)-298.2(expression)-300.9(of)-300.4(the)-300.9(3)]TJ /F9 1 Tf 13.1327 0 TD ()Tj /F5 1 Tf .2529 0 TD (UTR1956)Tj /F6 1 Tf 4.3375 0 TD (wnt5b)Tj /F5 1 Tf 2.8453 0 TD (shRNA)Tj -23.4137 -1.2203 TD [(relative)-254.9(to)-249.8(a)-252.4(control)]TJ /F6 1 Tf 8.7193 0 TD (YFP)Tj /F5 1 Tf 2.0739 0 TD [(shRNA)-250.6(\(Fig.)-249.7(2Ab\).)-255.3(Expression)-253.4(of)-256.2(the)]TJ -10.7932 -1.2203 TD [(guide)-360(strand)-362.5(of)-357.3(the)-364.2(siRNA,)-361.1(which)-359(would)-360.1(bind)-362.1(to)-357.3(the)]TJ /F6 1 Tf 24.1156 0 TD (wnt5b)Tj /F5 1 Tf -24.1156 -1.2266 TD [(RNA)-214.3(target,)-210.7(was)-217.4(conrmed)-211.9(by)-211.2(northern)-223.4(analysis)-212.5(\(Fig.)-211.8(2Ac\).)-212.2(A)]TJ 0 -1.2203 TD [(strong)-373.5(decrease)-376.6(in)]TJ /F6 1 Tf 8.5106 0 TD (wnt5b)Tj /F5 1 Tf 2.9212 0 TD [(RNA)-372.3(observed)-379.5(by)-369.2(qPCR)-376.9(was)-369.2(also)]TJ -11.4318 -1.2203 TD [(seen)-308(by)-299.7(whole)-308.4(mount)]TJ /F6 1 Tf 9.8448 0 TD [(in)-309.8(situ)]TJ /F5 1 Tf 2.9654 0 TD [(hybridization)-304.1(analysis)-307.4(at)-303.5(12)-169.7(hpf)]TJ -12.8102 -1.2267 TD [(and)-442.2(24)-163.4(hpf)-447.8(\(Fig.)-445.7(2B\).)-442.2(Some)-441.3(RNA)-448.2(remained)-443.8(in)-443.2(both)-447.3(assays,)]TJ 0 -1.2203 TD [(possibly)-332.5(reecting)-330.7(incomplete)-334.2(expression)-332.5(of)-325.7(the)-332.5(shRNA)-332.8(due)]TJ T* 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(=)Tj /F5 1 Tf .7208 0 TD [(29,)-406.6(62%)-405.3(normal)-405.8(embryos\))-409.9(\(Fig.)]TJ -12.7343 -1.2266 TD (2Cc-c)Tj /F9 1 Tf 2.409 0 TD ()Tj /F5 1 Tf .4047 0 TD [(\).)-199.4(The)]TJ /F6 1 Tf 2.6114 0 TD (wnt5b)Tj /F5 1 Tf 2.7505 0 TD [(LOF)-200.2(phenotype)-206.6(and)-195.6(specicity)-205.8(was)-198.5(further)]TJ -8.1755 -1.2203 TD [(explored)-304.2(by)-299.7(examining)-301.9(expression)-300.9(of)]TJ /F6 1 Tf 16.585 0 TD (myoD)Tj /F5 1 Tf 2.7758 0 TD [(RNA)-302.8(that)-301(marks)]TJ -19.3608 -1.2203 TD [(the)-326.2(developing)-332.4(somites,)-332.3(using)]TJ /F6 1 Tf 13.5816 0 TD [(in)-328.8(situ)]TJ /F5 1 Tf 3.0097 0 TD [(hybridization)-329.4(at)-328.8(12)-163.4(hpf)]TJ -16.5913 -1.2203 TD [(\(Fig.)-249.7(2Da-c)]TJ /F9 1 Tf 4.7359 0 TD ()Tj /F5 1 Tf .2529 0 TD [(\).)-243.7(While)-249.6(expression)-256.6(of)-243.5(the)]TJ /F6 1 Tf 11.2927 0 TD (YFP)Tj /F5 1 Tf 2.0676 0 TD [(control)-253.2(shRNA)-250.6(did)]TJ -18.3491 -1.2266 TD [(not)-291.2(alter)-288.6(somite)-290.1(formation)-289.4(\(Fig.)-287.6(2Da,a)]TJ /F9 1 Tf 16.5597 0 TD ()Tj /F5 1 Tf .2466 0 TD (,)Tj /F6 1 Tf .5374 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(15,)-292.8(100%)-284.7(normal\),)]TJ -18.779 -1.2203 TD (after)Tj /F6 1 Tf 2.2762 0 TD (wnt5b)Tj /F5 1 Tf 2.82 0 TD [(shRNA)-275.9(expression,)-278.5(the)-275.6(somites)-278.8(were)-274(compressed)]TJ -5.0963 -1.2203 TD [(\(Fig.)-218.1(2Db,b)]TJ /F9 1 Tf 4.7991 0 TD ()Tj /F5 1 Tf .2466 0 TD [(,)-217.9(white)-217.6(bracket,)]TJ /F6 1 Tf 6.8477 0 TD (n)Tj /F9 1 Tf .7145 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(15,)-216.9(20%)-215.6(normal)-216.1(embryos\).)-216.8(This)]TJ 14.6692 20.7771 TD [(indicates)-285.8(a)-277.7(convergence)-279.2(and)-277.8(extension)-288.7(abnormality,)-275.7(and)-277.8(was)]TJ 0 -1.2266 TD [(prevented)-363.4(by)-350.3(simultaneous)-357.4(injection)-362.7(of)]TJ /F6 1 Tf 17.7547 0 TD (wnt5b)Tj /F5 1 Tf 2.9085 0 TD [(RNA)-353.4(and)-353.7(the)]TJ -20.6633 -1.2203 TD [(shRNA)-219(construct)-224(\(Fig.)-218.1(2Dc,c)]TJ /F9 1 Tf 12.2854 0 TD ()Tj /F5 1 Tf .2466 0 TD (,)Tj /F6 1 Tf .4679 0 TD (n)Tj /F9 1 Tf .7145 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(15,)-216.9(73%)-215.6(normal)-216.1(embryos\).)-216.8(A)]TJ -14.4352 -1.2203 TD (similar)Tj /F6 1 Tf 3.3511 0 TD (myoD)Tj /F5 1 Tf 2.8074 0 TD [(phenotype)-333.1(was)-331.2(observed)-328.9(in)]TJ /F6 1 Tf 12.6016 0 TD (wnt5b)Tj /F5 1 Tf 2.8769 0 TD (morphants,)Tj -21.637 -1.2203 TD [(although)-335(compression)-336.4(of)-338.4(the)-332.5(somites)-329.3(was)-337.6(more)-329.7(pronounced)]TJ 0 -1.2266 TD [(than)-525.2(after)-525.9(shRNA)-528.8(expression)-528.5(\(Fig.)-521.6(2Dd,d)]TJ /F9 1 Tf 19.1774 0 TD ()Tj /F5 1 Tf .2529 0 TD [(,)-521.4(white)-521.1(bracket,)]TJ /F6 1 Tf -19.4303 -1.2203 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(15,)-280.2(13%)-278.9(normal)-279.4(embryos\).)]TJ -.3288 -1.2203 TD [(Together,)-365.2(these)-359.1(data)-362.9(indicate)-360.4(that)-357.9(expression)-364.1(of)-357.3(zebrash)]TJ /F6 1 Tf -1.1128 -1.2267 TD (wnt5b)Tj /F5 1 Tf 2.8706 0 TD [(can)-312(be)-314.1(inhibited)-322.3(by)-312.3(shRNAs.)-318(Specicity)-316.8(of)-319.4(the)-313.6(pheno-)]TJ -2.8706 -1.2203 TD [(type)-512(was)-514.6(indicated)-508.8(by)-514.7(similarity)-511.3(to)-509.1(the)-509.6(published)]TJ /F6 1 Tf 23.7109 0 TD (pipetail)Tj /F5 1 Tf -23.7109 -1.2203 TD [(mutant)-388.5(and)]TJ /F6 1 Tf 5.6337 0 TD (wnt5b)Tj /F5 1 Tf 2.9402 0 TD [(morphant,)-385.1(and)-385.3(by)-388.2(the)-383.1(ability)-386.1(of)-388.9(cognate)]TJ -8.5739 -1.2266 TD [(mRNA)-287.7(to)-281.4(prevent)-278.3(shRNA-associated)-293.3(phenotypes.)]TJ /F10 1 Tf 0 -3.054 TD [(shRNAs)-335.8(target)]TJ /F2 1 Tf 6.8793 0 TD (zDisc1)Tj /F10 1 Tf 3.3195 0 TD [(effectively)-332.5(and)-335.7(specically)]TJ /F5 1 Tf -9.086 -1.8336 TD [(The)-284.5(second)-287.9(gene)-285.8(targeted)-286.6(was)]TJ /F6 1 Tf 13.5374 0 TD (zDisc1,)Tj /F5 1 Tf 3.3069 0 TD [(for)-282.1(which)-289.4(we)-286.1(previ-)]TJ -17.9571 -1.2203 TD [(ously)-512.9(reported)-514.6(phenotypes)-511.4(of)-509.1(a)-511.7(morphant)-508.7(and)-511.7(a)-505.3(mutant,)]TJ T* (Disc1)Tj 6.7246 0 0 5.9768 334.6582 317.8204 Tm (fh291)Tj 8.9663 0 0 8.9663 350.8156 313.8519 Tm [(,)-426.6(demonstrating)-437.7(both)-434.7(brain,)-431.8(muscle)-439.2(segment,)-433.5(and)]TJ ET 62.022 731.735 486.198 .51025 re f BT /F7 1 Tf 8.9663 0 0 8.9663 62.022 719.7731 Tm [(FIG.)-292.5(3.)]TJ /F5 1 Tf 4.0467 0 TD [(Inhibition)-296.1(of)-287.8(endogenous)]TJ /F6 1 Tf 11.3749 0 TD (zDisc1)Tj /F5 1 Tf 3.0603 0 TD [(expression.)-291.1(The)-290.8(ORF2654)-288(shRNA)-288.5(\()]TJ /F6 1 Tf 15.2762 0 TD (zDisc1h\))Tj /F5 1 Tf 3.8759 0 TD (targeting)Tj /F6 1 Tf 4.249 0 TD (zDisc1)Tj /F5 1 Tf 3.0603 0 TD [(was)-287(expressed)-294.1(in)-285.1(Tol2)]TJ -44.9433 -1.1065 TD [(F0)-313.8(transgenic)-319.4(embryos,)-317.5(under)-318.2(control)-322.7(of)-313.1(the)]TJ /F6 1 Tf 19.7465 0 TD (b-actin)Tj /F5 1 Tf 3.0856 0 TD [(promoter,)-319(using)-317.5(the)-313.6(pTolDest-bactin-actin/intron-miR30-CFP-pA)-335.1(vector)]TJ -22.832 -1.1128 TD [(\(see)-349.3(Materials)-355(and)-347.3(Methods\).)]TJ /F7 1 Tf 13.2781 0 TD (\(Aa\))Tj /F5 1 Tf 2.2636 0 TD [(Vector)-353.7(schematic.)]TJ /F7 1 Tf 8.1313 0 TD (\(Ab\))Tj /F5 1 Tf 2.3711 0 TD [(Relative)-353.3(expression)-351.4(of)]TJ /F6 1 Tf 10.1167 0 TD (zDisc1)Tj /F5 1 Tf 3.1172 0 TD [(mRNA)-350.9(after)-348.8(inhibition)-354.4(by)-350.3(shRNA.)]TJ -39.2779 -1.1128 TD [(Expression)-322.9(was)-318.6(quantied)-322.1(by)-318.7(qPCR)-320(in)-316.7(24)-169.7(hpf)-321.4(embryos)-320.9(expressing)]TJ /F6 1 Tf 29.4964 0 TD (zDisc1h)Tj /F5 1 Tf 3.5914 0 TD [(relative)-318.1(to)-319.4(embryos)-320.9(expressing)-321.5(a)-322(control)-322.7(shRNA,)]TJ /F6 1 Tf -33.0878 -1.1065 TD (YFPh)Tj /F5 1 Tf 2.3205 0 TD [(.)-325.4(Data)-329.7(was)-324.9(normalized)-323.2(to)-325.7(endogenous)]TJ /F6 1 Tf 17.2489 0 TD (b-actin)Tj /F5 1 Tf 3.0919 0 TD (RNA.)Tj /F7 1 Tf 2.8327 0 TD (\(Ac\))Tj /F5 1 Tf 2.1814 0 TD [(Northern)-327.4(blot)-327.3(analysis)-326.4(of)-325.7(RNA)-321.8(isolated)-330.2(from)-324(24)-169.7(hpf)-321.4(embryos)]TJ -27.6754 -1.1128 TD [(demonstrating)-336.6(expression)-338.8(of)-338.4(the)-332.5(21)-334.1(base)]TJ /F6 1 Tf 18.2353 0 TD (zDisc1)Tj /F5 1 Tf 3.0982 0 TD [(\(antisense\))-342.4(guide)-334.7(strand)-337.2(\()]TJ /F6 1 Tf 11.2927 0 TD [(lane)-335.1(1)]TJ /F5 1 Tf 2.4786 0 TD [(\).)-332.2(The)-335.1(control)-335.4(is)-333.6(a)-334.6(synthetic)]TJ /F6 1 Tf 12.4372 0 TD (zDisc1)Tj /F5 1 Tf 3.1046 0 TD (antisense)Tj -50.6465 -1.1128 TD [(strand)-286.6(DNA,)-283.9(run)-290(in)-285.1(parallel)-283.5(on)-284.4(the)-288.3(Northern)-289.5(blot)-283.1(\()]TJ /F6 1 Tf 22.2377 0 TD [(lane)-284.5(2)]TJ /F5 1 Tf 2.428 0 TD (\).)Tj /F7 1 Tf .8599 0 TD (\(B\))Tj /F5 1 Tf 1.5934 0 TD [(Whole)-286.8(mount)]TJ /F6 1 Tf 6.3419 0 TD [(in)-284.5(situ)-285.7(hybridization)]TJ /F5 1 Tf 8.5423 0 TD [(analysis)-288.4(of)]TJ /F6 1 Tf 4.9572 0 TD (zDisc1)Tj /F5 1 Tf 3.054 0 TD (expression)Tj -50.0142 -1.1128 TD [(in)-240.9(24)-163.4(hpf)-239.2(embryos)-238.7(expressing)]TJ /F6 1 Tf 12.9177 0 TD (YFPh)Tj /F5 1 Tf 2.5545 0 TD [(\(a,)-243.2(a)]TJ /F9 1 Tf 1.8147 0 TD ()Tj /F5 1 Tf .2466 0 TD .2407 Tc [(\)o)240.7(r)]TJ /F6 1 Tf 1.7262 0 TD 0 Tc (zDisc1h)Tj /F5 1 Tf 3.5029 0 TD [(\(b,)-241.8(b)]TJ /F9 1 Tf 1.9158 0 TD ()Tj /F5 1 Tf .2529 0 TD [(\).)-237.3(\(a-b)]TJ /F9 1 Tf 2.5039 0 TD ()Tj /F5 1 Tf .2529 0 TD [(\))-234.4(Lateral)-240.6(views,)-237.7(anterior)-244.9(to)-237.2(the)-237.7(left;)-241.2(\(a)]TJ /F9 1 Tf 16.0096 0 TD ()Tj /F5 1 Tf .2466 0 TD (,b)Tj /F9 1 Tf .803 0 TD ()Tj /F5 1 Tf .2529 0 TD [(\))-234.4(higher)-243(magnication)]TJ -45.0002 -1.1065 TD (views.)Tj /F7 1 Tf 3.0729 0 TD (\(C\))Tj /F5 1 Tf 1.625 0 TD [(Phenotype)-269.9(of)-262.5(shRNA-expressing)-269(embryos)-264(and)]TJ /F6 1 Tf 20.5684 0 TD (zDisc1)Tj /F5 1 Tf 3.035 0 TD [(morphants)-263.4(and)-265.1(mutants.)-259.9(\(a-a)]TJ /F9 1 Tf 12.7407 0 TD ()Tj /F5 1 Tf .4047 0 TD [(\))-259.7(2)0(4)-169.7(hpf)-264.5(control)]TJ /F6 1 Tf 6.8477 0 TD (YFPh)Tj /F5 1 Tf 2.5861 0 TD (embryos)Tj -50.8805 -1.1128 TD [(coinjected)-408.5(with)]TJ /F6 1 Tf 7.1828 0 TD (mcherry)Tj /F5 1 Tf 3.7368 0 TD [(RNA.)-406.9(\(b-b)]TJ /F9 1 Tf 4.6663 0 TD ()Tj /F5 1 Tf .4047 0 TD [(\))-405.1(2)0(4)-169.7(hpf)]TJ /F6 1 Tf 3.7874 0 TD (zDisc1h)Tj /F5 1 Tf 3.6799 0 TD [(embryos)-403.1(coinjected)-408.5(with)]TJ /F6 1 Tf 11.3749 0 TD (mcherry)Tj /F5 1 Tf 3.7368 0 TD [(RNA.)-406.9(\(c-c)]TJ /F9 1 Tf 4.4387 0 TD ()Tj /F5 1 Tf .4047 0 TD [(\))-405.1(2)0(4)-169.7(hpf)]TJ /F6 1 Tf 3.7874 0 TD (zDisc1h)Tj /F5 1 Tf 3.6736 0 TD (embryos)Tj -50.8741 -1.1128 TD [(coinjected)-320(with)]TJ /F6 1 Tf 7.0058 0 TD (hDisc1)Tj /F5 1 Tf 3.1488 0 TD [(RNA.)-318.4(\(d-d)]TJ /F9 1 Tf 4.679 0 TD ()Tj /F5 1 Tf .4047 0 TD [(\))-316.6(2)0(4)-163.4(hpf)-321.4(embryos)-314.6(injected)-317.9(with)]TJ /F6 1 Tf 13.7397 0 TD (zDisc1)Tj /F5 1 Tf 3.0856 0 TD [(MO)-318(together)-315.5(with)-318.8(the)]TJ /F6 1 Tf 9.9902 0 TD (p53)Tj /F5 1 Tf 1.8147 0 TD (MO)Tj 6.7246 0 0 5.9768 473.5558 622.6581 Tm (7)Tj 8.9663 0 0 8.9663 479.792 620.1636 Tm [(\(see)-317.7(Materials)-317.1(and)]TJ -46.5935 -1.1128 TD [(Methods\).)-253.3(\(e-e)]TJ /F9 1 Tf 6.266 0 TD ()Tj /F5 1 Tf .4047 0 TD [(\))-253.4(2)0(4)-163.4(hpf)]TJ /F6 1 Tf 3.4776 0 TD (zDisc1)Tj /F5 1 Tf 6.7246 0 0 5.9768 177.8456 613.1904 Tm (fh291)Tj 8.9663 0 0 8.9663 196.2141 610.1857 Tm [(mutant)-249.4(embryos.)]TJ 6.7246 0 0 5.9768 263.2251 614.2109 Tm (31)Tj 8.9663 0 0 8.9663 272.1826 610.1857 Tm [(\(a,)-249.5(b,)-247.6(c,)-248.8(d,)-246.2(e\),)-249.9(lateral)-247.4(view)-246.1(of)-249.8(the)-250.4(head;)-249(\(a)]TJ /F9 1 Tf 17.2615 0 TD ()Tj /F5 1 Tf .2529 0 TD .2432 Tc (,b)Tj /F9 1 Tf 1.0496 0 TD 0 Tc ()Tj /F5 1 Tf .2466 0 TD .2495 Tc (,c)Tj /F9 1 Tf .9358 0 TD 0 Tc ()Tj /F5 1 Tf .2529 0 TD .2432 Tc (,d)Tj /F9 1 Tf 1.1002 0 TD 0 Tc ()Tj /F5 1 Tf .2466 0 TD .2495 Tc (,e)Tj /F9 1 Tf .9674 0 TD 0 Tc ()Tj /F5 1 Tf .2529 0 TD [(\),)-243.7(dorsal)-251.1(view)-246.1(of)-249.8(the)]TJ -46.0055 -1.1065 TD [(head)-435.8(after)-437.3(brain)-435.2(ventricle)-441.4(injection;)-441.4(\(a)]TJ /F9 1 Tf 17.173 0 TD ()Tj /F5 1 Tf .4047 0 TD .4329 Tc (,b)Tj /F9 1 Tf 1.2393 0 TD 0 Tc ()Tj /F5 1 Tf .4047 0 TD .4329 Tc (,c)Tj /F9 1 Tf 1.1192 0 TD 0 Tc ()Tj /F5 1 Tf .4047 0 TD .4392 Tc (,d)Tj /F9 1 Tf 1.2899 0 TD 0 Tc ()Tj /F5 1 Tf .411 0 TD .4329 Tc (,e)Tj /F9 1 Tf 1.1508 0 TD 0 Tc ()Tj /F5 1 Tf .4047 0 TD [(\),)-439.7(lateral)-430.8(view)-442.1(of)-433.2(whole)-434.9(embryo.)]TJ /F7 1 Tf 15.2509 0 TD (\(D\))Tj /F5 1 Tf 1.9032 0 TD [(Forebrain)-436.6(neurons)-442.8(stained)-434.8(for)]TJ -41.1558 -1.1128 TD [(ac)20.2(et)18.6(yl)17.3(a)13.1(t)0(e)18.6(d)-281.2(t)12.2(u)0(b)19.5(u)0(l)20.4(i)0(n)-272.5(i)0(n)-266.2(3)13.1(6)-157.6(h)0(p)14.2(f)-272.4(em)15.2(b)14.6(r)0(y)13.9(o)0(s)-263.3(e)0(x)16.9(p)0(r)16.9(e)12.8(ss)16.3(in)18.4(g)-276.3(t)12.2(he)-268.8(fo)15.7(ll)18.2(ow)20.9(in)18.4(g)-276.3(R)0(N)21.1(A)0(s)16.3(:)]TJ /F6 1 Tf 30.1223 0 TD [(YF)16.6(Ph)]TJ /F9 1 Tf 2.7378 0 TD (+)Tj /F6 1 Tf .9927 0 TD [(mc)18.9(he)18.8(rr)17.6(y)]TJ /F5 1 Tf 3.5535 0 TD [(RN)21.1(A)-279.6(\()12.2(a\))19(,)]TJ /F6 1 Tf 4.1605 0 TD -.0146 Tc [(zD)-14.6(is)-14.6(c)5(1)-1.5(h)]TJ /F9 1 Tf 3.6483 0 TD 0 Tc (+)Tj /F6 1 Tf .999 0 TD [(mc)18.9(h)13.1(e)0(r)17.6(r)0(y)]TJ /F5 1 Tf 3.5471 0 TD [(\(b)14.1(\))12.2(,)]TJ /F6 1 Tf 1.6945 0 TD [(zD)18.3(is)16.4(c1)20.8(h)]TJ /F9 1 Tf -51.4242 -1.1128 TD (+)Tj /F6 1 Tf .999 0 TD [(hD)18.1(is)16.4(c)14(1)]TJ /F5 1 Tf 3.0666 0 TD [(\()12.2(c)0(\))19.2(,)-281.1(in)18.4(j)14(e)0(c)13.5(t)12.2(ed)-268.5(wi)20.1(th)]TJ /F6 1 Tf 7.4357 0 TD -.0146 Tc [(zD)-14.6(is)-14.6(c)5(1)]TJ /F5 1 Tf 2.9971 0 TD 0 Tc [(MO)-273.7(t)12.2(o)0(g)18.1(e)12.8(th)15.1(e)12.8(r)-285.2(wi)20.1(t)12.2(h)-281.6(t)12.2(h)0(e)]TJ /F6 1 Tf 9.7183 0 TD -.0131 Tc [(p5)-13.1(3)]TJ /F5 1 Tf 1.7578 0 TD 0 Tc [(MO)-267.4(\(d)15.5(\))-278.7(o)0(r)]TJ /F6 1 Tf 4.6916 0 TD [(zD)18.3(is)16.4(c1)]TJ /F5 1 Tf 6.7246 0 0 5.9768 361.5873 584.3337 Tm [(fh)25.6(29)22(1)]TJ 8.9663 0 0 8.9663 379.8991 580.3085 Tm [(\()12.2(e)0(\))18.6(.)-281.1(La)22.8(te)18.6(ra)18.8(l)-281.7(v)0(i)21(e)0(w)17.4(s)0(,)-269.8(a)13.1(nt)15.1(e)12.7(r)0(i)14.7(o)0(r)-269(t)12.2(o)-281(l)0(e)15.5(f)12.2(t.)]TJ /F7 1 Tf 14.6186 0 TD [(\(E)15.5(\))]TJ /F5 1 Tf 1.5112 0 TD [(Mu)18.8(sc)18.4(le)]TJ -51.5823 -1.1065 TD [(segments)-299.1(stained)-295.6(with)-299.9(phalloidin)-293.1(in)-297.8(36)-163.4(hpf)-296.1(embryos)-301.9(expressing)-296.2(the)-294.6(following)-297.9(RNAs:)]TJ /F6 1 Tf 37.4822 0 TD (YFPh)Tj /F9 1 Tf 2.7821 0 TD (+)Tj /F6 1 Tf 1.0117 0 TD (mcherry)Tj /F5 1 Tf 3.6293 0 TD (\(a\),)Tj /F6 1 Tf 1.6882 0 TD (zDisc1h)Tj /F9 1 Tf 3.7305 0 TD (+)Tj /F6 1 Tf 1.0117 0 TD (mcherry)Tj /F5 1 Tf -51.3357 -1.1128 TD (\(b\),)Tj /F6 1 Tf 1.6882 0 TD (zDisc1h)Tj /F9 1 Tf 3.6799 0 TD (+)Tj /F6 1 Tf .9548 0 TD (hDisc1)Tj /F5 1 Tf 3.0729 0 TD [(\(c\),)-243.4(injected)-248.4(with)]TJ /F6 1 Tf 7.4547 0 TD (zDisc1)Tj /F5 1 Tf 3.0097 0 TD [(MO)-242.1(together)-246(with)-243(the)]TJ /F6 1 Tf 9.6867 0 TD (p53)Tj /F5 1 Tf 1.7451 0 TD [(MO)-235.8(\(d\))-244.2(or)]TJ /F6 1 Tf 4.6157 0 TD (zDisc1)Tj /F5 1 Tf 6.7246 0 0 5.9768 408.8125 564.3778 Tm (fh291)Tj 8.9663 0 0 8.9663 427.1243 560.4093 Tm [(\(e\).)-244(Shape)-242.9(of)-237.2(muscle)-243.2(segments)-248.5(is)]TJ -40.7196 -1.1128 TD [(indicated)-312.8(by)]TJ /F6 1 Tf 5.7981 0 TD [(dotted)-316.1(white)-315.2(lines)]TJ /F5 1 Tf 7.1575 0 TD (.)Tj /F6 1 Tf .5627 0 TD [(Black)-311.9(bracket)]TJ /F5 1 Tf 5.2986 0 TD [(:)-312.7(narrow)-312.2(brain)-315(ventricles;)]TJ /F6 1 Tf 11.4508 0 TD [(black)-318.8(asterisk)]TJ /F5 1 Tf 5.3365 0 TD [(:)-312.7(bent)-311.7(tail;)]TJ /F6 1 Tf 4.7359 0 TD [(black)-312.5(dotted)-316.1(lines)]TJ /F5 1 Tf 6.9678 0 TD [(:)-312.7(region)-311.9(between)]TJ -47.308 -1.1128 TD [(dotted)-433.2(lines)-429(was)-426.1(used)-429.2(for)-427.6(muscle)-432.9(segment)-424.3(analysis)-433.8(by)-426.1(phalloidin)-432.2(staining;)-427.1(ac,)-425.3(anterior)-434.6(commissure;)-431.9(poc,)-423.6(postoptic)-437(com-)]TJ 0 -1.1065 TD [(missure;)-394.3(sot,)-393.2(supraoptic)-397.4(tract;)-395.4(tpc,)-396(tract)-392.5(of)-395.3(posterior)-399.2(commissure;)]TJ /F6 1 Tf 29.547 0 TD [(white)-397.4(asterisk)]TJ /F5 1 Tf 5.6021 0 TD [(:)-388.6(defective)-399.2(supraoptic)-397.4(tract,)-395.4(region)-394.1(between)]TJ -35.1491 -1.1128 TD [(dotted)-338.4(lines)-334.1(was)-331.2(used)-340.7(for)-332.7(muscle)-338.1(segment)-335.7(analysis)-332.7(by)-331.3(phalloidin)-337.4(staining.)]TJ /F11 1 Tf 11.9551 0 0 11.9551 541.7006 727.9935 Tm ()Tj ET 62.022 298.318 486.198 .51022 re f BT /F7 1 Tf 8.9663 0 0 8.9663 62.022 286.4125 Tm [(FIG.)-260.9(4.)]TJ /F5 1 Tf 4.015 0 TD [(Tissue-specic)-265.2(inhibition)-259.6(of)]TJ /F6 1 Tf 12.2348 0 TD (zDisc1)Tj /F5 1 Tf 3.0287 0 TD [(expression.)-265.8(The)-252.9(ORF2654)-256.3(shRNA)-263.2(targeted)-261.3(to)]TJ /F6 1 Tf 19.8287 0 TD (zDisc1)Tj /F5 1 Tf 3.0287 0 TD (\()Tj /F6 1 Tf .3225 0 TD (zDisc1h)Tj /F5 1 Tf 3.2689 0 TD [(\))-259.7(was)-255.4(expressed)-262.5(in)-253.5(I-)]TJ -45.7273 -1.1128 TD [(SceI)-246.4(\(meganuclease\)-derived)-249.4(F0)-244.2(transgenic)-249.9(embryos,)-248(under)-242.3(control)-246.9(of)-249.8(the)]TJ /F6 1 Tf 31.9686 0 TD (mir124)Tj /F5 1 Tf 3.1678 0 TD (or)Tj /F6 1 Tf 1.1697 0 TD (ntl)Tj /F5 1 Tf 1.391 0 TD [(promoter)-246.5(\(see)-248.2(Materials)-247.5(and)-239.8(Methods\).)]TJ /F7 1 Tf -37.6972 -1.1128 TD (\(Aa\))Tj /F5 1 Tf 2.3015 0 TD [(Diagram)-379.7(of)-382.6(the)-389.5(I-SceI-mir124-miR30-GFP-pA)-389.3(vector,)-386.2(where)-384.9(shRNA)-383.4(expression)-389.4(is)-384.2(driven)-384.3(by)-381.9(the)-383.1(CNS-specic)]TJ /F6 1 Tf 49.1606 0 TD (miR124)Tj /F5 1 Tf -51.4622 -1.1065 TD (promoter.)Tj /F7 1 Tf 4.6157 0 TD (\(Ab\))Tj /F5 1 Tf 2.2699 0 TD [(Relative)-258.4(expression)-256.6(of)]TJ /F6 1 Tf 9.8258 0 TD (zDisc1)Tj /F5 1 Tf 3.0224 0 TD [(mRNA)-256.1(after)-247.6(inhibition)-259.6(by)-249.1(shRNA.)-253.5(Expression)-259.7(was)-249.1(quantied)-258.9(by)-249.1(qPCR)-250.4(in)-253.5(24)-169.7(hpf)]TJ -19.7338 -1.1128 TD [(embryos)-308.3(expressing)]TJ /F6 1 Tf 9.0671 0 TD (zDisc1h)Tj /F5 1 Tf 3.5724 0 TD [(relative)-305.5(to)-306.7(embryos)-308.3(expressing)-308.8(a)-303(control)-310.1(shRNA,)]TJ /F6 1 Tf 21.8014 0 TD (YFPh)Tj /F5 1 Tf 2.3205 0 TD [(.)-306.4(Data)-304.4(was)-306(normalized)-304.3(to)-306.7(endogenous)]TJ /F6 1 Tf 17.1288 0 TD (b-)Tj -53.8902 -1.1128 TD (actin)Tj /F5 1 Tf 2.3395 0 TD (RNA.)Tj /F7 1 Tf 2.858 0 TD (\(Ac\))Tj /F5 1 Tf 2.2004 0 TD [(Whole)-356.4(mount)]TJ /F6 1 Tf 6.4683 0 TD [(in)-354.1(situ)-348.9(hybridization)]TJ /F5 1 Tf 8.7446 0 TD [(analysis)-351.6(of)]TJ /F6 1 Tf 5.0836 0 TD (GFP)Tj /F5 1 Tf 2.2257 0 TD [(expression)-351.4(in)-348.4(in)-348.4(48)-169.7(hpf)-353(embryos)-346.2(expressing)]TJ /F6 1 Tf 19.5631 0 TD (zDisc1h.)Tj /F7 1 Tf 3.8696 0 TD (\(B\))Tj /F5 1 Tf -53.3527 -1.1065 TD [(Phenotype)-453.2(of)-452.2(embryos)-453.7(expressing)-454.3(shRNA)-452.9(from)-450.4(the)]TJ /F6 1 Tf 23.8943 0 TD (miR124)Tj /F5 1 Tf 3.6546 0 TD [(promoter.)-451.8(\(a-a)]TJ /F9 1 Tf 6.4557 0 TD ()Tj /F5 1 Tf .411 0 TD [(\))-449.4(2)0(4)-163.4(hpf)-454.1(control)]TJ /F6 1 Tf 7.4041 0 TD (YFPh)Tj /F5 1 Tf 2.7758 0 TD [(embryos.)-450.3(\(b-b)]TJ /F9 1 Tf 6.2407 0 TD ()Tj /F5 1 Tf .4047 0 TD [(\))-449.4(2)0(4)-163.4(hpf)]TJ /F6 1 Tf -51.2409 -1.1128 TD (zDisc1h)Tj /F5 1 Tf 3.5408 0 TD [(embryos.)-273.2(\(a,)-268.5(b\),)-267(lateral)-272.7(view)-271.4(of)-268.8(the)-275.6(head;)-268(\(a)]TJ /F9 1 Tf 18.7221 0 TD ()Tj /F5 1 Tf .2529 0 TD .2685 Tc (,b)Tj /F9 1 Tf 1.0686 0 TD 0 Tc ()Tj /F5 1 Tf .2529 0 TD [(\),)-268.9(dorsal)-270.1(view)-271.4(of)-268.8(the)-275.6(head)-271.4(after)-272.9(brain)-270.8(ventricle)-270.7(injection;)-277.1(\(a)]TJ /F9 1 Tf 25.3675 0 TD ()Tj /F5 1 Tf .4047 0 TD .2685 Tc (,b)Tj /F9 1 Tf 1.0686 0 TD 0 Tc ()Tj /F5 1 Tf .411 0 TD [(\),)-269(lateral)]TJ -51.0891 -1.1128 TD [(view)-284(of)-287.8(whole)-283.1(embryo.)]TJ /F7 1 Tf 10.4644 0 TD (\(C\))Tj /F5 1 Tf 1.644 0 TD [(Differentiated)-294.8(neurons)-284.7(stained)-289.3(for)-282.2(acetylated)-288.2(tubulin)-283.6(in)-285.1(36)-169.7(hpf)-283.4(embryos)-283(expressing)]TJ /F6 1 Tf 36.5148 0 TD (YFPh)Tj /F5 1 Tf 2.605 0 TD [(\(a,)-281.1(b\))-283.1(or)]TJ /F6 1 Tf -51.2282 -1.1128 TD (zDisc1h)Tj /F5 1 Tf 3.5914 0 TD [(\(c,)-325.1(d\))-325.9(under)-324.5(control)-322.7(of)-325.7(the)]TJ /F6 1 Tf 11.8112 0 TD (miR124)Tj /F5 1 Tf 3.5282 0 TD [(promoter.)-325.3(\(a,)-325.4(c\))-322.2(lateral)-323.3(view)-328.3(of)-319.4(the)-326.2(head;)-324.9(\(b,)-324(d\))-319.6(dorsal)-327(view)-321.9(of)-325.7(the)-326.2(hindbrain.)]TJ /F7 1 Tf 34.2702 0 TD (\(D\))Tj /F5 1 Tf -53.201 -1.1065 TD [(Muscle)-326.6(segments)-324.4(stained)-320.9(with)-325.2(phalloidin)-324.7(in)-323.1(36)-163.4(hpf)-327.7(embryos)-320.9(expressing)]TJ /F6 1 Tf 31.7726 0 TD (YFPh)Tj /F5 1 Tf 2.643 0 TD [(\(a\))-322.9(or)]TJ /F6 1 Tf 2.7125 0 TD (zDisc1h)Tj /F5 1 Tf 3.5914 0 TD [(\(b\))-321.5(under)-324.5(control)-329.1(of)-319.4(the)]TJ /F6 1 Tf 10.7426 0 TD (miR124)Tj /F5 1 Tf -51.4622 -1.1128 TD [(promoter.)-306.3(Shape)-299.8(of)-306.7(muscle)-300.1(segments)-305.4(is)-302(indicated)-306.5(by)-299.7(a)]TJ /F6 1 Tf 24.1282 0 TD [(dotted)-303.5(white)-308.9(line.)]TJ /F7 1 Tf 7.303 0 TD (\(Ea\))Tj /F5 1 Tf 2.0486 0 TD [(Diagram)-303.8(of)-300.4(the)-300.9(vector)-307.4(I-SceI-ntl-miR30-CFP-pA,)]TJ -33.4798 -1.1128 TD [(driven)-415.9(by)-413.5(the)-414.7(mesendoderm)-413.7(specic)]TJ /F6 1 Tf 16.9264 0 TD (ntl)Tj /F5 1 Tf 1.5554 0 TD (promoter.)Tj /F7 1 Tf 4.7801 0 TD (\(Eb\))Tj /F5 1 Tf 2.2636 0 TD [(Relative)-416.5(expression)-421(of)]TJ /F6 1 Tf 10.3127 0 TD (zDisc1)Tj /F5 1 Tf 3.1804 0 TD [(mRNA)-414.2(after)-418.4(inhibition)-417.7(by)-413.5(shRNA.)]TJ -39.0187 -1.1128 TD [(Expression)-405.1(was)-394.5(quantied)-404.3(by)-394.5(qPCR)-402.2(in)-398.9(24)-163.4(hpf)-403.6(embryos)-396.8(expressing)-403.7(the)]TJ /F6 1 Tf 31.975 0 TD (zDisc1h)Tj /F5 1 Tf 3.6673 0 TD [(relative)-400.3(to)-395.3(embryos)-403.1(expressing)-403.7(the)-395.8(control)]TJ -35.6423 -1.1065 TD (shRNA,)Tj /F6 1 Tf 3.8317 0 TD (YFPh)Tj /F5 1 Tf 2.3268 0 TD [(.)-331.7(RNA)-334.4(levels)-337.7(were)-337.2(normalized)-335.9(to)-332(endogenous)]TJ /F6 1 Tf 20.6886 0 TD (b-actin)Tj /F5 1 Tf 3.1045 0 TD (RNA.)Tj /F7 1 Tf 2.839 0 TD (\(Ec\))Tj /F5 1 Tf 2.0233 0 TD [(Whole)-337.4(mount)]TJ /F6 1 Tf 6.4367 0 TD [(in)-335.1(situ)-336.3(hybridization)]TJ /F5 1 Tf 8.7003 0 TD [(analysis)-332.7(of)]TJ /F6 1 Tf -49.951 -1.1128 TD (CFP)Tj /F5 1 Tf 2.0992 0 TD [(expression)-275.6(in)-272.5(in)-278.8(24)-163.4(hpf)-277.1(embryos)-276.6(expressing)]TJ /F6 1 Tf 19.1142 0 TD (zDisc1h)Tj /F5 1 Tf 3.5408 0 TD (\()Tj /F7 1 Tf .3225 0 TD (F\))Tj /F5 1 Tf 1.1508 0 TD [(Phenotype)-276.2(of)-275.1(embryos)-276.6(expressing)-277.2(shRNA)-275.9(from)-273.4(the)]TJ /F6 1 Tf 8.9663 0 0 8.9663 500.3148 136.97 Tm (ntl)Tj /F5 1 Tf 1.4227 0 TD (promoter.)Tj -50.3051 -1.1128 TD (\(a-a)Tj /F9 1 Tf 1.644 0 TD ()Tj /F5 1 Tf .411 0 TD [(\))-285(2)0(4)-169.7(hpf)-289.7(control)]TJ /F6 1 Tf 6.9173 0 TD (YFPh)Tj /F5 1 Tf 2.6114 0 TD [(embryos.)-292.2(\(b-b)]TJ /F9 1 Tf 6.0763 0 TD ()Tj /F5 1 Tf .411 0 TD [(\))-285(2)0(4)-169.7(hpf)-289.7(zDisc1h)-286.1(embryos.)-292.2(\(a,)-287.4(b\),)-286(lateral)-291.7(view)-284(of)-294.1(the)-288.3(head;)-286.9(\(a)]TJ /F9 1 Tf 26.1832 0 TD ()Tj /F5 1 Tf .2529 0 TD .2874 Tc (,b)Tj /F9 1 Tf 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1 Tf 19.5252 0 TD (YFP)Tj /F5 1 Tf 2.2067 0 TD [(shRNA)-389.7(ex-)]TJ -21.7318 -1.2266 TD [(pression)-330.4(\()]TJ /F6 1 Tf 4.3249 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(10,)-324.4(100%)-329(normal\).)-327(This)-321.1(tract)-329.2(is)-327.3(similarly)-321.1(thin)-333.2(in)]TJ /F6 1 Tf -5.7602 -1.2203 TD (zDisc1)Tj /F5 1 Tf 3.1488 0 TD [(mutants)-377.1(or)-382.8(morphants)]TJ 6.7246 0 0 5.9768 180.5102 302.3432 Tm (31)Tj 8.9663 0 0 8.9663 190.6015 298.3748 Tm [(\(Fig.)-376.2(3Dd,e,)-382.7(white)-375.6(asterisk\).)]TJ -14.3404 -1.2203 TD [(The)-752.4(axon)-747.6(tract)-746.5(phenotype)-756.7(was)-748.6(prevented)-749.1(by)-748.6(injection)]TJ 0 -1.2266 TD (of)Tj /F6 1 Tf 1.195 0 TD (hDisc1)Tj /F5 1 Tf 3.1615 0 TD [(RNA,)-331(but)-328.7(not)-329.1(by)-331.3(injection)-331(of)]TJ /F6 1 Tf 13.0631 0 TD (mcherry)Tj /F5 1 Tf 3.661 0 TD [(mRNA,)-328.6(con-)]TJ -21.0806 -1.2203 TD [(rming)-404.6(specicity)-414.4(of)-401.6(the)-408.4(shRNA-mediated)-416(phenotype)-409(\(Fig.)]TJ T* [(3Dc\))-481.2(\()]TJ /F6 1 Tf 2.839 0 TD (n)Tj /F9 1 Tf .7145 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(10,)-482.5(90%)-481.2(normal)-481.7(embryos\).)-482.4(Another)-486.2(characteristic)]TJ -4.2743 -1.2203 TD [(phenotype)-326.8(after)]TJ /F6 1 Tf 7.3283 0 TD (zDisc1)Tj /F5 1 Tf 3.0982 0 TD [(LOF)-320.3(is)-321(presence)-328.2(of)-325.7(U-shaped)-326.5(muscle)]TJ -10.4265 -1.2266 TD [(segments,)-327.3(likely)-322.8(linked)-327.6(to)-325.7(abnormal)-327.5(convergence)-329.8(and)-322(exten-)]TJ 0 -1.2203 TD (sion.)Tj 6.7246 0 0 5.9768 80.5039 225.6377 Tm (31)Tj 8.9663 0 0 8.9663 88.8944 221.6693 Tm [(Relative)-182.5(to)]TJ /F6 1 Tf 4.7801 0 TD (YFP)Tj /F5 1 Tf 2.0044 0 TD [(shRNA)-187.4(controls)-184.9(\(Fig.)-180.2(3Ea\))-185.9(\()]TJ /F6 1 Tf 11.6847 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(10,)-185.3(100%)]TJ -22.9016 -1.2203 TD [(normal\),)-270.1(U-shaped)-275.9(muscle)-274.8(segments)-273.8(were)-274(observed)-272(at)-271.9(36)-163.4(hpf)]TJ 0 -1.2267 TD [(after)-380.4(expression)-383.1(of)]TJ /F6 1 Tf 8.6497 0 TD (zDisc1)Tj /F5 1 Tf 3.1488 0 TD [(shRNA,)-380(when)-381.4(segments)-381.3(were)-375.2(vi-)]TJ -11.7986 -1.2203 TD [(sualized)-251.9(by)-242.8(phalloidin)-248.8(staining)-253.5(\(Fig.)-249.7(3Eb\))-247.7(\()]TJ /F6 1 Tf 18.5008 0 TD (n)Tj /F9 1 Tf .7145 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(10,)-248.5(10%)-247.2(normal)]TJ -19.9361 -1.2203 TD [(embryos\).)-374.9(Abnormal)-367.4(muscle)-376(shape)-372.2(was)-369.2(prevented)-376.1(by)-369.2(injec-)]TJ 0 -1.2266 TD [(tion)-237.8(of)]TJ /F6 1 Tf 3.054 0 TD (hDisc1)Tj /F5 1 Tf 3.0666 0 TD [(mRNA)-237.1(together)-239.6(with)-236.6(the)-231.4(shRNA)-237.9(construct,)-239.6(but)]TJ -6.1206 -1.2203 TD [(not)-525.1(by)-527.3(injection)-527(of)]TJ /F6 1 Tf 9.2125 0 TD (mcherry)Tj /F5 1 Tf 3.857 0 TD [(mRNA)-521.7(\(Fig.)-527.9(3Ec\),)-523.7(conrming)]TJ -13.0695 -1.2203 TD [(specicity)-471.3(of)-471.1(the)-471.6(shRNA-mediated)-479.2(phenotype)-472.2(\()]TJ /F6 1 Tf 21.6749 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(10,)-469.8(90%)]TJ -23.1166 -1.2266 TD [(normal)-222.5(embryos\).)-223.1(Similar)-221.8(phenotypes)-226.9(are)-221.4(observed)-227.7(for)]TJ /F6 1 Tf 23.8943 0 TD (zDisc1)Tj /F5 1 Tf -23.8943 -1.2203 TD [(mutant)-281(and)-284.1(morphant)-281(\(Fig.)-281.3(3Ed,e\).)]TJ 6.7246 0 0 5.9768 199.2755 126.9921 Tm (31)Tj 8.9663 0 0 8.9663 72 112.0819 Tm [(Together,)-485.4(the)-484.3(data)-483(demonstrate)-489.5(that)-490.7(shRNAs)-485.8(can)-482.7(effec-)]TJ -1.1128 -1.2203 TD [(tively)-442.6(mediate)]TJ /F6 1 Tf 6.8667 0 TD (zDisc1)Tj /F5 1 Tf 3.212 0 TD [(LOF,)-437(resulting)-442.2(in)-443.2(a)-442.1(brain)-435.2(phenotype)]TJ -10.0787 -1.2266 TD [(indistinguishable)-366(to)-363.6(that)-357.9(seen)-364.9(in)-361(the)-357.8(mutant,)-366.1(with)-356.8(a)-359.9(weaker)]TJ 0 -1.2203 TD [(brain)-270.8(phenotype)-276.2(than)-272.3(seen)-270.1(in)-266.2(the)-275.6(morphant.)-271.3(Muscle)-269.7(segment)]TJ T* [(phenotypes)-252.2(were)-242.4(identical)-250.1(after)-241.3(shRNA)-250.6(expression)-244(to)-249.8(mutant)]TJ 0 -1.2267 TD [(and)-315.7(morphant.)]TJ 6.7246 0 0 5.9768 121.2094 61.285 Tm (31)Tj 8.9663 0 0 8.9663 130.7905 57.2598 Tm [(The)-316.1(rescue)-312.3(of)-313.1(these)-321.1(phenotypes)-315.4(by)-312.3(injected)]TJ /F6 1 Tf 20.3282 74.5598 TD (hDisc1)Tj /F5 1 Tf 3.2247 0 TD [(RNA)-391.3(demonstrates)-402.3(specicity)-389.1(of)-395.3(shRNA)-396(effects)-393.8(and)]TJ -3.2247 -1.2203 TD [(indicates)-393.3(that)-395.9(minimal)-388(off-target)-395.4(effects)-393.8(are)-392.1(associated)-390(with)]TJ /F6 1 Tf 0 -1.2266 TD (zDisc1)Tj /F5 1 Tf 3.054 0 TD [(shRNA)-282.2(expression.)]TJ /F10 1 Tf -3.054 -3.054 TD [(Tissue-specic)-338.9(targeting)-331.8(of)]TJ /F2 1 Tf 12.3044 0 TD (zDisc1)Tj /F10 1 Tf 3.3195 0 TD (expression)Tj /F5 1 Tf -14.5111 -1.8336 TD [(A)-355.5(key)-356.5(use)-363(for)-351.7(shRNAs)-365.7(in)-354.7(the)-357.8(zebrash)-359.5(is)-352.6(to)-357.3(drive)-362(tissue-)]TJ -1.1128 -1.2203 TD [(specic)-342.8(LOF,)-342.2(and)-341(so)-339.2(dene)-338(the)-345.2(regional)-340.2(activities)-341.1(of)-344.7(a)-334.6(gene.)]TJ 0 -1.2266 TD [(Previous)-233(data,)-226.7(using)]TJ /F6 1 Tf 9.1113 0 TD (zDisc1)Tj /F5 1 Tf 2.9971 0 TD [(RNA)-226.9(specically)-227.3(expressed)-230.8(in)-221.9(the)]TJ -12.1084 -1.2203 TD [(CNS)-384.3(in)-380(a)-378.9(M)0(O)-381.2(rescue)-381.8(assay,)-382(suggested)-382.8(that)-383.2(the)-383.1(axonal)-377.6(phe-)]TJ T* [(notype)-600.3(was)-603.1(associated)-598.6(with)]TJ /F6 1 Tf 13.6891 0 TD (zDisc1)Tj /F5 1 Tf 3.3701 0 TD [(activity)-600(in)-594.9(the)-598.1(neur-)]TJ -17.0592 -1.2203 TD (ectoderm.)Tj 6.7246 0 0 5.9768 352.1196 609.2219 Tm (31)Tj 8.9663 0 0 8.9663 361.8708 605.2534 Tm [(In)-340.6(contrast,)-339.6(the)-338.9(muscle)-338.1(segment)-342.1(phenotype)-339.4(was)]TJ -5.444 -1.2266 TD [(associated)-586(with)-578.1(mesendodermal)-581.1(and,)-577.9(less)-575.1(so,)-576.1(with)-578.1(neur-)]TJ 0 -1.2203 TD [(ectodermal)-291.2(activity)-283.8(also)-285.3(required)-285.4(for)-288.5(convergence)-285.6(and)-284.1(exten-)]TJ T* (sion.)Tj 6.7246 0 0 5.9768 331.5968 576.34 Tm (31,34)Tj 8.9663 0 0 8.9663 350.2487 572.3715 Tm [(We)-390.9(therefore)-400.2(tested)-390.7(the)-395.8(tissue-specic)-401.7(activities)-391.7(of)]TJ /F6 1 Tf -4.1478 -1.2266 TD (zDisc1,)Tj /F5 1 Tf 3.4017 0 TD [(by)-375.6(expressing)-378.4(the)]TJ /F6 1 Tf 8.2577 0 TD (zDisc1)Tj /F5 1 Tf 3.1488 0 TD [(ORF2654)-370.2(shRNA)-383.4(from)-374.6(the)]TJ -14.8083 -1.2203 TD (CNS-specic)Tj /F6 1 Tf 5.8993 0 TD (miR124)Tj /F5 1 Tf 3.4902 0 TD (promoter)Tj 6.7246 0 0 5.9768 434.1542 554.3998 Tm (35)Tj 8.9663 0 0 8.9663 443.3951 550.4313 Tm [(or)-288(the)]TJ /F6 1 Tf 2.858 0 TD (ntl)Tj /F5 1 Tf 1.429 0 TD (promoter)Tj 6.7246 0 0 5.9768 518.74 554.3998 Tm (36)Tj 8.9663 0 0 8.9663 527.981 550.4313 Tm [(that)-288.4(is)]TJ -23.9702 -1.2203 TD [(active)-724.2(in)-715.1(the)-724.6(early)-716.2(mesendoderm)-723.5(and)-720.4(axial)-716.4(mesoderm)]TJ 0 -1.2266 TD [(\(Fig.)-281.3(4Aa,)-281.6(4Ea\).)]TJ 1.1128 -1.2203 TD [(After)-407.6(expression)-402(of)]TJ /F6 1 Tf 8.9912 0 TD (zDisc1)Tj /F5 1 Tf 3.1741 0 TD [(shRNA)-402.3(from)-406.2(the)]TJ /F6 1 Tf 7.9416 0 TD (miR124)Tj /F5 1 Tf 3.6041 0 TD (pro-)Tj -24.8237 -1.2203 TD (moter,)Tj /F6 1 Tf 3.0413 0 TD (zDisc1)Tj /F5 1 Tf 2.9654 0 TD [(mRNA)-199.2(levels)-198.6(decreased)-196.2(by)-198.5(an)-192.6(average)-200.7(of)-192.9(75%)-196.7(at)]TJ -6.0068 -1.2203 TD [(24)-169.7(hpf)-504.7(\(Fig.)-508.9(4Ab\))-505.3(\()]TJ /F6 1 Tf 8.5992 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(3)-499(independent)-514.4(experiments,)-512.6(46)-504.9(total)]TJ -10.0408 -1.2266 TD [(control)-386(and)-379(39)-384.7(total)-379.8(experimental)-385(embryos)-384.1(examined\).)-385.4(GFP)]TJ 0 -1.2203 TD [(expression,)-310.1(reecting)]TJ /F6 1 Tf 9.5982 0 TD (mir124)Tj /F5 1 Tf 3.231 0 TD [(promoter)-297.1(activity,)-305.7(was)-299.6(uniform)]TJ -12.8292 -1.2203 TD [(in)-323.1(the)-319.9(CNS)-321.1(with)-318.8(no)-322.3(detectable)-320.2(ectopic)-322.7(expression)-326.2(\(Fig.)-319.3(4Ac\).)]TJ 0 -1.2266 TD [(Relative)-334.3(to)-325.7(controls)-324.1(expressing)]TJ /F6 1 Tf 13.8851 0 TD (YFP)Tj /F5 1 Tf 2.1435 0 TD [(shRNA)-332.8(from)-324(the)]TJ /F6 1 Tf 7.714 0 TD (mir124)Tj /F5 1 Tf -23.7425 -1.2203 TD [(promoter)-322.4(\(Fig.)-319.3(4Ba-a)]TJ /F9 1 Tf 9.1366 0 TD ()Tj /F5 1 Tf .4047 0 TD .3166 Tc (\)\()Tj /F6 1 Tf .9611 0 TD 0 Tc (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(84,)-318.1(93%)-316.8(normal\),)-314.4(after)-323.5(expression)]TJ -11.9377 -1.2203 TD (of)Tj /F6 1 Tf 1.214 0 TD (zDisc1)Tj /F5 1 Tf 3.1172 0 TD [(shRNA,)-354.7(a)-347.3(brain)-346.7(and)-347.3(weak)-346.9(tail)-348.5(phenotype)-352.1(was)-350.2(ob-)]TJ -4.3312 -1.2266 TD [(served)-283.8(\(Fig.)-275(4Bb-b)]TJ /F9 1 Tf 7.9542 0 TD ()Tj /F5 1 Tf .4047 0 TD .2787 Tc (\)\()Tj /F6 1 Tf .9231 0 TD 0 Tc (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(78,)-280.2(21%)-278.9(normal)-279.4(embryos\).)-280(Forebrain)]TJ -10.7173 -1.2203 TD [(and)-416.9(hindbrain)-416.9(axon)-412.5(tracts)-419.1(were)-413.1(thin)-415.4(when)-419.3(visualized)-414.3(with)]TJ T* (anti-acetylated)Tj /F12 1 Tf 6.9678 0 TD (a)Tj /F5 1 Tf .4995 0 TD [(-tubulin)-511.7(at)-505.8(36)-163.4(hpf)-511(\()]TJ /F6 1 Tf 8.8394 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(10,)-501.5(0%)-507(normal)-507(em-)]TJ -17.7484 -1.2266 TD [(bryos\))-444.1(relative)-438.2(to)-433.2(normal)-437.4(tracts)-438(seen)-440.8(after)]TJ /F6 1 Tf 19.424 0 TD (YFP)Tj /F5 1 Tf 2.2573 0 TD [(shRNA)-440.3(ex-)]TJ -21.6813 -1.2203 TD [(pression)-292.5(\()]TJ /F6 1 Tf 4.2869 0 TD (n)Tj /F9 1 Tf .7145 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(10,)-280.1(100%)-284.7(normal\))-286.2(\(Fig.)-281.3(4Ca-d\).)-290.6(This)-283.1(is)-283.1(similar)-281.6(to)]TJ -5.7222 -1.2203 TD [(the)-288.3(phenotype)-295.2(seen)-282.7(in)-291.5(the)-282(mutant)-293.6(and)-284.1(morphant,)]TJ 6.7246 0 0 5.9768 507.4014 368.107 Tm (31)Tj 8.9663 0 0 8.9663 516.6423 364.1385 Tm [(and)-290.4(after)]TJ -22.7056 -1.2203 TD (ubiquitous)Tj /F6 1 Tf 5.033 0 TD (zDisc1)Tj /F5 1 Tf 3.0729 0 TD [(shRNA)-307.5(expression)-307.2(\(Fig.)-306.6(3\).)-300.1(These)-304.7(data)-306(are)]TJ -8.106 -1.2266 TD [(consistent)-281.4(with)-274.6(the)-275.6(majority)-273.2(of)]TJ /F6 1 Tf 13.6448 0 TD (zDisc1)Tj /F5 1 Tf 3.0413 0 TD [(expression)-281.9(in)-272.5(the)-275.6(brain)]TJ -16.6862 -1.2203 TD [(and)-271.5(spinal)-278.2(cord.)-267.9(In)-271.1(contrast,)-276.4(muscle)-268.5(segments)-273.8(were)-274(normally)]TJ T* [(shaped)-356.2(after)-361.5(neurectodermal)-359.7(LOF)-351.9(\(Fig.)-350.9(4Da,b\))-356(\()]TJ /F6 1 Tf 21.2956 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(10,)-356(100%)]TJ -22.7309 -1.2266 TD [(normal)-329.9(embryos;)-323.8(as)-317(also)-329.6(observed)-322.6(after)]TJ /F6 1 Tf 17.7864 0 TD (YFP)Tj /F5 1 Tf 2.1435 0 TD [(shRNA)-326.5(expres-)]TJ -19.9298 -1.2203 TD (sion:)Tj /F6 1 Tf 2.3458 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(10,)-280.2(100%)-278.4(normal\).)]TJ -2.6683 -1.2203 TD [(Conversely,)-335.9(when)]TJ /F6 1 Tf 8.3146 0 TD (zDisc1)Tj /F5 1 Tf 3.1045 0 TD [(shRNA)-332.8(was)-331.2(expressed)-332(from)-330.3(the)]TJ /F6 1 Tf -12.532 -1.2266 TD (ntl)Tj /F5 1 Tf 1.429 0 TD [(promoter,)-287.4(a)-277.7(50%)-278.9(average)-282.9(decrease)-288(in)-278.8(RNA)-283.8(levels,)-283.7(relative)]TJ -1.429 -1.2203 TD [(to)-344.7(total)]TJ /F6 1 Tf 3.5219 0 TD (b-actin)Tj /F5 1 Tf 2.7694 0 TD [(,)-344.4(was)-343.9(observed)-347.9(at)-341.4(12)-169.7(hpf,)-343.3(when)]TJ /F6 1 Tf 14.2329 0 TD (ntl)Tj /F5 1 Tf 1.4922 0 TD (expression)Tj -22.0164 -1.2203 TD [(is)-548.6(maximal)-540.8(\(Fig.)-546.9(4Eb\))-544.9(\()]TJ /F6 1 Tf 10.9576 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(3)-543.3(independent)-552.3(experiments,)-550.5(51)]TJ -12.3929 -1.2203 TD [(total)-379.8(control)-373.3(and)-372.6(49)-372.1(total)-373.5(experimental)-372.3(embryos)-371.5(examined\).)]TJ 0 -1.2266 TD [(This)-479.1(lower)-476.4(level)-475.5(of)-477.5(knockdown)-480.3(likely)-474.6(reects)-478.8(residual)-474.4(ex-)]TJ 0 -1.2203 TD [(pression)-564.4(of)]TJ /F6 1 Tf 5.659 0 TD (zDisc1)Tj /F5 1 Tf 3.3259 0 TD [(in)-557(the)-560.2(developing)-560(brain)-561.6(that)-560.3(was)-558.9(not)]TJ -8.9849 -1.2203 TD [(targeted)-419.4(by)-419.8(the)]TJ /F6 1 Tf 7.3409 0 TD (ntl)Tj /F5 1 Tf 1.5618 0 TD [(promoter.)-413.8(CFP)]TJ /F6 1 Tf 7.0437 0 TD [(in)-417.3(situ)]TJ /F5 1 Tf 3.1867 0 TD [(hybridization)-417.9(re-)]TJ -19.1331 -1.2267 TD [(vealed)-487.1(expression)-484.2(specically)-480.2(in)-481.1(the)-484.3(notochord,)-483(indicating)]TJ 0 -1.2203 TD (localized)Tj /F6 1 Tf 4.3755 0 TD (ntl)Tj /F5 1 Tf 1.625 0 TD [(promoter)-486.8(activity)-479.9(\(Fig.)-483.7(4Ec\).)-479.4(After)]TJ /F6 1 Tf 16.1171 0 TD (zDisc1)Tj /F5 1 Tf 3.25 0 TD (ex-)Tj -25.3675 -1.2203 TD [(pression)-317.8(from)-305(the)]TJ /F6 1 Tf 8.0933 0 TD (ntl)Tj /F5 1 Tf 1.4543 0 TD [(promoter,)-312.7(brain)-308.7(morphology)-308.2(and)-309.4(axon)]TJ -9.5476 -1.2266 TD [(tracts)-608.7(were)-602.8(indistinguishable)-606.3(from)-602.2(controls)-608.6(\()]TJ /F6 1 Tf 21.049 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(10,)-602.6(100%)]TJ -22.4843 -1.2203 TD [(normal)-323.6(embryos;)-323.8(after)-323.5(expression)-319.8(of)-325.7(the)]TJ /F6 1 Tf 18.0013 0 TD (YFP)Tj /F5 1 Tf 2.1435 0 TD [(shRNA:)-323.1(n)]TJ /F9 1 Tf 4.5588 0 TD (=)Tj /F5 1 Tf .7145 0 TD (10,)Tj -25.4181 -1.2203 TD [(100%)-373.2(normal\))-374.7(\(Fig.)-369.8(4Fa,a)]TJ /F9 1 Tf 11.1852 0 TD ()Tj /F5 1 Tf .2466 0 TD (,b,b)Tj /F9 1 Tf 1.606 0 TD ()Tj /F5 1 Tf .6196 0 TD [(and)-372.6(Fig.)-369.4(4G\).)-368.4(In)-372.2(contrast,)-371.2(the)]TJ -13.6575 -1.2266 TD [(tail)-386.4(was)-388.1(truncated)-387.4(\()]TJ /F6 1 Tf 8.8205 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(63,)-381.3(22%)-386.4(normal)-386.8(embryos\))-384.6(relative)-387.7(to)]TJ -10.2621 -1.2203 TD [(embryos)-409.4(after)]TJ /F6 1 Tf 6.6011 0 TD (YFP)Tj /F5 1 Tf 2.2257 0 TD [(shRNA)-408.7(expression)-408.4(\()]TJ /F6 1 Tf 9.0354 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7208 0 TD [(75,)-400.3(87%)-405.3(normal\))]TJ -19.3039 -1.2203 TD [(\(Fig.)-755.5(4Fa)]TJ /F9 1 Tf 4.268 0 TD ()Tj /F5 1 Tf .4047 0 TD (,b)Tj /F9 1 Tf .803 0 TD ()Tj /F5 1 Tf .4047 0 TD [(\).)-749.5(Consistently,)-761(thin)-756.9(and)-752(U-shaped)-756.4(muscle)]TJ -5.8803 -1.2203 TD [(segments)-463.5(were)-457.4(observed)-461.7(\(Fig.)-458.4(4H\))-455.2(\()]TJ /F6 1 Tf 16.4585 0 TD (n)Tj /F9 1 Tf .7208 0 TD (=)Tj /F5 1 Tf .7145 0 TD [(10,)-457.2(0%)-456.4(normal)-456.4(em-)]TJ -17.8938 -1.2266 TD [(bryos\))-431.4(relative)-425.6(to)-426.9(effects)-431.8(of)]TJ /F6 1 Tf 12.6585 0 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