Background:

Zebrafish,ortheteleostfishDaniorerio,isarapidlydevelopingorganismthatisa

popularspeciesforstudyingvertebratedevelopment.CleavageintheZebrafishonlyoccursin

theblastodisc,aregionofcytoplasmintheanimalcapoftheegg.Thistypeofmeroblastic

cleavageiscalleddiscoidal.Celldivisionsarerapidinthezebrafish,withdivisionstaking

aboutfifteenminuteseach.Gastrulationisusuallycompleteafteralittleovertenhoursfrom

fertilization.About24hoursafterfertilization,theembryohasformedmostofitstissueand

organprimordiaanddisplaysthetadpolelikeform.

Thisexperimentwilllookatthedevelopmentofmotorneuronsinzebrafishatthe24

hourstage.Motorneuronsarisefromneuronsattheventrolateralmarginoftheneuraltube.

ThesecellsarefirstsignaledbySonichedgehogfromthenotocordtoinstructthemtobecome

ventralneurons,andlaterinstructedbySonichedgehogfromthefloorplatecellstobecome

motorneuronsinsteadofaninterneuron.Furthermotorneuronspecificationisrequired,

sincetheymustinnervateallthevariouspartsofthebody.Inmammals,motorneuronsare

groupedintothreelargecolumnsaccordingtotheirtarget.MotorneuronsinthecolumnofTerni

(CT)projectintothesympatheticganglia,thoseinthelateralmotorcolumn(LMC)extendto

thelimbmusculature,andthoseofthemedialmotorcolumns(MMC)projecttotheaxial

muscles.Themigrationpatternsandproliferationofthesemotorneuronsisthoughttobe

regulatedbythecell"sagewhenitlastdivides.

Inaddition,thedevelopmentofsomitesina24houroldzebrafishwillalsobe

examined.Somitesgiverisetothecellsthatformthevertebrae,ribsandtheskeletalmuscles

oftheback.Theydeterminethemigrationpathwaysofneuralcrestcellsandspinalnerveaxons.

Theyfirstappearintheanteriorportionofthetrunk,andformasafunctionofthe

developmentalrateoftheembryo.Therefore,itispossIBLetousethenumberofsomites

presenttogaugehowfartheembryohasdeveloped.

Toexaminehowthoroughlythemotorneuronsandsomiteshavedevelopedbythe24

hourstage,thetechniqueofwholemountantibodystainingwillbeused.TheZNP-1antibody

willspecificallybindtotheprimarymotorneurons.SinceZNP-1willnotbindtothesomites,

anotherantibody,F6,willbeusedtobindtothesomiteboundaries.TheZNP-1andF6

treatedembryoswillbetreatedwithasecondaryantibody,FluorescentGoatanti-mouseIgG,

sothatthestainedneuronsandsomitescanbeobservedunderafluorescentmicroscope.

Procedure:

1.Dechorionate24hourzebrafishembryosinthepharyngulastagewith2fineforceps.

2.Fixapproximatelyoftheembryoswith4%,andtheotherhalfin1%paraformaldehydeinPBSforonehour.

3.Washembryos3xin5mlPBStoremovefixative

4.IncubateembryosinPBSwithgoatserumand0.2%saponin

5a.Addtheprimaryantibody,ZNP-1at1/2000tohalfthecellswith4%paraformaldehyde,keeptheotherhalfasacontrol,incubatefor24hours.

5b.Addtheprimaryantibody,F6at1/500tohalfthecellswith1%paraformaldehyde,keeptheotherhalfasacontrol,incubatefor24hours.

6.Washembryoswithseveralchangesof5mlPBStoremoveantibody

7.Incubatewiththesecondaryantibody,FluorescentGoatanti-mouseIgG+for45minutes

8.WashwithseveralchangesofPBS

9.Mountwithdepressionslidesandobservestainedembryosusingthefluorescentmicroscope.

10.Lookforindividualbrightlystainedcellsinthetailregion.

Figure1.

DechorionatedZebrafishembryoatapproximately24hours

Results

Allfourgroupsofembryoswereobservedunderafluorescentmicroscope.Thetrunkregionwasobservedforareasofbrightercells.InpictureA,verticallinesofcellsareslightlybrighterthansurroundingcells.Thisiswherethemotorneuronswilldevelop.InpictureC,weseeevidenceofarowofsomitesdevelopingalongthetrunkregion.Thecontrolsshowlessevidenceofdevelopingmotorneuronsorsomites.

Figure2.

Picturesoffixedzebrafishembryosunderafluorescentmicroscope

A-Trunkregionofa24hourzebrafishembryotreatedwithmotorneuronspecificZNP-1antibodies

B-Trunkregionofacontrol,unstained24hourzebrafishembryo

C-Trunkregionofa24hourzebrafishembryotreatedwithsomiteboundaryspecificF6antibodies

D-Trunkregionofacontrol,unstained24hourzebrafishembryo

DiscussionandConclusion

Thestainingofthemotorneuronsandthesomitesinthedevelopingzebrafishisadifficulttask.Thezebrafishisaquicklydevelopingorganism,usuallyresemblingtheadultzebrafishafteroneday.Theantibodyforthemotorneurons,willtheoreticallybindspecificallytothedevelopingmotorneuroncellsinthetail.However,giventhesmallnumberofcellswhicharepresentatagiventime,theresultingembryodoesnotshowclearsignsofthestainedcells.Inaddition,abiggerfactormaybetheefficiencyoftheauto-fluorescentsecondaryantibodyused.Thecellsofthezebrafishembryooftenexhibitsomeautofluorescencewithoutanystimulation,soitmaybedifficulttodifferentiatethestainedmotorneuronsfromthesurroundingcells,whichalsoappearbrightlystained.

Thesomitesarealsolocatedinthetailregion.Theywilleventuallygiverisetothevertebraeandtheskeletalmusclesintheback.ThecellspecificF6antibodywasusedtostaintheembryoforsomiteboundaries.However,littlestainingisobservablebecauseoftherelativelysimilarappearancebetweenthestainedsomitecellsandsurroundingcells,duetothesecondaryantibodyresultinginstainedcellswhichresembledsurroundingcells.

TheuseofZNP-1andF6coupledwiththesecondaryantibody.FluorescentGoatanti-mouseIgG+,tospecificallystainthemotorneuronsandsomiteswerenotassuccessfulasexpected.Thestainonlyresultedinfewcellsexhibitingasomewhatbrightercolorinthetailregion,andwasnotconclusive.AnalternateprocedureusingahorserADIshperoxidase-conjugatedsecondaryantibody,althoughmoretimeconsuming,resultedinabettersignal-to-noiseratioandclearerstaining.

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