Developmental Biology (351L .

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NOTEBOOKSHOULD:1. Haveyournameandthecoursetitleonthefront.2. Anaccuratetableofcontents3. 4. D:1. ofyourlabpartner(s).2. /flowchart.3. eriments.4. htsintoparticularprocessesbasedownyourresults.5. imizetheuseyournotebook.

)cellsatthetwo- onsofNa nalorexternalsourcesofCa2 ngseaurchinembryogenesisat20 ecFusionofspermandeggmembrane20- ‐60secCorticalgranuleexocytosis30- ‐50secVitellinemembranebeginstoraise60- toharden120- ‐135minFirstcelldivision(Cleavage)1stCleavage esofexercises,youwilltesthow[Na oreceptorsontheeggsurface.Normallythe[Na gsisapproximately- fuseswiththeeggsurface,thecytoplasmic[Na tentialtoapproximately econtributionofCa2 lowblocktopolyspermyislong- stheegg,cytosolic[Ca2 calgranulesandplasmamembrane.WavesofCa2 thenpropagateacrosstheegg,beginningatthesiteof

spermentry.ThisfluxofCa2 activatesaseriesofeventssuchaseggcellcyclere- ‐entry,NADP eratureshoveraround20 .Wewillculturetheurchinlarvaeat15 er(ASW)485mM10mM8mMNa- ‐freeASW010mM8mMCa- determinetherelationshipbetween[Na lyoninternalorexternalstoresofCa2 .stanford.edu/group/inquiry2insight/cgi- ‐bin/vu- ‐r1a/vu.php?view ontheleft- echniquedown).Choosevarious[Na esimplyC1V1 olutionsfortesting[Na ]dependenceofthefastblock.Desired[Na ]ASWNa- ‐freeASWEggsNa- ul0ul100ul30ul400ul100mMNa Example:94ul176ul0ul100ul30ul400ul150mMNa Practice:270ul0ul100ul0ul30ul400ul485mMNa 2

hm.ac.uk/research- ‐Na ,andASWminusCa .curation/research/projects/echinoid- beabletoseeKCl- nsWITHOUTeggsorsperm.TubesofASWandNa- ‐freeASWareinthe15 gthecalculatedamountsofASWandNa- ofugetubes,obtain ntoftheroom)into1.0mlofRSW.Mixbyvortexingfor1- nice.(3)Performthe485mM[Na ionmembranes.- nimmediatelypipette25ulontoa3

embraneson 75%offtheeggs,putyourtubeinthe15 (thissecondadditionofspermwon’tchangethe[Na ]concentrationofthepractice485mM[Na yyourtubeonitssideinthe15 rminginvitrofertilizationsin485mM[Na h50mM[Na ].Completestepsa- ‐e(above)foreach[Na ntration.- ‐Collect 0.5mlofNa- ‐freeASWeggsimmediatelybeforeyouneedthem.- llcomebacktoscorethesuccessofthefastblock dshouldnotbecounted.Recordyourresultsasnumberof2- opasttheirfirstcleavage(2- isanormal4- rolsgettothe4- H13- .Whatcanyouconcludeabouttherelationshipbetween[Na lowconcentrationsofNa onofyourresultsinyourlabnotebook.4

externalCa2 .Sperm- ‐eggfusioninitiatesanincreaseincytosolicCa2 omembranesandallowingCa2 a2 (RSWandASW 8mM;cytoplasm 10- ‐4mM;andendoplasmicreticulumstores 2 whichyoushoulddefine.Treatments:(A) 150ulASWeggs 6ulcalcimycin(2mM)(B) ASWcontrol (C) 150ulCa- ‐freeASWeggs 6ulcalcimycin(2mM)(D) Ca- ‐freeASWcontrol - ionbeforemovingontothenexttreatment.(1) Add150uleggsuspensiontoamicrofugetube.(2) exthetubefor1sec.(3) dsofview.Donotcounteggsthathaveexploded.(4) Recordyourdata.(5) exingproceduresothatyoumixfor1secorless.(6) izeandconfirmrepeatability.5

(7) dwateranddrythem.(8) toelicitfertilizationmembraneformation?Chi- inderH- ‐29.YoucanalsoperformtheChi- rewe’lluseforculturingseaurchinembryos:1. Onthefirstnight,removespermwaterusingthenylon- ‐meshcontraptionandreplacewithRSW.2. Ateachtimepoint,remove 530ulofembryosfromthebeaker.Observe water.Replacewith 500ulof100%icecoldMeOH(storedinthe- ‐20 Cfreezer).3. ou’reunsureofthestageoftheembryos.4. Placelabeledtubeinrackin- ‐20 my?6

ingbodyplanorganization,cell- ophilaresearchers–EdLewis,ChristianeNüsslein- bisthree- anismbyexaminingwild- nwild- agesoverthecourseof10- http://flymove.uni- ‐muenster.de/.Atwhichsuppressrecombination25 nstarlarvarequire bblybristlesontheirbacks.

andsealwithwax.TungstenmicroscalpelsCuta willinvestigategeneexpressionpatternsinbothwild- ‐typeandmutantemb

Developmental Biology (351L) ExpectationsandGuidelinesforLabNotebooks WHY LAB NOTEBOOKS? Laboratorynotebooksarediariesforscientists .

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