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  • Development of an Isolated, in Vitro C. elegans Gonad PreparationAdam Broslat

    Advisor: Dr. Kevin Strange Professor of Anesthesiology and Pharmacology

  • What are C. elegans ? nematode ~1 mm longhermaphroditiccompletely sequenced genome (97Mb or 19K distinct genes)simple body plan (959 somatic cells)

  • Design ProjectGoal:design an isolated, in vitro C. elegans gonad preparation protocol.

  • Purposes for Projectfor the purpose of characterizing the molecular mechanisms of heterologous cell-to-cell communication using quantitative microscopy This includes: Voltage sensitive dyes, pH sensitive dyes, Ca+ sensitive dyes, etc.

  • Research DescriptionWe recently identified a ClC-type anion channel encoded by clh-3 that is functionally expressed in C. elegans oocytes. CLH-3 is activated uring oocyte meiotic maturation suggesting that the channel plays a role in meiotic cell cycle progression, ovulation, fertilization, and/or early development. Disruption of channel expression by RNA interference has little effect on various reproductive events. However, in worms injected with clh-3 dsRNA, we observed that ovulatory contractions of the gonadal sheath cells were initiated prematurely This suggests that CLH-3 functions in inhibitory signaling pathways that modulate sheath cell contractile activity (Rutledge et al., Curr. Biol.11: 161-170, 2001). Oocytes are coupled to sheath cells by gap junctions (Hall et al., Dev. Biol. 212:101-123, 1999) indicating that the two cell types may communicate via electrical and chemical signals. We postulated that activation of CLH-3 during meiotic maturation depolarizes the oocyte and electrically-coupled sheath cell plasma membranes. We also postulated that depolarization modulates sheath cell contractile activity by regulating calcium influx via receptor-activated calcium channels that are triggered by depletion of IP3-sensitive intracellular calcium stores. To begin testing this model, we have developed an isolated gonad preparation.

  • Project PhasesPhase 1Physical extraction of gonadWhere, when, and howPhase 2Functional buffer development allow normal function Phase 3ImagingStaging setup for gonad positioning and stabilization

  • 1st Phase Micro-dissection procedureThe nematode's gonad will be isolated in such a way not to harm the physiology of the gonad.Gonad operates independently of the worm. Problems:The intestines cloud the view of gonad after dissection. Gonad does not completely remove itself without manipulation.Transport of isolated gonad is difficult.

  • Solutions to DateDissection is made in the scope chamber filled with bufferIncisions made with a modified injection needle (guillotined at red line)Gonad is half extracted through depressurizationThe other half is forced by suction using micropipettes or cutting past spermatheca in uterus

  • 2nd Phase Functional BufferThe worm and/or gonad must be placed in a buffer solution that promotes normal gonad function while being observed.

    Problems:Worms are extremely active in buffer.Buffer allows floating and movement.Buffer evaporates from scope chamber

  • Solutions to Date.1% Tricaine and .01% Tetramasole anesthetic was added chilled buffer for stabilization.Veterinary glue was used on the glass of perfusion chamber to secure gonad, but very hard to use.

  • Buffer Recipes

    Sheet1

    Lockery & GoodmanRichmond & JorgensenDavis et. al.Egg BufferModified

    Methods in Enzym.Nature NeuroscienceJ. of NeuroscienceEgg Buffer

    (mM)(mM)(mM)(mM)(mM)

    NaAcxxx110xxx

    NaCl14523140118118

    KCl5564848

    CaCl216322

    MgCl255122

    Glucose2011xxxxxx15

    HEPES10552510

    pH7.27.27.47.37.3

    mOsm315-325~330xxx335-340335-341

    Notes:extracellular salineAscuris Ringer'sforDent's saline forEgg Buffer usedGonad Buffer

    for neuronselectrophysiologymuscle isolationnow(new)

    experiment

    Sheet2

    Sheet3

  • Worm Response to Buffer Any of the previous listed buffers would sustain function to a point.The worm gonad needs the the salts to mimic the interstitial fluid of the worm.The gonad must also have an energy source . . .this is where the glucose comes in.

  • Actual Response to Buffer

    Sheet1

    Lockery & GoodmanRichmond & JorgensenDavis et. al.Egg BufferModified

    Methods in Enzym.Nature NeuroscienceJ. of NeuroscienceEgg Buffer

    Normal

    Contractionyesyesyesyesyes

    Length of normal

    contractile activity

    Trial 1x38 minutes49 minutes58 minutes96 minutes

    Trial 2x45 minutes49 minutes55 minutes88 minutes

    Trial 3x44 minutes44 minutes64 minutes85 minutes

    Trial 4xxx66 minutes107 minutes

    Notes:extracellular salineAscuris Ringer'sforDent's saline forEgg Buffer usedGonad Buffer

    for neuronselectrophysiologymuscle isolationnow(new)

    experiment

    Sheet2

    Sheet3

  • 3rd Phase - ImagingGonad stabilizationDIC image acquisitionImaging with argon laser confocal microscope (time allowing)Tie the process together and formalize protocol

  • 3rd Phase ProblemsGlue mentioned earlier does not work wellPrevents further slide useImmediately solidifies under liquid bufferAny movement under 63x DIC scope causes focal plane change or field changeProtocol must work for various microscope setups (DIC, laser confocal, etc)

  • 3rd Phase SolutionsUsing similar micro-pipettes as in dissection, gonad can be held on both ends.Pipettes must be mounted on both sides and allow for x,y, and z motion for initial placement to accommodate gonad. Pipette tip must be modified to restrict complete entry of gonad

  • Stage / Rig SetupPipette HolderY- motion Translation stageDampened post X- motion Translation stageZ- motion adjustable platformMagnetic Base