Development of an Isolated, in Vitro C. elegans Gonad Preparation

1. Development of an Isolated, in Vitro C. elegans Gonad Preparation Adam Broslat Advisor: Dr. Kevin Strange Professor of Anesthesiology and Pharmacology

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

3. Design Project Goal: design an isolated, in vitro C. elegans gonad preparation protocol.

4. Purposes for Project • for 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.

5. Research Description We 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.

6. Project Phases • Phase 1 • Physical extraction of gonad • Where, when, and how • Phase 2 • Functional buffer development • allow normal function • Phase 3 • Imaging • Staging setup for gonad positioning and stabilization

7. 1st Phase – Micro-dissection procedure • The 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.

8. Solutions to Date • Dissection is made in the scope chamber filled with buffer • Incisions made with a modified injection needle (guillotined at red line) • Gonad is half extracted through depressurization • The other half is forced by suction using micropipettes or cutting past spermatheca in uterus

9. 2nd Phase – Functional Buffer • The 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

10. 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.

11. Buffer Recipes

12. 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.

13. Actual Response to Buffer

14. 3rd Phase - Imaging • Gonad stabilization • DIC image acquisition • Imaging with argon laser confocal microscope (time allowing) • Tie the process together and formalize protocol

15. 3rd Phase Problems • Glue mentioned earlier does not work well • Prevents further slide use • Immediately solidifies under liquid buffer • Any movement under 63x DIC scope causes focal plane change or field change • Protocol must work for various microscope setups (DIC, laser confocal, etc)

16. 3rd Phase Solutions • Using 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

17. Stage / Rig Setup Pipette Holder X- motion Translation stage Y- motion Translation stage Z- motion adjustable platform Dampened post Magnetic Base