[
Worm Breeder's Gazette,
1982]
We have done an in situ hybridization analysis on dissected male tissues by using a 720bp Xba genomic fragment that is complementary to 15K mRNA as a probe. Hybridization was confined to a specific region of the male gonad in the mid-proximal arm beginning approximately one fourth the distance past the loop region. This is the same region that the sperm-specific protein 15K is first detected by immunocytochemistry.Hybridization was sensitive to RNase pre-treatment and was not observed in spermatids or in any other tissues of the male. DNase pre-treatment had no effect on hybridization. The conditions for in situ hybridization were as follows: 1) Tissues were dissected from adult males in M9 salt solution on gelatinized glass slides and fixed by the addition of 45 acetic acid 2) Tissues were flattened (not squashed) by the pressure of a cleaned coverslip; coverslips were subsequently removed by dipping in liquid nitrogen 3) Post fixation was in 3:1 ethanol: acetic acid 4) Slides were dehydrated to 100% ethanol at -20 C, air dried and baked at 80 C for 2 hrs in a vacuum oven. 5) Hybridization conditions were the same as those used by Thomas ( PNAS 77, 5201-5205, 1980) for the Northern blot. Pre-treatment was in 50% formamide, 5 X SSC, 50mM NaP04 pH 6.5, 250mg/ml sheared E. coli DNA, 0.02% w/v BSA, 0.02% w/v Ficoll, 0.02% w/v polyvinylpyrolidone at 42 C for 1 hr. Hybridization solution consisted of pre-hybridization solution plus 10% dextran sulfate, 2 mg/ml of Poly A and the labeled probe ([3H]p lambda8 1 X 10+E6 cpm/slide; 0.5-1.0 X 10+E7 cpm/ug). Hybridization was carried out overnight at 42 C by adding 35-40ml of hybridization solution to each slide, covering with a cleaned coverslip and sealing with Sanford's Rubber contact cement. 6) Post-Hybridization washes were carried out by first gently removing the coverslip and washing in 2 changes of prehybridization buffer without DNA for 10 minutes each at 42 C followed by four washes in 2 X SSC for five minutes each at room temperature. The slides were then washed twice in 0.1 X SSC at 50 C for 15 minutes each followed by washing in distilled water, washing in 5% TCA at 0 C and finally in distilled water and air dried. Squashing the tissue caused severe tissue destruction and a great increase in background probably due to the leakage of the cellular contents (mRNA?) by the gonad. Pre-treatment with proteinase-K caused an increase in the loss of tissue even after baking at 80 C. Washing in 0.1 X SSC at 50 C after hybridization was necessary to reduce background. Sealing the coverslip with rubber cement was suggested by Dr. W. Jeffery and was necessary to prevent evaporation. Removal of the sealed coverslip after hybridization often caused tissue loss due to the rubbing of the coverslip on the tissue. This was prevented by suspending the coverslip over a single layer of Scotch brand tape surrounding the tissue area.
[
Worm Breeder's Gazette,
1993]
SAMPLE PREPARATION-Egg Preps The Bennett laboratory uses the same protocol Ascaris or Caenorhabditis, although we currently have a lot more experience with Ascaris. We use the hypochlorite solution (40% Chlorox, 50% 1 N NaOH. 10% water) for gravid C. elegans adults or synchronous cultures of Ascaris eggs. When adults have dissolved and eggs have been stripped of their proteinaceous coat, rinse 3-4 times in M9 (or water). Transfer egg pellet to 1.5ml eppendorf tube. Add equal volume of 6:3:1 (methanol:acetic acid:chloroform) to permeabilize vitelline membrane. Invert. Immediately dilute in M9 or water and spin down eggs. Rinse several times. Fix in 4% paraformaldehyde in M9 or PBS (in DEPC water) on ice for 15 min. Rinse 2-3 times. A few embryos should be checked with DAPI at this point to assure they are intact and permeabilized. Mix fixed egg pellet with ~1/3 volume of O.C.T. embedding medium for frozen specimens (Tissue-Tek brand). Freeze the egg mixture on a microtome chuck (or a quarter) which has been pre-chilled on a block of dry ice. These egg blocks are stored at -80 C. until cutting. We have stored blocks for several weeks. SLICING EGGS We set the microtome for 12-16 m sections at -15 C. We fill a slide with sections and let the sections adhere to the slide on a 37 C hot plate for 30 min. After that the slides are fixed two more times in 4% paraformaldehyde (made fresh) in DEPC M9 or PBS + Mg. The first fixation is on ice. The second is at room temperature with the addition of 0.1% deoxycholate and 0.1% Triton-X100. All fixations are done in glass containers which have been baked in the "hot oven"(250 C for >4 hrs.) to remove RNases. Both fixations are for 15 min. The cellular RNA is acetylated in acetic anhydride/triethanolamine solution. (freshly made and used within 30 minutes) at room temp. for 5 min. 100mM Triethanolamine-HCl pH 7.5 250ml 250 ml DEPC-H(2)0 4.64g Triethanolamine-HCL (Sigma T-1502) 560 I 1ON NaOH add 625 l acetic anhydride immediately before adding to the slides Slides are then rinsed 3 times in 1xPBS+Mg or M9 .Slides are dehydrated in 30%,60%,80%,95% ETOH made in DEPC-H(2)0. We air dry them O/N. Store at -80 C until ready for hybridization. TRANSCRIPTION-ANTISENSE The transcription and hybridization protocols are from Dr. Etsuko Wada, courtesy of Dr. Sandra Petersen, UMC. Some comments may be useful. The lack of any cold nucleotide to supplement the hot label makes the reaction suboptimal in terms of molarity, and causes the probe to be very small (probably 50-75nt). However incorporation is often 95% or better and this small probe doesn't need hydrolysis. Dry down 250 uCi [35S]-CTP in speed-vac (CTP may also be used, cold UTP would then be added.) ADD: 1. 2ul 5X Transcription Buffer (we use that which comes with SP6 ,T7 or T3 polymerase) 2. 1 l 100mM DTT (Made fresh) 3. 0.5 l RNasin 40U/ l 4. 2 l 1OmM ATP 5. 2 l 1OmM UTP (or CTP when using hot UTP) 6. 2 l 1OmM GTP 7. 1 l Linearized DNA Template 1 g/ l 8. 0.5 l RNA Polymerase 20U/ l Gently mix. (Tap tube with finger.) Incubate 30min at 37 C. Add 0.5 l Polymerase and incubate another 30min. Add 89.5 l nuclease-free water. Check % incorporation with PEI chromatography paper. ([35S] count in fluor) Add: 1. 5 l 1 M Tris-HCL pH 8 2. 1 l 1M MgCl(2) 3. 0.5 l tRNA 25 g/ l 4. 0.5 l RNasin 40U/ l 5. 2ul DNase I 1U/ l Gently mix. (DNase is very labile. Do not vortex.) Incubate 30min at 37 C. Run Sephadex G-50 spin column to remove unincorporated counts.
[
Worm Breeder's Gazette,
1994]
Of the 14 neuron types in the pharynx, MC is the most important for muscle excitation since when it is killed, pharyngeal pumping rate is reduced 4-5 told. Two mechanisms could explain how MC excites the pharynx, a myogenic mechanism (M) and a neurogenic mechanism (N). In the myogenic mechanism, timing of pumps is controlled by the muscle and MC modulates the excitability of the muscle. In the neurogenic mechanism, timing of pumps is controlled by MC directly. M: The rhythm of the pharynx is clearly myogenic. MC just makes the muscle more excitable. MC is to the pharynx what sympathetic neurons are to the mammalian heart. They make the heart beat faster by modulating pacemaker currents in the muscle. Like the mammalian heart, the pharynx can pump without its nervous system so it must function myogenically. N: Your logic is flawed. The capability for a myogenic rhythm doesn't prove that the rhythm is myogenic when the nervous system is present. The leech heart tube rhythm is an example of a system that can be myogenic when its innervation is severed but is controlled by neuronal activity in the intact animal. In the C. elegans pharynx, it's likely that the myogenic mechanism is just a backup for MC, and that the rhythm is normally neurogenic. The timing of pumps is controlled by excitatory postsynaptic potentials (EPSPs) transmitted by MC. This is like the mammalian motor system where EPSPs transmitted by motor neurons trigger skeletal muscle contractions. M: Doesn't look like I'll convince you with behavioral data. What about elecrophysiology? N: Glad you brought that up! The EPG (electropharyngeogram) consists of four phases: E, P, R, and I. The E-phase and R-phase spikes are caused by pharyngeal muscle depolarization and repolarization respectively and the P-phase spikes are caused by inhibitory synaptic transmission between pharyngeal neuronM3 and the muscle. The I-phase spikes are key here. The I- (for interpump) phase has rare positive spikes that don't correlate with perceptible pharyngeal motion. Since the polarity of these spikes is positive, in the direction of muscle depolarization, they must represent synaptic transmission between MC and the muscle. Indeed, when MC is killed, these rare I-phase spikes are eliminated. Also, the first E-phase transient is greatly reduced or eliminated when you kill MC. This makes perfect sense. The rare I-phase transient is an ineffective MC EPSP, one that failed to trigger a pump, and the I-phase is a combination of an effective MC EPSP and the depolarization of a muscle action potential that it triggered. M: Not convinced. Your reasoning works just as well for a myogenic mechanism--Just replace 'MC" with "muscle pacemaker cell". The rare I-phase transient is an ineffective action potential in a pacemaker muscle cell, one that failed to trigger a pump, and the E-phase is a combination of an effective action potential in the pacemaker muscle cell and the depolarization phase of the synchronous action potentials in other pharyngeal muscle cells. MC just modulates the activity of the muscle pacemaker cell. The only way to convince me that the rhythm is neurogenic is to record from MC and show that its activity correlates with the I-phase spikes. N: That's cheap. You know we can't record from MC. Ah, I almost forgot! What about the EPG ofsnt-1 ,a mutant which lacks synaptotagmin? In this EPG, between the rare pumps, there are periodic bursts of I-phase spikes. MC is necessary and sufficient for these spikes. Since synaptotagmin is expressed in neurons at synapses, these bursts must be caused by the defective synaptic activity of MC. This synaptic transmission is extremely inefficient at triggering pumps. You're very quiet. Are you finally convinced? M: Well, OK. I guess it would be difficult to explain thesnt-1 EPG with a myogenic mechanism. But wouldn't you buy that even with MC intact, a myogenic rhythm is used sometimes? For instance, the EPG of a pharynx that is pumping slowly looks just like the EPG of an MC worm: there are no I-phase spikes and the first E-phase spike is small or absent. I'll use the heart as an analogy again. Usually the heartbeat is initiated in the SA node but sometimes ectopic beats can originate in other parts of the heart. I n this analogy "SA node" = MC, and "other parts of the heart" = pharyngeal muscle. N: I'll buy that without recording from MC, there's no way to rule that out. M: I still have two problems with MC being the pacemaker for the muscle. How do you explain the fact that it synapses on marginal cells and not on muscle cells? And how are the activities of the two MCs coordinated without gap junctions between them? N: Both are good questions. To answer the first, it's possible that marginal cells are gap functioned to muscle cells so that EPSPs in marginal cells are also effective in the muscle cells. Indeed, David Hall saw gap junctions between marginal cells and muscle cells. To answer the second, I could speculate blindly but will hold off until we know more.