[
Worm Breeder's Gazette,
1988]
We have exploited some of the unique features of embryos of the parasitic roundworm, Ascaris des to observe transcriptional patterns in nematode early embryogenesis. In contrast to the free-living nematode Caenorhabditis ble to obtain large synchronous populations of Ascaris embryos at various stages of development. It has been reported in C. elegans that transcription is detectable at approximately the 100-cell stage. [Hecht, et al., (1981). Dev. Biol. 83:374-379]. More recent work in C. elegans suggests that transcription is occurring at or before the 30-cell stage. [Schauer, et al., WBG 10:1, 72-73.]. Our laboratory has recently reported that Ascaris embryos in the 5-6th cleavage stage are actively transcribing. [Dalley, et al., WBG 10:1, 76.]. From staged embryos we have isolated nuclei to determine run-on transcription characteristics. Initial studies performed on nuclei isolated by the method of Dixon et al. [WBG 9:3, 73-74.] resulted in contamination of the embryonic preparations with mitochondria, as seen by microscopic examination after diamidinophenylindole (DAPI) staining, as well as a strong hybridization signal using a mitochondrial probe. Subsequent purification of nuclei on 50% Percoll gradients has yielded about a 100-fold visual reduction in mitochondria, although a hybridization signal can still be detected. Run-on transcription assays were performed on nuclei isolated from staged embryos of 4-8 cell, 24-30 cell, ~60 cell, and 10 day embryos (about 600 cells). Parallel assays were carried out with and without alpha-amanitin present at 1 g/ml a level which inhibits RNA polymerase II activity. The results are shown on Fig. 1 as counts of [3H]-UTP incorporated per 10 l reaction. Total levels of incorporation were typically 10-35 fold over background, which has been subtracted from each of the values shown. The darkened portion of each bar represents the alpha-amanitin sensitive (RNA polymerase II) fraction of total transcription. We consistently detect RNA polymerase II activity as early as the 4-8 cell stage and by the 30 cell stage active mRNA transcription is occurring. When DNA content of preparations was measured by fluorimetric assay the highest level of incorporation per pg DNA was at the 30 cell stage. Similar transcription reactions were carried out with [32P]-UTP, with the labelled RNAs used to probe duplicate dot blots containing cold DNA probes. Results from the 60-cell stage showed no hybridization to the SP6 vector or the Ascaris sperm- specific MSP cDNA. Ascaris ribosomal and actin probes showed significant hybridization (Fig. 2). When the labelling was carried out in the presence of 1 I/ml alpha-amanitin, the actin signal was eliminated, while the ribosomal signal was retained as expected. This shows that we are able to detect specific mRNA transcription occurring in staged embryos. Similar studies are being carried out with each of the mentioned cell stages to establish if specific messages, i.e., actin for now, can be detected at the 4-8 cell stage. We propose that differential library screening using staged early vs. Iate RNA populations will yield transcripts unique to the early embryo. The following two references have been valuable for run-on transcriptions: J. R. Nevins, Methods in Enzymology, (1987), 172:235-241. M. E. Greenberg, Current Protocols in Molecular Biology, (1987), 4. 10.1-4.10. 7
[
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.