Worm Breeder's Gazette 10(3): 19
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have been trying to identify animals in which some cells are XX and others are XO. The question we wanted to address was whether or not the reading of the X/A ratio is cell autonomous. Animals in which the X/A ratio is close to the edge between male and hermaphrodite ( some 3A;2X;Dp animals [Madl and Herman 1979] or 3A;2X animals carrying mutations in dpy-21 or dpy-27 [Hodgkin 1987]) are frequently intersexual, and such intersexes have mixtures of male and female parts, as if different cells or groups of cells can make different assessments of the X/A ratio. On the other hand, P. Schedin and W. Wood have shown that in mosaic intersexes produced in this way, the patches of intestine exhibiting vit expression are not cell clones and are invariably in the anterior intestine, as if there are cell nonautonomous effects in going from X/A ratio to sexual differentiation. All of our attempts at generating XX-XO animals have been variations of the following general scheme. unc-93(e1500)/unc-93(0) III; sup- 10/+ X zygotes are generated by mating. Such animals normally show the Unc-93 (rubber band) phenotype conferred by e1500 (Greenwald and Horvitz 1980), because e1500 is dominant to its null allele; sup-10 is an excellent suppressor of e1500 but it is recessive to sup-10(+). The action of sup-10 is specific to muscle, so that if the sup-10(+) bearing X chromosome were lost at P1, the resulting XX-XO animal ought to show a wild-type phenotype, which is easy to pick out of an Unc-93 background. Those of you who are familiar with some previous work on genetic mosaics produced by somatic loss of free duplications, will here recognize that RKH is seeking entry to Hershey heaven (where you may keep repeating your experiments and they always work); unfortunately, he has not yet succeeded. If the X/A ratio is assessed cell autonomously, we would expect that the XX-XO animals we are looking for would have male gonads, germ line and muscle (all derived from P1) and hermaphrodite tails (derived from AB). We have screened over 50,000 zygotes of the above general genotype and found no non-Unc creatures answering this description (or any other clear intersexual description). Why not? One possibility is that we haven't screened enough animals. The frequency of spontaneous XX-XO gynanders in Drosophila is about 10+E-4, and the frequency of mitotic chromosome loss in yeast is 10+E- 5-10+E-4 per chromosome per cell division. Many Drosophila mutations that affect meiosis lead to enhanced X chromosome loss at the early cleavage divisions (and hence high frequencies of gynanders). And so we have in fact tried to enhance our chances by making use of one or another him mutation, including him-1, him-5, him-10, mn164 and him(it13) (from Ken Kemphues), in most of our experiments. In some cases (him-5, 4) the males used to generate the zygotes were him, as follows (cross 1): unc-93(0); him; sup-10(+)/0 x unc-93( e1500); sup-10 hermaphrodites; here we looked for loss of the paternal X chromosome. In other cases (him-5 again, as well as the other hims), the hermaphrodite parent was him and we looked for loss of the maternal X chromosome (cross 2): unc-93(e1500); sup-10/0 x unc-93(0); him; unc-3 hermaphrodites. We also did some experiments in which the hermaphrodite parent in cross 2 was not him but was treated with gamma rays before mating. A second possibility is that the X/A ratio is not read cell autonomously and the XX-XO animals we want would look either like normal hermaphrodites or like normal males. If they looked like hermaphrodites, however, they would have XO germ lines and would either be sterile or generate lots of XO and OO self progeny (as do XO her-1 animals), and we have seen no such animals. (We recovered rare fertile wild-type hermaphrodites that appeared to be due either to mutation [of either unc-93(e1500) or sup-10(+)] or to the fusion of diplo-X sperm and nullo-X ova produced by meiotic X chromosome nondisjunction [in cross 2, above]). What about the occurrence of non- Unc males? No such animals turned up in cross 1, above, but there were of course many in cross 2, owing to the fusion of haplo-X sperm and nullo-X ova. But in this case, the males should all be Daf (and FITC ) , whereas a male generated by loss of the maternal X chromosome at P1 would be FITC+. We have stained 281 non-Unc males in various experiments, and all were FITC . We did not stain all non-Unc males we saw, often because they crawled off the plate before we got to them (as Daf animals tend to do); overall, we estimate that there were no male-appearing XX-XO gynanders among about 40,000 XX zygotes. A third possibility is that the XX-XO mosaics we sought all died, owing to problems with dosage compensation. In principle, dosage compensation problems could arise in two different ways. First (and perhaps less probable), if the X/A reading were irrevocably assessed before the first cleavage, then the subsequent loss of an X chromosome in the P1 lineage would leave all P1 descendants with a single underexpressing X chromosome (according to this scenario, only animals with ambiguous X/A ratios would show the indecisive choices of sexual differentiation noted in the first paragraph). Second, if dosage compensation were established somewhat later but not cell autonomously according to X/A ratio, then either some XX cells could be overexpressing or some XO cells could be underexpressing those genes that are subject to dosage compensation. We note that on the basis of their work with sdc-1, Villeneuve and Meyer suggested in the last WBG that the X/A ratio may indeed not be assessed cell autonomously. If the XX-XO mosaics we have sought are inviable owing to dosage compensation problems, a conceivable, but difficult, alternative strategy (discussed some time ago with Bill Wood) might be to generate animals with an X/A ratio put slightly over the edge on the hermaphrodite side by a sup-10(+)-bearing free duplication (as, for example, in certain 3A;2X;DP animals); one would then look for mosaics in which the duplication had been lost at P1 and investigate the consequences for sexual differentiation. (The guess in this case would be that dosage compensation would be put in some intermediate mode or that every cell, with roughly the same intermediate X/A ratio, could manage in either the male mode or the hermaphrodite mode.)