THE INFLUENCE OF SOME CROSSVEINLESS-LIKE GENES

Transcription

1 THE INFLUENCE OF SOE CROSSVEINLESS-LIKE GENES ON THE CROSSVEINLESS PHENOCOPY SENSITIVITY IN DROSOPHILA ELANOGASTER' J. D. OHLERZ Department of Zoology, Syracuse University, Syracuse, New York, and Oregon State Uniuersity, Corvallis Received September 2, 1964 is true for any character, a phenocopy is a product of genotype-environment Asinteractions. Phenocopy studies provide information, admittedly not on gene action at the primary level, but certainly on gene-controlled processes at derived levels. This paper is a genetical comparison of a crossveinless-like (cvl) strain with a wild-type strain primarily in terms of their responses to a heat shock that induces crossveinless phenocopy. It demonstrates that (1) this cvl line is especially susceptible to the induction of the crossveinless phenocopy, (2) the genetic basis of the phenocopy in this cvl line is probably the same as that of the spontaneous phenotype, and (3) the different regions along the crossvein respond differently to phenocopy induction. ATERIALS AND ETHODS The cvl strain was cvl-6b, a product of direct selection. The phenotypic difference between cvl-6b and wild-type or marker strains depends upon a major allelic difference localized on the X chromosome and polygenic modifiers distributed over all major chromosomes (OHLER 1965). Some lines of cvl-6b have extreme expression, but they do not have enough posterior crossvein to measure the crossveinlessness induced by heat treatment. For this reason a subline of moderate penetrance and expressivity was used in these experiments. The wild-type strain was ILKAN'S highly inbred Oregon-R (Ore-R). The crossveinless phenocopy is induced by heat treating pupae that are about one day old. The details of the response depend upon the treatment and the condition of the pupae (ILKAN 1962b). The following conditions of culture, of aging, and of treating the pupae were kept constant. The larvae grew at 25 C in the standard cornmeal-agar-molasses-moldex medium, supplemented with massive quantities of fresh yeast. From these cultures, white pupae were collected and placed onto the walls of shell vials, which were then plugged with moistened cotton. At the appropriate age, measured in hours following collection, treatments were made at 405 C in a Precision water bath. Rrom collection to treatment the pupae were kept at 23 C; at the end of the treatment they were returned to 25 C to complete development. Controls received identical handling, except that no treatments were made. Transfer from the 23" bath to the 25" incubator was made at 25 hours of age for the controls. Survival of the pupae was quite satisfactory; more than 90 percent of the flies emerged as adults whether untreated or treated for 20 minutes. I 'Technical and clerical assistance were provided through Public Health Service Grant G to DR. ROGER ILKAN. This work was done during the tenure of a sabbatical leave from Oregon State University and of a Public Health Service Traineeship under the aegis of Syracuse University. Present address: Department of Zoology, Oregon State University, Corvallis. Genetics 51 : arch 1965.

2 330 J. D. OHLER For each adult fly that emerged, its posterior crossveins were described according to the position of break, if any, and its estimated amount. The quantitative estimate is in units based upon fifths of crossvein length. Summing the missing fractions of both posterior crossveins yields a rating (rl0) for the fly. This rating is similar to ILKAN S, except that his, based on sixths of the crossvein length, has a maximum value of 12. Breaks in the anterior crossvein were not scored. In order to have sufficient numbers of pupae, the cross3s rcquired larg- numbers of parents. In each case 100 to 200 virgins and males were distributed among three to six bottles. Frequent subculturing minimized the crowding effects. The presumptive virgins were pre-aged for two or three days in cultures separated from the males. Failure of the eggs to hatch in these cultures confirmed the virginity of the females and thus the successful control of mating. The inbred nature of Oregon-R insures the uniformity of its contribution to the progeny. The cvl-6b line was homozygous for the major gene of the X, but probably not for autosomal modifiers. Problems that could arise out of the uncontrolled genotypic differences are of two sorts. The first is that of chance genetic variation between experiments. Since the number of parents in each cross was so large, chance differences in parental modifier frequencies were probably small. The second problem is the possibility of a regular bias introduced by phenotypic selection of parents. To avoid this the parents were collected without reference to their individual phenotypes. The standard practice of writing the female parent first is followed. The hybrid progeny are designated, for example, as F, OR/6b with the original female parent preceding the slant. The four backcrosses to cvl-6b are distinguished as female backcrosses -F, females x parental males, and male backcrosses -parental females x F, males. The backcross progenies are designated by the notations Bc OR/6b 0 and Bc 6b/OR 9 for the two female backcrosses and by the notations Bc OR/6b 8 and Bc 6b/OR 8 for the two male backcrosses. Backcrosses to Oregon-R were not made. RESULTS The results are presented in three parts. First, quantitative responses of cvl-6b and F, OR/6b are described with the emphasis upon age-response studies. Next, the genetic basis of the phenocopy response is analyzed and compared with that of the spontaneous cvl. Finally, qualitatively different responses along the length of the crossvein are recognized in certain genotypes. The quantitative responses: The untreated cvl-6b flies developed crossveinlessness with mean ratings (rlo) of 3.4 in females and 52.2 in males. Over the range of pupal ages between 16 and 28 hours, this strain responded to 20 minute, 40.5 C heat shocks by developing greater degrees of crossveinlessness. The response was not constant for all ages; rather there was a peak sensitivity at 20 to 21 hours of age. The detailed information is given as age-response curves in Figure 1 (upper pair of lines). ost points represent the mean rating of about 100 animals; a very few points are of only 50 to 70 individual flies. Within this range of ages from 16 through 28 hours, Oregon-R flies are also sensitive to heat shock with two important differences (ILKAN 1962b, 1963). In the first place, Oregon-R responds weakly, if at all, to 20 minute treatments of 40.5 C at any age. The 20 minute treatment, effective on cvl-6b, is subthreshold on Oregon-R. Secondly, given a high enough dose to get response, Oregon-R has two periods of sensitivity, a major one at 24 to 25 hours and a smaller one at 18 to 19 hours of age, with a valley at 20 to 21 hours. Thus, the age when cvl-6b responds maximally is for Oregon-R a period of resistance to heat shock.

3 CROSSVEINLESS PHENOCOPY SENSITIVITY 33 1 \ 0 Q I c 60-0 Y 50- t 4 Dz IO cvl cvl 6 d F, Ore R / cvl F, Ore R I cvl 6 d FIGURE 1.-Age-response curves of cvl-6b (upper curves)and of F, OR/6b (lower curves) treated for 20 minutes at 40.5"C. The ordinate is the rating (rl0) of crossveinlessness; the abscissa is the pupal age at treatment. The ratings of untreated flies are 3.4 in females and 2.2 in males of cvl-6b; the ratings of untreated flies are negligible in the F, OR/6b. Another feature of the 25 hour response of Oregon-R is the linear increase of crossveinlessness with increasing doses above threshold. The shape of the doseresponse curve of cvl-6b is not now known in detail; however, it is likely that the points on the cvl-6b age-response curves of Figure 1 are near maximal response for the ages they represent. For example, another 20 minutes at 25 hours increased the rating from 6.7 to 7.3 for females and that for males from 6.8 to 7.9. The effect of the last 20 minutes was only 1/5 the effect of the first 20 minutes of treatment. The untreated offspring of Oregon-R x cvl-6b had almost no cvl (4 among 106 females; none in 103 males). The lower pair of lines in Figure 1 depicts the hybrid responses to heat shock. First of all, there was a response; one or more of the genes introduced from cvl-6b lowered the threshold. oreover, where cvl-6b had its peak the hybrid had the valley typical of Oregon-R. In the hybrid, however, it was the first peak, not the 25 hour peak, that was greater. A similar result was reported by ILKAN (1961) for hybrids between Oregon-R and his cve strain. The hybrids had a reduced threshold and the earlier peak of the ageresponse curve was larger than the second peak at 25 hours. It is worth emphasis

4 332 J. D. OHLER that the selection of cvl-6b was conducted entirely under normal, physiological conditions of temperature. In making the cve strain selection for sensitivity to heat shock was included. It could be expected that the cve strain would have genes altering the heat sensitive processes in its Oregon-R hybrid. It is apparent that cvl-6b must have similar genes, even though no deliberate attempt to include them was made. The genetics: Two treatment ages were employed in the genetics experiments; a complete age-response study of each generation would have been impractical. The first choice was the treatment at 20 hours; at this age the difference between the responses of Oregon-R and of cvl-6b is large and represents the peak us. valley effect recognized in comparing the age-response curves. The other choice was the treatment at 25 hours, for it is at this age that the crossveinless phenocopy in Oregon-R has been so thoroughly characterized. As already noted, the genetic differences between cvl-6b and wild type for the cvl phenotype are a major genepolygenic modifier system having the major gene on the X chromosome. This system was originally defined by the genetic differences between cvl-6b and non-cvl marker strains for the spontaneous cvl. It is shown below that the same TABLE 1 Penetrance (percent) and expressiuity (rl0) of untreated flies compared with the degree of crossveinlessness in heat treated flies Treated, 20 min, 40.5"C Untreated at 20 hrs at 25 hrs - Progeny Sex n % rlo n '10 n 50 Oregon-R (OR) F cvl-6b (6b) F F, OR/6b F F, 6b/OR F F, OR/6b F F, 6b/OR F Bc OR/6b 8 F ' Bc 6b/OR 8 F Bc OR/6b Q F Bc 6b/OR 0 F < <O.l The standard errors of mean differences lie in the range betweep Ifr0.2 and 40.3; a difference as large as 0.6 between any preselected pair of means is significant at the 5 percent level.

5 CROSSVEINLESS PHENOCOPY SENSITIVITY 333 kind of system is recognized in the genetic differences between cvl-6b and Oregon- R, not only for the spontaneous cvl but also for the phenocopy responses. The crossvein phenotypes of heat treated flies and of their untreated sibs are in Table 1. The results of all crosses in all treatment series are consistent with the expectations of sex-linkage. Critical demonstration of sex-linkage is found among the progenies of the reciprocal crosses and the male backcrosses. The reciprocal cross F, males differed from each other both in their phenotypes and in their genotypes exactly as expected for sex-linkage. Generally where only homozygous or hemizygous cvl-6b X chromosomes were present, spontaneous cvl flies occurred with high penetrance (>70 percent) and moderate expressivity (rlo>l.5). Where only the Oregon-R X chromosomes were present, no spontaneous cvl flies occurred. Where all females were heterozygous, spontaneous cvl flies occurred with low penetrance (2 to 6 percent) and low expressivity (rlo = 0.1). Quite similar results are apparent for the ratings obtained by heat treatment at either treatment age. Of course, the response to the heat treatment is the increase (Ar,,) over the spontaneous level. The Ar's of the critical progenies are plotted in Figure 2. Large responses at both ages occurred where homozygous or hemizygous cvl-6b X chromosomes were present. Little or no response occurred where only the Oregon-R X chromosomes were present. Intermediate responses of heterozygous females occurred in the series of treatments at 20 hours, but not consistently in the 25 hour series. The age difference here is probably another form of the fact that in the hybrids the earlier peak has the greater magnitude. FEALES X-CHROOSOES OR/OR -OR/6b- -6b/6br 1 Arlo 4 '1 U 2 0- Ft FI Bc Bc Cross OR ORX6b 6bXOR OR/6bb 6b 6b/ORd ALES X-CHROOSOE -OR- -6b 1 r 1 " FI FI Be Bc Cross OR ORZ6b 6b 6bTOR OR/6b6 6b/OR6 FIGURE 2.-Crossveinless phenocopy responses of specific X-chromosome genotypes from different sources. The open bars are the responses at 20 hours; shaded bars are the responses at 25 hours. The ordinate (Arlo) is the difference between the treated (20 min, 40.5"C) and the untreated series.

6 334 J. D. OHLER The F, and female backcross progenies, segregating for their X chromosomes, were accordingly halfway between Oregon-R and cvl-6b in expression of the crossveinless phenotype in all series (see Table 1 ). The effect on expressivity of autosomal differences can be tested as follows. The relevant comparisons are first among the flies having only the cvl-6b X chromosome. If it is assumed that Oregon-R alleles, being unselected as modifiers of expressivity, are more likely to lower the rating, then the order of ratings would be among males: P cvl-6b > either male backcross progeny > F, 6b/OR; and among females: P cvl-6b > Bc 6b/OR8. Among these predicted differences, only that between the males of cvl-6b and of the male backcrosses did not obtain (see Table 1). For the rest, statistically significant differences were found as expected in all series, untreated and the two treated. It is meaningful that the increase due to heat shock is constant in these comparisons (Figure 2). The heat shock, therefore, revealed no more variation than that already made apparent by the presence of the cvl-6b major gene. One other relevant comparison, free of X-chromosomal differences, is between the males of Oregon-R and of F, OR/6b. It is not possible here to look for modifiers of expressivity; any variation under normal, physiological conditions is entirely subthreshold. It is possible to look for increased sensitivity to heat shock. If it is assumed that cvl-6b alleles act to cause greater sensitivity, the ratings of males treated as 20 hour pupae should be higher in the hybrids. A difference need not obtain at 25 hours, because of the smaller peak at this age in the hybrids. At 20 hours, a statistically significant increase was induced by the heat shock (Table 1 and Figure 2). It appears that heat shock can be substituted for the cvl-6b X chromosome to reveal the modifier differences in the form of differences in phenocopy sensitivity. Does the X-chromosome difference segregate as if it were a single locus difference, or is it easily reduced by recombination? If there is but a single locus, each X chromosome from a heterozygous female will have one or the other parental effect. If there are two or more loci of accumulative effect, some X chromosomes, being recombinants, will have intermediate effects. Given a single locus, the rating distribution in segregating progeny would be predicted by the rating distributions of the parental chromosomes in nonsegregating progenies. Given recombination in heterozygous females, the distribution would be shifted toward an intermediate mode. Of course, a test of this kind is limited; two or more closely linked loci may not recombine with sufficient frequency to permit measurement of the shift. In the data at hand (treatment at 20 hours) the male backcrosses provide the predicted phenotypic distribution determined by segregating parental chromosomes. This is compared in Table 2 with the actual phenotypic distribution from the female backcrosses. The expectations for the mean effects of autosomal factors are identical in female backcrosses and in male backcrosses. Likewise, the expectations for the mean effects of environmental factors are identical under the conditions of simultaneous handling and treatment of all genotypes. Only if there is frequent crossing over among sex-linked cvl genes should prediction and observation differ. Prediction and observation are very

7 CROSSVEINLESS PHENOCOPY SENSITIVITY 335 TABLE 2 The distributions by rating of the females in the backcross progenies. (Treated 20 min, 40.5 C, 20 hours) Ratings of individuals Female progeny n lo Genotype - (XI ale backcrosses: BcOR/6b$ all OR/6b Bc6b/ORg all 6b/6b Pooled ?h OR/6b, /z 6b/6b Female backcrosses: BcOR/6bQ expect Bc 6b/OR Q ?h 1 /2 6b/6b Pooled J similar (x2 = 9.71 for 10 d.f.; 0.5 > P > 0.25). As a first approximation, then, the segregation of the X-chromosome genes is consistent with assumptions that confine the difference (s) to some small part of the length of the X. The phenotype: All three ways in which the posterior crossvein might be broken can be found among the flies of cvl strains. The break may be at the fourth longitudinal vein (L4 type) or at the fifth (L5 type) or in the middle of the crossvein. Combinations of these also occur so that a variety of crassvein types may be encountered in a sample of cvl flies. Different cvl strains may even be distinguished by the relative frequency of the different types of missing crossvein (OHLER 1965). TIOF~EFF-RESSOVSKY (1934), who described this kind of phenotypic variation in terms of specificity, considered it to be complementary to penetrance and expressivity. In the instance of the cvl-6b stock used here, the L4 type is much less common than that at L5. Both occur with sufficient frequency that the combination (L4, 5 type) is seen in one or both wings of many flies. (The occurrence of breaks in the middle is so rare that they may be ignored.) The phenocopy induced by treating Oregon-R pupae occurs primarily at L4. The age of treatment does make some difference. The phenocopies induced at 20 hours include rare breaks at L5; the phenocopies induced at 25 hours never are the L5 type. The phenocopy response of cvl-6b was also primarily at the L4 end and was differentiated with age of treatment. At 20 hours nearly all the flies had breaks at both longitudinal veins. At 125 hours nearly all had breaks at the L4 end, but only some of the flies had the L5 type of break. The difference in responses at the two ages may be quantitative in any case. The difference between the spontaneous cvl phenotype and the phenocopy is probably qualitative. The application of the latter difference to the analysis of the phenocopy reveals that the quantitative effect (e.g., expressivity) of heat shock is the resultant of two opposing responses. These two responses are differentially expressed adjacent to the two longitudinal veins and both are altered by varying the genotype.

8 336 J. D. OHLER 100- I I I I I U 0 P w 0 ar 40 - I I I I I REGION OF CROSSVEIN =- UNTREATED Bc 0 0- UNTREATED Ec d IN.,25 HR.,40.5'C, Ec P 20 IN.,25 HR.,40.5'C, Be d FIGURE 3.--Crossveinless phenotyp-s, spontaneous (lower linzs) and phenocopy (upper lines), of a backcross progeny with the cvl-6b X chromosome. The abscissa corresponds to the linear crossvein which is arbitrarily subdivided into five regions. Region 5 is adjacent to L4; region 1 is adjacent to L5. The ordinate (percent cvl) is the percent of the wings that have breaks in the indicated region. The differential expression of two opposing responses is amply illustrated by Figure 3. Here the crossvein is imagined to be divided into five numbered regions of equal length. The phenotypes, both treated (at 25 hours) and untreated, of the progeny from cvl-6b x F, 6b/OR are plotted as the percent, among wings, of breaks in each region. Crossveinlessness adjacent to L4 (regions 5,4, 3) was increased by the heat shock. But crossveinlessness at the 5th longitudinal vein (region 1) was decreased. The heat shock, which severely restricted the ability for crossvein formation in one place, actually restored this ability in another region of the crossvein. The effect of genotypic variation is shown in Table 3. Here, males with the cvl-6b X chromosome are described by the percent, among wings, of the two types of crossveinlessness in untreated and treated series. There were in each case a few more (1 to 3 percent of the wings) L4,5 types than expected assuming independence of the two simple types of break; still, in no case is the difference statistically significant. Taken together, the frequences of each type define the distribution. Three different genetic backgrounds are ordered according to the fraction of the autosomal heredity presumed to come from Oregon-R. Though the previously described Arlo of treatment is constant for all genotypes, the autosomal modifier differences do affect the qualitative nature of the induced crossveinless-

9 CROSSVEINLESS PHENOCOPY SENSITIVITY 337 TABLE 3 The types of induced breaks in the crossueirzs of cul-66 X chromosome males ale progeny A. Treatment at 25 hours P cvl-6b ale backcrosses F, 6b/OR B. Treatment at 20 hours P cvl-6b ale backcrosses F, 6b/OR observed induced observed induced observed induced observed induced observed induced observed induced Treated Percent IA. type Percent L.5 type Untreated Treated Untreated ness. To the extent that the heat-induced change in percent is equal to response, the crossvein-limiting response at L4 is increased by autosomal genes from Orgeon-R. At the same time the crossvein-restoring response at L5 is increased, especially after treatment at 25 hours, by autosomal genes from Oregon-R. It can also be seen in Table 3 how the responses change quantitatively with age. DISCUSSION The existence of genetic variation affecting phenocopy responses is not in doubt. Though there are exceptional experiences ( BERTSCHANN 1955 ; ILKAN 1962a), the basic observation has been that different strains give different results ( GOLDSCHIDT 1935; GOLDSCHIDT and FITERNICK 1957; LANDAUER 1957; OHLER 1956; SANG and CDONALD 1954; BURNET and SANG 1964a, b). The logical expectation that such differences are based in heredity has been fulfilled in the repeated observation that selection can be employed to cause strain differences in phenocopy response (see the review by WADDINGTON, 1961 ). Even so, something more in the way of classical identification and localization of segregating alleles is desired, not just to add proof of the genetic basis of phenocopy differences, but to have breeding control of individual allelic differences. Only when it can be stated which individual has which genotype will analysis of genotypeenvironment interaction as the basis of the phenocopy character become critical. For the combined reasons that the variation is subthreshold and that it is probably polygenic ( WADDINGTON 1961), frontal approaches to control of the individual allelic differences have been avoided. The favored approach has been to study the ways in which known genetic differences already under control may affect the phenocopy response. Examples include the SANG and CDONALD (1954) study of the eyeless phenocopy induced by borate, the current BURNET

10 338 J. D. OHLER and SANG (1964a, b) studies of melanotic tumors induced nutritionally, and the LANDAUER (1957) studies of phenocopies induced in chickens by insulin. In the case of the crossveinless phenocopy, ILKAN (personal communication) tested several well known mutants, such as crossveinless (cu, ) and crossveinless-c (cu-c, ) among others, and did not find any with effects on the crossveinless phenocopy response. The cvl-6b major gene is the first instance in which a single allelic difference can be recognized to vary the crossveinless phenocopy response. The coincidence of sex-linkage is the primary evidence that the large phenocopy response and the spontaneous cvl are pleiotropic effects of one major gene. Any demonstration of pleiotropic differences obtained by selection requires that the multiple effects are rigorously localized to a common site. The major gene of spontaneous cvl is localized midway between forked (f, ) and carnation (car, ) (OHLER 1965). In a trial experiment, the sex-linked difference of the phenocopy effect is localized to the right end of the X chromosome (OHLER, unpublished). The latter localization is not more precise since the response phenotypes are overlapping. A single, common locus is likely, but the measure of the coincidence does need refinement. For the smaller effects of the autosomal genes there is yet very little certainty that the spontaneous cvl variation and the phenocopy variation share a single genetic system. Even so, the coincidence in magnitude of effect is difficult to interpret in terms of two separate genetic systems in the autosomes. The total is a polygenic system with cumulative effects in response to heat shock, but with epistatic effects (of the sex-linked wild-type allele) under conditions of physiological temperature. This kind of interpretation of major gene-modifier systems is familiar. There are two selection programs by which spontaneous cvl lines can be produced. The first of these, direct selection, depends upon the occurrence of low frequencies of the crossveinless-like flies under normal conditions. The second program starts with selection of the crossveinless phenocopy. During phenocopy selection the heat shock serves first as an agent for recognizing subthreshold genetic differences. Since the heat shock can recognize differences only at those loci with functions interacting with heat shock, it serves also as an agent for selecting among the available cvl genes. When the program begins with direct selection, any major genes will be fixed early since they have the greatest likelihood to cause crossveinlessness. Then the major gene serves as the dual agent for recognizing subthreshold genetic differences interacting with its function and for selecting among the available cul genes. Thus spontaneous cvl lines produced by phenocopy selection must have genes effecting increased susceptibility to induction of the crossveinless phenocopy. If only few of the cvl determining genes are relevant for the phenocopy, those lines produced by direct selection would rarely have many such genes. Actually, ILKAN ( 1962a, 1964) demonstrated that several lines produced by direct selection had no increased susceptibility to heat shock. However, the directly selected line of this study, cvl-6b7 does have,increased susceptibility to heat shock. Furthermore, hybrids of Oregon-R and cvl-6b show the kind of age-response curve previously thought to be unique for

11 CROSSVEINLESS PHENOCOPY SENSITIVITY 339 hybrids of Oregon-R and a phenocopy selection strain (cve). The major gene of cvl-6b, unlike its counterparts in most other directly selected cvl lines, determines increased susceptibility to the induction of the crossveinless phenocopy. imicking the effects of heat shock, the major gene of cvl-6b served in its place to select other phenocopy sensitive alleles from among the available cvl genes. A final consideration is the meaning of the differential response in the two ends of the crossvein. ost of the principal facts about crossveinless phenocopy induction are explained in a simple scheme devised by ILKAN (1963). This scheme supposes a protein that is essential for crossvein formation and that when treated with high temperature undergoes a series of changes in tertiary structure. The final structure, following prolonged treatment, is one that will not support crossvein formation. Some of the facts are not explained by the scheme in its present form. Particular attention can be called to the valley of insensitivity in the ageresponse curves and to the reversal of effect in the dose-response curves with prolonged 36.5"C treatments. In this second example as the dose (time at 36.5"C) increases, the response increases to a maximum and then falls off. To these is added the restoration of missing crossvein by treatment at 40.5"C. The restoration of missing crossvein (or crossvein-making response) in these experiments is greater when alleles from Oregon-R are present in the genotype. The crossvein-restoring response must be, then, a part of the system of responses that the homozygous Oregon-R has. Is this, perhaps, why the maximum mean rating is 10 (on a scale of 12) following treatment of 25 hour Oregon-R pupae? The valley of insensitivity is present when alleles from Oregon-R are present in the genotype. Is the crossvein-restoring response at 25 hours the same phenomenon, in quantitatively diminished form, as the valley of insensitivity at 21 hours? Is the crossveinrestoring response related to the reversal of effect with 36.5"C? These questions about the phenocopy process now arise out of the recognition of the two opposing responses to heat shock; similarly, a question about the effect of the cvl genes now arises. How is the lower threshold of cvl-6b obtained-by reduction in concentration of the heat sensitive protein or by reduction of the opposing response to heat shock or by something else? Speculation and detailed explanation are yet premature. Anyway, the simplicity of the posterior crossvein is deceiving. This short, thin tube must have a developmental history of considerable complexity, dependent as it is upon diverse gene-controlled processes that show opposite responses to a single environmental influence. The technical assistance of SHARON ABRAS, the graphical assistance of ALLEN AYRES, and the clerical assistance of AREN BROWN are gratefully acknowledged. I want to thank DR. ROGER ILKAN for his advice and for criticism of the paper. SUARY A crossveinless-like strain (cvl-6b) was examined for its responses to heat shock of the kind that induces crossveinless phenocopy in Oregon-R wild type. The cvl-6b strain has a lower response threshold than Oregon-R; the F, hybrids

12 1964b 340 J. D. OHLER have an intermediate threshold. Within the common temperature effective period, the cvl-6b strain has a single peaked age-response curve with maximal response at a time corresponding to the valley in the two peaked age-response curve of Oregon-R. The F, hybrids have a two peaked age-response curve with B larger first peak than that of Oregon-R. The genetic analysis of subsequent generations suggests strongly that the genetic differences of phenocopy expression are the same as the genetic differences of the spontaneous expression.-when treated and untreated phenotypes of cvl-6b are compared, the spontaneous expression is predominantly at the fifth longitudinal vein, but the phenocopy expression is at the fourth longitudinal vein with normal crossvein at L5. It may be that there are two opposing responses to heat shock differentially expressed at the two ends of the crossvein. LITERATURE CITED BERTSCHANN,., 1955 Versuche zur phanokopierenden Wirkung von Chemikalien (nitrogenmustard U.A.) bei Drosophila melanogaster. Z. Ind. Abst. Vererb. 87: BURNET, B., and J. H. SANG, 1964a Physiological genetics of melanotic tumors in Drosophila melanogaster. 11. The genetic basis of response to tumorigenic treatments in the tuk and tu bw; st su-tu strains. Genetics 49: ~ Physiological genetics of melanotic tumors in Drosophila melanogaster Phenocritical period in relation to tumor formation in the tu bw; st su-tu strain. Genetics 49: GOLDSCHIDT, R. B., 1935 Gen und Ausseneigenschaft (Untersuchen an Drosophila) I. Z. Ind. Abst. Vererb. 69 : GOLDSCHIDT, R. B., and L. K. PITERNICK, 1957 The genetic background of chemically induced phenocopies in Drosophila. I. J. Exptl. Zool. 135: LANDAUER, W., 1957 Phenocopies and genotype, with special reference to sporadically occurring developmental variants. Am. Naturalist 91 : ILKAN, R. D., 1961 The genetic basis of natural variation Developmental lability and evolutionary potential. Genetics 46: a The genetic basis of natural variation. IV. On the natural distribution of cue polygenes of Drosophila melanogaster. Genetics 47: b Temperature effects on day old Drosophila pupae. J. Gen. Physiol. 45: On the mechanism of some temperature effects on Drosophila. J. Gen. Physiol. 46: The genetic basis of natural variation. V. Selection for crossveinless polygenes in new wild strains of Drosophila melanogaster. Genetics 50: OHLER, J. D., 1956 Genotype and phenocspy frequency in Drosophila melanogaster. Genetics 41: Preliminary genetic analysk of crossveinless-like strains of Drosophila melanogaster. Genetics 51 (in press). SANG, J. H., and J.. CDONALD, 1954 Production of phenocopies in Drosophila, using salts, particularly sodium metaborate. J. Genet. 52 : TIOFEEFF-RESSOVSKY, N. W., 1934 Uber den Einfluss des genotypischen ilieus und der Aussenbedingungen auf die Realisation des Genotyps. Genmutation uti (venae transuersae incompletae bei Drosophila funebris. Nachr. Ges. Wissensch. Gottingen, ath.-physik. Kl., Biol., N.F. 1 : WADDINGTON, C. H., 1961 Genetic assimilation. Advan. Genet. 10: