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Russian Journal of Genetics, Vol. 40, No. 3, 2004, pp. 271281. Translated from Genetika, Vol. 40, No.

3, 2004, pp. 353365. Original Russian Text Copyright 2004 by Chadov, Chadova, Kopyl, Khotskina, Fedorova.

GENERAL GENETICS

Genes Controlling Development: Morphoses, Phenocopies, Dimorphs, and Other Visible Expressions of Mutant Genes
B. F. Chadov, E. V. Chadova, S. A. Kopyl, E. A. Khotskina, and N. B. Fedorova
Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090 Russia; e-mail: chadov@bionet.nsc.ru
Received May 22, 2003

AbstractWe studied facultative dominant lethal mutations obtained earlier in Drosophila melanogaster. In some genotypes, these mutations were expressed as lethals, but in other genotypes they lacked this expression. The mutations were maintained in the following cultures: (1) females Muller-5 heterozygous for the mutation; (2) males crossed to attached-X females; and (3) females and males homozygous for the mutation. During culturing, many mutations were found to give rise to phenotypically abnormal progeny. Generally, these abnormalities were morphoses involving various body parts; they were mostly asymmetric and nonheritable. Maternal and paternal effects in the formation of morphoses were observed. In four cases, dimorphic mutations were recorded: a female homozygous for the mutation had mutant phenotype whereas its male counterpart was phenotypically normal. The mutations were recessive with regard to the norm. New phenotypes behaving as mutations with incomplete penetrance arose during culturing. In cultures of mutant homozygotes phenocopies would appear en masse; they would persist for one or two generations and disappear. One wave of phenocopies succeeded another. Visible phenotypes appeared, which further behaved as ordinary recessive mutations. We concluded that these visible manifestations are characteristic for regulatory mutations controlling ontogeny. Their appearance is explained by the activation of new regulatory scenarios caused by blocking standard regulatory pathways.

INTRODUCTION Any genic mutation is realized only in the process of development. However, only some of these mutations are involved in developmental control. Apparently, these are mutations that concurrently possess two properties: they can arrest development and alter its progression. The former implies a particular importance of the gene for the implementation of ontogeny, the latter conrms the regulatory nature of the gene. These properties at rst glance seem to be mutually exclusive. However, this is not so. In 2000, an attempt was made to nd in Drosophila melanogaster a special kind of mutation, which would be lethal in one genotype and nonlethal in another [1, 2]. The mutations of this type were indeed found and termed facultative dominant lethals. In some genotypes, such mutation behaved as a dominant lethal, i.e., all the progeny bearing this mutation died as early as at the embryonic stage. In other genotypes, this mutation was not lethal, i.e., the progeny were viable [3, 4]. Soon it was found that individuals with various (sometimes severe) developmental malformations occurred among the normal progeny of mutant parents [4, 5]. Thus, the same mutation could either cause lethality or disturb individual development. Mutations having properties predicted for ontogeny-controlling mutations in fact existed.

So far, we have developed three techniques of isolation of facultative (also called conditional) lethals. According to the rst of them, a potential mutation is obtained in the X chromosome of male D. melanogaster and then this male is crossed to yellow females. The mutation is determined by the absence of female progeny [1, 2]. The second technique is used to isolate conditional dominant lethals in autosome 2 of D. melanogaster [6]. The third method, like the rst one, is used for isolating mutations in the X chromosome, but in the reverse order. First, lethal mutations are obtained using the Muller-5 technique. These mutations manifest their lethality in crosses between females In(1)Muller-5/lethal with males In(1)Muller-5 (absence of male lethal carriers in the progeny). Then, out of these lethal mutations, the lethal are selected that do not manifest lethality in crosses of the same females In(1)Muller-5/lethal with males of genotypes different from In(1)Muller-5 [7]. Lethality is the main property of these mutations: they were detected by means of a test for lethality. In the process of their maintenance in culture we have found that their presence is manifested as the appearance of morphoses in the progeny. However, morphoses were not an only manifestation of these mutations. In addition to morphoses, we have found (1) dimorphs, i.e., mutant forms with mutation manifesting only in one sex; (2) forms manifesting as visible mutations with varying penetrance; (3) mass modications; and

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(4) forms manifesting as visible mutations with complete penetrance. This article is devoted to phenotypic description of the visible manifestation of the conditional lethal mutations. MATERIALS AND METHODS Isolation of Mutations Protocol 1. Males of D. melanogaster are exposed to -irradiation and crossed to females carrying attached-X chromosomes. Sons from this cross bear an irradiated X chromosome and a haploid set of irradiated autosomes. Each son is individually crossed to a yellow female. The male that did not produce daughters is supposed to carry an X-chromosomal mutation [13]. Protocol 2. Males of D. melanogaster are exposed to -irradiation and crossed to females carrying inversion Curly in autosome 2. Sons from this cross bearing an irradiated autosome 2 and autosome Curly are individually crossed to a yellow female. The male that did not produce normal (not Curly) daughters and sons is supposed to carry an autosome 2 mutation [3, 6]. Protocol 3. Recessive X-chromosomal lethals are obtained in D. melanogaster using the classic Muller-5 method. The lethals are maintained by crossing In(1)Muller-5/lethal females to In(1)Muller-5 males. These crosses do not yield normal male progeny. In crosses of the females with wild-type males, cultures with appearing normal males are isolated. These cultures contain the sought mutation [7]. Maintenance of Mutations X-chromosomal mutations were maintained in stock using two methods. In the rst method, mutations were carried by a female with a mutant X chromosome (+) and an inverted chromosome Muller-5, B wa. In culture, these females were mated to males bearing a mutant or inverted X chromosome. The progeny that inherited the mutant chromosome from their mother consisted of daughters +/B wa and wild-type sons (+). The progeny that did not inherit the mutant chromosome from their mother consisted of daughters B wa/B wa and sons B wa. In the second method, wild-type males carrying the mutation were mated with females carrying attached-X chromosomes (females C(1)DX, y w f/Y). In these cultures, males inherit the mutant X chromosome paternally (fathers phenotype +) and daughters inherit attached-X chromosomes (mothers phenotype y w f). Mutations in autosome 2 were maintained in cultures of ies that carried mutation in one autosome 2 and complex inversion In(2LR)CyO, in the other. During culturing, we succeeded in isolating some of the X-chromosomal mutations in a homozygous state. In addition to the above methods, these mutations were maintained in a homozygous state in males and females.

Examination of the cultures revealed novel phenotypes [4, 5, 7], which were either stable or unstable. Using a digital video camera, we obtained the pictures of the mutant phenotypes and stored them as computer les. RESULTS Morphoses Morphoses result from developmental disturbances of different degree of severity, which do not prevent eclosion, existence and mating of their carriers; these ies even sometimes produce progeny. Our collection contains several hundreds of colored video pictures of morphoses, some of which are presented in Fig. 1. Morphological defects involved all parts of the y body and sometimes were extremely severe (the absence of half of the head or thorax; one, two, three or four legs; one wing; external genitalia). Some of the new formations were similar to homeotic ones: a doubled arista; the seventh leg; a tarsus on the abdomen; protrusions on eyes, thorax, etc.; melanomalike neoplasms of various localization. The distribution of morphoses over the y body was presented earlier [4]. In contrast to visible mutations, morphoses (1) appear in part of the progeny at a frequency varying from several percent to several tens percent; (2) are not inherited; (3) typically unilateral; (4) often put the individual at the brink of extinction, but even in severe cases may involve satisfactory viability and fertility. In culturing X-chromosomal mutations, two types of progeny appear: those bearing and not bearing the mutant X chromosome. The morphose formation is characterized by matro- and patrocliny. A morphose may appear not only in the progeny that inherited the mutant parental chromosome but also in the progeny that did not. Obviously, the presence of the mutation in a parent is a necessary condition for the morphose formation in the progeny. Maternal effect. Maternal effect appeared in the progeny of females carrying a mutant X chromosome (+) and an inverted chromosome Muller-5, B wa. In the cultures, these females mated to males carrying a mutant or inverted X chromosome. The progeny that received a mutant chromosome from the mother, consisted of daughters +/B wa and wild-type sons (+). The progeny that had not received the mutant chromosome consisted of daughters B wa/B wa and sons B wa. In a group of seven X-chromosomal mutations, obtained in a separate experiment (protocol 1), all mutants produced morphoses in their progeny. Five mutations exhibited maternal effect on the morphose formation. Twenty-six progeny of these mutants (daughters B wa/B wa and sons B wa) showed malformations despite the fact that they did not inherit a mutant X chromosome from their mothers. In a group of 24 mutations, isolated following protocol 3, morphoses were recorded in the progeny of all mutant females Muller-5, B wa/+. Seventy-three (46%)
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Fig. 1. Endogenous morphoses in D. melanogaster: (a) Sack-like protrusion on the median line of the lower thorax part; (b) absent right wing, a necrosis focus at the wing base; (c) additional third metathoracic leg; (d) absent right thorax half; (e) cut right wing; (f) absent femur and tibia part of the left metathoracic leg; (g) absent bristles and hair at the left thorax half, absent left wing; (h) absent four legs, deformed leg and wing; (i) tarsus on the abdomen; (j) circular melanoma on the abdomen; (k) left head half substituted by a modied arista; (l) head split in two; (m) rounded short right wing; (n) enlarged wing with a bubble at the right side; (o) split thorax and disrupted abdominal tergite pattern; (p) additional wing as a protrusion on the right side.

out of 157 progeny with morphoses did not receive a mutant X chromosome from their mothers. They included 30 daughters and 43 sons. Thus, the formation of a morphose in progeny and the presence in it of a mutant chromosome are independent events. The formation of morphoses without inheriting maternally a
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mutant chromosome was recorded in special crosses of mutant females +/Muller-5, B. Paternal effect. A paternal effect was found in cultures of attached-X females (females C(1)DX, y w f/Y) and mutant wild-type males (+). In these cultures, sons
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receive a mutant X chromosome from fathers, and daughters receive attached X chromosome without mutations from mothers. The paternal effect was expressed in the appearance of daughter with morphoses. In the group of seven mutations mentioned above, the paternal effect manifested in four mutations (22 morphoses). The paternal effect was observed also in crosses of mutant males with other females carrying attached-X chromosomes. These females were daughters with morphoses, which did not receive any mutant X chromosomes. In two mutations out of seven, both maternal and paternal effects were observed. In the group of 29 mutations isolated following protocol 1, which produced morphoses in the progeny, a one-time examination of the progeny revealed morphoses in the daughters. As noted above, the daughters did not inherit mutant X chromosomes from their fathers. The experimental data convincingly show that for the mutations examined, the formation of pathological phenotype (morphoses) without the inheritance of a mutant gene is a rule rather than an exception. Dimorphs: Mutation Expressed in One Sex Several mutant-like forms having unusual, sexdependent expression appeared in the progeny of mutants carrying conventional lethals. The mutational nature of these forms is demonstrated by the possibility to isolate them into cultures having 100 percent manifestation and stable inheritance in generations. We have found four such mutations: short-legged, bubbly wing, and two interrupted vein mutations (Fig. 2). (1) short-legged (Fig. 2, top row). Males are phenotypically normal. In females, wings are obliquely cut and dropped at sides. At eclosion of females, the upper and lower blades of their wings are dissociated at the median part and lled with hemolymph (balloons). After hemolymph resorption, circular defects are left on the wing. On both wings of females, longitudinal vein L2 is interrupted (similar to a well-expressed radius incompletus mutation). The tarsus is altered in all six legs having one rather than ve parts. This part ends in two bristles. Females look short-legged because of the absence of four tarsus parts. They are viable and fertile but get easily stuck in the medium. The mutation is not expressed in heterozygous females. It was isolated shortly after nding using protocol 1. In cultures (females Muller-5/mutation males mutation and males Muller-5) homozygous B+ females having this mutant phenotype appeared in addition to heterozygous B/+ females. The mutation is maintained in homozygous state by crossing with phenotypically normal males. The penetrance in females is 100 percent. Immediately after the isolation of the culture, 198 phenotypically normal sons and 168 short-legged daughters in the scored progeny of 366 ies were observed. During a year of culturing, the appearance of one shortlegged male was recorded. This male left no progeny.
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Mutant cultures with phenotypically normal homozygous females were not served. (2) bubbly wing (Fig. 2, bottom row). Males are phenotypically normal. Females have narrow wings with upturned outer edge. At eclosion, balloons lled with hemolymph occur on the median wing parts. After one to two days, they disappear leaving circular defects with impaired venation. A case of the appearance of a male with the bubbly wing phenotype was recorded. We failed to obtain a culture with manifestation of the character in both sexes. The bubbly wing culture is maintained in a homozygous and heterozygous variants. Heterozygous do not exhibit the bubbly wing phenotype. A bubbly wing female appeared in a culture (females Muller-5/mutation males mutation and males Muller-5), which contained homozygous B/+ females with the above phenotype in addition to heterozygous B+ females. In this culture, phenotypically normal females occurred together with the bubbly wing females. The former were used to obtain a sister culture, in which both sexes are phenotypically normal. Processes resulting in formation of a new phenotype occur in the bubbly wing culture. Flies with impaired tergites and short bristles appeared in it (Fig. 2b, bottom row). This phenotype occurs in both sexes. Some ies have small eyes (Fig. 2d, bottom row). The work on xing these new characters in a new derivative culture is now in progress. (3) Interrupted longitudinal vein (mutations 1 and 2). Males are phenotypically normal. All females lack the second longitudinal vein (radius incompletus, ri). The size of the veinless area varies in progeny produced in crosses with different strains but it is stable in the mutant itself. Mutants carrying mutation 1 (Fig. 2b, middle row) display only a short proximal fragment of the vein, whereas in the case of mutation 2, the distal L2 fragment is preserved. The mutations are expressed symmetrically on both wings. They are not expressed in heterozygote. Females with impaired venation (mutation 1) were detected immediately after obtaining homozygotes for a conditional lethal. Out of 1205 progeny, there was 450 ri daughters and 755 ri+ sons in the dimorphic mutation 1 strain immediately after its isolation. In subsequent scores carried out in four generations approximately six months after obtaining the strain, yielded the counts of 126 ri daughters and 278 ri+ sons. Mutation ri no. 1 has a sister strain containing the same lethal but lacking the interrupted longitudinal vein phenotype. All females and males in this strain are phenotypically normal (ri+). During culturing of the original strain Muller-5/mutation 1 (phenotype ri+), two B/+ females with the interrupted longitudinal vein phenotype. They founded a strain, in which both sexes have visible interrupted longitudinal vein phenotype. In one of derivative strains, penetrance of the ri pheno2004

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Fig. 2. Dimorphic mutations in D. melanogaster. Top row: mutation short-legged: (a) at right, a male without visible manifestation of the mutation; at left, a female with the mutant phenotype (the absence of four tarsus members, obliquely cut, bubbly wings); (b) at left, a heterozygous for the mutation female with normal 5-member tarsi; at right, a homozygous for the mutation female with short tarsi; (c) total view of a female homozygous for the mutation. Middle row: mutation interrupted L2 vein: (a) a male without manifestation of the mutation; (b) absent L2 on both sides in a female homozygous for the mutation; (c) reduction of L4 and L5 in a male (a new phenotype). Bottom row: mutation bubbly wings: at left, a male without the manifestation of the mutation; at right, a female with bubbly wings; (b) new phenotype defective tergites, short bristles (male above, female below); (c) new phenotype small eyes, a male).

type is complete; in another, it is lower, i.e., phenotypically normal ies appeared. In the dimorphic strain, males with attenuated vein L4 started to appear; then, this vein disappeared in previously phenotypically normal males (Fig. 2c, middle row). Then we obtained females with the same phenotype. In one of the strains, the combination of phenotypes is as follows: all females have the interrupted longitudinal vein phenotype, and all males, the interrupted longitudinal vein together with the L4 phenotype. In this case, we also observe sexual dimorphism but for the character absence of L4. In the case of dimorphic mutation ri (mutation 2), homozygotes for the lethal had been for a long time phenotypically normal. Then, two ri females appeared among them; they were used to found the second dimorphic ri culture. The expression of the mutation was somewhat different: the gap in the vein was

smaller, and the distal vein fragment was present. Unlike mutation 1, cultures no further phenotypic change was observed in mutation 2. The absence of the mutant phenotype in males was not related to selective death of mutant males in the cultures. The visible mutant manifestation in females was recessive: females bearing the mutation in one X chromosome and inversion Muller-5 in the other, were phenotypically normal. Long-Term Modications Each of the obtained mutations has been maintained in culture in ve to ten vials. Each new generation is scored under a binocular microscope to select parents for the next generation. The lethal mutations obtained were characterized by phenotypic alterations of the modication type. This was clearly seen in homozygous cultures, in which all
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ies have the same normal phenotype. Scoring of each new generation suddenly revealed up to ten ies with altered phenotypes. We observed ies with dark-colored body similar to those carrying the known black mutation; light-colored body similar to yellow-2 phenotype (yellow cuticle, dark bristles), cut Notch-like wings; altered form and order of abdominal tergites (analogous to abnormal abdomen); reduced eyes (analog of eyeless); obliquely cut dumpy-like wings. Waves of the appearance of the brown phenotype (brown instead of red eyes) were observed and massive formation of dark melanoma-like spots. These ies were individually placed into vials to start new derivative cultures. In most cases, the number of ies having the new phenotype increased in the next generation. In derivative strains, the number of individuals with the new phenotype was even higher. The expression level also enhanced. However, after several generations, the number of ies having the new phenotype started to decrease in both the original culture and the derivative strain. Attempts to make the mutation homozygous failed. In next rounds, ies with unusual phenotypes generally ceased to appear. For instance, in the case of melanomas, the penetrance of the character in the derivative strain reached 100 percent. In spite of that, ies with melanoma-like spots disappeared from the cultures in three to four generations. Flies with a special phenotype characteristically appeared at once in several vials. The most enduring of the modications observed was biased sex ratio. In some vials where the so-called recessive sex-linked lethal in the X chromosome obtained by protocol 3 was maintained, males stopped to appear after one of the transfers. The females to males ratio attained 200 : 1 and then, after four to ve generations, returned to 1 : 1. In some derivative strains, however, the sex ratio remained biased: females outnumbered males approximately by a factor of 2. Another remarkable event was the appearance of small males in the cultures. These males were 2- to 2.5-fold smaller in size than the normal males of the same brood. The small males were fertile, but we failed to obtain their culture though the number of their appearance over all cultures was at least six. One wave of the appearance of a particular modication could be succeeded by a wave of another modication, and then by a wave of still other one. Thus, the wave of biased sex-ratio was followed by that of the Notch-like phenotype (cuts at wing tips). The Notchlike phenotype appeared at once in seven vials in different derivative strains maintained for monitoring sexratio change. The Notch-like ies form the progeny were mated to one another. Only a few ies with this phenotype were observed among several hundreds of the progeny. In the next generation, no ies with the Notch-like phenotype were found. The receding Notch wave was followed by massive appearance of males with the yellow-2 phenotype (yelRUSSIAN JOURNAL OF GENETICS Vol. 40 No. 3

low cuticle, dark bristles). About twenty males appeared among several hundreds of the progeny. In the next generation, no ies of either sex with the yellow-2 phenotype were found. The appearance of mutant phenotypes described here as modications differed from that of morphoses. Unlike morphoses, the appearance of a new phenotype occurred massively and the phenotype was precisely reproduced during several generations. Its expression was the same on both the left and right sides of the body. No severe and drastic, morphose-like disturbances were recorded. In general, the appearing phenotypes imitated known mutations. They could be classied as phenocopies [8], but the latter are induced by extraordinary external factors. In our case, they were induced by the presence of a conditional lethal. Taking into account the transmission of the mutant phenotypes in generations, these phenotypic alterations should be referred to as long-term modications. Visible Mutations with Incomplete Penetrance Visible mutations with low penetrance often appeared in the cultures of the lethal mutations. For instance, a new ri mutation arose in the culture that produced the riri+ dimorphic mutation. In contrast to the dimorphic mutation, both males and females had the ri phenotype but some of ies of either sex were phenotypically normal (ri+). The ri+ ies again produced ri+ and ri progeny. In six cultures homozygous for the conditional lethal, we observed other abnormal phenotypes caused by low-penetrance mutations. These visible mutations were introduced in culture. Their phenotypes are inherited according to the rules typical for low-penetrance mutations. Phenotypically they include tergite defects (two abnormal abdomen-like variants), impaired wing shape (two variants), brown eyes (of the brown type), and bubbly wings. Visible Mutations with Complete Penetrance Visible mutations appeared in the cultures at a rate higher than spontaneous but were not specically studied. A derivative strain with such mutation could be easily obtained. DISCUSSION The problem of individual development, which is clearly dened in biological studies, is not as clear when addressed at the genetic level. In a sense, all or nearly all genes are involved in ontogeny because all of them function in the process of individual development. Geneticists classify as ontogenetic genes controlling development, such as homeotic genes [9, 10] or genes of early development [11, 12]. There is yet no specic criterion to distinguish among other genes those controlling development.
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Fig. 3. Genetic model of ontogeny. (a) Genes and signal pathways. The genome of an individual consists of structural genes (small black circles) and regulatory genes of different ranks (large open circles, squares, and sectors). A regulatory ontogenetic gene is represented by a set of cis-alleles (division of symbols into sectors). The genome is activated through a regulated system of signaling pathways (lines between genes, arrows). The signaling pathways are terminated by switching of structural genes. Ontogeny is a process of consecutive switching of regulatory genes of different rank according to the relay principle. A transition from the previous to the next ontogenetic stage involves switching off of regulatory genes functioning at the former stage and structural genes providing the formation of presumptive structures (hatched circles); (b) ontogeny at a latter stage (t4). Switched off genes and inactive signaling pathways are shown by a dotted line. Most structural genes and some regulatory genes with similar time of switching (regulatory genes of stem cells) are still in operation.

A discovery of regulatory genes [13] rst in lower, and then in higher organisms [14], motivated development of ontogenetic schemes in the form of hierarchic systems of regulatory genes. In these schemes, structural genes are presented as completing elements [1519]. However, the deployment of the developmental program occurs in real live structures that require functioning of structural genes at all ontogenetic stages. To depict the process of ontogeny in a systemic way, structural and regulatory genes should be combined in a logical scheme. In our graphic scheme of ontogeny (Fig. 3), structural genes are located at the ends of the regulatory pathways. The main space is occupied by numerous of hierarchically organized regulatory genes. Biological characters are placed beyond the scheme outside the structural genes. The characters are controlled by an array of hierarchically connected regulatory genes, which are ultimately meant to activate in the right place at the right time particular structural genes supplying specic cell families with their products [20]. In the proposed scheme, the development is shown as a relay of activity passing through regulatory genes of different rank. The signal pathways are terminated by structural genes. Structural genes do not pass signals but their activity is also of sequential wavelike character. They could be regarded as developmental genes

both judging from their switching nature and from their impact on development. However, in essence, they do not control development. Mutations studied in the present work affect regulatory genes. The scheme shows why mutations of regulatory developmental genes are lethals with incomplete penetrance. On the one hand, mutations of genes combined in functional chains must arrest whole directions of development, which causes lethality. On the other hand, the lethal effect of the mutations can be suppressed because of the existence of parallel signaling pathways. Our scheme provides for this possibility. The regulatory genes on it are divided in sectors. A possibility of existence of several rather than one link between the genes is stipulated. These multiple links are not depicted in the gure for the reasons of simplicity. The arguments in favor of the complicated structure of regulatory genes (cis-alleles) were discussed earlier [5]. The visible manifestation of lethals is an unexpected property of genes responsible for individual development. The forms of visible manifestation can be arranged according to the degree of reproducibility of the visible phenotype: in the immediate progeny and in generations. The common visible mutations rank rst in this series; mutations with sex-dependent manifestation and visible mutations with varying expression rank second and third, respectively; the fourth place in
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the series is occupied by variations of non-mutation nature: modications and morphoses. These forms of morphological variation have been known to geneticists and described in a similar fashion. The formation of morphoses commonly occurs when a developing organism is exposed to extreme environmental factors [2125]. Visible mutations with low penetrance have been known since classic studies of Timofeev-Ressovsky on D. funebris [2628]. Phenocopies and modications have been described in many organisms including Drosophila [8]. Mutations with clearly dimorphic manifestations, apparently, are rst described in the present work although small sexdependent shifts in mutation expressions have been repeatedly reported. However, the point is that all these events have a common cause. They are all caused by mutations of a particular category: conditional dominant lethals. These mutations affect genes controlling ontogeny. Investigation of visible manifestation of ontogeny genes is still restricted to observation. However, we can fairly conclusively state that each of the mutations have multiple manifestations. Each mutation is expressed at least in three ways: as a (1) dominant lethal, (2) morphose, and (3) no visible manifestation (norm). The rst and third form of manifestation allowed us to detect the mutations. Morphoses in the progeny have been recorded in the overwhelming majority of mutants. Such visible manifestations of mutations as morphoses, modications, and low-penetrance mutations are characterized by low penetrance. Presumably, the main property of ontogeny controlling genes is their ability to assume two states: active and inactive. Since these genes are of a paramount importance for development, they must be present in multiple copies. This conclusion has been already reached earlier, as a result of analysis of mutation lethality, and reected in the regulatory gene model [5]. In the model, regulatory genes are represented as cassettes of alleles in cis-position (cis-alleles) [5]. Chromosome rearrangements and sex can alter the activity of the cis-alleles [3, 5]. The visible gene manifestations conrm the conclusion on the presence of the ontogeny genes in multiple copies and on the activity alternation of the copies (cisalleles) in different individuals of the same species. Bilateral asymmetry in morphose manifestation can be also interpreted as resulting from multiple copies of the ontogeny genes. One cis-allele (e.g., the normal one) can act on the one side of the body while the other allele (e.g., the mutant one) acts on the other side. As a result, a morphose appears at the side of the mutant allele but is absent at the other side of the body. This explanation is in agreement with the general view on the asymmetry mechanism advanced by Astaurov [29]. A strict determination of the regulatory gene order in a signal chain seems to be slacker when selecting a cis-allele of a concrete gene.
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From the theoretical viewpoint, the presence of multiple copies of ontogeny genes facilitates the construction of subprograms of development. Experimental data indicate in Drosophila the existence of two such subprograms: male and female programs of ontogeny. The very fact of existence of dimorphic mutations testies to this. The female ontogenetic subprogram includes implementation of phenotypes short-legged, incomplete longitudinal vein, and bubbly wings. The male subprogram implements the normal variants of these structures. We would like to emphasize that we speak about phenotypic traits in Drosophila that do not have sex distinctions. Because of this, the proposed male and female programs of development are more general than the existing concepts of sex determination and differentiation [30] as a genetic basis of formation of sex differences. We believe that these are separate and independent programs of individual development. The results on dimorphs indicate that the changes in the female subprogram of the mutants were of a permanent nature: each of the Drosophila females has a mutant phenotype. However, no radical switch to another female program occurred. The mutant phenotype was expressed in homozygous females but lacked expression in heterozygous ones. Normally, the female and male developmental programs are invariant: both males and females keep their sexual appearance regardless of the presence of specic mutations. The change of allele functioning in a dimorphic mutation occupies an intermediate position between a normal (structural) gene and a regulatory gene in the ontogenetic program. Figure 4 presents a hypothetical scheme explaining the existence of dimorphic mutations at regulatory genes. Earlier it was suggested that in the normal program of development, high-order regulatory genes function according to the principle of allele exclusion [5]. They are switched off in the opposite homolog (dashed box in the gure). Normally, a signal is transmitted from the cis-allele (d) at regulatory gene A to the cisallele (a) at gene B. In the case of dimorphic mutations, in addition to the normal cis-allele (a), a mutant allele (1) appears, which functions as the normal one. Consequently, there are two variants of manifestation in homozygote (a/a and 1/1) and one variant (1/), in heterozygote. In the dimorphs, all three variants were observed. A regulatory gene in principle works as a structural gene, i.e., both trans-alleles function. For a transition of the dimorphic manifestation of a regulatory gene to a monomorphic one (typical for a regulatory ontogenetic gene) requires next to nothing: suppression of one of the two opposite trans-alleles in the process of female ofr male gametogenesis (formation of a non-alternative program of individual development [4]). A transition of functioning of gene B from cis-allele a to cis-allele b leads to the complete disappearance of the mutant manifestation of the regulatory gene. This phenomenon is specially considered for lethal expression of mutations [5]. If the transition is unstable, a typ2004

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Fig. 4. Putative mechanism of operation of a dimorphic mutation. A and B are regulatory ontogenetic genes of the same rank. The signal (horizontal arrow) passes from gene A to gene B. The downward chain of the regulatory genes of lower ranks leading to the structural genes (black circles) is shown by a vertical arrow. Each of the regulatory genes is represented by a set of cis-alleles (small squares). In the given organism, one cis-allele is functioning. Genes A and B function according to the rule of allele exclusion. Genes of the top homolog are switched off (dotted boxes). Normally, the signal is transmitted along the (A)d(B)a pathway. In the dimorphic mutant, both the normal (B) a and the mutant (B) 1 alleles are active. Thus, three allele combinations (/, 1/, and 1/1) become possible.

ical mutation with incomplete penetrance appears. This was characteristic for some derivatives of mutation ri (incomplete longitudinal vein), which is a classic case of incomplete penetrance of a visible mutation. Judging from their relative frequency, the norm and lethality are the most common forms of manifestation of the mutations studied. Morphoses are rare. The frequencies suggest that the normal development follows a standard pathway upon signal transmission through the chain of regulatory genes. A cis-allele mutation on this pathway arrests development (is lethal). In rare cases, the signal circumvents the hurdle, and the developmental arrest does not happen. However, in this case the development is impaired due to a defect in the pathway going down to the structural genes (Fig. 4, lateral branches of the central pathway). The collection of video images of various Drosophila morphoses that we obtained in screening the mutants attains several hundred. The number of scored morphoses is even higher. However, despite their diversity, the number of morphose types seems to be restricted. Some of them are more common and standard in appearance. One of the standard features is the absence of a body part on one side of the y; half thorax, a wing, leg, haltere, or half tergite may be absent. Apparently, as noted above, blocking the signal on a standard pathway may result in its switching to another pathway. The number of scenarios of abnormal devel-

opment is limited. A mutation in a regulatory developmental gene provokes the expression of hidden developmental scenarios, which were formed in the organism but did not manifest. It sufces to compare the existing variants of wing alterations (wing morphoses) to be condent that they reect denite developmental variants of wing development rather than chaotic distortions of this organ. The morphose formation occurred according to the parental effect rule: the presence of a mutation in a parent is sufcient for the appearance of a morphose in the progeny. The presence of the mutation in the progeny having the corresponding morphose was not obligatory. Earlier, parental effect was reported for lethal expression of mutations [35]. This effect allowed us to formulate a property, which principally distinguishes genes of ontogeny from other regulatory genes. This property is initiation of cell division by ontogeny genes. An elementary ontogenetic event is a transition from one gene controlling ontogeny to another one through cell division [7]. Each step of the gene-to-gene transition of activity is backed up by the formation of cell population of a strictly denite number. Thus is formed and maintained the chain reaction of three interconnected developmental processes: activation of genetic information, augmentation of cell mass, and distribution of the activated information in the cell [7].
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The main result of this study is a discovery of a series of visible manifestations of genes controlling ontogeny. The manifestation forms are diverse and completely dissimilar to the form of mutation expression known from classic literature. It is likely that these are manifestations of regulatory ontogenetic genes, which is primarily indicated by the formation of morphoses. The mechanisms of the formation of each manifestation type are still vague. Genetic research of these mutations is required. ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research, grant no. 01-01-48899. REFERENCES
1. Chadov, B.F., Chadova, E.V., Kopyl, S.A., and Fedorova, N.B., A New Class of Mutations in Drosophila melanogaster, Dokl. Akad. Nauk, 2000, vol. 373, no. 5, pp. 714717. 2. Chadov, B.F., Mutations in the Regulatory Genes of Drosophila melanogaster, Proc. Int. Conf. Biodiversity and Dynamics of Ecosystems in North Eurasia, Novosibirsk, 2000, pp. 1618. 3. Chadov, B.F., Mutations Able to Induce Speciation, Evolyutsionnaya biologiya: Materialy konferentsii Problema vida i vidoobrazovanie (Evolutionary Biology: Proc. Conf. The Problem of Species and Speciation), Stegnii, V.N., Ed., Tomsk: Tomsk. Gos. Univ., 2001, vol. 1, pp. 138162. 4. Chadov, B.F., Facultative Dominant Lethals: Genetics, Ontogenesis, and Phylogenesis, Evolyutsionnaya biologiya: Materialy II konferentsii Problema vida i vidoobrazovanie (Evolutionary Biology: Proc. II Conf. The Problem of Species and Speciation), Stegnii, V.N., Ed., Tomsk: Tomsk. Gos. Univ., 2002, vol. 2, pp. 118142. 5. Chadov, B.F., The Image of the Regulatory Gene in Experiments with Drosophila, Genetika (Moscow), 2002, vol. 38, no. 7, pp. 869880. 6. Chadov, B.F., Chadova, E.V., Kopyl, S.A., and Fedorova, N.B., Delayed Activation of the Maternal Genome in Early Development of Drosophila, Dokl. Akad. Nauk, 2001, vol. 378, no. 6, pp. 841845. 7. Chadov, B.F. and Fedorova, N.B., The Elementary Event in Ontogenesis, Dokl. Akad. Nauk, 2003, vol. 389, no. 3, pp. 408412. 8. Ashburner, M., Drosophila: A Laboratory Handbook, Cold Spring Harbor, New York: Cold Spring Harbor Lab., 1989. 9. Ouweneel, W.J., Developmental Genetics of Homoeosis, Adv. Genet., 1976, vol. 18, pp. 179248. 10. Garcia-Bellido, A., Homoeotic and Atavic Mutations in Insects, Am. Zool., 1977, vol. 17, pp. 613629. 11. Lewis, E.B., A Gene Complex Controlling Segmentation in Drosophila, Nature, 1978, vol. 276, pp. 565570. 12. Nusslein-Volhard, C., Maternal Effect Mutations That Alter Spatial Coordinates of the Embryo of Drosophila melanogaster, Determinants of Spatial Organization, Subtelny, S. and Konigsberg, I.R., Eds., New York: Academic, 1979, pp. 185214.
RUSSIAN JOURNAL OF GENETICS Vol. 40 No. 3

13. Jacob, F. and Monod, J., Genetic Regulatory Mechanisms in the Synthesis of Proteins, J. Mol. Biol., 1961, vol. 3, pp. 318356. 14. Lewin, B., Genes, New York: Oxford Univ. Press, 1990. 15. Korochkin, L.I., Vvedenie v genetiku razvitiya (Introduction to Developmental Genetics), Moscow: Nauka, 1999. 16. Davidson, E.H., Rast, J.P., Oliveri, P., et al., A Genomic Regulatory Network for Development, Science, 2002, vol. 295, pp. 16691678. 17. Panshin, I.B., A Heterochromatin Scheme of the Operon, in Uspekhi sovremennoi genetiki (Achievements of Modern Genetics), Moscow: Nauka, 1991, pp. 224268. 18. Ratner, V.A., Genetika, molekulyarnaya kibernetika: Lichnosti i problemy (Genetics, Molecular Cybernetics: Personalities and Problems), Novosibirsk: Nauka, 2002. 19. Altukhov, Yu.P., Geneticheskie protsessy v populyatsiyakh (Genetic Processes in Populations), Moscow: Akademkniga, 2003, 3rd ed. 20. Chadov, B.F., Chadova, E.V., Kopyl, S.A., et al., From the Genetics of Intraspecic Differences to the Genetics of Intraspecic Similarity, Aktualnye problemy genetiki: Materialy 2 konferentsii MOGiS, posvyashchennoi 115-letiyu so dnya rozhdeniya akad. N.I. Vavilova (Topical Problems of Genetics: Proc. 2nd Conf. of the Moscow Society of Geneticists and Breeders in Commemoration of the 115th Anniversary of Academician N.I. Vavilov), Moscow: Mosk. Skh. Akad., 2003, vol. 2, pp. 232233. 21. Frizen, G., Roentgenomorphoses in Drosophila, Biol. Zh., 1935, vol. 4, no. 4, pp. 687704. 22. Rapoport, I.A., Specic Morphoses Induced in Drosophila melanogaster by Chemical Compounds, Byull. Eksp. Biol. Med., 1939, no. 7, pp. 415417. 23. Goldschmidt, R., Theoretical Genetics, Berkley: Univ. California Press, 1957. 24. Goldschmidt, R. and Piternick, L., The Genetic Background of Chemically Induced Phenocopies in Drosophila: I, J. Exp. Zool., 1957, vol. 135, pp. 127202. 25. Goldschmidt, R. and Piternick, L., The Genetic Background of Chemically Induced Phenocopies in Drosophila: II, J. Exp. Zool., 1957, vol. 136, pp. 201228. 26. Timofeev-Resovskii, N.V., On the Phenotypic Expression of the Genotype: I. Genetic Variation of radius incompletes in Drosophila funebris, Zh. Eksp. Biol., Ser. A, 1925, vol. 1, nos. 34, pp. 93142. 27. Timofeeff-Ressovsky, N.V., Association between Genes and Phenotypic Characters, N.V. Timofeev-Resovskii: Izbrannye trudy (N.V. Timofeeff-Ressovsky: Selected Works), Gazenko, O.G. and Ivanov, V.I., Eds., Moscow: Meditsina, 1996, pp. 5984. 28. Timofeeff-Ressovsky, N.V. and Ivanov, V.I., Several Issues of Phenogenetics, in Aktualnye voprosy sovremennoi genetiki (Topical Problems of Modern Genetics), Moscow: Mosk. Gos. Univ., 1966, pp. 114130. 29. Astaurov, B.L., A Study of Hereditary Changes of the Halter in Drosophila melanogaster Schin., Zh. Eksp. Biol., Ser. A, 1927, vol. 3, nos. 12, pp. 161. 30. Cline, Th.W., Primary Events in the Determination of Sex in Drosophila, The Origin and Evolution of Sex, Halvorson, H.O. and Monroy, A., Eds., New York: Alan Liss, 1985, pp. 301327.
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