- Email: [email protected]
Gene interactions in germ differentiation of Drosophila
GENE
INTERACTIONS DIFFERENTIATION DROSOPHILA
IN
GERM OF
WOLFGANG HENNIG Friedrich-Miescher-Laboratorium der Max-Planck-GeseUschaft, D-7400 Tiibingen, Germany
INTRODUCTION
Our knowledge of genome organization in eukaryotic organisms has been considerably extended during the past decade. Very little new information has however been obtained on gene regulation and gene function in higher organisms, although this might be of critical importance for understanding the events preceding and accompanying cellular differentiation and development as far as they concern the level of genome activity. This might in part be due to the limited range of experimental techniques in developmental biology which are suited to provide insight into molecular processes. In microorganisms mutants were very useful in studies of morphogenetic events, as for example in the study on T4 head assembly. Mutations which affect cell development in such a way as to enable the analysis of a distinct differentiation process are however rare in eukaryotic systems. An elucidation of the various elements involved in a defined differentiation process might be facilitated by a substitution of genes or gene groups containing related but not identical information. Resulting modifications in the differentiation process might enable one to determine the particular function of the substituted genetic information. Such alterations could of course be attained by mutations, but often it is very difficult to select for such mutations. An alternative approach is the construction of species hybrids; this is possible in some organisms. We hope to employ this type of experimental approach for our investigations on sperm differentiation in Drosophila. We use Drosophila hydei, a species which offers a much greater wealth of cytological detail in the cell types relevant for differentiation (i.e. spermatocyte nuclei) than D. melanogaster. One of the sibling species with which genetic hybrids can be constructed is D. neohydei.We first investigated the extent to which genes of D. neohydei can be regulated if they are introduced into the genome of D. hydei and whether the neohydei gene products can replace the corresponding products of the hydei genome. My presentation will summarize some general aspects and the results of these experiments which will be published in detail elsewhere. 363
364
WOLFGANG HENNIG GERM
CELL
DEVELOPMENT
First we must recapitulate the various stages of the male germ cell development; these are summarized in Figure 1. A series of mitotic divisions take place to provide a sufficient number of spermatogonia. These undergo a considerable increase in size during the first meiotic prophase ("primary spermatocyte stage"). As a result of the two following meiotic divisions each primary spermatocyte gives rise to four spermatids. These cells which are initially undifferentiated morphologically undergo drastic morphological modifications; within two days they are converted into structurally fully developed sperm. During this time of morphological differentiation the genome does not manifest any detectable activity. During the subsequent phase (4 to 5 days) the sperm undergo final maturation which is not accompanied by significant morphological changes. The primary spermotocyte stage of the male germ cells accounts for a large proportion of the entire period of sperm differentiation. Spermatocyte nuclei are cytologically most remarkable because of their extraordinary voluminous 1 2 3 4 5 6 7 8 9 10111213141516171819 I
I
I
I
I
I
I
I
I
I
I
I
I
I
III
I
I
I
I
I
I
I
Spermstogonia / ] Spermatids Sl:~'matoeyies I II
I
Days I
Stages of Spermatogenesis
•
Sperm
t
Fertility
Meiotic divisions FIG. 1. Stages of spermatogenesis in Drosophila hydei and their time course at 23°C (modified from 4).
intranuclear structures which are derived from the Y chromosome and represent lampbrush loops of active Y chromosomal genes(I,2). Deletion mapping allowed the identification and approximate localization of five loop forming loci which occur along the entire length of the Y chromosome (3). It is interesting that such loops are only produced by the Y chromosome although other chromosomes are also active during spermatogenesis (see 4). Complementation studies together with cytology and electron microscopy suggest that the loop forming regions of the Y chromosome carry information essential for spermatogenesis (5, 6). This is in agreement with the occurrence of fertility genes in the Y chromosome of D. melanogaster (7, 8). The formation of such Y chromosomal lampbrush loops seems to be a general feature of Drosophila spermatogenesis since they have been observed in all species so far studied including D. melanogaster (1,9). The number and morphology of the loops are nevertheless considerably different in various species; even such closely related
GENE INTERACTION IN DROSOPHILA
365
species as D. hydei and D. neohydei have loops with distinctly different morphologies (Fig. 2). FERTILITY
G E N E S IN D. H Y D E I AND D. N E O H Y D E I It is remarkable that, in spite of these substantial interspecific morphological differences between the Y chromosomal lampbrush loops, male species hybrids (as female hybrids) are fertile irrespective of the origin of the Y chromosome. An obvious question is whether the Y chromosome of one species will still be functional when it is introduced into a genome completely homozygous for the autosomes of the other species. In order to answer this question we carried out repeated backcrosses of species hydrids with females of the maternal genotype. Various combinations of autosomes and X chromosomes were brought together with either the hydei or the neohydei Y chromosome. The fertility of the resulting individuals was studied (see Tables 1 and 2). As can be seen, the number of offspring varies considerably depending upon the sex chromosome constitution of the hybrids. Species hybrids containing a hydei X chromosome and a neohydei Y in the male (Table 2) are obtained much more readily than those with the inverse sex chromosome constitution (i.e. neohydei X and hydei Y, Table 1). If male hybrids of both constitutions are backcrossed with females of the respective maternal genotype the number of offspring is, however, much larger for males with a neohydei X and a hydei Y (Table 1). The distribution of genotypes in the offspring of both backcrosses is rather similar, and Tables 1 and 2 show that some genotypes occur with an extremely low frequency (lines I 1 and 12; autosome constitutions: A 2 H/A2 H ; A3 H/A3 N ; A4 H/A 4 H ; As H/As N ; A6?/A6 ?, and A2H/A2H; A3H/AaN; A4H/A4H; AsH/AsH; A6?/A62). One could argue that this is due to a non-random segregation of certain autosomes in hybrids, but more likely it results from a reduced viability of hybrids with these genetic constitutions. Differences in the viability exist obviously also between males and females of certain autosome constitutions, particularly in the presence of a hydei X chromosome (Table 2). It can moreover be seen that most genotypes are 'sterile. In particular, chromosome 4 plays an important role in determining the fertility of these hybrids, since all fertile males carried this autosome in a heterozygous constitution. However, since not all of the males with a heterozygous chromosome 4 are fertile, and the fertility varies even between the different genotypes (cf. lines 4 and 5 or 6), genetic sites in different chromosomes must interact. This clearly shows that non-Y chromosomal genes contribute essential information to spermiogenesis. Genes of this type are also located in the X chromosome as can be seen from Table 1 : males with neohydei X chromosome and all of the other chromosomes derived from hydei (line 16) are completely sterile. Independent evidence in support of the conclusion that non-Y chromosomal genes supply information to spermiogenesis was obtained from mutagenesis studies; during a study of 3000 mutagenized X chromosomes
W O L F G A N G HENNIG
366
T A B L E 1. F R E Q U E N C Y AND F E R T I L I T Y OF HYBRIDS WITH V A R I O U S AUTOSOME CONSTITUTIONS Sex c h r o m o s o m e constitution: xxhydei/yhy dei in females ~-ffeohydei/yhydei in males
No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Autosome constitution
+ -
+ +
+ + +
+ +
+ + +
+ -
-
+
- + -+ + +
+ + - + + + + + + + + + + + + +
Total offspring
Female~ number %
Males number
%
105 108 95 135 132 118 124 119 109 105 0 3 92 95 93 115
6.8 7.0 6.1 8.7 8.5 7.6 8.0 7.7 7.0 6.8 0 0.2 5.9 6.1 6.0 7.4
88 118 91 96 102 112 83 114 90 86 3 10 82 88 87 69
6.7 9.0 6.9 7.3 7.7 8.5 6.3 8.6 6.8 6.5 0.2 0.8 6.2 6.7 6.6 5.2
1548
100
1319
100
Fertile Sterile males 39 24 13 50 2 2 7 0 0 0 0 0 0 0 0 0
11 26 47 25 48 70 67 77 50 50 1 6 50 50 61 53
" + " indicates an a u t o s o m e c o n s t i t u t i o n h o m o z y g o u s for D. h y d e i c h r o m o s o m e s , " - " indicates a h e t e r o z y g o u s a u t o s o m e constitution (D. hydei/D, neohydei). The sequence o f the c h r o m o s o m e s is always c h r o m o s o m e 2, 3, 4 and 5. A u t o s o m e markers are: c h r o m o s o m e 2: st/st (st = scarlet), c h r o m o s o m e 3: p x / p x (px = plexus), c h r o m o s o m e 4: h t / h t (ht = heart), c h r o m o s o m e 5: sea/sea (sea = scabrous), c h r o m o s o m e 6: no marker. T h e stock was kindly supplied by Dr. H. Kobel, Geneva. T h e crosses were carried o u t as follows: 61 bottles with 20 females o f D. hydei (constitution: X X (y m ch)/Y; st/st; p x / p x ; h t / h t ; sea/sea) and 20 D. n e o h y d e i wildtype males gave a total o f 360 female and 376 male offspring. Hybrid males were backcrossed with females o f the maternal genotype. F r o m this baekcross a total o f 1548 female and 1319 male offspring was obtained which were listed above according to their respective genotypes. For fertility tests at least 50 males o f each constitution (as far as available) were crossed in single crosses with 3 females of the maternal g e n o t y p e each and transferred at least twice to fresh vials. Dead females were replaced. Fertility was indicated only if adult offspring were obtained. Sterility was assertained by cytologically checking for motile sperm. F r o m males o f t h e g e n t o y p e s in line 5 and 6 few offspring were obtained. Generally t h e fertility is extremely low whenever m o r e t h a n one a u t o s o m e pair is h o m o z y g o u s for hydei. In contrast to the cross summarized in Table 2, the n u m b e r o f hydrids obtained in t h e first hybrid regeneration is very low in this cross with attached-X females while the n u m b e r o f offspring increased in the baekeross generation. of D. hydei mutants
we isolated
were
unpublished).
selected
121 m a l e s t e r i l e m u t a n t s in
chromosome
2
and an additional
(Hennig,
It can of course not be excluded
Leoncini
and
15 s u c h Rohde,
that male sterility in some of
t h e s e m u t a n t s is d u e t o p l e i o t r o p i c e f f e c t s . T h e e x i s t e n c e o f m a l e f e r t i l i t y g e n e s in all c h r o m o s o m e s
is w e l l e s t a b l i s h e d
GENE INTERACTION IN DROSOPHILA
367
TABLE 2. FREQUENCY AND FERTILITY OF HYBRIDS WITH VARIOUS AUTOSOME CONSTITUTIONS Sex chromosome constitution: xhydei/xhY dei in females xhydei/yneohy dei in males
No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Autosome constitution + - + + + + + + + +
+ +
-
+ + -
+ + + + + - + + -+ + + + +
Total offspring
+ + + + + + +
Females number
%
Males number %
19 12 25 22 26 25 14 23 6 34 0 1 2 24 27 21
6.8 4.3 8.9 7.8 9.3 8.9 5.0 8.2 2.6 12.1 0 0.4 0.7 8.5 9.6 7.5
24 25 26 17 29 26 28 23 26 30 0 0 28 31 27 27
6.5 6.8 7.1 4.6 7.9 7.1 7.6 6.3 7.1 8.2 0 0 7.6 8.4 7.4 7.5
281
100
367
100
Fertile males? yes yes no yes yes no yes no no yes no no no no
Symbols and autosome markers as deser~ed for Table 1. The crosses were carried out as follows: 43 bottles with 20 females of D. hydei (constitution: X+/X÷; st/st; px/px; ht/ht; sca/sca) and 20 wild type males of D. neohydei gave a total of 1375 female and 1383 male offspring. Hybrid males were backcrossed with females of the maternal genotype. From this backeross a total of 281 female and 367 male offspring was obtained which were listed above according to their respective genotypes. The fertility tests were carried out as descr~ed for Table 1. In contrast to the cross summarized in Table 1, the number of offspring is very large in the first hybrid generation but is very low in the backcrosses. for D. m e l a n o g a s t e r . In D. h y d e i s u c h studies h a v e so far o n l y b e e n u n d e r t a k e n b y L i f s c h y t z (10, 11) w h o i s o l a t e d s o m e X c h r o m o s o m a l m u t a n t s w h i c h a f f e c t s p e r m a t o g e n e s i s . This is in a g r e e m e n t w i t h o u r o b s e r v a t i o n s , b u t it is at the p r e s e n t difficult t o d i s t i n g u i s h p l e i o t r o p i c effects f r o m specific effects o n s p e r m a t o g e n e s i s . T h e use o f species h y b r i d s for c o m p l e m e n t a t i o n m a y also p r o v e useful in this r e s p e c t . Details c o n c e r n i n g t h e e f f e c t o f d i f f e r e n t a u t o s o m a l c o n s t i t u t i o n s o n s p e r m a t o g e n e s i s are, h o w e v e r , n o t o f d i r e c t relevance for t h e p r o b l e m s discussed in this c o m m u n i c a t i o n a n d will b e p u b l i s h e d elsewhere. F r o m t h e e x p e r i m e n t a l d a t a o f e x p e r i m e n t s as s h o w n in T a b l e s 1 a n d 2 s o m e general c o n c l u s i o n s c a n b e d r a w n . First, it is clear t h a t a u t o s o m a l a n d X c h r o m o s o m a l genes c o n t r i b u t e essential i n f o r m a t i o n t o g e r m cell d i f f e r e n t i a t i o n , as is i n d i c a t e d b y t h e d e p e n d e n c e o f male fertility o n the a u t o s o m a l a n d X c h r o m o s o m e c o n s t i t u t i o n in h y b r i d s . S e c o n d , it is e v i d e n t t h a t several o f t h e s e n o n - Y c h r o m o s o m a l fertility genes are species specific in spite o f t h e fact t h a t
368
WOLFGANGHENNIG
both species are closely related. Moreover it appears that the neohydei Y chromosomal lampbrush loops can be induced to transcribe in an environment which mainly consists of hydei autosomes, since cytological observations show that they were formed in spermatocytes from hybrids with all genetic constitutions which can be obtained. It appears, on the other hand, that the gene products from these loops cannot be utilized in any possible autosome constitution in the hybrids since backcross hybrids with increased homozygosity of hydei autosomes are sterile (cf. line 16, Table 2). Another remarkable observation should be mentioned in this context. The main constituents of the lampbrush loops are proteins, as is shown by histochemical investigations (4, 12). It is so far unclear whether these proteins are coded for by the Y chromosomal genes or in the remainder of the genome. They are most probably synthesized in the cytoplasm as earlier pulse-chase labeling experiments indicate (4). The morphology of the loops is rather different in D. hydei and D. neohydei, although the number of loops is approximately the same (13, 14). However if a neohydei Y chromosome is introduced in a genetic environment mainly composed of hydei chromosomes (Fig. 3), then the typically rather diffuse morphology of the neohydei loops is altered and the loops become more compact. The morphology of some of the loops is more reminiscent of the morphology of hydei loops. The most probable interpretation of this observation is that at least some of the loop proteins are not coded for by the Y chromosome; although probably species specific, they
T PcP N
N D.h.ydei
D.neohydei
FIG. 4. Maps of the Y chromosomallampbrush loops in (a) D. hydei (accordingto Hess (3)) and (b) D. neohydei (preliminary data from our laboratory: I. Hennig, B. Link and G. Jacob, unpublished). The comparison o f both maps reveals considerable differences in the loop positions in the Y chromosomes between both species in the course of evolution. This is in contrast to the situation found in the autosomal and X chromosomal euchromatin where extreme conservatism in the giant chromosome banding patterns of both species was observed (13). Designation of the loops: (a) D. hydei. Th: threads, Pc: cones, P: pseudonueleoins, T: tubular ribbons, C: clubs, No: nooses (from ref. 3). (b) D. neohydei, pl: proximal loops, dl: distal loops, c: clubs, th: threads, dn: diffuse nooses, no: nooses (modified from 13 and unpublished data of B. Link and I. Hennig). N: nucleolus (location in D. hydei according to ref. 16, in D. neohydei according to Jacob and Hennig, in preparation).
GENE INTERACTION IN DROSOPHILA
369
might be similar enough to interact with the foreign Y chromosomal genes. Definite experimental data substantiating these assumptions might considerably enhance our understanding of the interaction between active genetic loci and proteins.
SUBSTITUTION OF Y C H R O M O S O M A L G E N E S Backcrossing of genetic hybrids with a neohydei Y chromosome to hydei females results in sterility of most of the male offspring (Table 2). It was one of our questions whether at least some fertility genes of the neohydei Y chromosome can functionally substitute the respective hydei genes. In order to answer this question a different experimental approach had to be employed. We chose to translocate fragments of the neohydei Y chromosome to a hydei X chromosome by conventional genetic techniques. This experimental approach was successful, and a series of neohydei Y chromosomal lampbrush loops could be transferred into a hydei genome (14, 15). In agreement with the conclusions drawn from observations in hybrid males (see p. 367) the transcription of the neohydei genes was shown to be regulated in the host genome (15). This could be concluded from the observation that the transferred loops appear in primary sPermatocyte nuclei of hydei males carrying the Xhydei-yneohydei translocation chromosome. The question as to whether neohydei gene products can functionally replace hydei Y chromosomal genes during spermiogenesis was, however, difficult to answer with the genetic material available. Partially deficient hydei Y chromosomes were only available as X-Y or autosome-Y translocation stocks (see 5). Most combinations of such chromosomes with Xhydei-yne°hy dei translocation chromosomes were sterile (13, I. Hennig, in preparation). Nevertheless, early in the course of these experiments one fertile combination was obtained(15). The reason for this scarcity of fertile combinations became apparent as soon as we had sufficient experimental data to construct a preliminary map of the neohydei Y chromosome. In males which have different Xhydei.yneohydei translocation chromosomes but neither a free Y nor a normal X chromosome ("X/O" males), distinct neohydei loops in the primary spermatocytes can be correlated with the region of the neohydei Y chromosome in which they are located. The same method has been used to construct a map of the loops of the hydei Y chromosome. Both maps are shown in Figure 4. The comparison of both maps shows that the distribution of loops is quite different in the Y chromosome of the two species. For example no loop of the neohydei Y chromosome appears to occupy a position comparable to that of the "threads" in D. hydei. Two explanations are possible. Either no site homologous to the "threads" exists in the neohydei Y chromosome, or it occurs in a different position. Our complementation data suggest that the latter is true. This illustrates the reason why it is often not possible to obtain complementation with translocation chromosomes. If the neohydei loops are
370
WOLFGANG HENNIG
arranged in a sequence fundamentally different from the sequence of their homologs in the hydei Y chromosome, then the combination of shorter fragments of the Y chromosomes of both species - for example the distal third of the long arm of the neohydei Y with the short arm and a proximal portion of the long arm of the hydei Y chromosome - are certain not to result in a completely functional Y and a fertile individual: in this example the locus corresponding to the loops "threads" in hydei is absent. Similar conclusions hold true for other loci. The analysis of such interspecific translocations was facilitated by several recessive temperature sensitive mutations in the hydei Y chromosome which were isolated in our laboratory (Leoncini, in preparation). The Xhydei-yneohydei translocation chromosomes could then be put into genomes with free hydei Y chromosomes non-functional in single genetic loci at the restrictive temperature. In these experiments it was demonstrated that at least two different loci of the hydei Y chromosome can be replaced by homologous sites of the neohydei Y chromosome without loss of fertility. The morphology of sperm in individuals with interspecific gene complementation is currently being studied. It is of some general interest in view of recently developed techniques of genetic engineering that genes can be transferred between species and remain functional, even though cytological and cytogenetic studies show that their location in the genome and their transcriptional properties (i.e., loop morphology) have been altered in the course of evolution. Our studies may moreover provide some information on the stability of regulatory systems in eukaryotes. SUMMARY With the aid of species hybrids between two Drosophila species it is shown that cooperative interactions between species specific genes occur during spermatogenesis. It is however possible to incorporate fertility genes of one species into the genome of the other species and to functionally substitute homologous genes of the host species. Thus, the foreign genes can still be regulated and in some cases their gene products remain functional although considerable evolutionary changes can be observed between both species. Such changes include alterations in the positions of the respective genes in the genome as well as transcriptional properties (i.e., the morphological structure of lampbrush loops). ACKNOWEDGEMENTS I am mostly obliged to Miss Georgette Rohde for her excellent technical assistance in parts of this work. The Deutsche Forschungsgemeinschaft is thanked for financial support.
GENE INTERACTION IN DROSOPHILA
371
REFERENCES 1. G. F. MEYER, O. HESS and W. BEERMANN, Phasenspezifische Funktionsstrukturen in Spermatocytenkernen yon Drosophila melanogaster und ihre Abh~ngigkeit vom Y-Chromosom, Chromosoma (8erl.) 12,676-716 O961). 2. G. F. MEYER, Die Funktionsstrul
INTERACTIONS DIFFERENTIATION DROSOPHILA
IN
GERM OF
WOLFGANG HENNIG Friedrich-Miescher-Laboratorium der Max-Planck-GeseUschaft, D-7400 Tiibingen, Germany
INTRODUCTION
Our knowledge of genome organization in eukaryotic organisms has been considerably extended during the past decade. Very little new information has however been obtained on gene regulation and gene function in higher organisms, although this might be of critical importance for understanding the events preceding and accompanying cellular differentiation and development as far as they concern the level of genome activity. This might in part be due to the limited range of experimental techniques in developmental biology which are suited to provide insight into molecular processes. In microorganisms mutants were very useful in studies of morphogenetic events, as for example in the study on T4 head assembly. Mutations which affect cell development in such a way as to enable the analysis of a distinct differentiation process are however rare in eukaryotic systems. An elucidation of the various elements involved in a defined differentiation process might be facilitated by a substitution of genes or gene groups containing related but not identical information. Resulting modifications in the differentiation process might enable one to determine the particular function of the substituted genetic information. Such alterations could of course be attained by mutations, but often it is very difficult to select for such mutations. An alternative approach is the construction of species hybrids; this is possible in some organisms. We hope to employ this type of experimental approach for our investigations on sperm differentiation in Drosophila. We use Drosophila hydei, a species which offers a much greater wealth of cytological detail in the cell types relevant for differentiation (i.e. spermatocyte nuclei) than D. melanogaster. One of the sibling species with which genetic hybrids can be constructed is D. neohydei.We first investigated the extent to which genes of D. neohydei can be regulated if they are introduced into the genome of D. hydei and whether the neohydei gene products can replace the corresponding products of the hydei genome. My presentation will summarize some general aspects and the results of these experiments which will be published in detail elsewhere. 363
364
WOLFGANG HENNIG GERM
CELL
DEVELOPMENT
First we must recapitulate the various stages of the male germ cell development; these are summarized in Figure 1. A series of mitotic divisions take place to provide a sufficient number of spermatogonia. These undergo a considerable increase in size during the first meiotic prophase ("primary spermatocyte stage"). As a result of the two following meiotic divisions each primary spermatocyte gives rise to four spermatids. These cells which are initially undifferentiated morphologically undergo drastic morphological modifications; within two days they are converted into structurally fully developed sperm. During this time of morphological differentiation the genome does not manifest any detectable activity. During the subsequent phase (4 to 5 days) the sperm undergo final maturation which is not accompanied by significant morphological changes. The primary spermotocyte stage of the male germ cells accounts for a large proportion of the entire period of sperm differentiation. Spermatocyte nuclei are cytologically most remarkable because of their extraordinary voluminous 1 2 3 4 5 6 7 8 9 10111213141516171819 I
I
I
I
I
I
I
I
I
I
I
I
I
I
III
I
I
I
I
I
I
I
Spermstogonia / ] Spermatids Sl:~'matoeyies I II
I
Days I
Stages of Spermatogenesis
•
Sperm
t
Fertility
Meiotic divisions FIG. 1. Stages of spermatogenesis in Drosophila hydei and their time course at 23°C (modified from 4).
intranuclear structures which are derived from the Y chromosome and represent lampbrush loops of active Y chromosomal genes(I,2). Deletion mapping allowed the identification and approximate localization of five loop forming loci which occur along the entire length of the Y chromosome (3). It is interesting that such loops are only produced by the Y chromosome although other chromosomes are also active during spermatogenesis (see 4). Complementation studies together with cytology and electron microscopy suggest that the loop forming regions of the Y chromosome carry information essential for spermatogenesis (5, 6). This is in agreement with the occurrence of fertility genes in the Y chromosome of D. melanogaster (7, 8). The formation of such Y chromosomal lampbrush loops seems to be a general feature of Drosophila spermatogenesis since they have been observed in all species so far studied including D. melanogaster (1,9). The number and morphology of the loops are nevertheless considerably different in various species; even such closely related
GENE INTERACTION IN DROSOPHILA
365
species as D. hydei and D. neohydei have loops with distinctly different morphologies (Fig. 2). FERTILITY
G E N E S IN D. H Y D E I AND D. N E O H Y D E I It is remarkable that, in spite of these substantial interspecific morphological differences between the Y chromosomal lampbrush loops, male species hybrids (as female hybrids) are fertile irrespective of the origin of the Y chromosome. An obvious question is whether the Y chromosome of one species will still be functional when it is introduced into a genome completely homozygous for the autosomes of the other species. In order to answer this question we carried out repeated backcrosses of species hydrids with females of the maternal genotype. Various combinations of autosomes and X chromosomes were brought together with either the hydei or the neohydei Y chromosome. The fertility of the resulting individuals was studied (see Tables 1 and 2). As can be seen, the number of offspring varies considerably depending upon the sex chromosome constitution of the hybrids. Species hybrids containing a hydei X chromosome and a neohydei Y in the male (Table 2) are obtained much more readily than those with the inverse sex chromosome constitution (i.e. neohydei X and hydei Y, Table 1). If male hybrids of both constitutions are backcrossed with females of the respective maternal genotype the number of offspring is, however, much larger for males with a neohydei X and a hydei Y (Table 1). The distribution of genotypes in the offspring of both backcrosses is rather similar, and Tables 1 and 2 show that some genotypes occur with an extremely low frequency (lines I 1 and 12; autosome constitutions: A 2 H/A2 H ; A3 H/A3 N ; A4 H/A 4 H ; As H/As N ; A6?/A6 ?, and A2H/A2H; A3H/AaN; A4H/A4H; AsH/AsH; A6?/A62). One could argue that this is due to a non-random segregation of certain autosomes in hybrids, but more likely it results from a reduced viability of hybrids with these genetic constitutions. Differences in the viability exist obviously also between males and females of certain autosome constitutions, particularly in the presence of a hydei X chromosome (Table 2). It can moreover be seen that most genotypes are 'sterile. In particular, chromosome 4 plays an important role in determining the fertility of these hybrids, since all fertile males carried this autosome in a heterozygous constitution. However, since not all of the males with a heterozygous chromosome 4 are fertile, and the fertility varies even between the different genotypes (cf. lines 4 and 5 or 6), genetic sites in different chromosomes must interact. This clearly shows that non-Y chromosomal genes contribute essential information to spermiogenesis. Genes of this type are also located in the X chromosome as can be seen from Table 1 : males with neohydei X chromosome and all of the other chromosomes derived from hydei (line 16) are completely sterile. Independent evidence in support of the conclusion that non-Y chromosomal genes supply information to spermiogenesis was obtained from mutagenesis studies; during a study of 3000 mutagenized X chromosomes
W O L F G A N G HENNIG
366
T A B L E 1. F R E Q U E N C Y AND F E R T I L I T Y OF HYBRIDS WITH V A R I O U S AUTOSOME CONSTITUTIONS Sex c h r o m o s o m e constitution: xxhydei/yhy dei in females ~-ffeohydei/yhydei in males
No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Autosome constitution
+ -
+ +
+ + +
+ +
+ + +
+ -
-
+
- + -+ + +
+ + - + + + + + + + + + + + + +
Total offspring
Female~ number %
Males number
%
105 108 95 135 132 118 124 119 109 105 0 3 92 95 93 115
6.8 7.0 6.1 8.7 8.5 7.6 8.0 7.7 7.0 6.8 0 0.2 5.9 6.1 6.0 7.4
88 118 91 96 102 112 83 114 90 86 3 10 82 88 87 69
6.7 9.0 6.9 7.3 7.7 8.5 6.3 8.6 6.8 6.5 0.2 0.8 6.2 6.7 6.6 5.2
1548
100
1319
100
Fertile Sterile males 39 24 13 50 2 2 7 0 0 0 0 0 0 0 0 0
11 26 47 25 48 70 67 77 50 50 1 6 50 50 61 53
" + " indicates an a u t o s o m e c o n s t i t u t i o n h o m o z y g o u s for D. h y d e i c h r o m o s o m e s , " - " indicates a h e t e r o z y g o u s a u t o s o m e constitution (D. hydei/D, neohydei). The sequence o f the c h r o m o s o m e s is always c h r o m o s o m e 2, 3, 4 and 5. A u t o s o m e markers are: c h r o m o s o m e 2: st/st (st = scarlet), c h r o m o s o m e 3: p x / p x (px = plexus), c h r o m o s o m e 4: h t / h t (ht = heart), c h r o m o s o m e 5: sea/sea (sea = scabrous), c h r o m o s o m e 6: no marker. T h e stock was kindly supplied by Dr. H. Kobel, Geneva. T h e crosses were carried o u t as follows: 61 bottles with 20 females o f D. hydei (constitution: X X (y m ch)/Y; st/st; p x / p x ; h t / h t ; sea/sea) and 20 D. n e o h y d e i wildtype males gave a total o f 360 female and 376 male offspring. Hybrid males were backcrossed with females o f the maternal genotype. F r o m this baekcross a total o f 1548 female and 1319 male offspring was obtained which were listed above according to their respective genotypes. For fertility tests at least 50 males o f each constitution (as far as available) were crossed in single crosses with 3 females of the maternal g e n o t y p e each and transferred at least twice to fresh vials. Dead females were replaced. Fertility was indicated only if adult offspring were obtained. Sterility was assertained by cytologically checking for motile sperm. F r o m males o f t h e g e n t o y p e s in line 5 and 6 few offspring were obtained. Generally t h e fertility is extremely low whenever m o r e t h a n one a u t o s o m e pair is h o m o z y g o u s for hydei. In contrast to the cross summarized in Table 2, the n u m b e r o f hydrids obtained in t h e first hybrid regeneration is very low in this cross with attached-X females while the n u m b e r o f offspring increased in the baekeross generation. of D. hydei mutants
we isolated
were
unpublished).
selected
121 m a l e s t e r i l e m u t a n t s in
chromosome
2
and an additional
(Hennig,
It can of course not be excluded
Leoncini
and
15 s u c h Rohde,
that male sterility in some of
t h e s e m u t a n t s is d u e t o p l e i o t r o p i c e f f e c t s . T h e e x i s t e n c e o f m a l e f e r t i l i t y g e n e s in all c h r o m o s o m e s
is w e l l e s t a b l i s h e d
GENE INTERACTION IN DROSOPHILA
367
TABLE 2. FREQUENCY AND FERTILITY OF HYBRIDS WITH VARIOUS AUTOSOME CONSTITUTIONS Sex chromosome constitution: xhydei/xhY dei in females xhydei/yneohy dei in males
No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Autosome constitution + - + + + + + + + +
+ +
-
+ + -
+ + + + + - + + -+ + + + +
Total offspring
+ + + + + + +
Females number
%
Males number %
19 12 25 22 26 25 14 23 6 34 0 1 2 24 27 21
6.8 4.3 8.9 7.8 9.3 8.9 5.0 8.2 2.6 12.1 0 0.4 0.7 8.5 9.6 7.5
24 25 26 17 29 26 28 23 26 30 0 0 28 31 27 27
6.5 6.8 7.1 4.6 7.9 7.1 7.6 6.3 7.1 8.2 0 0 7.6 8.4 7.4 7.5
281
100
367
100
Fertile males? yes yes no yes yes no yes no no yes no no no no
Symbols and autosome markers as deser~ed for Table 1. The crosses were carried out as follows: 43 bottles with 20 females of D. hydei (constitution: X+/X÷; st/st; px/px; ht/ht; sca/sca) and 20 wild type males of D. neohydei gave a total of 1375 female and 1383 male offspring. Hybrid males were backcrossed with females of the maternal genotype. From this backeross a total of 281 female and 367 male offspring was obtained which were listed above according to their respective genotypes. The fertility tests were carried out as descr~ed for Table 1. In contrast to the cross summarized in Table 1, the number of offspring is very large in the first hybrid generation but is very low in the backcrosses. for D. m e l a n o g a s t e r . In D. h y d e i s u c h studies h a v e so far o n l y b e e n u n d e r t a k e n b y L i f s c h y t z (10, 11) w h o i s o l a t e d s o m e X c h r o m o s o m a l m u t a n t s w h i c h a f f e c t s p e r m a t o g e n e s i s . This is in a g r e e m e n t w i t h o u r o b s e r v a t i o n s , b u t it is at the p r e s e n t difficult t o d i s t i n g u i s h p l e i o t r o p i c effects f r o m specific effects o n s p e r m a t o g e n e s i s . T h e use o f species h y b r i d s for c o m p l e m e n t a t i o n m a y also p r o v e useful in this r e s p e c t . Details c o n c e r n i n g t h e e f f e c t o f d i f f e r e n t a u t o s o m a l c o n s t i t u t i o n s o n s p e r m a t o g e n e s i s are, h o w e v e r , n o t o f d i r e c t relevance for t h e p r o b l e m s discussed in this c o m m u n i c a t i o n a n d will b e p u b l i s h e d elsewhere. F r o m t h e e x p e r i m e n t a l d a t a o f e x p e r i m e n t s as s h o w n in T a b l e s 1 a n d 2 s o m e general c o n c l u s i o n s c a n b e d r a w n . First, it is clear t h a t a u t o s o m a l a n d X c h r o m o s o m a l genes c o n t r i b u t e essential i n f o r m a t i o n t o g e r m cell d i f f e r e n t i a t i o n , as is i n d i c a t e d b y t h e d e p e n d e n c e o f male fertility o n the a u t o s o m a l a n d X c h r o m o s o m e c o n s t i t u t i o n in h y b r i d s . S e c o n d , it is e v i d e n t t h a t several o f t h e s e n o n - Y c h r o m o s o m a l fertility genes are species specific in spite o f t h e fact t h a t
368
WOLFGANGHENNIG
both species are closely related. Moreover it appears that the neohydei Y chromosomal lampbrush loops can be induced to transcribe in an environment which mainly consists of hydei autosomes, since cytological observations show that they were formed in spermatocytes from hybrids with all genetic constitutions which can be obtained. It appears, on the other hand, that the gene products from these loops cannot be utilized in any possible autosome constitution in the hybrids since backcross hybrids with increased homozygosity of hydei autosomes are sterile (cf. line 16, Table 2). Another remarkable observation should be mentioned in this context. The main constituents of the lampbrush loops are proteins, as is shown by histochemical investigations (4, 12). It is so far unclear whether these proteins are coded for by the Y chromosomal genes or in the remainder of the genome. They are most probably synthesized in the cytoplasm as earlier pulse-chase labeling experiments indicate (4). The morphology of the loops is rather different in D. hydei and D. neohydei, although the number of loops is approximately the same (13, 14). However if a neohydei Y chromosome is introduced in a genetic environment mainly composed of hydei chromosomes (Fig. 3), then the typically rather diffuse morphology of the neohydei loops is altered and the loops become more compact. The morphology of some of the loops is more reminiscent of the morphology of hydei loops. The most probable interpretation of this observation is that at least some of the loop proteins are not coded for by the Y chromosome; although probably species specific, they
T PcP N
N D.h.ydei
D.neohydei
FIG. 4. Maps of the Y chromosomallampbrush loops in (a) D. hydei (accordingto Hess (3)) and (b) D. neohydei (preliminary data from our laboratory: I. Hennig, B. Link and G. Jacob, unpublished). The comparison o f both maps reveals considerable differences in the loop positions in the Y chromosomes between both species in the course of evolution. This is in contrast to the situation found in the autosomal and X chromosomal euchromatin where extreme conservatism in the giant chromosome banding patterns of both species was observed (13). Designation of the loops: (a) D. hydei. Th: threads, Pc: cones, P: pseudonueleoins, T: tubular ribbons, C: clubs, No: nooses (from ref. 3). (b) D. neohydei, pl: proximal loops, dl: distal loops, c: clubs, th: threads, dn: diffuse nooses, no: nooses (modified from 13 and unpublished data of B. Link and I. Hennig). N: nucleolus (location in D. hydei according to ref. 16, in D. neohydei according to Jacob and Hennig, in preparation).
GENE INTERACTION IN DROSOPHILA
369
might be similar enough to interact with the foreign Y chromosomal genes. Definite experimental data substantiating these assumptions might considerably enhance our understanding of the interaction between active genetic loci and proteins.
SUBSTITUTION OF Y C H R O M O S O M A L G E N E S Backcrossing of genetic hybrids with a neohydei Y chromosome to hydei females results in sterility of most of the male offspring (Table 2). It was one of our questions whether at least some fertility genes of the neohydei Y chromosome can functionally substitute the respective hydei genes. In order to answer this question a different experimental approach had to be employed. We chose to translocate fragments of the neohydei Y chromosome to a hydei X chromosome by conventional genetic techniques. This experimental approach was successful, and a series of neohydei Y chromosomal lampbrush loops could be transferred into a hydei genome (14, 15). In agreement with the conclusions drawn from observations in hybrid males (see p. 367) the transcription of the neohydei genes was shown to be regulated in the host genome (15). This could be concluded from the observation that the transferred loops appear in primary sPermatocyte nuclei of hydei males carrying the Xhydei-yneohydei translocation chromosome. The question as to whether neohydei gene products can functionally replace hydei Y chromosomal genes during spermiogenesis was, however, difficult to answer with the genetic material available. Partially deficient hydei Y chromosomes were only available as X-Y or autosome-Y translocation stocks (see 5). Most combinations of such chromosomes with Xhydei-yne°hy dei translocation chromosomes were sterile (13, I. Hennig, in preparation). Nevertheless, early in the course of these experiments one fertile combination was obtained(15). The reason for this scarcity of fertile combinations became apparent as soon as we had sufficient experimental data to construct a preliminary map of the neohydei Y chromosome. In males which have different Xhydei.yneohydei translocation chromosomes but neither a free Y nor a normal X chromosome ("X/O" males), distinct neohydei loops in the primary spermatocytes can be correlated with the region of the neohydei Y chromosome in which they are located. The same method has been used to construct a map of the loops of the hydei Y chromosome. Both maps are shown in Figure 4. The comparison of both maps shows that the distribution of loops is quite different in the Y chromosome of the two species. For example no loop of the neohydei Y chromosome appears to occupy a position comparable to that of the "threads" in D. hydei. Two explanations are possible. Either no site homologous to the "threads" exists in the neohydei Y chromosome, or it occurs in a different position. Our complementation data suggest that the latter is true. This illustrates the reason why it is often not possible to obtain complementation with translocation chromosomes. If the neohydei loops are
370
WOLFGANG HENNIG
arranged in a sequence fundamentally different from the sequence of their homologs in the hydei Y chromosome, then the combination of shorter fragments of the Y chromosomes of both species - for example the distal third of the long arm of the neohydei Y with the short arm and a proximal portion of the long arm of the hydei Y chromosome - are certain not to result in a completely functional Y and a fertile individual: in this example the locus corresponding to the loops "threads" in hydei is absent. Similar conclusions hold true for other loci. The analysis of such interspecific translocations was facilitated by several recessive temperature sensitive mutations in the hydei Y chromosome which were isolated in our laboratory (Leoncini, in preparation). The Xhydei-yneohydei translocation chromosomes could then be put into genomes with free hydei Y chromosomes non-functional in single genetic loci at the restrictive temperature. In these experiments it was demonstrated that at least two different loci of the hydei Y chromosome can be replaced by homologous sites of the neohydei Y chromosome without loss of fertility. The morphology of sperm in individuals with interspecific gene complementation is currently being studied. It is of some general interest in view of recently developed techniques of genetic engineering that genes can be transferred between species and remain functional, even though cytological and cytogenetic studies show that their location in the genome and their transcriptional properties (i.e., loop morphology) have been altered in the course of evolution. Our studies may moreover provide some information on the stability of regulatory systems in eukaryotes. SUMMARY With the aid of species hybrids between two Drosophila species it is shown that cooperative interactions between species specific genes occur during spermatogenesis. It is however possible to incorporate fertility genes of one species into the genome of the other species and to functionally substitute homologous genes of the host species. Thus, the foreign genes can still be regulated and in some cases their gene products remain functional although considerable evolutionary changes can be observed between both species. Such changes include alterations in the positions of the respective genes in the genome as well as transcriptional properties (i.e., the morphological structure of lampbrush loops). ACKNOWEDGEMENTS I am mostly obliged to Miss Georgette Rohde for her excellent technical assistance in parts of this work. The Deutsche Forschungsgemeinschaft is thanked for financial support.
GENE INTERACTION IN DROSOPHILA
371
REFERENCES 1. G. F. MEYER, O. HESS and W. BEERMANN, Phasenspezifische Funktionsstrukturen in Spermatocytenkernen yon Drosophila melanogaster und ihre Abh~ngigkeit vom Y-Chromosom, Chromosoma (8erl.) 12,676-716 O961). 2. G. F. MEYER, Die Funktionsstrul