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Homoeosis in Drosophila: The Lethal Syndrome of the Regulator of bithorax (or trithorax) Locus and Its Interaction With Other Homoeotic Loci

Takashi Sate*" and Robin E. Denellt *Yakult Central Institute for Microbiological Research, I796 Yaho, Kunitachi, Tokyo 186, Japan, and +Division of Biology,

Kansas State University, Manhattan, Kansas 66506 Manuscript received October 16, 1986 Revised copy accepted March 12, 1987

ABSTRACT Regulator of bithorax (Rg-bx)- [or trithorax (trx)-] lethal zygotes show anterior transformations of

various cuticular features of the larval thorax and abdomen. The Rg-bx- lethal syndrome depends on the dosage of the bithorax gene complex (BX-C), and lack of Rg-bx+ function is antagonistic to posterior transformations displayed by Polycomb (Pc)- embryos. Significantly, when the BX-C is deleted, the Rg-bx- embryos disclose homoeosis of mesothoracic to prothoracic cuticular structures. This homoeotic transformation is due to a reduction in Antennapedia (Antp)+ gene activity and is consequently dependent on the dosage of the Antennapedia gene complex (ANT-C), suggesting that the Rg-bx+ activity is necessary for proper expression of the Antp+ gene. However, the functional relationship between the Rg-bx and Sex combs reduced (Scr) loci in embryogenesis is still to be established.

HE body of Drosophila melanogaster consists of a T series of segments, which are subdivided into an anterior and a posterior compartment (GARCIA-BEL- LIDO, RIPOLL and MORATA 1973; STEINER 1976; SZA- BAD, SCHUPBACH and WIESCHAUS, 1979; WIESCHAUS and GEHRING 1976) [also for reviews, see LAWRENCE (1981) and MAHOWALD and HARDY (1985)l. Devel- opment of the compartment-specific cuticular pattern depends on homoeotic loci like genes of the bithorax gene complex (BX-C) (LEWIS 1978, 1981, 1982) and the Antennapedia gene complex (ANT-C) (KAUFMAN 1983). Morphological analyses and molecular biolog- ical studies of mutants suggest that expressions of the BX-C, Antennapedia (Antp; a gene of the ANT-C) and Sex combs reduced (Scr; another member of the ANT- C) genes are spatially regulated along the body and that combinatory and differential expression of the BX-C and ANT-C genes is prerequisite for developmental decision of identities of the compartment (AKAM 1983; AKAM and MARTINEZ-ARIAS 1985; BEACHY, HELFAND and HOGNESS 1985; HARDING et al. 1985; KUROIWA et al. 1985; LEVINE et al. 1983; LEWIS 1978; MORATA et al. 1983; REGULSKI et al. 1985; SANCHEZ-HERRERO et al. 1985; SATO, HAYES and DENELL 1985; STRUHL 1981b, 1982, 1983; WHITE and WILCOX 1984, 1985). There are three classes of loci which are supposed to regulate the expression of the BX-C and ANT-C genes. The first class contains at least two segmentation gene fushi tarazu (ftz; also a member of the ANT-C) and hunchback (hb) which may be involved in initiation and establishment of spatial pattern of the expression of

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Genetics 116: 389-398 (July, 1987)

these genes (DUNCAN 1986; INGHAM and MARTINEZ- ARIAS 1986; WHITE and LEHMANN 1986). The second is some genes of the BX-C. For example, Ultrabithorax (Ubx) affects the expression of the Antp locus and Abdominal B does the expression of the Ubx (CARROLL et al. 1986; HARDING et al. 1985; STRUHL and WHITE 1985). The third is a group of homoeotic genes, normal activities of which seem necessary to maintain the correct expression of the BX-C and ANT-C genes.

One group within this third class includes extra sex combs ( e x ) (STRUHL 1981b), Polycomb (Pc) (PURO and NYGREN 1975; LEWIS 1978), Polycomblike (Pcl) (DUN- CAN 1982), and other genes (DURA, BROCK and SAN- TAMARIA 1985; INGHAM 1984; JURGENS 1985), while another group is represented by Regulator of bithorax (Rg-bx) (or trithorax (trx)) (CAPDEVILLA and GARCIA- BELLIDO 198 1 ; INGHAM 198 1 ; INGHAM and WHITTLE 1980). Mutations of genes of the former group show morphological transformations of various cuticular structures to resemble those of more posterior segments and most of these homoeotic transformations can be explained by indiscriminate expression of the BX-C and ANT-C gene (DENELL and FREDERICK 1983; DUNCAN 1982; DUNCAN and LEWIS 1982; SATO, HAYES and DENELL 1985; STRUHL 1981b, 1983). Recent findings that esc- and Pc- lethal embryos exhibit a significant alteration of the distribution of the Ubx+ gene product (BEACHY, HELFAND and HOG- NESS 1985; STRUHL and AKAM 1985; WEDEEN, HARD- ING and LEVINE 1986) endorse this interpretation.

The Rg-bx gene is a haplo-insufficient locus, and the decrease of the function results in anterior transformation of the cuticular pattern as well as other cuticular malformation (CAPDEVILLA and GARCIA-BEL-

390 T. Sato and R. E. Denell

LIDO 198 1 ; DUNCAN and LEWIS 1982; INGHAM 198 1, 1985; INGHAM and WHITTLE 1980). Because the anterior transformation of adult cuticle mimics some aspects of the BX-C mutants of a loss-of-function type and depends on the dosage of the BX-C, the lack of the Rg-bx+ function seems to cause hypomorphic expression of the BX-C (CAPDEVILLA and GARCIA- BELLIDO 198 1). CAPDEVILLA and GARCIA-BELLIDO (1 98 1) have proposed that this locus may be involved in an inducer of the BX-C and interact specifically with the Pc gene. However, details of the interaction of this locus with Antp and Scr genes remain unclear. We have examined cuticular morphology of lethal zygotes bearing the Rg-bx- mutation under various genetic background to analyze its functional relationship with other homoeotic genes. We report here that, a t least in embryogenesis, the impact of the Rg-bx- mutation is antagonistic to that of the Pc- mutation in relationship with the BX-C and Antp genes.

MATERIALS A N D METHODS

Genetic variants: Flies were reared on the standard corn meal-molasses-yeast-agar medium at 25". Canton S was used as a wild type. Single mutations of the Regulafor ofbithorax locus (3-54.2 f 0.4) (INCHAM and WHIITLE 1980). Rg-bx (CAPDEVILLA and GARCIA-BELLIDO 1981) and trx3 (INCHAM 198 1) were analyzed in the hemizygous condition over Df(3R)~ed" '~ (which deletes polytene chromosome bands 88B 1 to B4) (CAPDEVILLA and GARCIA-BELLIDO 198 1). For the Polycomb (3-47.1) locus, the mutant allele Pc3 (LEWIS 1978) was studied. Homozygotes of Df(3R)P 9 (89E1 ;E5) (LEWIS 1978) lack the entire HX-C (9-58.8). whereas dupli- cationsDp(3;3)P5 (89E1-2;90A) (DUNCAN and LEWIS 1982), Dp(3;f)P ff5 (89B;E6; inserted into the heterochromatin of the X chromosome) (MORATA ef al. 1983), and Dp(3; f )bxd '" (4D;89E3-4;90B) (LEWIS 198 1) provide the BX-C function necessary to make the sterile Df(3R)P9 heterozygotes fertile. A deficiency and a duplication of the ANT-C, Df(3R)Scr (84A1-2;B1-2) (LEWIS et al. 1980a) and Dp(3;3)Df (84A;85A) (DUNCAN and LEWIS 1982), respectively, were used for construction of embryos with varying dosages of the ANT-C. For a p arent amorphic alleles of genes within the ANT-C, Anfp"'R(LEWIS et al. 1980b) of Antennapedia, Scr"" (LEWIS et al. 1980b) of Sex combs reduced, andftz"'" (WAKIMOTO and KAUFMAN 198 1 ; WAKIMOTO, TURNER and KAUFMAN 1984) offushi farazu loci were analyzed. Symbols for deficiencies and duplications are abbreviated (e.g., Dfp9) throughout the text. Although most chromosomes used in this study were marked with irrelevant mutations, we omitted the description for convenience. For other mutations and chromosomes, see LINDSLEY and GRELL (1968) and LINDSLEY and ZIMM (1 985). Morphology: Crosses conducted in this study are sum-

marized in APPENDIX. Lethal embryos were prepared from unhatched eggs collected for 24 h r and incubated further for 26 h r as described previously (SATO. HAYES and DENELL 1984). With respect to crosses generating n o lethal embryos, eggs collected were dechorionated with sodium hypochlorite and incubated in distilled water as described elsewhere (SATO and DENELL 1986). Lethal embryos and hatched first instar larvae were mounted in nine parts lactic acid and one part 95% ethanol (LEWIS 1978), incubated a t least overnight at 45", and observed microscopically with phase contrast

A

PRO

B

FIGURE I .-Segment-specific rnorphologv of the ventral denticle belts (VDBs). A. A ventral aspect of the first instar larva of a wild type is viewed under dark field microscopy (X43). Each segment except for the head (H) displays a characteristic pattern of the VDB on its anterior edge. Prothorax (PRO) shows two belts (also see panel B). and mesothorax (MS) and metathorax (MT) show the belts of very fine denticles. The abdominal first (ABI) to eighth (AB8) segment display more coarse denticles on the belts. The VDB of the ABS is rectangular whereas the belts of other abdominal segments are trapezoidal. B. An enlarged aspect of thoraces through phase contrast optics (X210). Note the difference of denticle size between PRO and MS and the second belt of the PRO (indicated by an arrow).

optics. T h e cuticular morphology of the first instar larva of the wild type has been described (Figure 1) (HAYES, SATO and DENELL 1984; LOHS-SCHARDIN, CREMER and NUSSLEIN- VOLHARD 1979). Pc- embryos show generally posterior transformation of cuticular features to various extents. For semiquantitative comparison of the extent of the posterior transformation, we scored the abdominaliiation of the ventral denticle belt (VDB) of mesothorax (MS) of these embryos according to SATO and DENELL (1985). Embryos of class 1 show completely or nearly wild-type VDB, and class 2 embryos show some hooked abdominal denticles but less than four anteriorly facing denticles on the anterior-most row. In embryos of class 3, the anterior-most row of the VDB has more than four anteriorly oriented denticles, but is not complete. T h e VDB of class 4 embryos is trapezoidal and resembles that of the normal middle abdominal segment. Class 5 embryos show the rectangular VDB which is similar to that of the normal abdominal eighth segment (ABS).

RESULTS

Lethal syndrome: We found the hemizygotes of the Rg-bx and trx3 alleles display very similar phenotypes under the conditions utilized. Thus, both mutations are subsequently referred to as Rg-bx unless otherwise specified .

T h e Rg-bx- zygotes, which die as larvae, show subtle but distinct anterior transformations of the VDBs located on the anterior edges of larval segments (INGHAM 1983). We have noted additional aspects of homoeotic transformations of the larval cuticles (Fig-

Regulator of Bithorax Syndrome 39 1

. . @ . . .. A . . . I . ’ . : - c.:~:,

FIGURE 2.-Lethal Hg-bx- syndrome. Several aspects of the cuticular morphology of i~ wild type (A. C. E and C) and a Rg-bx/ DfredP” (B, D, F and H) first instar larva were viewed with phase contrast microscopy. A and B. The dorsal spinule patterns of the abdominal first segment. Note in the Rg-bx- larva the absence of hooked spinules (indicated by triungles) typical of the normal a b dominal first segment (X 139). C and D. The dorsal spinule patterns of the abdominal fifth segment. The Rg-bx- larva displays a “win- dow” of naked cuticle (flanked by asterisk) on the lawn of spinules (X139). E and F, The ventral views of the caudal segment. The ventral denticle belt (vdb) o f the abdominal eighth segment of the Rg-bx- larva is trapezoidal and a tiny chitinized plate (indicated by an arrow) appears anteriorly to the anal pads (ap) (X347). C and H, Lateral aspects of the telson. Note a posterior shift of the hair sensillum designated number four in the Rg-bx- larva. Other sensilla are also indicated by their respective number according to the nomenclature of DENELL and FREDERICK (1983). Filzkorper and anal pads are symbolized by fk and ap, respectively (X 139).

ure 2): development of the small pieces of chitinized material between the VDB of AB8 and the anal pads in 20-30% of the Rg-bx- zygotes (panels E and F), appearance of patches of naked cuticle typical of the normal MS on the lawn of dorsal spinules of the metathorax (MT) through the middle abdominal segments (panels C and D), and disappearance of dorsal hooked spinules of the posterior part of the first abdominal segment (ABl ) (panels A and B). These phenotypes, which are similar to those of zygotes with overlapping deletions removing the Rg-bx locus (DUN- CAN and LEWIS 1982), suggest that Rg-bx and trx’ are amorphic alleles, and resemble the cuticular morphology of zygotes missing a distal portion of the BX- C as suggested previously (DUNCAN and LEWIS 1982).

In addition, as shown in Figure 2 (panels G and H), Rg-bx- zygotes display a posterior shift of a hair- sensilla on the telson denoted number four according to the nomenclature of DENELL and FREDERICK (1 983) [also see SATO and DENELL (1 986)].

Rg-bx DjP9/DfredP5’ larvae bearing one dose of the BX-C show more extreme anterior transformations of the cuticular features than the Rg-bx/DfredPS2 zygotes with two copies of the BX-C, as described previously (DUNCAN and LEWIS 1982). Rg-bx DpP5/DfredP5’ larvae with an extra copy of the BX-C exhibit a s u p pressed Rg-bx- lethal syndrome. For example, like wild-type larvae, the Rg-bx- larvae with three doses of the BX-C display a wild-type pattern of dorsal spinules on the abdominal segments rather than the more thoracic-like pattern described above for Rg-bx- lar- vae wild type for the BX-C. Dependence of the Rg-bx phenotypes on the dosage of the BX-C has been reported for the adult flies as well (CAPDEVILLA and GARCIA-BELLIDO 198 l), and further supports the no- tion that the absence of the Rg-bx+ gene function results in a decreased level of BX-C functions.

Cuticular morphologies of Pc- Rg-bx’ embryos: Pc- embryos show transformations of cuticular structures to resemble those of more posterior segments (DENELL and FREDERICK 1983; DUNCAN and LEWIS 1982; LEWIS 1978; STRUHL, 198 1 b, 1983). In Pc’ Rg- bx/Pc’ Dfred”’ embryos, such posterior transformations a re globally suppressed as shown previously for est- lethal embryos homozygous for trx’ (INGHAM 1983); head involution is less incomplete, the VDBs of thoracic segments show fewer abdominal denticles, and the posterior transformation of the VDBs of abdominal segments is less extreme. In addition, Pc’ Rg-bx/Pc3 DfredP5’ embryos show more frequently a second VDB on the MS and M T than do Rg-bx+ controls (Table 1, lines IV and V). We have made similar observations for the MS of e x - Rg-bx- embryos derived from esc- mother (data not shown). In addition to its morphology, the prothoracic nature of this second VDB is suggested by its less frequent appearance in Pc’ ScrWI7 Rg-bx/Pc’ DfScr DfredP5’ embryos (Table 1, lines IV and VII). Evidently, the extent of abdominaliiation of the mesothoracic VDB and fre- quency of development of the second VDB on the MS of Pc- embryos are negatively correlated and dependent on the genotype of the Rg-bx locus. We have observed a similar negative correlation between anterior and posterior transformations of mesothoracic structures in Pc- embryos of various BX-C genotypes (SATO and DENELL 1985). Accordingly, hypomorphic activities of the BX-C genes associated with the Rg- bx- condition can account for such a negative correlation. However, such an interpretation does not exclude the possible influences of the Rg-bx- condition on the expression of other homoeotic genes, particu-

392 T. Sat0 and R. E. Denell

TABLE 1

Anterior and posterior transformations of the mesothorax of Pc- Rg-bx embryos

Embryos showing the class of abdominalization (%) 2nd No. of

No. Genotype“ VDB’ embryos 1 2 3 4 Total Mean‘

I Pc’/Pc’

I1 Pc3 Rg-bx/ PI?

111 PE’/ Pc’ DfredP5’

Pc’ DfredP5’

PI? DfredPs2

Pc’ DfScr DfredP5”

Pc’ DfScr Dfred”’

I V Pc’ Rg-bx/

v Pc’trx’l

VI Pc’ Rg-bx/

VI1 Pc’ ScrWI7 Rg-bxl

94 21 61 17 59 34 39 48 29 20 16 51 43 21

0.9 60.0 19.2 1.7 81.8 2.27 1.7 10.4 6.1 18.2 2.24

19.2 50.0 9.0 78.2 1.87 9.0 12.8 21.8 1.59

21.5 40.9 1 . 1 63.5 1.68 17.2 19.3 36.5 1.53 35.6 9.2 44.8 1.21 49.4 5.8 55.2 1.10 44.9 14.3 59.2 1.24 34.7 6.1 40.8 1.15 19.4 4.5 23.9 1.19 58.2 17.9 76.1 1.24 56.3 10.9 67.2 1.16 32.8 32.8 1 .oo

a For crosses, see APPENDIX. The presence or absence of prothoracic second VDB is indicative of anterior transformation of mesothoracic cuticle. In some genotypes,

the second VDB contains also denticles significantlv longer than the normal prothoracic denticles of the second VDB. In those genotypes, U , -

the prothorax also displays such longer denticles in the second belt. Mean of classes of abdominalization.

larly of those involved in homoeosis between prothoracic and mesothoracic cuticular features.

Cuticular morphology of Rg-bx- BX-C- embryos and effects of the ANT-C mutations: HAFEN, LEVINE and GEHRING (1 984) have reported that expression of Antp gene is enhanced by the absence of the BX-C in larval abdomen. Therefore, the animals deleting the BX-C are appropriate for tests of the relationship between the ANT-C and Rg-bx gene, because we can exclude any possible effect on the ANT-C expression by the BX-C which is regulated by the Rg-bx gene. The cuticular pattern of the Rg-Bx Dfp9/DfredP5’ Dfp9 embryos is similar to the DjP9 homozygotes with one exception. The Dfp9 homozygotes lacking the BX-C show a reiterating cuticular pattern on the MS through abdominal seventh segment (AB7) which is predominantly mesothoracic anteriorly and prothoracic posteriorly (HAYES, SATO and DENELL 1984; SATO, HAYES and DENELL 1985), though a prothoracic second VDB very rarely appears on the anterior portion of these segments (Table 2, Line I). In the Rg-bx Dfp9/DfredP52 DjP9 embryos, however, the MS through AB7 display the prothoracic second VDB much more often than in the Rg-bx+ control (Table 2, lines I1 and III), and prothoracic denticles occasionally mingle with mesothoracic denticles on the first VDB of the MS through AB7.

We have analyzed the cuticular morphology of the Rg-bx- BX-C- embryos of various genotypes at the ANT-C to examine possible interactions between the Rg-bx and ANT-C genes, for an involvement of the ANT-C genes is a reasonable explanation for such a transformation of mesothoracic cuticular features into

prothoracic ones. Results summarized in Table 2 (lines IV, V and VI) show dependence of anterior transformations of mesothoracic cuticular structures of the Rg-bx- BX-C- embryos on the dosage of the ANT-C. A negative correlation between the number of the ANT-C dose and probability of anterior transformation is evident (Table 2, lines IV, V and VI). The ANT-C dosage affects not only appearance of the second VDB which was scored in Table 2 but also the morphology of the first VDB. Segments, particularly posterior abdominal ones, of the Rg-bx Dj2’9I DfScr Df~-ed‘~~ DjP9 embryos very often elaborate prothoracic denticles on the first VDB and look prothoracic rather than mesothoracic in extreme cases (Figure 3, panel E). Hypomorphic activities of the ANT-C genes appear responsible for the anterior transformations of mesothoracic cuticular structures of the Rg-bx DjP9lDfScr DfredP5’ DjP9 embryos with one dose of the ANT-C, since segments of the DpDl Rg-bx DjP9IDfScr Df~ed’~~ DjP9 embryos with two copies of the ANT-C show mesothoracic first VDB (Figure 3, panel J) and develop prothoracic second belt at lower frequencies comparable with the Rg-bx DjP9/DfredPZ2 Dfp9 embryos (Table 2, line VII). Thus, development of prothoracic denticles on both VDBs is obviously related with the decrease of the dosage of the ANT-C in Rg-bx- BX-C- embryos. However, prothoracic denticles do not develop on the VDBs of the Rg-bx+ BX-C- embryos with one dose of the ANT-C (SATO, HAYES and DENELL 1985). T o specify further which gene of the ANT-C is involved in this homoeosis, we have inspected the morphology of the Rg-bx- BX-C- embryos heterozygous for

Regulator of Bithorax Syndrome

TABLE 2

Development of prothoracic second VDBs on the MS through AB7 of BX-C- embryos

393

No. of em-

Segment showing prothoracic second VDB (%)

No. Genotype" bryos Penetrance' MS M T AB1 AB2 AB3 AB4 AB5 AB6 AB7

I I1

111 IV V

VI

VI1

VI11

IX

X

XI

DF9IDjP9 DjP9/Dfredps2 DjP9 t r g DjP9/DfredPs2 DjP9 Rg-bx DjP9/Dfredps2 DjP9 Rg-bx DjP91DfScr DfredPs2 DjP9

DfredPs2 DfP9

DfScr DfredPSZ DjP9 Scr"I7 Rg-bx DjP9/ DfredP5' DjP9 Antp"" Rgbx DjP9/ Dfredps2 DjP9 Scr"I7 Antp"" Rg-bx DjP9/Dfredpsz DjP9 Scr"I7 Rg-bx DjP9/ DfScr Dfred"' DjP9

DpDI Rg-bx DjP9/

DpDl Rg-bx DjP9/

62 65 23 44 32

35

39

34

30

30

26

1.6 1.6 3.1 1.5 3.1

21.7 4.3 4.3 4.3 8.7 72.7 6.8 13.6 29.5 11.4 25.0 6.8 22.7 43.2 38.6 96.9 25.0 28.1 75.0 68.8 71.9 68.8 71.9 71.9 75.0

5.7 2.9 2.9

46.2 2.6 5.1 5.1 10.3 5.1 5.1 17.9 20.5

79.4 8.8 8.8 11.8 32.4 41.2 26.5 29.4 35.3 14.7

86.7 26.3 46.7 56.7 50.0 60.0 56.7 46.7 66.7 70.0

96.7 10.0 16.7 63.3 53.3 66.7 56.7 66.7 73.3 80.0

88.5 11.5 19.2 57.5 57.5 50.0 46.2 53.8 50.0 61.5

a DjP9 homozygotes of various genetic backgrounds were generated by crosses summarized in APPENDIX. Percentage of embryos showing the prothoracic second ventral denticle belt (VDB) on any segment from the mesothorax (MS) through

abdominal seventh segment (AB7).

amorphic alleles of Scr and Antp loci. The prothoracic denticles appear on the first and second VDBs of the MS through AB7 of the Antp"" Rg-bx DjP9/DfredP5' DjP9 or ScrWl7 Antp"" Rg-bx DjP9/DfredP5' DjP9 embryos as frequently as the Rg-bx DjP9IDfScr DfredP5' DjP9 (Table 2, lines VI, IX and X; Figure 3, panels C, D and E), whereas the ScrWl7 Rg-bx DjP9/DfredP5' DjP9 embryos display the prothoracic second VDB as frequently as the Rg-bx DfP9/Dfred "' embryos (Table 2, lines IV and VIII) and mesothoracic first VDB (Figure 3, panel B). Moreover, theftrwZ0 Rg-bx DjP9/ DfredP5' DfP9 embryos heterozygous for an amorphic allele of the fushi tururu locus (another gene of the ANT-C) show the prothoracic second VDB much less frequently than the Rg-bx DjP91DfScr DfredP5' DjP9 embryos and the first VDB is mesothoracic (data not shown). These results evidently indicate that the hypomorphism of the Ant$+ gene is responsible for homoeotic conversion of mesothoracic to prothoracic cuticular features in the Rg-bx DjP9/DfScr DfredP5' DjP9 embryos, though we can not exclude completely the possibility of involvement of other genes of the ANT-C than those tested here. Because D~P9lDp9 Dfscr embryos are morphologically similar to the DjP91DjP9 embryos which do not usually display the prothoracic denticles (SATO, HAYES and DENELL 1985), the lack of the Rg-bx+ gene activity renders the Antp+ gene functionally hypomorphic.

The cuticular morphology of the corresponding hemizygotes of Antp"" and ScrWl7 were also analyzed to evaluate the extent to which the activity of the Antp gene is impaired in the Rg-bx DP9/DfredP5' DfP9

embryos. Antp"" Rg-bx DfP9/DfScr Dfred p52 DjP9 embryos show cuticular morphology basically similar to that of the Rg-bx+ counterpart except for occasionally poor sclerotization of the cuticle; the prothorax is anteriorly normal but posteriorly bears "vertically ridged cuticle" (STRUHL 1983) on the ventral side and chitinized plates on the dorsal side. The MS through AB7 show anteriorly prothoracic VDBs which are usually split into two parts and posteriorly chitinized plates (Figure 3, panel K) (SATO, HAYES and DENELL 1985). Obviously, the cuticular phenotype of either Antp"" Rg-bx DjP9/DfredP52 DjP9 or Rg-bx DjP9/ DfScr DfredP5' DjP9 embryos is not so extreme as the Antp"" DjP9lDfScr DjP9 or Antp"" Rg-bx DjP91DfScr DfredPS2 DjP9 embryos (Figure 3, panels C and K). It is therefore suggested that the absence of the Rg-bx+ gene function lowers the level of the Antp+ gene activity but does not completely eliminate it. This conclusion is also supported by the cuticular morphology of the ScrWl7 Rg-bx DjP91DfScr DfredP5' DjP9 embryos. We have previously reported that the cuticular phenotype of Scr- BX-C- embryos depends on the genotype of the Antp+ gene; the VDBs of the MS through AB7 are mesothoracic in Scr- BX-C- embryos when they are Antp+, whereas they are prothoracic in Scr- Antp- BX-C- embryos (SATO, HAYES and DENELL 1985). When compared with the Rg-bx DjP9/ DfScr DfredP5' DjP9 embryos, ScrWl7 Rg-bx DjP91DfScr DfredP5' DjP9 embryos exhibit nearly mesothoracic VDB on the MS through AB7 (Figure 3, panels E and F) and development of the prothoracic second VDBs on these segments is somewhat suppressed (Table 2,


I I G L R K :<.-CuticiiI;ir morphologv o f Kg-hx- BX-<:- embryos of various :\ST-C gcrlotypcs. l ' t i o ~ o ~ i i i ~ r o ~ ~ ~ ~ ~ t i ~ nwc c.iLcn through the phase contrast optics for the ventral aspects of lethal embryos. The abdominal segments (except for thc. eighth one of the IIpY homoi!gotes lacking the entire BX-C) display thoracic cuticular structures such as a mesothoracic ventral denticle belt (I'DR) on the anterior edge, sensory papillae [or ventral pits in LEWIS (1978): indicated by open triangles], and the Keilin's organs (indicated by closed triangles). Arrows and arrowheads indicate rudimentary second VDBs and sclerotized cuticular materials, respectively. For crosses generating embnos. see APPENDIX

(X810 for panels G , H, and 1. and X396 for other panels). A, T h e abdominal fifth segment (AB5) of the Rg-bx DfP9/DfredP" Dfp9. B, The AB5 of ScrWJ7 Rg-bx D f p 9 / D f r ~ d ~ ' ~ Dfp9. C . T h e AB5 of Antp"'" Rg-bx Dfp9/Dfredp'z Dfp9. Note prothoracic denticles on the first VDB and several denticles of the rudimentary prothoracic second VDB. D, The AB5 of the SCT""~ Antp"'" Rg-bx D f p 9 / D f r ~ d " ' ~ Dfp9. E, T h e AB5 of the Rg-bx Dfp9lDfScr Dfred"' Dfp9. F, T h e abdominal third segment of the S C ~ " ' ~ Rg-bx DfPP/l)fScr DfrcdP'2 Dfp9. C , A rudimentary prothoracic second belt magnified from panel C. H. A magnified view of a rudimentary prothoracic second belt of panel D. I , A rudimentary prothoracic second belt of panel E in magnification. J, T h e abdominal seventh segment of the D p D I Rg-bx Dfp9/Df.Scr DfredP" Dfp9. K. The AB5 of the Antp"" Rg-bx Dfp9/DfScr Dfred"" Dfp9. T h e abdominal segments of embryos shown in this and following panels show prothoracic denticles anteriorly and chitinized plates (indicated by arrowheads) posteriorly on the ventral sides. L, The abdominal fourth segment of the Scr"" Antp"' Rg-bx Djl'9/DfScr Dfred"'' Dfp9.

lines V and XI). Therefore, the cuticular phenotype of S C P * ' ~ Rg-bx Dfp9lDfScr DfredP5' Dfp9 embryos is consistent with the speculation that the Antp+ gene is still somewhat active, if not fully, in the Rg-bx Dfp9/ DfredP5' DfP9 embryos. T h e SCI"''~ Antp"'" Rg-bx Dfp9lDfScr Dfred"' Dfp9 embryos exhibit almost sim-

ilar morphology to the Rg-bx+ counterpart except for heavier sclerotization of chitinized plates on the dorsal and ventral sides of the segments.

In summary, the absence of the Rg-bx+ function causes directly or indirectly low level expression of the Antp and BX-C genes.

Regulator of Bithorax Syndrome 395

DISCUSSION

The cuticular morphology of the Rg-bx- larvae is represented by homoeotic transformations of cuticular features to resemble those of more anterior segments. Results presented in this paper and INGHAM (1983) are consistent in that there is no aberrant cuticular morphology in the head and prothorax (PRO) of the Rg-bx- embryos. INGHAM (1983) also reported homoeosis of the VDB of the MT to resemble that of the MS, and of the VDBs of posterior abdominal segments to that of the AB3 or AB4 in the hemizygotes of the trx2 or trx’ allele. We have made similar observations on hemizogytes of Rg-bx or trx’. However, the pattern of the central nervous system analyzed by GHYSEN, JAN and JAN (1985) disclosed patchy transformations of abdominal segments to the pattern of thoracic or the abdominal first segment in the trx’ viable homozygotes without parallel effect on the epidermal pattern. By careful examination of the dorsal spinule pattern of the Rg-bx- larvae, we have also noted the parallel homoeosis in the cuticular pattern; transformation of the M T and anterior abdominal segments to the MS (Figure 2, panel D). Thus, homoeotic transformation is variegated in abdominal segments of the Rg-bx- larvae; transformations to mesothoracic pattern on the dorsal side and to the pattern of the AB3 and AB4 on the ventral side. It could be possible that requirement for the Rg- bx+ function and activities of other homoeotic genes influenced by the absence of the Rg-bx+ activity is regionally variable within a segment.

The caudal segment also displays abnormal morphology in the Rg-bx- embryos. Tiny chitinized plates which may be homologous to the mouth parts appear just anteriorly to the anal pads in the Rg-bx- embryos. Discrepancy between our and INGHAM’S (1 983) results with this regard might be due to the difference of the genetic background or of procedures to yield the zygotes. In addition, in Rg-bx- embryos a hair sensillum designated number four is located at a subtly but significantly more posterior position. This sensillum recently has been assigned to the cryptic abdominal ninth segment (AB9) (DAMBLY-CHAUDIERE and GHY- SEN 1986; SATO and DENELL 1986; WHITTLE, TIONG and SUNKEL 1986). A posterior shift of the sensillum number four is associated with an anterior transformation of the AB9 in some cases like lethal embryos of Pc- embryos with one dose of the BX-C (T. SATO, unpublished data). We favor the possibility that the posterior shift of the number four sensillum in the Rg-bx- larvae indicates an anterior transformation of the AB9. Thus, the cuticular morphology of the M T through the telson is affected and largely transformed anteriorly in the Rg-bx- embryos.

In contrast with the Rg-bx- lethal syndrome in larvae, the adult Rg-bx- phenotype is more variable

(CAPDEVILLA and GARCIA-BELLIDO 198 1 ; INGHAM 1981, 1985, INGHAM and WHITTLE 1980). The adult Rg-bx- clones show anterior transformation of cuticular patterns of the metathoracic leg, haltere, posterior wing, and female genitalia, and these transformations may be comparable to the anterior transformation in the Rg-bx-. However, the Rg-bx- clones of the antenna, eye, proboscic, prothoracic leg and anterior wing show other types of transformation, which have no corresponding counterpart in the Rg-bx- lar- val homoeosis. The type of these morphological transformations is very diverse, and interactions of the Rg- bx+ locus with many loci could be involved in imaginal cells. The expression and function of the Rg-bx gene might not be identical in imaginal and larval cells. Interestingly abdominal segments except for the AB8 show a kind of allele specificity in adult phenotypes. Homozygotes or hemizygotes of hypomorphic trx’ show anterior transformation of tergites of abdominal segments while clones which are homozygous for Rg- bx or are trx2/trx3, and which should be nearly or completely lacking Rg-bx+ activity, are morphologically normal in abdominal segments (CAPDEVILLA and GARCIA-BELLIDO 198 1 ; INGHAM 198 1, 1985; LNGHAM and WHITTLE 1980). As discussed in INGHAM (1 985), these paradoxical results could be explained by the growth dynamics of abdominal histoblast cells which do not divide through larval stages when the Rg-bx- clones are induced.

It is the consensus that the insufficient expression of the BX-C is brought about by the absence of the Rg-bx+ function. Morphological resemblance to the BX-C mutants and dependence on the dosage as well as on the genotype of the BX-C in adult and larval Rg-bx mutants are compatible with this conjecture. We have found that the phenotype of Rg-bx- larvae indicates an apparent reduction in Antp+ gene function. In contrast with the BX-C, however, neither adult Rg-bx heterozygotes nor Rg-bx- clones display any morphology corresponding to the hypomorphism of the Antp+ gene. In adult flies, the lack of the Antp+ function provokes development of antennal tissue on the mesothoracic leg, but indiscriminate expression of the Antp+ results in homoeosis of antennae to the mesothoracic leg (STRUHL 198 1 a). In fact, Rg-bx- and trx2/trx3 clones show rather antenna to leg transformation (CAPDEVILLA and GARCIA-BELLIDO 198 1 ; INGHAM 1985) which could result from the expression of Antp+ gene in the antenna, where it is normally repressed. Thus, mode of interactions between the Rg-bx and Antp genes may not be identical in larval and imaginal cells. However, it is not yet guaranteed that antenna to leg transformation in the Rg-bx- tis- sues is based on the hypermorphism of the Antp gene, and accordingly, modulation of other gene activities could be responsible for this homoeosis. We have


shown that the Antp' gene is not rendered quiescent but retains its activity, though not fully, in the Rg-bx- BX-C- embryos. Therefore, a residual activity of the Antp' gene might be sufficient to support the normal growth of the mesothoracic legs in imaginal Rg-bx- clones. In this context, growth of mesothoracic legs in place of antennae might be based on the alteration of expression of other genes than the Antp gene. That this homoeosis is rather of the spineless-aristapedia type than Antennapedia is compatible with this possibility (INGHAM 1985). There is no evidence available for the interaction between the Scr and Rg-bx genes in embryogenesis. We have found also no conclusive evidence in the literature for such an interaction in adult phenotypes, though the Scr and Rg-bx mutants share a common phene (INGHAM and WHITTLE 1980). Thus, the nature of Scr gene expression in the Rg-bx mutant is still open to question.

The Antp and BX-C (particularly Ubx) genes share several common genetic factors controlling their expression. The ftz+ gene function is required for initiation and establishment of their expression in early embryogenesis (DUNCAN 1986; INGHAM and MARTINEZ-ARIAS 1986). Their expression is also regulated by genes within the BX-C (CARROLL et al. 1986; HAFEN, LEVINE and GEHRING 1984; STRUHL and WHITE 1985). The Antp and BX-C genes are subjected to regulation by a group of homoeotic loci like Pc and esc which is necessary to maintain proper expression of the Antp and BX-C genes. We have added to the third group another genetic loci, the Rg-bx, controlling expression of the Antp and BX-C genes, though it is not still certain that the Rg-bx regulates expression of the Antp and BX-C genes in an identical way. On the assumption that the Antp and BX-C genes are identically regulated by the Rg-bx, the role of the Rg- bx gene seems to be required for maintenance of optimal expression of those genes as suggested by INGHAM (1985) and in this paper. However, the Rg- bx + function is seemingly dispensable, because the VDBs of Pc- Rg-bx- or esc- Rg-bx- embryos derived from esc- mother are nearly normal, suggesting the differential expression of the BX-C genes in the absence of the Rg-bx+ function (INGHAM 1983) (T. SATO, unpublished data).

CAPDEVILLA and GARCIA-BELLIDO ( 1 98 1) reported in detailed analysis of flies with varying dosages of Pc and Rg-bx genes that the Rg-bx gene is antagonistic to the Pc gene in function. Cuticular morphology of Pc- Rg-bx- lethal embryos is consistent with this interpretation. Rg-bx- mutations also exert similar influences on Polycomblike adult phenotype (T. SATO, unpublished data) and esc- embryos (INGHAM 1983). How- ever, these results do not ensure the possibility that the Rg-bx+ gene product interacts directly with the product of Pc+ and other similar genes. It has been

indicated that the BX-C, Antp, and Scr genes are indiscriminately expressed in lethal embryos of mutations of Pc, esc and other genes (DUNCAN 1982; SATO, HAYES and DENELL 1985; STRUHL 1981b, 1983). Therefore, the resulting phenotypes of Pc- Rg- bx- or esc- Rg-bx- embryos could be the balance of the opposing effects of the Rg-bx and either Pc or other related genes on the expression of the BX-C and other genes.

We are grateful to P. W. INGHAM, T . C. KAUFMAN and E. B. LEWIS for sharing mutations and deletions with us. We also appre- ciate A. GHYSEN providing unpublished informations. The initial phase of this work was conducted at Division of Biology, Kansas State University. This work was supported in part by a National Science Foundation grant (PCM-840525 1) to R.E.D.

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Communicating editor: W. M. GELBART

APPENDIX

Crosses Generating Zygotes Analyzed

Genotypes"

Zygotesb Female parents Male parents

DfredP5'/Rg-bx DfredP"/trx3 DfredP5'/Rg-bx DjP9 DfredP5'/trx3 DjP9 DfredP5'/Rg-bx DpP5 DfredPi2/trx3 DpP5

Pc' Rg-bx/Pc' Pc3 DfredP5'/Pc3 Pc3 DfredP3'/Pc3 Rg-bx Pc' DfredP5'/Pc3 trx3 PC' DfScr DfredP5'/Pc3 Rg-bx

Pc3/Pc3

Pc3 DjScr DfredP5'/Pc3 Scr"I7 Rg-bx DfP9lDfP9 DfredPJz DjP9IDJP9 DfredPJz DjP9/trx3 DjP9 Dfred"' DjP9/Rg-bx DjP9 DfScr DfredP5' DjP9/Rg-bx DjP9 Dfred''' DjP9/DpDI Rg-bx DjP9 DfScr DfredP5' DjP9/DpDI Rg-bx DjP9 DfredP5' DjP9/Scr"I7 Rg-bx DjP9 DfredP5' DjP9/AntpW" Rg-bx DjP9 Dfred"' DjP9/ftzWzo Rg-bx DjP9 DfredP5' DjP9/Scr"I7 Antp"" Rg-bx DjP9 DfScr Dfredp'2 DjP9/ScrWJ7 Rg-bx DjP9 DfScr DfredP5' DjP9/AntpW" Rg-bx DjP9 DfScr DfredP12 DfP9/Scr"I7 Antp"" Rg-bx DjP9

Dfred p52/+ Dfred p52/+

Dfred p52/+ DfredPJ2/+ Dfred "'/+ Dfred 'Ii2/+

Pc ' Rg-bx/TM I Pc3 Dfred p5z/TMI Pc3 DfredP5'/TM1 Pc DfredP5'/TM I Pc3 DfScr Dfred""/TMI Pc' DfScr DfredP5'/TMI DjP9lDpP5 Dpbxd"'/+; DfredP-" DjP9ITMI Dpbxd I " / + ; Dfred"-" DjP9ITM 1 Dpbxd"'/+; Dfred"" DjP9ITMI Dpbxd"'/+; DfScr DfredPs2 DjP9ITMl Dpbxd"'/+; Dfred"' DjP9ITML Dpbxd"'/+; DfScr Dfred"' DjP9ITMI Dpbxd"'/+; DfredPSZ DjP9ITMI Dpbxd"'/+; DfredP-" DjP9ITMI Dpbxd"'/+; DfredP5' DjP9ITMI Dpbxd"'/+; Dfred"" DjP9ITM I Dpbxd"'/+; DfScr Dfred"' DfP9ITMI Dpbxd"'/+; DfScr Dfred'" DjP9ITMl Dpbxd"'/+; DfScr DfredPi2 DjP9ITMI

Pc3/+

Rg-bx /+ trx'/+ Rg-bx DjPYIDpP5 trx3DjP9/DpP5 Rg-bx DpP5I-k trx3 DpP5/+ Pc;'/+ Pc3/+ Pc3/+ Pc3 Rg-bx/+ PC' trx3/+ PE' Rg-bx/+ Pc3 Scr"I7 Rg-bx/+ DjPYlDpP5 DjP9lDpP5 trx ' DjPYIDpP5

Rg-bx DjPYlDpP5

DpDl Rg-bx DjPYIDpP5 Scr"I7 Rg-bx DjP9IDpP5 Antp"" Rg-bx DjP9IDpP5 flzWzo Rg-bx DjP9IDpP5 Scr"I7 Antp"" Rg-bx DjP9/DpP5 Scr"I7 Rg-bx DjPYIDpP5 Ant$"" Rg-bx DjPYIDpPS Scr"I7 Antp"" Rg-bx DfP9/DpP5

Rg-bx DjP9/DpP5

DpDl Rg-bx DjP9/DpP5

~ ~~~

a Most chromosomes listed in this table are marked with irrelevant mutations, description of which is omitted for convenience. * Hemizygotes of Rg-bx or trx' with varying doses of the BX-C are larval lethal. Larvae of these genotypes were identified when compared

with larvae from the control crosses (e.g., DfredP5'/+ X Rg-bx DjP9/DpP5 with +/+ X Rg-bx DjP9/DpP5). Derivatives of Pc' homozygotes are lethal classes from the corresponding crosses. DjP9 homozygotes are easily identified on the basis of typically reiterating pattern of segments in abdomen.

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