Structural Changes in the Antennapedia Complex of ... · Antennapedia Complex Evolution 32 1 1984). The probes were made using the random primer a’* P method (FEINBERG and VOGELSTEIN

Copyright 0 1993 by the Genetics Society of America

Structural Changes in the Antennapedia Complex of Drosophila pseudoobscura

Filippo M. Randazzo,’ Mark A. Seeger,* Catherine A. HUSS, Margaret A. Sweeney, Jeffrey K. Cecil and Thomas C. Kaufman’

Howard Hughes Medical Institute, Institute for Cellular and Molecular Biology, and Programs in Genetics and Cellular, Molecular and Developmental Biology, Department of Biology, Indiana University, Bloomington, Indiana 47405

Manuscript received October 16, 1992 Accepted for publication January 27, 1993

ABSTRACT The discovery of the striking positional conservation between the Antennapedia and Bithorax

homeotic gene complexes (ANT-C and BX-C) in Drosophila melanogaster and the murine Hox and human HOX clusters has had a substantial impact on our understanding of the evolution of development and its genetic regulation. Structural differences do exist among the mammalian Hox complexes and the ANT-C in D. melanogaster. To gain further insight into the evolutionary changes among these complexes, the ANT-C was cloned in the closely related species, Drosophila pseudoobscura. The overall structure of the ANT-C in D. pseudoobscura is highly similar to its D. melanogaster counterpart; however, two differences in the organization of the ANT-C have been identified. First, the 2 2 gene, a member of the ANT-C in D. melanogaster, is not present in the D. pseudoobscura ANT- C and is possibly absent from the D. pseudoobscura genome. Second, the orientation of the Deformed gene is inverted in D. pseudoobscura, providing it with a 5’to 3‘ direction of transcription identical to the remaining ANT-C homeobox genes with the exception of fushi tarazu. These differences demonstrate that subtle changes can occur in ANT-C structure during relatively short periods of evolutionary divergence, although the fundamental organization of the complex is conserved. These - - observations and others suggest that the complex is have maintained this organization of genes for some

T HE discoveries of the Antennapedia Complex (ANT-C) and the Bithorax Complex (BX-C) in

Drosophila melanogaster (KAUFMAN, LEWIS and WAKI- MOTO 1980; LEWIS 1978) and the subsequent isolation of cognate Hox complexes in mammals (DUBOULE and DOLLE 1989; GRAHAM, PAPLOPULU and KRUMLAUF 1989; BONCINELLI et al. 1988) have contributed significantly to an understanding of the evolution of the developmental process and its genetic control. It has become increasingly clear that not only are genes that encode metabolically important products conserved across phyla, but also loci that encode developmentally important gene products. Among the developmentally important genes, homeobox genes have been isolated from a large array of eucaryotic species (see SCOTT, TAMKUN, and HARTZELL 1989 for a review). Perhaps more significantly, clustered sets of homeobox genes have been discovered in humans (BONCI- NELLI et al. 1988), mice (DUBOULE and DOLLE 1989; GRAHAM, PAPLOPULU and KRUMLAUF 1989; KAPPEN, SCHUGHART and RUDDLE 1989), chickens (WEDDEN,

Avenue, Toronto, Ontario M5G 1x5, Canada. ’ Present address: Mount Sinai Hospital Research Institute, 600 University

* Present address: Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104.

’Present address: Howard Hughes Medical Institute, Department of Biology, Indiana University, Bloomington, Indiana 47405.

Genetics 133: 319-330 (May, 1993)

v

not absolutely rigid but that selective pressures Functional reason that remains elusive.

PANG and EICHELE 1989; IZPISUA-BELMONTE et al. 199 l), and the German cockroach Blattella germanica (Ross and TANAKA 1988) and the red flour beetle Tribolium castaneum (STUART et al. 1991). Genetic analysis of Tribolium has uncovered a combined ANT-C/BX-C-like gene complex referred to as the HOM-C (BEEMAN et al. 1989). Its member genes are functionally homologous and similarly aligned to the ANT-C and BX-C homeotic genes of Drosophila (BEE- MAN et al. 1989). Furthermore, four duplicated sets of genes with high sequence similarity to the ANT-C/ BX-C genes has been found in the mouse (Hox) and in humans (HOX) (see AKAM 1989 for review). These observations taken together indicate that the homeobox must have been present early on in the evolution of eucaryotes, and a combined ANT-C/BX- C-like complex must have been present before protostomes and deuterostomes diverged over 500 million years ago.

The structure of the ANT-C and the BX-C in D. melanogaster is characterized by the unusual proximity of a significant number of developmentally important genes and by the collinearity between homeotic gene position on the chromosome and their respective functional domains along the anterior-posterior axis of the animal. The numerous homeobox-containing genes

320 F. M. Randazzo et al.

of both these complexes presumably arose through a series of duplication and divergence events (LEWIS 1978). In contrast to the clustered configuration of these genes, inversion events normally tend to rear- range genes over evolutionary time (STURTEVANT and TAN 1937; DOBZHANSKY and STURTEVANT 1937). Thus, the extreme proximity of the homeobox genes infers some form of evolutionary constraint on change. At least two hypotheses could explain the existence of the homeotic gene complexes: (A) these genes cannot function o r do not function as well outside the domain of the complexes, e.g., because of possible chromatin structure requirements; (B) the juxtaposition of the member genes and their complex cis-regulatory regions makes separation by random genetic processes highly unlikely or even impossible, thereby maintaining the ancient configuration.

T h e unique characteristics of the ANT-C in D. melanogaster necessitate evolutionary analysis in more closely related species to determine the plasticity of the ANT-C structure. The ANT-C contains five homeotic genes that specify head and thoracic segmental identity, Antennapedia (Antp), Sex combs reduced (Scr), Deformed (Dfd ) , proboscipedia ( p b ) and labial (lab) [see MAHAFFEY and KAUFMAN 1987 or KAUFMAN, SEEGER and OLSEN 1990 for review]. The ANT-C differs from the BX-C [see DUNCAN 1987 and PEIFER, KARCH and BENDER 1987 for review] and from the murine Hox (DUBOULE and DOLLE 1989; GRAHAM, PAPLOPULU and KRUMLAUF 1989; KAPPEN, SCHUCHART and RUD- DLE 1989) and human HOX (BONCINELLI et al. 1988) complexes in that it carries some unique homeobox genes involved in establishing the embryonic body plan, namely zen,f tr and bcd. T h e pair rule genefushi tarazu ( f t z ) acts to divide the embryo into segments (WAKIMOTO, TURNER and KAUFMAN 1984; WEINER, SCOTT and KAUFMAN 1984), the maternal effect gene bicoid (bcd) encodes a morphogen important in establishing anterior-posterior polarity (FRIGERIO et al. 1986; BERLETH et al. 1988), and the zygotic gene zerknullt (ren) specifies dorsal embryonic structures (WAKIMOTO, TURNER and KAUFMAN 1984; RUSHLOW et al. 1987). In addition, the z2 gene, which is located just proximal to Zen, contains a zen-like homeobox (RUSHLOW et al. 1987; PULTZ et al. 1988). It is ex- pressed in a pattern similar to that of zen, but has no detectable genetic function (PULTZ et al. 1988). The ANT-C also carries several other transcription units that do not contain homeoboxes- amalgam (ama) a member of the immunoglobulin superfamily (SEEGER, HAFFLEY and KAUFMAN 1988), and a cluster of eight genes that show sequence similarity to cuticle protein genes (FECHTEL et al. 1988; Fechtel, Fristrom and FRISTROM 1989; PULTZ et al. 1988; PULTZ 1988) . The presence of these nonhomeotic genes is one differen- tiating characteristic of the ANT-C from the BX-C,

the murine HOX complexes and the human HOX complex.

To address the questions pertaining to the rigidity of the ANT-C organization and the unique presence of nonhomeotic genes within its boundaries, the ANT-C in Drosophila pseudoobscura was cloned. Pre- vious studies have documented numerous chromosomal changes in D. pseudoobscura when compared with D. melanogaster, indicating the potential for changes in the ANT-C (STURTEVANT and TAN 1937; DOBZHANSKY and STURTEVANT 1937; STEINEMANN, PINSKER and SPERLICH 1984). The cloning of ANT- C from a second Drosophilid species, estimated to have diverged approximately 46 million years ago (BEVERLY and WILSON 1984), and the previous cloning of homologous homeobox gene clusters in two mammalian species (mouse and human), estimated to have diverged roughly 68 million years ago (BENTON 1990), presents an opportunity to explore patterns in the more recently evolutionary history of homeobox gene complexes.

MATERIALS AND METHODS

Cloning ANT-C The chromosome walk was performed using techniques described in SCOTT et al. (1983). Both lambda EMBL-4 (gift of C. LANGLEY, University of Califor- nia, Davis) and lambda DASH-2 (gift of T. HAZELRIGG, Columbia University) D. pseudoobscura genomic libraries were used. The Ayala strain was used to make the EMBL-4 library. All clones except for p191, p195, p474, p475 and p488 were obtained from the EMBL-4 library. Prehybridi- zation and hybridization were performed using either 4X SSC, 5X Dendhardt's solution, 0.1% (w/v) SDS and 0.1% (w/v) carrier calf thymus DNA at 65" (interspecies and intraspecies hybridizations) or 5X SSC, 5X Denhardts, 0.1 % (w/v) SDS and 0.1 % (w/v) calf thymus DNA plus either 30% (w/v) formamide (intraspecies) or 50% (w/v) formamide (interspecies) at 42". Intraspecies hybridizations were followed by 0.1X SSC, 0.1 % (w/v) SDS, 0.1% (w/v) sodium py.rophosphate washes (IX wash at room temperature for 4 mln followed by 2 X washes for 40 min at 65'). The same conditions were used for Southern hybridizations. Probes were made by the nick translation method using Amersham kits (Arlington Heights, Illinois). All lambda genomic clones recovered in this study were mapped with five restriction enzymes (BurnH1, EcoRI, HindIII, Sal1 and XbaI). Cuticle- like genes were identified by Southern blot hybridization using a pIBI5 1 RX 1.5 (a known cuticle gene) subclones as probes (FECHTEL et al. 1988; FECHTEL, FRISTROM and FRIS- TROM 1989) WITH THE METHODS AND CONDITIONS AS DE- SCRIBED ABOVE.

Genomic Southern blot hybridizations: D. melanoguster Oregon R DNA was a gift of S. Horikami, while D. pseudoobscura DNA was extracted using methods described in SCOTT et al. (1983). Genomic DNA was blotted on Nytran filters (Schlechier and Schuell, Keene, New Hampshire). Prehybridization and hybridization were performed using 5x SSC, 5x Denhardt's solution, 250 Pg/ml sonicated and boiled calf thymus DNA, 50 mM Nap04 pH 7.0, 0.1% (w/ V) SDS, and 43% (w/v) deionized formamide at 37" (Mc- GINNIS et al. 1984). Washing was performed first at room temperature for 5 min ( 3 X ) and then at 50" for 15 min (2X) using 2x SSC, 0.1% (w/v) SDS solution (MCGINNIS et al.

Antennapedia Complex Evolution 32 1

1984). The probes were made using the random primer a’* P method (FEINBERG and VOGELSTEIN 1983).

RESULTS

Cloning the ANT-C from D. pseudoobscura: The ANT-C in D. pseudoobscura (psANT-C) was cloned to determine the extent of evolutionary constraints on the structure of the ANT-C in another Drosophilid species. A number of separate regions of the psANT- C were cloned by hybridizing a D. pseudoobscura genomic lambda phage library with D. melanogaster Antp, ftz, pb and lab derived probes under low stringency hybridization conditions (see MATERIALS AND METH- ODS). Standard chromosome walking techniques were used to fil l in the gaps between the cloned genomic entry points. Overlaps between steps in the walk were confirmed by Southern blot cross-hybridization and by restriction site mapping. All lambda genomic in- serts were mapped using five restriction enzymes- EcoRI, BamHI, HindIII, Sal1 and XbaI. A few restriction site polymorphisms among overlapping genomic clones were identified, and we suspect that they reflect heterogeneity in the nonisogenic flies used to generate the genomic libraries. The relative positions of D. pseudoobscura homologs of the ANT-C coding regions were first identified by Southern blot hybridization. This was done using D. melanogaster cDNAs and corresponding genomic probes and low stringency conditions (final wash of 2x SSC, 65”; data not shown). The identity of restriction fragments similar to the D. melanogaster gene probes were then confirmed by sequencing selected D. pseudoobscura subclones. The DNA sequences were then compared with existing ANT-C gene sequences from D. melanogaster.

Gene order remains conserved for ANT-C in D. pseudoobscura: The ANT-C in D. pseudoobscura has been cloned from a position downstream of the labial gene to the Antp homeobox. Except for the cuticle- like genes that were identified by Southern blot homology alone (data not shown, see MATERIALS AND

METHODS), diagnostic DNA sequences identified ANT-C gene homologs (Figure 1; SEECER and KAUF- MAN 1990 for bcd sequence) and their precise location within the psANT-C and the chromosomal positions of their D. melanogaster homologs is summarized in Figure 2. The results demonstrate that Antp,ftZ, Scr, Dfd, ama, bcd, zen, pb, the culticle-like gene cluster, and lab are clustered and identically ordered in both Drosophila species.

A 22 homolog is apparently absent from D. pseudoobscura: One difference between the two species is the absence of z 2 from the bcd-pb interval in D. pseudoobscura (Figure 3). Southern blots of D. pseudoobscura genomic clones that encompass the pb-bcd interval, p300 and p3 10, were first hybridized with D. melanogaster genomic DNA clones containing either

the 3’ or 5‘ half of z 2 and then washed under low- stringency conditions (data not shown). The 3’ probe consists entirely of open-reading frame sequences located directly downstream of the homeobox, while the 5‘ probe contains 300 bp of coding sequence, including the homeobox, and 140 bp of transcribed, noncoding sequence, as determined from previous structural analysis (RUSHLOW et al. 1987). The 12 3’ probe hybridized to the predicted restriction fragments from the D. melanogaster genomic clones and to itself, but not to any DNA within the pb-bcd interval of D. pseudoobscura (data not shown). A Southern blot probed with the z2 5’ homeobox-containing fragment showed hybridization to the 22-containing restriction fragments of D. melanogaster genomic DNA and to the 22 5’ positive control. There were no strongly hybridized bands in the lanes containing the D. pseudoobscura genomic phage (p300 and p3 10) from the pb-bcd interval. There were however several weakly hybridizing bands from these latter phages that represent cross-hybridization to zen (data not shown). A z 2 homolog should hybridize more strongly to a z 2 homeobox-containing probe than the zen homolog. Thus, D. melanogaster z2 probes fail to detect a z 2 homolog in the pb-bcd interval of D. pseudoobscura.

The question remains, however, whether z2 is present elsewhere in the D. pseudoobscura genome. T o address this question, D. pseudoobscura genomic Southern blots were hybridized separately with (A) the zen cDNA, (B) the 3’ half of z2, and (C) the 5‘ half of z2 (all of which were derived from D. melanogaster) and then washed under very low-stringency conditions (Figure 4). The zen cDNA probe, a positive control, hybridized to the expected zen-containing restriction fragments in D. melanogaster-7.2 kb BamHI, 1.2 kb BamHI, 5.9 kb HindIII and 1.4 kb HindIII (Figure 4A; see Figure 3 for restriction map). Consistent with our previous Southern hybridization and DNA sequence date in D. pseudoobscura, the zen cDNA probe hybridizes to restriction fragments that conform to the predicted sizes of zen-containing fragments in D. pseudoobscura-7.4 kb BamHI, 4.7 kb BamHI, 6.0 kb HindIII, 10.4 kb EcoRI and 1.2 kb EcoRI restriction fragments. Also evident are a number of other DNA fragments with weaker homology in both D. melanogaster and D. pseudoobscura (Figure 4A). Due to the low stringency conditions used, homeobox cross-hybridization is the most likely explanation for the numerous secondary bands.

No apparent homology to the 3‘ portion of D. melanogaster 22 (22 3’) is detectable in D. pseudoobscura ( z 2 3’ contains the downstream 47% of the 22 open- reading frame (Figure 4B)). The z 2 3’ probe detects D. melanogaster z2, since the D. melanogaster control lanes display strong hybridization to the predicted 9.6 kb BamHI fragment. In the D. melanogaster HindIII

322 F. M . Randazzo et al.

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ends 1 FIGURE 1.-DNA sequence comparisons between D. melanogaster (m.; above) and D. pseudoobscura (p.; below) from selected regions in the

D. pseudoobscura chromosome walk. The DNA sequence was aligned using the "bestfit" program of the GCG software package (DEVEREUX, HAEBERLI and SMITHIES 1984). All alignments use a gap weight of 5.00 and a gap length weight of 0.30. Vertical bars betwen D. melanogaster and D. pseudoobscura DNA sequence indicate nucleotide identity. Vertical arrows indicate intron/extron junctions. The highly conserved AG sequence immediately preceding all 3' splice sites and the highly conserved G T sequence that immediately

Antennapedia Complex Evolution 323

lane, however, strong hybridization signals are observed at 12 kb and at 4.7 kb, but not at the predicted 6.7 kb HindIII fragment. This discrepancy is most likely due to a strain polymorphism. The absence of detectable z 2 3‘ homology in D. pseudoobscura indi- cates that the 3‘ half of z 2 has diverged beyond detectability or that z 2 does not exist in D. pseudoobscura.

The 5’ z 2 probe (z2 5’), which contains a homeobox, hybridizes weakly to an apparently single genomic locus in D. pseudoobscura (Figure 4C) rather than zen (see below). The origin of this signal at 14 kb (BamHI) , 5.0 kb (HindI I I ) and 2.8 kb (EcoRI) is unclear (Figure 4C). No comparable band is observed when D. pseudoobscura genomic DNA, digested with EcoRI, is probed with the zen cDNA (Figure 4A). The z 2 5‘ probe also produces a weak signal at the predicted zen positions in D. pseudoobscura, and this prob- ably derives from homeobox cross-hybridization (Fig- ure 4C). Taken together, the absence of detectable z 2 3’ homology and the presence of weak z2 5’ homology suggests at least two alternative possibilities. The first is that z2 is no longer present in the D. pseudoobscura genome, but another related gene with detectable homeobox homology to D. melanogaster z 2 resides within the genome. Alternatively, a 22 homolog is present outside of the pb-bcd interval of D. pseudoobscura, but has diverged significantly in nearly half of its coding region. Given the high degree of DNA sequence conservation found outside of the homeobox for the other ANT-C genes ( A n t p , f t z , Scr, Dfd, bcd, zen, pb and lab) in both protein coding and nontranslated regions (Figure 1 ; SEECER and KAUF- MAN 1990 for bcd) and given that homology to the 3’ half of 22 is undetectable in D. pseudoobscura, it seems more likely that a 22 homeobox subfamily member exists in D. pseudoobscura rather than a true z 2 hom-

The D. pseudoobscura Dfd gene is inverted with respect to its homolog in D. melanogaster: One interesting aspect of ANT-C organization in D. melanogaster is the common 5’ to 3‘ orientation of each of the member homeobox genes except for Dfd and f t z .

olog.

However, in contrast, in D. pseudoobscura the 5’ to 3’ orientation of the Dfd gene is in phase with the other homeobox-containing genes of the psANT-C (Figure 2). It seems that an inversion between ama and Scr has occurred in one of these two species. This is demonstrated by Southern hybridization data using separate D. melanogaster exon-l/exon-2 and exon-3/ exon-4 probes derived from Dfd (data not shown) and by DNA sequence from p431 and p475.

In contrast to Dfd, the remaining psANT-C genes remain oriented as in D. melanogaster. DNA sequence analysis from phage p 15 reveals that D. pseudoobscura ftz remains in the same 5’ to 3’ orientation relative to the complex as its homolog in D. melanogaster (Figure 1). Additionally, DNA sequence data demonstrate that the orientations of the remaining genes in psANT-C are unchanged with respect to their homologs in D. melanogaster (Figure 1). Since only Southern blot analysis was performed on the cuticle-like genes in the psANT-C, their 5’ to 3’ orientations are unknown.

Intergenic distances and large intron lengths are conserved across species: One defining characteristic of the ANT-C is the proximity of its member genes. Has the distance between adjacent genes changed over 46 million years of evolution? Measurements of the distances between selected positions (sequenced regions are identified in Figure 2) within adjacent genes demonstrate that there is < lo% difference in the size of the complexes in the two species (data not shown). Even the distance between pb and zen has remained relatively constant despite the apparent absence of z2 from this interval in D. pseudoobscura. Additionally, the relative positions of the outer boundaries of the cuticle-like gene homology are similar to those in D. melanogaster (Figure 2).

An unusual property of the D. melanogaster homeotic genes is the large size of the transcription unit compared with the processed mRNA product. This discrepancy is due to the presence of one or more large introns within these genes (see Figure 2). T o determine whether intron length has changed between these two species, the distances between se-

follows all 5’ splice sites are darkened. Asterisks overlie D. melanogaster intronic sequences. Amino acid sequences overlie the D. melanogaster open-reading frame (ORF). The conceptually translated D. pseudoobscura amino acid sequence is identical to D. melanogaster unless otherwise designated below. The D. melanogaster sequences were obtained from the following sources: Antp SCHNEUWLY et al. 1986 (from EMBL- DROANTPE5);ftz. LAUCHON and SCOTT 1984; Scr, LEMOTTE et al. 1989; Dfd RECULSKI et al. 1987 (from EMBL or GENEBANK- DROANTDFD); ren, RUSHLOW et al. 1987; pb, CRIBB~ et al. 1992; lab, DIEDERICH et al. 1989). The genomic clones and subclones from which the D. pseudoobscura sequences were obtained are indicated above each comparison set. The located of these sequences on the molecular map are indicated in Figure 2. All of the sequence comparisons show high DNA sequence identity and high amino acid identity. Most of the nucleotide changes occur at a third position within codons and therefore usually do not result in amino acid changes (A)-(K). Listed for each DNA sequence comparison is the origin of the D. melanogaster sequence, followed by the origin of the D. pseudoobscura seuqence: (A) lab (exon-1 region) X p543 1.1 kb EcoRIIBamHI; (B) lab (exon-2 region) X p503 0.6 kb EcoRI; (C) pb (exon-2 region) X p300 1.4 kb HindIII; (D) pb (exon-8 region) X p202 1.3 kb EcoRI; (E) ren (exon-2 region) X p3 10 1.2 kb BamHI/HindIII; (F) ama (exon-2 region) X p401 0.75 kb BamHI/EcoRI (this sequence from SEECER 1989); (C) Dfd (exon-4 region) X p 431 1.0 kb HincIII; (H) Dfd (exon-2 region) p 474 1.1 kb BamHI; (I) Scr (exon-2 region) X p69 3.5 kb EcoRI; (J) Scr (exon-3 region) X p73 2.3 kb EcoRI and p90 1.7 kb EcoRI; (K)& (exon-1 region) X p15 2.3 kb XbaI/ClaI; (L) Antp (exon-8 region) pl105 3.0 kb EcoRI.

324 F. M. Randazzo et al.

(4 3'

cuticlelike cluster

+5 5

FIGURE 2.-Molecular map of ANT-C in D. melunoguster with corresponding D. pseudoobscuru genomic clones. The molecular map of ANT-C in D. melunoguster, depicted above the coordinate line (Scorn et ul. 1983; SEEGER, HAFFLEY and KAUFMAN 1988; DIEDERICH et ul. 1989), is divided into four segments, (a)-(d). The first segment (a) starts at the telomere-proximal end of the complex and the last segment (d) ends after the 3' most exon of Antennupediu. Exons are depicted as shaded boxes. Shown below the coordinates (kb of DNA) are the D. pseudoobscuru genomic clones obtained from the chromosome walk. Hind111 restriction sites found within the genomic clones are depicted as thin vertical bars. The thicker vertical bars at the ends of each clone are lambda phage cloning sites (see MATERIALS AND METHODS for description). Lines connecting the D. melanoguster molecular map to the D. pseudoobscuru clones indicate locations of homology as determined by DNA sequence analysis. The hatched bar located directly below the coordinates in (a) delineate the region of Southern blot homology in D. pseudoobscuru to D. melunoguster (ANT-C cuticle gene-like probes). The bcd and umu positions are from SEECER and KAUFMAN (1990) and S E E G E R ( ~ ~ ~ ~ ) .


H

9.6 B 3.1 B 128 ?.6 B 3.8R 3.S R 2.4 R S2R 8.3 R

(b) D. pseudoobscunr

lkb - 7.1 R 10.4 R 1 2 R 4 2 R

1.6 H 1.0 H 8.7 H 28H &OH 1.SH H H H R H H R R HR H

8.5 B B a

6.6 B 4.7 B 7.4 B ”_ e

FIGURE 3.-Restriction maps of the pb-zen interval in D. rnelano- gaster and D. pseudoobscura. Restriction map of the pb-ren interval in (a) D. melanogaster and (b) D. pseudoobscura (SEEGER 1989), (B) BamHI, (H) Hind111 and (R) EcoRI. The stippled boxes represent exon-2 of pb, the striped box represents the 22 transcription unit, the black boxes represent Zen, and the white box represent bcd. Drawn, with arrows, above the restriction maps are the boundaries of the genomic library clones from the respective chromosome walks. Note that an artifical EcoRI restriction site lies at each of those boundaries.

quenced positions within exons that border large introns were compared (Figure 2). Dramatic changes are not observed for Scr (1 %), Dfd (2%) and lab (7%). On the other hand, a moderate change is observed for pb (1 3%). Clearly, gross changes in intron size and intergenic spacing have not occurred during the divergence of these two species.

DISCUSSION

We report here the cloning of the ANT-C from Drosophila pseudoobscura. The homologs to the D. melanogaster Antp, ftz, Scr, Dfd, ama, bcd, zen, pb, and lab genes, but not z2 are all present in the D. pseudoobscura complex (psANT-C), in the same linear order and similarly spaced along the chromosome. The similar arrangement of ANT-C homeotic genes in organisms ranging from D. melanogaster to D. pseudoobscura to beetles, mice and humans suggests a functional importance to this organization of genes that natural selection has maintained.

A small chromosomal inversion reveals some flexibility in ANT-C structure: The extraordinary positional conservation of homeotic genes within the murine Hox complex, Drosophila ANT-C and BX-C,

and the beetle HOM-C suggests that the structures of these complexes are relatively rigid and incapable of undergoing rearrangements that survive evolutionary selection. The observed inversions of the Dfd gene in D. pseudoobscura and the f t z transcription unit in Dro- sophila hydei (relative to D. melanogaster) demonstrate that subtle rearrangements can occur without compromising gene function (this study and MAIER, PREISS and POWELL 1990). Whether these observed inversions indicate the potential for other rearrangements that would split the complex is more difficult to determine. It is quite possible that cis-regulatory sequences are rearranged by these inversions, but these alterations have little effect due to the ability of enhancer elements to work 5’ or 3’ of a gene. Thus, a translocation with an identical breakpoint could disrupt proper gene regulation and result in lethality. Since the cis-regulatory elements for Dfd have not been fully defined and the precise extents of these inversions are not known, the impact of these inversions on the rigidity of the ANT-C is difficult to assess completely.

Interestingly, the inversion of Dfd and ftz, in D. pseudoobscura and D. hydei, respectively, likely represents the orientation of these genes in an ancestral complex. The homeobox genes of the ANT-C presumably arose from a series of duplication and divergence events that must have occurred before the ancestral split between mammals and arthropods. Consistent with this gene duplication and divergence model, one characteristic of the mouse Hox-2 complex is the identical direction of transcription for all the homeobox-containing member genes (RUBOCK et al. 1990). In D. melanogaster all the homeobox containing genes are transcribed in the same 5’ to 3’ direction except for Dfd andftz (KAUFMAN, SEEGER and OLSEN 1990). After the duplication events that gave rise to Dfd andftz, separate inversion events most likely resulted in the reversal of orientation of these two genes somewhere in the D. melanogaster lineage. Whether there is a Drosophilid species that has all ANT-C homeobox containing genes with the same direction of transcription, as is seen in the mouse Hox- 2 complex, remains unknown and would require the analysis of additional species.

The nonhomeotic genes of the ANT-C are conserved in D. pseudoobscura: A number of nonhomeotic genes reside within the ANT-C of D. melanogaster. These include the homeobox-containing gene Zen, bcd and f t z , and the nonhomeobox-containing genes ama and the cuticle-like gene cluster. The origin of the homeobox containing genes zen, z 2 (see below for discussion), bcd, and f t z presumably derives from duplication and divergence events within the ancestral complex. It has been postulated that the lab and Abd- B genes are among the original homeobox genes of

326 F. M. Randazzo el al.

(A) zen cDNA probe (B) z z 3' Probe (c) 22 5' probe 0. mel. 0. pso. 0. mel. 0. pso. D. me/. 0. pso. mG-2 mxm

1.2"

l=d 4.7- 5"> "1

2.0-->

the ancient complex (CRIBBS et al. 1992). If this hypothesis is correct, then zen, 22, bcd and f t z would represent more recent additions to the ANT-C.

All of these homeobox-containing genes (except 22 (see below)) are found in the psANT-C complex. In addition, bothftz and bcd have been identified in other Diptera: f t z in D. hydei (MAIER et al. 1990) and bcd in seven different Drosophilid species (MACDONALD 1990; SEEGER and KAUFMAN 1990) and in Musca domestica (SOMMER and TAUTZ 1991). The question whether any of these genes has a homolog outside the Diptera remains unanswered. Attempts to identify a

j z homolog in the red flour beetle and honeybees have been to date unsuccessful (STUART et al. 1991 ; WALLDORF, FLEIC and GEHRING 1989). It is also the case that clear homologs of these four genes have yet to be identified within the murine Hox and human HOX complexes (see MCGINNIS and KRUMLAUF 1992). Thus, it is tempting to speculate that these genes represent recent additions to the ANT-C and may be limited to the Diptera. However more extensive analyses of the nondipteran examples will be required to demonstrate this point.

The nonhomeobox containing genes of the ANT- C have been conserved in the psANT-C as well. Hom- ologs to ama and the cuticle-like gene cluster are found in the psANT-C at positions similar to their D. melanogaster counterparts. Although deficiencies re- moving the cuticle-like gene cluster or ama do not have major phenotypic consequences (PULTZ 1988; M. A. SEECER, T. C. KAUFMAN and C. GOODMAN, in preparation), the conservation of these genes in D.

FIGURE 4.-Genomic Southern hybridizations of D. melanogasterand D. pseduoobscura DNA: searching for 22 homology. Southern blot of restriction enzyme digested genomic D. melanogaster (D. mel.) Oregon R and D. pseudoobscura (D. pso) DNA-(B) BamHI. (H) Hindlll, (R) EcoRI. DNA size markers consisted of a 1 : l mixture of lambda phage DNA digested with Hind111 and with Hind111 and EcoRI. Random primed S2P-labeled D. melanogaster (A) Zen pB.2 cDNA (1.2 kb EcoRI), (B) 22 3' (0.262 kb EcoRI/ Sall) , and (C) 22 5' (0.445 kb Sall/XbaI) probes were hybridized to identical Southern blots for greater than 36 hr using the low stringency conditions of MCGINNIS et al. (1984). Specific activity of the probes: (A) 7.1 X IO8 cpm/pg DNA; (B) 5.2 X 10" cpm/pg DNA and (C) 5.6 X 10" cpm/pg DNA. After hybridization, the blots were washed under very low-stringency conditions (2X ssc, 50" (McCrNNlset a[. 1984). Low- stringency hybridization and wash conditions plus excess lambda marker DNA relative to any given genomic DNA sequence resulted in strong signals eminating from some of the size markers. Proper DNA transfer to the Nytran filters in the D. pseudoobscura lanes is evident from the equal and even smearing caused by nonspecific hybridization of probe to the genomic DNA.

pseudoobscura may indicate their functional importance. The presence of nonhomeobox genes has not been reported in the murine Hox or human HOX complexes or the beetle HOM-C. If they are truly absent from those complexes, it is likely that ama and the cuticle-like genes reside in the ANT-C as a result of recent insertion events and the insertion of these genes in the ANT-C may be another example of the disintegration of the homeotic gene complexes in Drosophila relative to other organisms (see MCGINNIS and KRUMLAUF 1992).

r2 gene evolution: The absence of 22 from the psANT-C, its apparent absence from the D. pseudoobscura genome, and the lack of a detectable function for 22 in D. melanogaster raises some interesting questions concerning the origin and function of the 22 gene. Because of the high degree of relatedness of Zen and 22 with respect to homeobox sequence (75% amino acid identity), gene structure (-1 0 10 bp vs. -1350 bp transcription unit with one small intron located just downstream of the ATG translation start site), and temporal and spatial regulation, these genes likely arose from a duplication of an ancestral gene followed by divergence in sequence (RUSHLOW et al. 1987). Since the nonhomeobox sequences of zen and 22 exhibit no amino acid sequence similarity, the divergence between these two genes has been substantial.

A number of possibilities could explain the apparent absence of 22 in D. pseudoobscura. The 22 gene could have existed before the divergence of the common ancestor to D. melanogaster and D. pseudoobscura, but


was then subsequently lost in the D. pseudoobscura lineage. Alternatively, 2 2 may have arisen as a zen duplication after the separation of the D. melanogaster and D. pseudoobscura lineages. Thus, 22 may never have been present in a D. pseudoobscura ancestor. This explanation is consistent with the absence of a detectable function for z 2 in D. melanogaster. Clearly an analysis of other intermediate Drosophilid species will be necessary to more fully understand the origin of the z2-zen gene pair.

Besides the zen-z2 gene pair, two other examples of adjacent and highly related homeobox genes have been described in Drosophila. The gooseberry (gsb) locus consists of two adjacent genes, gooseberry-proximal and gooseberry-distal, which possess highly related homeoboxes and other sequence similarities. Al- though the gsb-p gene product accumulates primarily in the central nervous system and the gsb-d gene product in the epidermis, the gsb genes do have some overlapping sites of expression. These genes presumably arose by a duplication and divergence event, as did zen and 22, but unlike the zen-22 situation, the gsb patterns of expression have diverged considerably. The engrailed and invected genes represent another set of adjacent homeobox genes that are highly related in sequence (COLEMAN et al. 1987). Similar to Len and z2, the functional contribution of each gene in these gene pairs has not been defined.

The creation of new genes to fulfill novel functions is an essential process in the evolution of more com- plicated organisms. The presence of these highly related and adjacent genes, like zen and 2 2 , most likely represent a phase in the evolutionary development of new genes and functions. Over time some of these duplicated genes will acquire novel functions and become important or essential to the organisms while others will be lost. The zen-z2 gene pair offers an excellent opportunity to study this process. The location of zen and z 2 in a defined region, the ANT-C, our knowledge about their functions in D. melanogaster, and the changes that have occurred during the divergence of D. melanogaster and D. pseudoobscura, make the analysis of the origin of zen and z 2 very accessible and attractive.

Why is the organization of the homeotic gene complexes conserved? The most striking feature of the homeotic gene complexes is the collinearity of gene order on the chromosome with their domains of expression along the anterior-posterior axis of the embryo. That this aspect of homeotic gene complex organization has been maintained from Drosophila to human, with over 500 million years of divergence between these species, is truiy remarkable. Experi- mental evidence that helps to explain this conserved organization is sparse. Several hypotheses, however, can be proposed.

First, important transcriptional regulatory elements might be shared or interspersed within the complex. These cis-acting elements could be traditional enhancer sequences that are interspersed among and used by multiple genes, or they could represent elements that organize chromatin structure throughout the complex (see MIRKOVITCH, MIRAULT and LAEM- MLI 1984; GASSER and LAEMMLI 1986; GYURKOVICS et al. 1990; KELLUM and SCHEDL 199 1). In the murine Hox complex, preliminary evidence suggests that regulatory elements may be interspersed between genes, thereby potentially restricting the fixation of inversion events (RUBOCK et al. 1990; RENUCCI et al. 1992). In D. melanogaster, theftz gene is embedded within Scr regulatory regions (PATTATUCCI, OTTESON and KAUF- MAN 199 I ) , and there is evidence that abd-A and Abd- B of the BX-C share some enhancer-like regions (CEL- INKER et al. 1990). However, further evidence for interspersed regulatory elements is lacking for other ANT-C and BX-C genes. In fact, the three homeotic genes of the BX-C can be separated genetically without detectably compromising functions (STRUHL 1984; TIONG, WHITTLE and GRIBBIN 1987; however, see KARCH et al. 1985 for contradictory evidence), and there are numerous inversions in the ANT-C that break within one gene but fail to affect other adjacent genes, suggesting that genes of the BX-C and ANT- C may not share regulatory elements (ABBOTT and KAUFMAN 1986; PULTZ et al. 1988; DIEDERICH et al. 1989). Additionally, p b and Zab null mutations have been rescued using transgenes that do not contain DNA derived from adjacent functional genes (RAN- DAZZO, CRIBBS and KAUFMAN 199 I ; CHOUINARD and KAUFMAN 1991). These data indicate that interspersed regulatory elements, if present, are not essential for proper gene functions, although they may contribute a fine-tuning role that increases the overall fitness of the organism.

The role of chromatin structure in the regulation of ANT-C or BX-C genes is not well defined. Chro- matin scaffolding attachment sites (SARS) have been experimentally defined for sequence surrounding the f t z locus (GASSER and LAEMMLI 1986), and consensus sequences characteristic of SARS have been identified in upstream regulatory regions of the labial locus (S. CHOUINARD and T. C. KAUFMAN, unpublished observations). In addition, synapsis dependent regulation (transvection effects) has been observed for a number of homeotic genes, including Scr in the ANT-C (PAT- TATUCCI and KAUFMAN 1991). In certain genetic back- grounds, the homologous chromosomes must be able to pair for proper Scr transcriptional regulation. Re- arrangements that prevent pairing disrupt Scr regulation. These observations reveal a nonessential re- quirement for transcriptional regulation that is de-


pendent on chromosome organization and possibly chromatin architecture.

The complexity of regulatory elements surrounding the homeotic genes in combination with the proximity of these cis-acting sequences to adjacent genes could contribute to the conservtion of the homeotic gene complexes. These two factors, complexity and proximity, would substantially increase the probability that a random chromosomal rearrangement would disrupt proper gene regulation and thus would be eliminated from the gene pool. The observation that in D. melanogaster Scr regulatory sequences extend minimally to the 3’ end and possibly within the Ant@ transcription unit, with the ftz locus also embedded in this region, is an excellent example of this complexity and proximity (PATTATUCCI, OTTESON and KAUFMAN 1991). Other regions of the ANT-C are not as “dense” (see Figure 2). For example, the distances from ama to Dfd and Dfd to Scr are seemingly quite extensive, although the extent of cis-regulatory sequences for these genes has not been precisely defined. A variation of this hypothesis, proposed by RANDAZZO, CRIBBS and KAUFMAN (1991) as the “prisoner effect,” suggests that rearrangement events that disrupt the complexes are unlikely due to potential deleterious position-effects on homeotic loci. Ectopic expression of homeotic genes often results in lethality. Unlike the first hypothesis, where the complexes are conserved for advantageous functional reasons, these latter structural hypotheses suggest that the conservation of the complexes is a function of their organization and the improbability of changing this organization without deleterious consequences.

What insights, with respect to these hypotheses, are gained from our knowledge of the structure of the ANT-C in D. pseudoobscura and the description of homeotic genes complexes in other more diverse species? Although the overall conservation of the homeotic complexes is consistent with the predictions of either hypothesis, the nature of this conservation does potentially suggest a distinction between the two ra- tionales. The feature of the homeotic complexes that is conserved in these different organisms is the colin- earity of homeotic gene order on the chromosome with their domains of expression in the embryo. Other aspects of the complexes, including the number of genes, types of genes, direction of transcription, gene size and spacing, are relatively dynamic features. Some of these features have changed in D. pseudoobscura ( e .g . , direction of transcription for Dfd and the absence of z2), wherreas others have changed only in more diverse species (e .g . , gene number and size in the Hox complexes). These dynamic aspects of the homeotic complexes are more consistent with the hypothesis that the complexes exist for some functional advantage and are less consistent with the suggestion that

the complexes are conserved as a consequence of their organizational complexity. Although it is likely that nonessential but advantageous regulatory elements are interspered within the homeotic complexes, offer- ing a selective advantage to those who maintain this organization, it is also possible that the complexity of cis-regulatory sequences and the deleterious consequences to ectopic expression contribute to the observed rigidity of the complexes as well. Of course, these evolutionary comparisons are merely suggestive, and the proof of these hypotheses will depend on further experimental analysis.

We are grateful to CHARLES LANGLEY and TULLE HAZELRIGG for the D. pseudoobscura genomic libraries. We thank DAVID CRIBBS for the pb exon-2 sequence in D. pseudoobscura sequence, DWAYNE JOHNSON for performinga number of phage preps, SANDI HORIKAMI for the D. melanogaster genomic DNA, KIM FECHTEL for the cuticle gene probes and DEE VEROSTKO for her priceless administrative work. We also thank the dissertation committee of F.M.R., a.k.a. THOMAS BLUMENTHAL, MARC MUSKAVITCH, ELIZABETH RAFF and SUSAN STROME, for critical reading of this work. This work was supported by a National Institutes of Health Predoctoral Fellowship (GMO7757) to F.M.R. and M.A.S. and an NIH grant (GM24299) to T.C.K. T.C.K. is an investigator of the Howard Hughes Medical Institute.

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Cornmunicating editor: R. E. DENELL

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Structural Changes in the Antennapedia Complex of ... · Antennapedia Complex Evolution 32 1 1984). The probes were made using the random primer a’* P method (FEINBERG and VOGELSTEIN