The homeotic genes of Drosophila control the differentiation of segments during development. Mutations in these genes cause one or more segments to develop structures normally found elsewhere in the organism. Several studies have shown that the spatial patterns of homeotic gene transcription are highly complex, and that these precise patterns of transcription are critical to normal development. The homeotic gene Antennapedia (Antp), a member of the Antennapedia Complex, is required for the correct differentiation of thoracic segments in both embryos and adults. The patterns of total Antp transcript and protein accumulation have been described in detail, but the contribution of each promoter to the overall pattern in embryos has not been reported. We have examined in detail the spatial distribution of transcripts from each of the Antp promoters in both embryo sections and whole embryos by in situ hybridization using promoter-specific probes. We show that the transcripts from each of the two promoters accumulate in distinct, but overlapping patterns during embryogenesis. The results demonstrate that the two Antp promoters are differentially regulated in embryos and provide a basis for examining the regulation of the two promoters and characterizing more fully the func tion of Antp during embryogenesis. In addition, we have examined the regulation of each of the Antp promoters by genes of the bithorax complex (BX-C). We show that in BX-C embryos both promoters are derepressed in the abdomen.

The segments of the Drosophila embryo differentiate along distinct developmental pathways, providing each segment with a unique set of structures appropriate to its position along the body axis. Genetic analyses have identified several classes of genes required for the normal development of embryonic segments (reviewed in Nüsslein-Volhard et al. 1987; Akam, 1987; Scott and Carroll, 1987; Ingham, 1988). Maternally acting genes are required for the establishment of the anterior-posterior and dorsal-ventral embryonic axes. A set of zygotically active genes also controls pattern formation along the dorsoventral axis. The zygotically active segmentation genes are required to make the correct number of segments, and the correct pattern and orientation of structures within each segment. In addition, some of the segmentation genes affect the identities of segments, i.e. which segments form head or thoracic or abdominal structures. Mutations in the ‘gap’ segmentation genes, for example, lead to a failure to form particular groups of segments, and, in some cases, the identities of remaining segments are altered as well. Segment-specific differentiation is also controlled by the homeotic genes; mutations in these genes cause one or more segments to follow the developmental pathway characteristic of another segment.

To learn how genes control differentiation, many of the homeotic genes and their products have been characterized. Two important conclusions have emerged from this work. One is that homeotic genes are transcribed in complex spatial patterns that change during development. Second, many homeotic genes encode transcriptional regulators. The array of homeotic genes expressed in a cell, in concert with other regulators, appears to determine the fate of the cell.

Many of the known homeotic genes are clustered in two complexes, the Antennapedia complex (ANT-C; Kaufman et al. 1980) and the bithorax complex (BX-C; Lewis, 1978). Different members of the ANT-C and BX-C are expressed, and function, in different regions along the anterior-posterior axis of the embryo (re views, op. cit.). There are also dorsal-ventral differ ences in where genes are active. The initial expression of the homeotic genes is spatially restricted; the pat terns are maintained and refined as development pro ceeds. It appears that the early patterns of expression of the homeotic genes are regulated by gap, pair-rule, and segment polarity segmentation genes (Ingham and Martinez-Arias, 1986; Duncan, 1986; White and Leh mann, 1986; Riley et al. 1987; Martinez-Arias and White, 1988; Irish et al. 1989). Later in development, the patterns are modified and maintained by interac tions among the homeotic genes. The transcription of those homeotic genes that are primarily expressed in more anterior domains appears to be repressed by the action of homeotic genes expressed in more posterior domains (Hafen et al. 1984; Harding et al. 1985; Struhl and White, 1985; Carroll et al. 1986; Wirz et al. 1986; Riley et al. 1987).

Several lines of evidence suggest that the protein products of homeotic genes are transcriptional regu lators (reviewed in Gehring, 1987; Levine and Hoey, 1988; Scott et al. 1989; Mitchell and Tjian, 1989). Many homeotic genes contain a conserved DNA sequence, the homeobox (McGinnis et al. 1984; Scott and Weiner, 1984), that encodes a protein motif, the homeodomain, with predicted structural homology to bacterial and yeast DNA-binding regulatory proteins (Laughon and Scott, 1984; Shepherd et al. 1984). In vitro, homeo-domain-containing proteins bind specific DNA se quences (Desplan et al. 1985, 1988; Hoey and Levine, 1988; Beachy et al. 1988; Laughon et al. 1988). Several Drosophila homeodomain-containing proteins also regulate transcription in cultured cells or yeast (Jaynes and O’Farrell, 1988; Thali et al. 1988; Fitzpatrick and Ingles, 1989; Han et al. 1989; Samson et al. 1989; Winslow et al. 1989; Krasnow et al. 1989); one has been shown to do so in embryos (Driever and Nusslein-Volhard, 1989) and several mammalian transcription factors have been found to contain homeodomains (Ko et al. 1988; Bodner et al. 1988; Ingraham et al. 1988; Sturm et al. 1988; Scheidereit et al. 1988). These findings strongly support the idea that homeodomain-containing proteins regulate transcription and suggest that the distribution of transcription factors in precise spatial arrays in the developing embryo is a major mechanism of developmental control. Differences be tween cells in the array of transcription factors they contain presumably lead to differences in developmen tal fate. Thus, in order to understand the role of homeotic genes in segment differentiation, it is import ant to understand how the patterns of homeotic gene expression are generated.

The Antennapedia (Antp) gene, which contains a homeobox and is located in the ANT-C, plays a major role in the differentiation of the thoracic segments. Dominant and recessive mutations in the Antp gene cause transformations involving thoracic structures. Most Antp mutations are recessive lethals; homozygous mutant embryos display transformations of the second and third thoracic segments toward the first thoracic segment (Denell et al. 1981; Wakimoto and Kaufman, 1981). The first metameric divisions of the embryo are parasegmental rather than segmental (Martinez-Arias and Lawrence, 1985). The precise features of the transformation in Antp embryos suggest that paraseg ment (PS) 4, which consists of the posterior compart ment of T1 and the anterior compartment of T2, is transformed into PS 3, and PS 5 into a hybrid with features of both PS 3 and PS 6 (Martinez-Arias, 1986). Certain dominant mutations activate the gene ectopically, resulting in antenna to leg transformations (Denell, 1973; Struhl, 1981; Hazelrigg and Kaufman, 1983; Frischer et al. 1986; Schneuwly et al. Y)Kla,b‘, Gibson and Gehring, 1988) or a transformation of dorsal head toward dorsal thorax (I. Duncan and E. Lewis, unpublished data; Scott et al. 1983).

The Antp locus has a complex structure that is reflected genetically as intragenic complementation. Molecular analyses have revealed that Antp exons are distributed over a 100 kb region, and that Antp tran scripts are initiated from two promoters, Pl and P2, separated by approximately 65 kb (Scott et al. 1983; Garber et al. 1983; Schneuwly et al. 1986; Stroeher et a/. 1986; Laughon et al. 1986) (Fig. 1). Transcripts initiated from either promoter contain the same protein-coding exons. Antp mutations that primarily affect one transcription unit apparently can complement Antp mu tations that affect the other transcription unit. Many Antp mutations are associated with chromosome re arrangements that have one breakpoint between the Pl and P2 promoters, thereby interrupting the Pl tran scription unit (Fig. 1; Scott et al. 1983; Garber et al. 1983). Such mutations complement the recessive allele Antpsl (Kaufman and Abbott, 1984; Abbott and Kauf man, 1986), a mutation in which a transposon is inserted into an exon that is only included in P2 transcripts (e.g. Antp73b, Fig. 1; Scott et al. 1983; Laughon et al. 1986). Thus, mutations that destroy the Pl transcription unit can provide the P2 function that is damaged by the Antpsl mutation, and the Antpsi allele can provide Pl transcription unit function. These results suggest that the Antp gene has at least two separable functions, which correspond to the Pl and P2 transcription units. In addition to the intragenic complementation, somatic clonal analyses suggest that the two promoters have different functions: the Pl promoter is necessary for proper dorsal thoracic development in adults, while the P2 promoter is necessary for proper leg development (Abbott and Kaufman, 1986). The comparative func tions of the two promoters in the embryo have not been determined.

Fig. 1.

Antennapedia gene structure and probes used for in situ hybridization. Antp transcripts are initiated at two promoters, Pl and P2. Exon C is spliced out of Pl transcripts but retained in P2 transcripts. Transcripts initiated at either promoter use the same protein-coding exons, E through H. The alternative 3’ end processing of Antp transcripts is indicated by the stippled region in exon H. The coordinates shown (in kb) are from Scott et al. (1983). The positions of the Antp13b inversion breakpoint and the Antpsl insertion are shown. Antplib is one of several inversion mutations at the Antp locus that separate the Pl promoter from the coding exons, yet complement the Antpsl allele (Abbott and Kaufman, 1986). Probes: The genomic locations of DNA fragments used to make probes for in situ hybridization are shown above the scale. Filled regions of the rectangles indicate exon sequence. Open regions indicate intron or nontranscribed sequence. The stippled portion of probe HB corresponds to sequence that is included in exon H only when the downstream polyadenylation signal is used. The cDNA probe used for whole-mount in situ hybridization is not shown (see Materials and methods).

Fig. 1.

Antennapedia gene structure and probes used for in situ hybridization. Antp transcripts are initiated at two promoters, Pl and P2. Exon C is spliced out of Pl transcripts but retained in P2 transcripts. Transcripts initiated at either promoter use the same protein-coding exons, E through H. The alternative 3’ end processing of Antp transcripts is indicated by the stippled region in exon H. The coordinates shown (in kb) are from Scott et al. (1983). The positions of the Antp13b inversion breakpoint and the Antpsl insertion are shown. Antplib is one of several inversion mutations at the Antp locus that separate the Pl promoter from the coding exons, yet complement the Antpsl allele (Abbott and Kaufman, 1986). Probes: The genomic locations of DNA fragments used to make probes for in situ hybridization are shown above the scale. Filled regions of the rectangles indicate exon sequence. Open regions indicate intron or nontranscribed sequence. The stippled portion of probe HB corresponds to sequence that is included in exon H only when the downstream polyadenylation signal is used. The cDNA probe used for whole-mount in situ hybridization is not shown (see Materials and methods).

In the imaginai discs, the primordia of adult tissues, the two Antp promoters are expressed in different patterns, implying that they are controlled by different regulators. Pl transcripts are located primarily along the anterior margins of the wing and mesothoracic leg imaginai discs, while P2 transcripts are more evenly distributed in these discs. Both promoters are also expressed in the prothoracic and metathoracic leg discs (Jorgensen and Garber, 1987). In embryos, Antp RNA has been detected in thoracic ectoderm and mesoderm, as well as in much of the ventral nervous system, using nucleic acid probes (Hafen et al. 1983; Levine et al. 1983; Hafen et al. 1984; Martinez-Arias, 1986; Harding et al. 1985; Wedeen et al. 1986; Ingham and Martinez-Arias, 1986) and antibody probes (Carroll et al. 1986; Wirz et al. 1986). However, the RNA localization studies have only examined expression of the Pl pro moter, or the sum of the Pl and P2 contributions, and the antibody probes cannot distinguish between the contributions of the Pl and P2 promoters in wild-type embryos.

Previous studies of Antp expression during embryo genesis have left several important questions un answered. When and where do Antp Pl and P2 tran scripts accumulate in the embryo? Do the expression patterns differ from those of Antp proteins, indicating posttranscriptional regulation? Do homeotic genes of the BX-C, which are known to negatively regulate Antp, act on one or both promoters? In this paper, we compare the separate tissue- and stage-specific expression patterns of the Pl and P2 promoters during embryogenesis. Tn addition, we describe the expression of the two promoters in embryos lacking the BX-C genes.

Construction of probes

Two different pairs of Antp promoter-specific probes were used in these experiments. One pair, P1A and P2A, consisted of a 4 kb EcoRl exon A-containing (Fig. 1) fragment from phage ASTI (Scott et al. 1983) cloned into pSP64, and a 1.9 kb EcoRl fragment from phage 448 (Scott et al. 1983) containing 657 bp of exon C cloned into pSP64. The other pair of probes, P1B and P2B, was constructed as follows: P1B: a 1216bp SacI-AccI fragment containing the Pl promoter and 835 bp of exon a was cloned into pGEM2 that had been cut with Fμndlll, partially filled in to accept the Accl sticky end, and then cut with Sacl. After cutting with Xhol, this subcloned DNA served as a template to produce T7-generated tran scripts containing 784 bases of exon A. The template for P1B DNA probes was a gel-purified Xhol-Accl fragment from a separate subclone of this region. P2B: a 1901 bp HindUl-Clal fragment containing the P2 promoter and 1283 bp of exon C was cloned into pGEM-1 that had been cut with Hind 1 II and Accl. After digestion with Avail, this subcloned DNA served as a template to produce T7-generated transcripts containing 796 bases of exon C. For DNA probes, we isolated the Avall-Xbal fragment containing the same 796bp of Pl sequence plus about 10bp of vector sequence.

The INTRON probe, containing intron sequences between Pl and P2 was constructed as follows: R4.6 is a 4.6 kb EcoRl fragment derived from phage 448 (Scott et al. 1983) and contains the P2 promoter and 663 bp of exon C (Laughon et al. 1986). R4.6p-35 is a deletion subclone of R4.6 generated for sequencing; it lacks the P2 promoter and exon C sequences, and contains a Earn Hl site, introduced during deletion construction, at position 520 in the sequence of Laughon et al. (1986), a 3.8kb EcoRl-BawHI fragment consisting of the entire R4.6p-35 insert was cloned into pGEM-2 that had been cut with BamHl and EcoRl. After digestion of the construct DNA with EcoRl, it was used as a template to produce T7-generated RNA transcripts. The HB probe was a 1.7 kb EcoRl fragment isolated from phage A2015 and the D-G probe template was a 3.1 kb EcoRl fragment isolated from phage A77 (Scott et al. 1983). The cDNA template was a 1.7 kb fragment containing the sequence between the Ball site at position 144 and the Bglll site at position 904 in the Antp sequence (Laughon et al. 1986). This fragment contains part of exon D, all of exons E, F, and G (long form; see Bermingham and Scott, 1989), and part of exon H.

RNAs were labelled by the incorporation of 35S-ribonucleotides during in vitro transcription using the T7 RNA polym erase. Preparation of embryo sections, hybridization and autoradiography were performed as described by Ingham et al. (1985). Sections were exposed for 3 to 8 days.

Digoxygenin-labelled DNA probes used for whole-mount in situ hybridization were labeled using the Boehringer-Mannheim ‘Genius’ kit. Digoxygenin conjugated dUTP is incorporated during random primed DNA synthesis. In some cases, the DNA template was digested with frequently cutting restriction enzymes to reduce the sizes of the labeled frag ments (C. Oh and B. Edgar, personal communication). Whole-mount in situ hybridization was performed essentially as described by Tautz and Pfeifle (1989), with some modifi cations. After treatment of embryos with heptane and formal dehyde, either while they are in methanol or while they are in 70 % ethanol, they are treated with 0.3 % hydrogen peroxide for 2-4 min. This step greatly reduces background (T. Yeh and D. Andrew, personal communication).

Antp transcript-specific probes

For in situ hybridization to Antp transcripts, we used labelled RNA to probe sectioned material and labelled DNA to probe whole embryos. Two different pairs of promoter-specific probes were used (Fig. 1, and see Materials and methods); they differ both in size and in their amounts of non-exon sequence. These probes are called PIA, P2A, P1B, and P2B. In addition, the following probes were used and are illustrated in Fig. 1: a probe designated HB, which contains the Antp homeobox sequence, that should detect both Pl and P2 transcripts; the D-G probe, which contains genomic sequence from exons D through G, that should detect both Pl and P2 transcripts; the INTRON probe, which contains sequence located between the Pl and P2 promoters, that should detect Pl-derived transcripts only; and the cDNA probe, which contains the entire protein-coding sequence from exons E through H, as well as some noncoding sequence from exons D, E, and H.

Initiation of Antp transcription at the blastoderm stage Pl promoter

Antp Pl promoter-specific transcripts are first detected early in nuclear cycle 14 (Foe and Alberts, 1983; stage 5 of Campos-Ortega and Hartenstein, 1985), as the blastoderm cells form. Pl-specific transcripts are detect able in a ring encircling the blastoderm embryo be tween about 40 % and 55 % egg length at the ventral surface, where 0% is the posterior end of the egg (Fig. 2A). The ring is not uniform in width, being somewhat narrower at the dorsal surface, broader at the ventral surface, and tapering uniformly in between the ventral and dorsal surfaces. This region contains the primordia of parasegments (PS) 4-6, which correspond to the posterior first thoracic segment (pTl) through the anterior first abdominal segment (aAl) (Martinez-Arias and Lawrence, 1985).

Fig. 2.

Promoter-specific whole-mount in situ hybridization. (A-C) Cellular blastoderm stage embryos probed with the P1B (panel A), P2B (panel B), or cDNA probe (panel C). The approximate position of PS 4 is indicated (panel A). The inferred positions of PS 4 and PS 6 are shown in B and C (see Fig. 4A). P2 expression in PS 14 is faint in sagittal views (panels B and C) but is readily visible in horizontal views (Fig. 3A). Ventral P2 expression is also shown in Fig. 3B and C. (D-F) Expression patterns during and just after germ band elongation. The embryo in panel D (stage 11) is somewhat older than embryos shown in panels E and F (stage 9), but expression patterns do not change dramatically between these two stages. Numbers and arrowheads indicate the positions of specific parasegments. At stage 9 the cDNA pattern closely resembles the P2 pattern. (G-l) Expression patterns during stage 12. The strongest signals are seen in the developing neuromeres. The positions of the T1 and A7 neuromeres are indicated. (J-K) Expression patterns after germ band retraction (stages 13, panels J and L; and 14, panel K). The positions of the T1 and A7 neuromeres are indicated.

Fig. 2.

Promoter-specific whole-mount in situ hybridization. (A-C) Cellular blastoderm stage embryos probed with the P1B (panel A), P2B (panel B), or cDNA probe (panel C). The approximate position of PS 4 is indicated (panel A). The inferred positions of PS 4 and PS 6 are shown in B and C (see Fig. 4A). P2 expression in PS 14 is faint in sagittal views (panels B and C) but is readily visible in horizontal views (Fig. 3A). Ventral P2 expression is also shown in Fig. 3B and C. (D-F) Expression patterns during and just after germ band elongation. The embryo in panel D (stage 11) is somewhat older than embryos shown in panels E and F (stage 9), but expression patterns do not change dramatically between these two stages. Numbers and arrowheads indicate the positions of specific parasegments. At stage 9 the cDNA pattern closely resembles the P2 pattern. (G-l) Expression patterns during stage 12. The strongest signals are seen in the developing neuromeres. The positions of the T1 and A7 neuromeres are indicated. (J-K) Expression patterns after germ band retraction (stages 13, panels J and L; and 14, panel K). The positions of the T1 and A7 neuromeres are indicated.

Fig. 3.

Whole-mount in situ hybridization. (A, B) The P2 pattern (probe P2B) in two different optical horizontal sections of the same cellular blastoderm stage embryo. Arrowheads indicate the locations of specific parasegments. B shows the ventral surface of the embryo to indicate the extent of P2 expression around the circumference of PS 6. (C) P2 expression in an early gastrula. Most of the PS 6 cells expressing P2 at high levels invaginate at the ventral furrow (arrows). (D-F) cDNA pattern in the lateral ectoderm. At stage 11 (panel D) Antp expression is detected in a small cluster of cells on the lateral surface of each segment from PS 4 to PS 14. The cells are approximately at the center of each parasegment and may be the pnmordia of peripheral neurons. P2 probes detect a similar pattern of RNA at stage 11, but PI probes do not (data not shown). During stages 12 (panel E) and 13 (panel F), ectodermal or subectodermal staining remains in a subset of cells within each segment from T2 through A9. A similar pattern is seen with P2 probes, but not with PI probes. (G) Horizontal view of a stage 14 embryo probed with the cDNA probe. The arrows indicate two patches of cells that express Antp. (H, I) Two horizontal, optical sections of the same stage 16 embryo probed with the cDNA probe. H shows Antp expression in mesodermal cells surrounding one of the midgut constrictions (arrow). The dark, diffuse signal at about T2 is due to ventral nervous system expression, which is out of focus.

Fig. 3.

Whole-mount in situ hybridization. (A, B) The P2 pattern (probe P2B) in two different optical horizontal sections of the same cellular blastoderm stage embryo. Arrowheads indicate the locations of specific parasegments. B shows the ventral surface of the embryo to indicate the extent of P2 expression around the circumference of PS 6. (C) P2 expression in an early gastrula. Most of the PS 6 cells expressing P2 at high levels invaginate at the ventral furrow (arrows). (D-F) cDNA pattern in the lateral ectoderm. At stage 11 (panel D) Antp expression is detected in a small cluster of cells on the lateral surface of each segment from PS 4 to PS 14. The cells are approximately at the center of each parasegment and may be the pnmordia of peripheral neurons. P2 probes detect a similar pattern of RNA at stage 11, but PI probes do not (data not shown). During stages 12 (panel E) and 13 (panel F), ectodermal or subectodermal staining remains in a subset of cells within each segment from T2 through A9. A similar pattern is seen with P2 probes, but not with PI probes. (G) Horizontal view of a stage 14 embryo probed with the cDNA probe. The arrows indicate two patches of cells that express Antp. (H, I) Two horizontal, optical sections of the same stage 16 embryo probed with the cDNA probe. H shows Antp expression in mesodermal cells surrounding one of the midgut constrictions (arrow). The dark, diffuse signal at about T2 is due to ventral nervous system expression, which is out of focus.

P2 promoter

The expression pattern of the Antp P2 promoter alone has been inferred from studies of mutant embryos. Antp protein in embryos lacking a functional Pl tran scription unit is distributed in cells that are a subset of those making Antp protein in wild-type embryos (Boulet and Scott, 1988), suggesting that the two promoters are expressed in different patterns in em bryos, as they are in imaginai discs. However, the analysis of protein in these mutants may not truly reflect the P2 transcription pattern for two reasons: First, mutations disrupting the Pl transcription unit may affect P2 function as well. Second, Antp transcripts could be subject to posttranscriptional regulation. Another limitation is that it is difficult to detect the Antp protein at the blastoderm stage.

Antp P2 promoter-specific transcripts are first detected at roughly the same time as Pl transcripts, at the blastoderm stage, but the two classes of transcripts are expressed in different patterns. P2 transcripts in itially accumulate in a ring three or four cells wide,

Fig. 3. Whole-mount in situ hybridization. (A, B) The P2 pattern (probe P2B) in two different optical horizontal sections of the same cellular blastoderm stage embryo. Arrowheads indicate the locations of specific parasegments. B shows the ventral surface of the embryo to indicate the extent of P2 expression around the circumference of PS 6. (C) P2 expression in an early gastrula. Most of the PS 6 cells expressing P2 at high levels invaginate at the ventral furrow (arrows). (D-F) cDNA pattern in the lateral ectoderm. At stage 11 (panel D) Antp expression is detected in a small cluster of cells on the lateral surface of each segment from PS 4 to PS 14. The cells are approximately at the center of each parasegment and may be the primordia of peripheral neurons. P2 probes detect a similar pattern of RNA at stage 11, but Pl probes do not (data not shown). During stages 12 (panel E) and 13 (panel F), ectodermal or subectodermal staining remains in a subset of cells within each segment from T2 through A9. A similar pattern is seen with P2 probes, but not with Pl probes. (G) Horizontal view of a stage 14 embryo probed with the cDNA probe. The arrows indicate two patches of cells that express Antp. (H, I) Two horizontal, optical sections of the same stage 16 embryo probed with the cDNA probe. H shows Antp expression in mesodermal cells surrounding one of the midgut constrictions (arrow). The dark, diffuse signal at about T2 is due to ventral nervous system expression, which is out of focus. centered around 56% egg-length (Figs2B, 3A, and 4A). These cells lie in PS 4, three or four cells anterior to the primordium of PS 6. The position of PS 6 was determined using the expression of the Ultrabithorax (Ubx) homeotic gene as a reference (Fig. 4A; Akam and Martinez-Arias, 1985). The segmentation gene fushi taraza (ftz) is expressed in PS 2, 4, 6, 8,10,12 and 14 (Carroll et al. 1988; Lawrence and Johnston, 1989); ftz expression was also used to localize the Antp P2 transcript in adjacent serial sections (data not shown).

Fig. 4.

Double labelling with Ubx and expression of Antp during germ band elongation. (A) Horizontal section of a blastoderm stage embryo probed with both the P2A probe and a Ubx probe. Ubx expression coincides with PS 6 (Akam and Martinez-Arias, 1986). (B) A slightly oblique section of a stage 8 embryo probed with both the P2A and Ubx probes. Based upon the location of the Ubx signal, the two domains of P2 expression in the mesoderm are inferred to coincide with PS 4 and PS 6. (C, D) Adjacent sections of a gastrulating embryo probed with the Pl A (panel C) or P2A (panel D) probe. Arrowheads indicate homologous positions at approximately the anterior border of PS 4. Open arrow in D indicates PS 14 expression. (E-H) Bright-field and corresponding dark-field images of alternate sections of a stage 8 embryo probed with the P1A probe (panels E and G) or the P2A probe (panels F and H). Arrowhead indicates the anterior boundaries of PS 4 and PS 13.

Fig. 4.

Double labelling with Ubx and expression of Antp during germ band elongation. (A) Horizontal section of a blastoderm stage embryo probed with both the P2A probe and a Ubx probe. Ubx expression coincides with PS 6 (Akam and Martinez-Arias, 1986). (B) A slightly oblique section of a stage 8 embryo probed with both the P2A and Ubx probes. Based upon the location of the Ubx signal, the two domains of P2 expression in the mesoderm are inferred to coincide with PS 4 and PS 6. (C, D) Adjacent sections of a gastrulating embryo probed with the Pl A (panel C) or P2A (panel D) probe. Arrowheads indicate homologous positions at approximately the anterior border of PS 4. Open arrow in D indicates PS 14 expression. (E-H) Bright-field and corresponding dark-field images of alternate sections of a stage 8 embryo probed with the P1A probe (panels E and G) or the P2A probe (panels F and H). Arrowhead indicates the anterior boundaries of PS 4 and PS 13.

Two other bands of P2 expression appear at the cellular blastoderm stage in the primordia of PS 6 and 14. The PS 6 band is limited to a strip of cells at the ventral surface of the blastoderm embryo (Figs 2B and 3B). This band probably corresponds to the mesoder mal primordium in PS 6, since most of the staining cells invaginate as part of the ventral furrow (Fig. 3C). Occasionally, very faint staining is seen at the dorsal surface of PS 6, although the dorsal signal is always much weaker than the ventral one (data not shown). The PS 14 stripe appears to encircle most or all of the embryo, being centered at about 12% egg length ventrally and 18% egg length dorsally. In whole em bryos, the signal is strongest in horizontal views (Fig. 3A) and is only very weak in sagittal views (Fig. 2B). Embryo sections often contain the PS 4 signal with only one of the other two signals, probably due to the plane of sectioning.

Pl intron-containing transcripts

The P2-specific probes could hybridize to unprocessed Pl transcripts, as well as to P2 transcripts, giving a misleading ‘P2-specific’ pattern. Serial sections of late cellular blastoderm (stage 5) embryos were hybridized with the P1B probe, the P2B probe, or the INTRON probe. The INTRON probe produced a signal like that produced by the P1B probe, but slightly weaker (data not shown). The similarity of the P1B and INTRON patterns suggests that the pattern of Pl transcript accumulation seen in the embryo is not due to spatially controlled RNA processing. The Pl pattern is barely detectable, if at all, on sections hybridized with the P2B probe, indicating that the P2 pattern is principally due to bona fide P2 transcripts.

Transcripts containing exons D through G

During nuclear cycle 14, the probes D-G and cDNA, which both hybridize to both Pl and P2 transcripts, detect transcripts only in a P2-like pattern (Fig. 2C), although the signal from the D-G probe is detected somewhat later in the cycle than the P2-specific signal, and it remains weak (data not shown). Because both the cDNA and D-G probes contain only sequences close to the 3’ end of the Antp gene, this result suggests that complete P2 transcripts appear before complete Pl transcripts. The Pl primary transcripts are about 64 kb longer than the P2 primary transcripts (100 kb versus 36kb), and, therefore, if transcription at both pro moters begins at about the same time, then the lag in appearance of Pl transcripts containing exons D-G is probably due to the extra time it takes to transcribe the Pl transcription unit relative to the P2 transcription unit.

Antp expression during gastrulation and early germ band elongation

Gastrulation and germ band elongation occur rapidly after blastoderm cell formation, and are accompanied by extensive changes in the patterns of Antp expression.

Adjacent sections of the same embryos were probed with Pl- or P2-specific probes (Fig. 4).

Pl promoter

During gastrulation and early germ band elongation, the posterior limit of Pl expression on the ventral side of the embryo is at approximately 17 % egg length, thus presumably including PS 12 (Fig. 4C). Pl transcripts are also detected in PS 5 of the forming mesoderm (data not shown). In stage 8 embryos, midway through germ band elongation, the Pl promoter is expressed princi pally in the ectoderm of PS 4, the ectoderm and mesoderm of PS 5 and anterior PS 6, and more weakly in the ectoderm from posterior PS 6 through PS 12 (Fig. 4E,G).

P2 promoter

During gastrulation and germ band elongation, P2 transcripts continue to be present at high levels in PS 4. PS 6 expression is only detected in the cells that invaginate as part of the ventral furrow (Fig. 3C), although ectodermal PS 6 expression is detected early during germ band elongation. As the pole cells move dorsally, so do the ventral PS 14 cells that express P2 (Fig. 4D, open arrow). The dorsal PS 14 expression is hard to follow and may simply disappear. As the germ band begins to elongate, P2 transcripts are detected in PS 3, as well as at low levels in some ectodermal cells of each PS from 5 through 14. P2 is strongly expressed in the ectoderm and mesoderm of PS 4 and in the mesoderm of PS 6 (Fig. 4D). In stage 8 embryos, the P2 promoter is strongly expressed in the ectoderm of PS 3 and PS 4, and in the mesoderm of PS 4 and PS 6 (Figs 2E and 4F,H). Additionally, the P2 promoter is expressed more weakly in the ectoderm and mesoderm of PS 5 and in the ectoderm from PS 6 through PS 14, with stronger signals in the even-numbered paraseg ments (see also Ingham and Martinez-Arias, 1986). For comparison, a stage 8 embryo probed with both a P2 probe and a Ubx probe is shown in Fig. 4B, confirming the identification of PS 4 and PS 6 as the locations of the strongest mesodermal P2 expression. The patterns of hybridization seen with the cDNA probe continue to resemble the P2 pattern during the early stages of germ band elongation.

Antp expression in extended germ-band embryos

The germ band begins to retract at the end of stage 11, approximately 7h 20min after fertilization. During stage 12, the germ band shortens, and the ventral nerve cord separates from the epidermis (Campos-Ortega and Hartenstein, 1985). At this stage, the homeobox probe detects a pattern that is the sum of Pl and P2 transcrip tion patterns, suggesting that the two promoters con tribute similar amounts of RNA to the pattern (for example, see wild-type embryos in Fig. 7).

Pl promoter

During stages 11 and 12, RNAs from Pl are found at high levels in most epidermal cells of PS 4, PS 5, and anterior PS 6, as well as the mesoderm of PS 5 and anterior PS 6 (Martinez-Arias, 1986; Figs 2D and 7A,B). Pl transcripts are detected in the developing VNS during stage 11 in PS 4 and 5. During stage 12, transcripts from Pl decrease in the epidermis and increase in the nervous system (Fig. 2G).

P2 promoter

During stage 11, the patterns of P2 expression in the epidermis are different from the patterns of P2 ex pression in the developing nervous system. In the epidermis, expression is restricted to a subset of cells in PS 3, PS 4 and PS 5 ventrally. Laterally, P2 transcripts are detected in a small cluster of epidermal or subepi dermal cells in the center of each PS from 4 to 14. Tlie pattern is visible with the cDNA probe in Fig. 3D (arrows). These cells may correspond to the primordia of sensory organs, since slightly later P2 tanscripts are detected in cells clearly located just below the epidermis (e.g. Fig. 6D). In the nervous system, transcripts from P2 accumulate in a subset of the neural cells within each parasegment from PS 3 to the anus (Fig. 7D,E). During stage 12, P2 transcripts also decrease in the epidermis and increase in the nervous system (Fig. 2H).

Antp expression after germ-band retraction

Soon after the end of germ-band retraction, head involution and dorsal closure of the embryo commence. Later, the gut encloses the yolk sac to form a closed tube. The final stages of embryogenesis are highlighted by constrictions of the midgut and contraction of the ventral nerve cord (Campos-Ortega and Hartenstein, 1985).

Pl promoter

After germ band shortening, transcription in the epi dermis decreases to undetectable levels (Fig. 2J), ex cept in a few cells in anterior PS 4, including or adjacent to the primordium of the anterior spiracle (Figs 2J and 6A,C). In the mature nervous system, Pl continues to be expressed at high levels in PS 4, at lower levels in PS 5, and at very low levels in PS 6 – 12 until hatching (Figs 2J and 5A,C,F).

The differentiation of somatic and visceral muscles during and after germ band shortening allows identifi cation of specific muscle sets expressing Antp. At the shortened germ band stage, all somatic muscles of T3 express Pl (Martinez-Arias, 1986; Fig. 6C). In the visceral mesoderm, at least two isolated groups of cells around the anterior midgut express Pl (Fig. 6A,C). At stage 17, Pl is also expressed in cells in or near the aorta (data not shown; Campos-Ortega and Hartenstein, 1985). In horizontal views of whole embryos probed with the P1B probe, the pattern is a narrow band of cells posterior to the lymph glands and just below and perpendicular to the dorsal midline.

P2 promoter

After germ band shortening, P2 transcription decreases to very low levels in the epidermis (Figs 2K and 6D). P2 is active in groups of cells below the epidermis within every segment from T1 to A8/A9 (Figs 5B and 6B,D; cDNA pattern shown in Fig. 3F). Based upon their location in the subepidermal cleft, these cells probably correspond to precursors of sensory organs (Campos-Ortega and Hartenstein, 1985). In the mature nervous system, P2 is expressed at a uniform low level in a subset of neurons in each parasegment from PS 3 to PS 14 throughout the later stages of development (Fig. 5B,D,G). Most or all of the somatic muscles of T2 and T3 express P2 (Fig. 6D). In the visceral mesoderm, P2, like Pl, is expressed in a group of cells around the anterior midgut. The P2 pattern is broader than the Pl pattern, and overlaps it (Fig. 6B,D). The cDNA pat tern in the visceral mesoderm is shown in Fig. 3 (G and H). The Antp expression in the visceral mesoderm cells appears to be responsible for the formation of the gut constriction (Fig. 3H) in that this constriction does not form in Antp embryos (R. Reuter and M.P.S., submit ted). Like Pl transcripts, P2 transcripts are detected in or adjacent to cells of the aorta in stage 17 embryos. The identities of the cells in this area expressing both Pl and P2 transcripts has not been determined unambiguously, nor is it known whether Pl and P2 transcripts are present in the same cells.

Fig. 5.

Promoter-specific expression after germ band shortening. (A, B) Alternate horizontal sections of a stage 15 or 16 embryo probed with P1A (panel A) or P2A (panel B). Arrowhead indicates the T2 segment. The posterior limit of Pl expression in the nerve cord is A7. P2 expression in the nerve cord extends from the T1 neuromere through the A9 neuromere. Subectodermal expression of P2 is visible in every segment from T2 to A9 (not visible in this section). (C, D) Adjacent sagittal sections of a stage 16 embryo showing Pl (panel C) and P2 (panel D) expression. Arrowheads indicate the approximate locations of the T2 neuromeres. (E) Sagittal section of an embryo probed with the HB probe. The arrowhead indicates the approximate location of the T2 neuromere. (F, G) Adjacent sagittal sections of a stage 17 embryo probed with Pl A (panel F) or P2A (panel G). Arrowheads indicate the locations of the T2 neuromeres.

Fig. 5.

Promoter-specific expression after germ band shortening. (A, B) Alternate horizontal sections of a stage 15 or 16 embryo probed with P1A (panel A) or P2A (panel B). Arrowhead indicates the T2 segment. The posterior limit of Pl expression in the nerve cord is A7. P2 expression in the nerve cord extends from the T1 neuromere through the A9 neuromere. Subectodermal expression of P2 is visible in every segment from T2 to A9 (not visible in this section). (C, D) Adjacent sagittal sections of a stage 16 embryo showing Pl (panel C) and P2 (panel D) expression. Arrowheads indicate the approximate locations of the T2 neuromeres. (E) Sagittal section of an embryo probed with the HB probe. The arrowhead indicates the approximate location of the T2 neuromere. (F, G) Adjacent sagittal sections of a stage 17 embryo probed with Pl A (panel F) or P2A (panel G). Arrowheads indicate the locations of the T2 neuromeres.

Hybridizations with the HB, D-G, and cDNA probes give signals that are the sum of the Pl and P2 patterns after germ band retraction, again suggesting the two promoters contribute comparable amounts of RNA to the total pattern (Figs 5E and 7G,H).

Fig 6.

Promoter-specific expression in the mesodermal derivatives. (A, B) Alternate horizontal sections of a stage 14 embryo probed with either Pl (panel A) or P2-specific (panel B) probes. Solid arrows indicate visceral mesoderm expression. Open arrow in B indicates ventral nerve cord expression in A8/A9. Pl expression in the gap between T1 and T2 (panel A) corresponds to cells at or near the anterior spiracle, as described previously (Martinez-Arias, 1986).

(C, D) Adjacent sections of a stage 14 embryo probed with Pl- (panel C) or P2-specific (panel D) probes. Solid arrows above the gut indicate expression in the visceral mesoderm. The Pl and P2 patterns overlap, but the P2 pattern extends farther anteriorly and posteriorly. Pl is expressed in somatic muscles of T3 (panel C). P2 expression is visible in clusters of cells below the epidermis in every segment from T2 to A9 (panel D). The open arrow in D indicates a signal that is also seen with both the cDNA and P2 probes in whole embryos (data not shown). The identity of these cells has not been determined unambiguously.

Fig 6.

Promoter-specific expression in the mesodermal derivatives. (A, B) Alternate horizontal sections of a stage 14 embryo probed with either Pl (panel A) or P2-specific (panel B) probes. Solid arrows indicate visceral mesoderm expression. Open arrow in B indicates ventral nerve cord expression in A8/A9. Pl expression in the gap between T1 and T2 (panel A) corresponds to cells at or near the anterior spiracle, as described previously (Martinez-Arias, 1986).

(C, D) Adjacent sections of a stage 14 embryo probed with Pl- (panel C) or P2-specific (panel D) probes. Solid arrows above the gut indicate expression in the visceral mesoderm. The Pl and P2 patterns overlap, but the P2 pattern extends farther anteriorly and posteriorly. Pl is expressed in somatic muscles of T3 (panel C). P2 expression is visible in clusters of cells below the epidermis in every segment from T2 to A9 (panel D). The open arrow in D indicates a signal that is also seen with both the cDNA and P2 probes in whole embryos (data not shown). The identity of these cells has not been determined unambiguously.

Fig. 7.

Regulation of Pl and P2 by the BX-C. Arrowheads indicate the boundary between PS 3 and PS 4 and, in some cases, the boundary between PS 12 and PS 13. (A, B) Bright-field (panel A) and dark-field (panel B) images of a sagittal section of a stage 11 embryo probed with the P1B probe. (C) Sagittal section of a Df(3R)P9 homozygote (lacking the BX-C) probed with the P1B probe. (D, E) Section adjacent to the one in A and B probed with the P2B probe. (F) Section adjacent to the one in C (i.e. BX-C) probed with the P2B probe. (G, H) Sagittal section of a stage 11 embryo probed with the HB probe. Note that the pattern generated by the HB probe is approximately the sum of the patterns generated by the Pl and P2 probes.

Fig. 7.

Regulation of Pl and P2 by the BX-C. Arrowheads indicate the boundary between PS 3 and PS 4 and, in some cases, the boundary between PS 12 and PS 13. (A, B) Bright-field (panel A) and dark-field (panel B) images of a sagittal section of a stage 11 embryo probed with the P1B probe. (C) Sagittal section of a Df(3R)P9 homozygote (lacking the BX-C) probed with the P1B probe. (D, E) Section adjacent to the one in A and B probed with the P2B probe. (F) Section adjacent to the one in C (i.e. BX-C) probed with the P2B probe. (G, H) Sagittal section of a stage 11 embryo probed with the HB probe. Note that the pattern generated by the HB probe is approximately the sum of the patterns generated by the Pl and P2 probes.

Antp expression in embryos that lack bithorax complex functions

In embryos lacking the entire BX-C, Antp expression is derepressed in the abdomen. Antp transcripts, as detected by cDNA probes that hybridize to both Pl and P2 transcripts, are present at high levels from the second thoracic segment through most of the abdominal segments (Hafen et al. 1984; Harding et al. 1985). Antp protein is abundant in the nerve cord of BX-C embryos from pTl through aA7 (PS 4 – 12; Carroll et al. 1986; Wirz et al. 1986). These observations indicate that genes in the BX-C negatively regulate Antp expression, either directly or indirectly. BX-C regulation of P2 has previously been examined in two somewhat indirect ways. First, in BX-C flies that carry a lacZ gene under the control of the P2 promoter, β-galactosidase ex pression is derepressed posteriorly through PS 14 (Boulet and Scott, 1988). These P2-lacZ fusions, how ever, may not contain all the cis-acting control elements required by the endogenous P2 promoter for proper regulation by BX-C genes or may respond abnormally. Second, Antp P2-derived protein is expressed in PS 3–14 in BX-C embryos (Boulet and Scott, 1988). However, this result was obtained from embryos that in addition to lacking the BX-C also carried the dominant Antp73b mutation, which was necessary to eliminate the Pl contribution to the protein pattern. Conceivably the P2 expression pattern in these embryos could be affec ted by the Antp73b mutation. A direct examination of P2 transcripts in BX-C embryos that are wild-type for Antp is therefore preferable. It has not been reported whether the Pl promoter is regulated by BX-C genes.

Pl promoter

In extended germ-band embryos that lack BX-C func tion, Pl transcripts are expressed at high levels from PS 4 to PS 12 (Fig. 7C). Therefore genes of the BX-C negatively regulate Pl expression. However, Pl tran scripts in BX-C embryos, as in wild-type embryos, are not detected in PS 13 and PS 14. The apparent absence of Pl transcripts in these parasegments suggests that either Pl is repressed by other genes in PS 13 and PS 14, or that, unlike P2, Pl does not contain the regulatory sequences required for activation in those paraseg ments.

P2 promoter

In BX-C embryos, P2 transcripts are expressed from PS 3 through PS 14, further posterior than Pl ex pression (Fig. 7F). Derepression of the P2 promoter is more subtle than derepression of the Pl promoter and is difficult to document convincingly. However, embryo sections displaying putative P2 derepression can be distinguished from sections of wild-type embryos on the basis of slightly more intense labelling of the nerve cord. Such identifications are possible only with good sagittal sections, and were confirmed as BX-C em bryos by the ectopic Pl expression seen in adjacent serial sections. The P2 derepression may be less ex treme than the Pl derepression for several possible reasons. First, P2 may be expressed more strongly in the nervous system in wild-type embryos, so its dere pression is less noticeable. Second, Pl could be repressed in more cells or tissues than P2. Third, within a given cell, Pl could be repressed more strongly than P2.

How do Antp and other homeotic genes control seg ment differentiation? Many of the homeotic genes encode transcription factors that presumably activate and/or repress different sets of ‘target’ genes that in turn generate the morphology of the fly. Because the ANT-C and BX-C homeotic genes are expressed in specific domains within the embryo, cells in different parts of the embryo may activate or repress distinct sets of target genes, or control the synthesis of particular ratios of target gene products, thus directing cells along particular developmental pathways. Ectopic expression of Antp or Dfd results in segment transformations (Denell, 1973; Struhl, 1981; Hazelrigg and Kaufman, 1983; Frischer et al. 1986; Schneuwly et al. 1987a,b\Gibson and Gehring, 1988; Kuziora and McGinnis, 1988b). Therefore the position-specific expression pat terns of the homeotic genes constitute a crucial aspect of their function in development. The initial patterns of expression of many homeotic genes are fairly simple, but as development proceeds, they become extremely complex: different tissues, and different cells within these tissues, express various combinations of homeotic gene products (e.g. Carroll et al. 1988). Such dynamic expression patterns result from complicated regulatory interactions, including interactions among the homeotic genes themselves.

In this paper, we have shown that the two Antp promoters are expressed in distinct spatial patterns during embryogenesis, as is summarized in Fig. 8. The patterns are tissue specific as well as position specific, and in several cases it is apparent, even with the limited resolution of in situ hybridization, that only some of the cells within a segment are expressing Antp. The obser vations are important for two reasons: First, the two Antp promoters must respond to different regulatory inputs during embryogenesis. Second, Antp transcripts with different 5’ untranslated sequences are made in different places during development. The two types of Antp transcript could have different stabilities and/or could be translated with different efficiencies.

Fig. 8.

Schematic summary of the Pl and P2 transcript patterns during embryogenesis. Solid pattern=moderate to high level expression. Stippled pattern=expression that is clearly a subset of cells in that tissue. Hatched pattern=low level expression. Abbreviations: PS, parasegment; meso, mesoderm; VNS, ventral nervous system; ecto, ectoderm. Expression of Pl and P2 after stage 15 is not diagrammed because of the complexity of the tissues and expression patterns at later stages.

Fig. 8.

Schematic summary of the Pl and P2 transcript patterns during embryogenesis. Solid pattern=moderate to high level expression. Stippled pattern=expression that is clearly a subset of cells in that tissue. Hatched pattern=low level expression. Abbreviations: PS, parasegment; meso, mesoderm; VNS, ventral nervous system; ecto, ectoderm. Expression of Pl and P2 after stage 15 is not diagrammed because of the complexity of the tissues and expression patterns at later stages.

The Antp Pl and P2 promoters perform different functions

Antp plays multiple roles in development. The pheno types of somatic clones of Antp cells, the intragenic complementation among Antp mutations, and the dis tinct lethal periods for individuals carrying different Antp mutations suggest that the two transcription units have different functions. The Pl transcription unit is required for anterior spiracle eversion and dorsal tho racic development, while the P2 transcription unit is required for embryonic viability and leg development (Kaufman and Abbott, 1984; Abbott and Kaufman, 1986).

The distributions of Pl and P2 transcripts corrobor ate, in part, the genetic inferences about promoter specific functions. Pl transcripts are observed in or near the developing anterior spiracles during embryogen esis, while P2 transcripts are not (Fig. 6). Pl transcripts accumulate in those parts of the wing discs destined to become dorsal thorax (Jorgensen and Garber, 1987). However, both Pl and P2 RNAs accumulate in both dorsal and ventral tissue (Jorgensen and Garber, 1987). The presence of Pl or P2 transcripts in tissues that are not affected by specific mutations that alter the Pl or P2 transcription unit suggest that Antp can be expressed in places where it does not function, or that the effects of Antp in these tissues are subtle and therefore have not yet been observed.

The Antp phenotypes that have been observed are likely to be just the most obvious external effects of the loss of Antp function. There are severe limitations to what it has been possible to learn about the detailed phenotypes of Antp mutants, particularly in internal tissues such as the nervous system. It is generally impossible, presently, to recognize the transformation of the fates of one or a few cells to those of similar cells. The complexity of the expression patterns suggests that Antp participates in a very large number of develop mental decisions, only some of which can currently be recognized and interpreted.

The pair of Antp promoters provide Antp with regulatory flexibility

It is becoming clear that the complicated expression patterns of the homeotic genes result from large arrays of czó-acting regulatory elements. In many cases, these regulatory elements are likely to be binding sites for proteins that regulate homeotic gene expression. For example, the transcription of the Ultrabithorax (Ubx) gene is controlled by regulatory elements that act over great distances on the Ubx promoter (Hogness et al. 1985; Bender et al. 1985; Peifer et al. 1987). It is likely that Antp is also regulated by a large number of regulatory elements as well, but, in contrast to Ubx, two promoters must be regulated. It is not yet clear whether any c/s-acting control elements act on both promoters, or whether each promoter has its own set of regulatory sequences. Many genes containing multiple promoters are expressed in complex temporal and position-specific patterns. In Drosophila, these genes include Alcohol dehydrogenase (Adh) (Benyajati et al. 1983), caudal (cad) (Mlodzik and Gehring, 1987), and hunchback (hb) (Tautz et al. 1987; Schroder et al. 1988). In each case, the different promoters are utilized in different stage- and tissue-specific patterns. At least one other homeotic gene, Abdominal B, uses multiple promoters (Sánchez-Herrero and Crosby, 1988; DeLorenzi et al. 1988; Kuziora and McGinnis, 1988a).

Genes that differentially regulate the Antp promoters

The different patterns of expression of the Antp pro moters suggests that they are controlled by different sets of genes. Homeotic gene expression is regulated, directly or indirectly, by maternally active genes, seg mentation genes, and other homeotic genes. Several of these genes appear to differentially regulate the two promoters. The results of in situ hybridizations of Antp promoter-specific probes to mutant embryos suggest that the maternally active gene oskar regulates P2 but not Pl (Irish et al. 1989). The zygotically active gap segmentation genes Krilppel, hunchback, and knirps also regulate Antp expression (Harding and Levine, 1988). Krilppel activates Pl but not P2, while hunch back activates P2 but not Pl (Irish et al. 1989). Antp expression is altered in embryos mutant for knirps, but the effect may be indirect, resulting from regulatory interactions among the gap genes themselves (Harding and Levine, 1988). The pair-rule genefushi tarazu (ftz) activates P2 but not Pl (Ingham and Martinez-Arias, 1986). The transcription factor DTF-1 activates the P2 promoter in vitro, but probably not Pl (Perkins et al. 1988). Therefore although many regulators of Antp remain to be identified, there is already ample evidence for promoter-specific regulation.

Both Pl and P2 are derepressed in embryos carrying mutations in the BX-C. However, in BX-C embryos Pl is derepressed only back through PS 12 while P2 is derepressed through PS 13-14. Therefore an activator of P2 is absent from PS 13 and 14, or an additional negative regulator of P2 exists in PS 13 and 14. Recently, a hybrid Deformed-Ubx protein, containing a Ubx homeodomain, has been expressed under heat shock promoter control in embryos and shown to activate Pl, but not P2, in the head (M. Kuziora and W. McGinnis, personal communication). Antp is not acti vated by Deformed protein under these conditions so the effect on Antp is presumably due to the binding of the hybrid protein to the Antp gene through the Ubx homeodomain. These results suggest that Ubx acts differentially upon the two Antp promoters.

Surprisingly, Antp transcripts are not detected in PS2, where ftz and hb, both activators of Antp P2 in some cells, are expressed. This observation suggests that a negative regulator of Antp P2, perhaps bicoid, prevents Antp expression in PS 2 (Irish et al. 1989).

The Antp Pl and P2 transcripts may be translated with different efficiencies

Recently it has been shown that a polyclonal anti-Anfp antiserum (Carroll et al. 1986) mainly detects protein translated from Pl transcripts, while a more sensitive monoclonal anti-Antp antiserum detects protein trans lated from both Pl and P2 transcripts (Boulet and Scott, 1988). These results indicate that Pl-derived Antp protein is more easily detected than P2-derived Antp protein. Three plausible explanations for this obser vation are: (1) Pl transcripts are much more abundant than P2 transcripts. Two observations indicate that this is not the case. First, probes that hybridize to both Pl and P2 transcripts do not predominantly detect the Pl pattern (for example, see Fig. 2). Second, SI nuclease protection analysis of RNA from 4–8 h embryos reveals that transcripts from the two promoters are present at similar steady-state levels (Bermingham and Scott, 1988). (2) Pl transcripts produce protein different from that produced from P2 transcripts. Although alternative RNA splicing permits four different (but closely re lated) Antp proteins to be made, there is no detectable linkage between alternative splicing and promoter usage (Bermingham and Scott, 1988; Stroeher et al. 1988). (3) Pl transcripts are translated more efficiently than are P2 transcripts. AUG codons followed by short open reading frames upstream of a larger open reading frame have been implicated in translational efficiency and regulation (Thireos et al. 1984; Hinnebusch, 1984; Hinnebusch, 1988). Therefore, it may be relevant that Pl transcripts contain 8 upstream AUGs while P2 transcripts contain 15 upstream AUG’s (Schneuwly et al. 1986; Stroeher et al. 1986; Laughon et al. 1986).

The authors wish to thank Diethard Tautz and C. Pfeifle for communicating their method for whole-mount in situ hybrid ization before publication; Joan Hooper, Tammie Yeh and Debbie Andrew for useful technical advice; Rolf Reuter for the use of some of his in situ hybridization data; and D. Andrew and John Tamkun for making useful suggestions on the manuscript. M.P.S. is an investigator of the Howard Hughes Medical Institute. The work was supported by N.I.H. grant no. 18163.

Abbott
,
M. K.
and
Kaufman
,
T. C.
(
1986
).
The relationship between the functional complexity and the molecular organization of the Antennapedia locus of Drosophila melanogaster
.
Genetics
114
,
919
942
.
Akam
,
M.
(
1987
).
The molecular basis for metameric pattern in the Drosophila embryo
.
Development
101
,
1
22
.
Akam
,
M. E.
and
Martinez-Arias
,
A.
(
1985
).
The distribution of Ultrabithorax transcripts in Drosophila embryos
.
EMBO J
.
4
,
1689
1700
.
Beachy
,
P. A.
,
Krasnow
,
M. A.
,
Gavis
,
E. R.
and
Hogness
,
D. S.
(
1988
).
An Ultrabithorax protein binds near its own and the Antennapedia Pl promoters
.
Cell
55
,
1069
1081
.
Bender
,
W.
,
Weiffenbach
,
B.
,
Karch
,
F.
and
Peifer
,
M.
(
1985
).
Domains of cw-interaction in the bithorax complex
.
Cold Spring Harbor Symp. quant. Biol
.
50
,
173
180
.
Benyajati
,
C.
,
Spoerel
,
N.
,
Haymerle
,
H.
and
Ashburner
,
M.
(
1983
).
The messenger RNA for alcohol dehydrogenase in Drosophila melanogaster differs in its 5’ end in different developmental stages
.
Cell
33
,
125
133
.
Bermingham
,
J. R.
, Jr
and
Scott
,
M. P.
(
1988
).
Developmentally regulated alternative splicing of transcripts from the Drosophila homeotic gene Antennapedia can produce four different proteins
.
EM BO J
.
7
,
3211
3222
.
Bodner
,
M.
,
Castrillo
,
J-L.
,
Theill
,
L. E.
,
Deernick
,
T.
,
Ellisman
,
M.
and
Karin
,
M.
(
1988
).
The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein
.
Cell
55
,
505
518
.
Boulet
,
A. M.
and
Scott
,
M. P.
(
1988
).
Control elements of the P2 promoter of the Antennapedia gene
.
Genes and Development
2
,
1600
1614
.
Campos-Ortega
,
J. A.
and
Hartenstein
,
V.
(
1985
).
The Embryonic Development of Drosophila melanogaster
.
Springer Verlag
:
Berlin, Heidelberg, New York
.
Carroll
,
S. B.
,
Dinardo
,
S.
,
O’Farrell
,
P. H.
,
White
,
R. A. H.
and
Scott
,
M. P.
(
1988
).
Temporal and spatial relationships between segmentation and homeotic gene expression in Drosophila embryos: distributions of the fushi taraza, engrailed, Sex combs reduced, Antennapedia, and Ultrabithorax proteins
.
Genes and Development
2
,
350
360
.
Carroll
,
S. B.
,
Laymon
,
R. A.
,
Mccutcheon
,
M. A.
,
Riley
,
P. D.
and
Scott
,
M. P.
(
1986
).
The localization and regulation of Antennapedia protein expression in Drosophila embryos
.
Cell
47
,
113
122
.
Delorenzi
,
M.
,
Ali
,
N.
,
Saari
,
G.
,
Henry
,
C.
,
Wilcox
,
M.
and
Bienz
,
M.
(
1988
).
Evidence that the Abdominal-B r element function is conferred by a trans-regulatory homeoprotein
.
EMBO J
.
7
,
3223
3231
.
Denell
,
R. E.
(
1973
).
Homoeosis in Drosophila. I. Complementation studies with revenants of Nasobemia
.
Genetics
75
,
279
297
.
Denell
,
R. E.
,
Hummels
,
K. R.
,
Wakimoto
,
B. T.
and
Kaufman
,
T. C.
(
1981
).
Developmental studies of lethality associated with the Antennapedia gene complex in Drosophila melanogaster
.
Devl Biol
.
81
,
43
50
.
Desplan
,
C.
,
Theis
,
J.
and
O’Farrell
,
P. H.
(
1985
).
The Drosophila developmental gene, engrailed, encodes a sequence specific DNA binding activity
.
Nature
318
,
630
635
.
Desplan
,
C.
,
Theis
,
J.
and
O’Farrell
,
P. H.
(
1988
).
The sequence specificity of homeodomain-DNA interaction
.
Cell
54
,
1081
1090
.
Driever
,
W.
and
Nüsslein-Volhard
,
C.
(
1989
).
The bicoid protein is a positive regulator of huchback transcription in the early Drosophila embryo
.
Nature
337
,
138
143
.
Duncan
,
I. M.
(
1986
).
Control of bithorax complex functions by the segmentation gene fushi tarazu of Drosophila melanogaster
.
Cell
47
,
297
309
.
Fitzpatrick
,
V. D.
and
Ingles
,
C. J.
(
1989
).
The Drosophila fushi tarazu polypeptide is a DNA-binding transcriptional activator in yeast cells
.
Nature
337
,
666
668
.
Foe
,
V. E.
and
Alberts
,
B.
(
1983
).
Studies of nuclear and cytoplasmic behavior during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis
.
J. Cell Sci
.
61
,
31
70
.
Frischer
,
L. E.
,
Hagen
,
F. S.
and
Garber
,
R. L.
(
1986
).
An inversion that disrupts the Antennapedia gene causes abnormal structure and localization of RNAs
.
Cell
47
,
1017
1023
.
Garber
,
R. L.
,
Kuroiwa
,
A.
and
Gehring
,
W. J.
(
1983
).
Genomic and cDNA clones of the homeotic locus Antennapedia in Drosophila
.
EMBO J
.
2
,
2027
2034
.
Gehring
,
W. J.
(
1987
).
Horneo boxes in the study of development
.
Science
236
,
1245
1252
.
Gibson
,
G.
and
Gehring
,
W. J.
(
1988
).
Head and thoracic transformations caused by ectopic expression of Antennapedia during Drosophila development
.
Development
102
,
657
675
.
Hafen
,
E.
,
Levine
,
M.
,
Garber
,
R. L.
and
Gehring
,
W. J.
(
1983
).
An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex
.
EMBO J.
2
,
617
623
.
Hafen
,
E.
,
Levine
,
M.
and
Gehring
,
W. J.
(
1984
).
Regulation of Antennapedia transcript distribution by the bithorax complex in Drosophila
.
Nature
307
,
287
289
.
Han
,
K.
,
Levine
,
M. S.
and
Manley
,
J. L.
(
1989
).
Synergistic activation and repression of transcription by Drosophila homeobox proteins
.
Cell
56
,
573
583
.
Harding
,
K.
and
Levine
,
M.
(
1988
).
Gap genes define the limits of Antennapedia and bithorax gene expression during early development in Drosophila
.
EMBO J
.
7
,
205
214
.
Harding
,
K.
,
Wedeen
,
C.
,
Mcginnis
,
W.
and
Levine
,
M.
(
1985
).
Spatially regulated expression of homeotic genes in Drosophila
.
Science
229
,
1236
1242
.
Hazelrigg
,
T.
and
Kaufman
,
T. C.
(
1983
).
Revenants of dominant mutations associated with the Antennapedia gene complex of Drosophila melanogaster
.
Genetics
105
,
581
600
.
Hinnebusch
,
A. G.
(
1984
).
Evidence for translational regulation of the activator of general amino acid control in yeast
.
Proc. natn. Acad. Sci. U.S.A
.
81
,
6442
6446
.
Hinnebusch
,
A. G.
(
1988
).
Novel mechanisms of translational control in Saccharomyces cerevisiae
.
Trends in Genetics
4
,
169
174
.
Hoey
,
T.
and
Levine
,
M.
(
1988
).
Divergent horneo box proteins recognize similar DNA sequences in Drosophila
.
Nature
332
,
858
861
.
Hogness
,
D. S.
,
Lipshitz
,
H. D.
,
Beachy
,
P. A.
,
Peattie
,
D. A.
,
Saint
,
R. A.
,
Goldschmidt-Clermont
,
M.
,
Harte
,
P. J.
,
Gavis
,
E. R.
and
Helfand
,
S. L.
(
1985
).
Regulation and products of the Ubx domain of the bithorax complex
.
Cold Spring Harbor Symp. quant. Biol
.
50
,
181
194
.
Ingham
,
P. W.
(
1988
).
The molecular genetics of pattern formation in Drosophila
.
Nature
335
,
25
34
.
Ingham
,
P.
,
Howard
,
K.
and
Ish-Horowicz
,
D.
(
1985
).
Transcription pattern of the Drosophila segmentation gene hairy
.
Nature
318
,
439
445
.
Ingham
,
P. W.
and
Martinez-Arlas
,
A.
(
1986
).
The correct activation of Antennapedia and bithorax complex genes requires the fushi tarazu gene
.
Nature
324
,
592
597
.
Ingraham
,
H. A.
,
Chen
,
R.
,
Mangalam
,
H. J.
,
Elsholtz
,
H. P.
,
Flynn
,
S. E.
,
Lin
,
C. R.
,
Simmons
,
D. M.
,
Swanson
,
L.
and
Rosenfeld
,
M. G.
(
1988
).
A tissue specific transcription factor containing a homeodomain specifies a pituitary phenotype
.
Cell
55
,
519
529
.
Irish
,
V. F.
,
Martinez-Arias
,
A.
and
Akam
,
M.
(
1989
).
Spatial regulation of the Antennapedia and Ultrabithorax homeotic genes during Drosophila early development
.
EMBO J
.
8
,
1527
1537
.
Jaynes
,
J. B.
and
O’Farrell
,
P. H.
(
1988
).
Activation and repression of transcription by homoeodomain-containing proteins that bind a common site
.
Nature
336
,
744
749
.
Jorgensen
,
E. M.
and
Garber
,
R. L.
(
1987
).
Function and misfunction of the two promoters of the Drosophila Antennapedia gene
.
Genes and Development
1
,
544
555
.
Kaufman
,
T. C.
and
Abbott
,
M. K.
(
1984
).
Homoeotic genes and the specification of segmental identity in the embryo and adult thorax of Drosophila melanogaster
.
In Molecular Aspects of Early Development
(eds
G. M.
Malacinski
and
W. H.
Klein
), pp.
189
218
.
Plenum Press
:
New York
.
Kaufman
,
T. C.
,
Lewis
,
R.
and
Wakimoto
,
B.
(
1980
).
Cytogenetic analysis of chromosome 3 in Drosophila melanogaster: the homoeotic gene complex in polytene chromosome interval 84A,B
.
Genetics
94
,
115
133
.
Ko
,
H-S.
,
Fast
,
P.
,
Mcbride
,
W.
and
Staudt
,
L. M.
(
1988
).
A human protein specific for the immunoglobulin octamer DNA motif contains a functional homeobox domain
.
Cell
55
,
135
144
.
Krasnow
,
M.
,
Saffman
,
E. E.
,
Kornfeld
,
K.
and
Hogness
,
D.
(
1989
).
Transcriptional activation and repression by Ultrabithorax proteins in cultured Drosophila cells
.
Cell
57
,
1031
1043
.
Kuziora
,
M. A.
and
Mcginnis
,
W.
(
1988a
).
Different transcripts of the Drosophila Abd-B gene correlate with distinct genetic sub functions
.
EMBO J
.
7
,
3233
3244
.
Kuziora
,
M. A.
and
Mcginnis
,
W.
(
1988b
).
Autoregulation of a Drosophila homeotic selector gene
.
Cell
55
,
477
485
.
Laughon
,
A.
,
Boulet
,
A. M.
,
Bermingham
,
J. R.
Jr
,
Laymon
,
R. A.
and
Scott
,
M. P.
(
1986
).
The structure of transcripts from the homeotic Antennapedia gene of Drosophila: Two promoters control the major protein-coding region
.
Mol. cell. Biol
.
6
,
4676
4689
.
Laughon
,
A.
,
Howell
,
W.
and
Scott
,
M. P.
(
1988
).
The interaction of proteins encoded by Drosophila homeotic and segmentation genes with specific DNA sequences
.
Development
104
(
Suppl
.).
75
83
.
Laughon
,
A.
and
Scott
,
M. P.
(
1984
).
Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins
.
Nature
310
,
25
31
.
Lawrence
,
P. A.
and
Johnston
,
P.
(
1989
).
Pattern formation in the Drosophila embryo: allocation of cells to parasegments by even-skipped and fushi taraza
.
Development
105
,
761
767
.
Levine
,
M.
,
Hafen
,
E..
Garber
,
R. L.
and
Gehring
,
W. J.
(
1983
).
Spatial distribution of Antennapedia transcripts during Drosophila development
.
EMBO J
.
2
,
2037
2046
.
Levine
,
M.
and
Hoey
,
T.
(
1988
).
Homeobox proteins as sequence specific transcription factors
.
Cell
55
,
537
540
.
Lewis
,
E. B.
(
1978
).
A gene complex controlling segmentation in Drosophila
.
Nature
276
,
565
570
.
Martinez-Arias
,
A.
(
1986
).
The Antennapedia gene is required and expressed in parasegments 4 and 5 of the Drosophila embryo
.
EMBO J
.
5
,
135
141
.
Martinez-Arias
,
A.
and
Lawrence
,
P. A.
(
1985
).
Parasegments and compartments in the Drosophila embryo
.
Nature
313
,
639
642
.
Martinez-Arias
,
A.
and
White
,
R. A. H.
(
1988
).
Ultrabithorax and engrailed expression in Drosophila embryos mutant for segmentation genes of the pair-rule class
.
Development
102
,
325
338
.
Mcginnis
,
W.
,
Levine
,
M.
,
Hafen
,
E.
,
Kuroiwa
,
A.
and
Gehring
,
W. J.
(
1984
).
A conserved DNA sequence in homeotic genes of the Drosophila Antennapedia and bithorax complexes
.
Nature
308
,
428
433
.
Mitchell
,
P. J.
and
Tjian
,
R.
(
1989
).
Transcriptional regulation in mammalian cells by sequence-specific DNA-binding proteins, submitted
.
Mlodzik
,
M.
and
Gehring
,
W. J.
(
1987
).
Expression of the caudal gene in the germ line of Drosophila: Formation of an RNA and protein gradient during early embryogenesis
.
Cell
48
,
465
478
.
Nüsslein-Volhard
,
C.
,
Frohnhofer
,
H. G.
and
Lehmann
,
R.
(
1987
).
Determination of anteroposterior polarity in Drosophila
.
Science
238
,
1675
1681
.
Peifer
,
M.
,
Karch
,
F.
and
Bender
,
W.
(
1987
).
The bithorax complex: control of segmental identity
.
Genes and Development
I
891
898
.
Perkins
,
K. K.
,
Daly
,
G. M.
and
Than
,
R.
(
1988
).
In vitro analysis of the Antennapedia P2 promoter: identification of a new Drosophila transcription factor
.
Genes and Development
2
,
1615
1626
.
Riley
,
P. D.
,
Carroll
,
S. B.
and
Scott
,
M. P.
(
1987
).
The expression and regulation of Sex combs reduced protein in Drosophila embryos
.
Genes and Development
1
,
716
730
.
Samson
,
M.-L.
,
Jackson-Grusby
,
L.
and
Brent
,
R.
(
1989
).
Gene activation and DNA binding by Drosophila Ubx and abd-A proteins
.
Cell
57
,
1045
1057
.
Sánchez-Herrero
,
E.
and
Crosby
,
M. A.
(
1988
).
The Abdominal-B gene of Drosophila melanogaster. overlapping transcripts exhibit two different spatial distributions
.
EMBO J
.
7
,
2163
2173
.
Scheidereit
,
C.
,
Cromlish
,
J. A.
,
Gerster
,
T.
,
Kawakami
,
K.
,
Balmaceda
,
C-G.
,
Currie
,
R. A.
and
Roeder
,
R. G.
(
1988
).
A human lymphoid-specific transcription factor that activates immunoglobulin genes is a homoeobox protein
.
Nature
336
,
551
557
.
Schneuwly
,
S.
,
Klemenz
,
R.
and
Gehring
,
W. J.
(
1987a
).
Redesigning the body plan of Drosophila by ectopic expression of the homoeotic gene Antennapedia
.
Nature
325
,
816
818
.
Schneuwly
,
S.
,
Kuroiwa
,
A.
,
Baumgartner
,
P.
and
Gehring
,
W.J.
(
1986
).
Structural organization and sequence of the homeotic gene Antennapedia of Drosophila melanogaster
.
EMBO J
.
5
,
733
739
.
Schneuwly
,
S.
,
Kuroiwa
,
A.
and
Gehring
,
W. J.
(
1987b
).
Molecular analysis of the dominant homeotic Antennapedia phenotype
.
EMBO J
.
6
,
201
206
.
Schrôder
,
C.
,
Tautz
,
D.
,
Seifert
,
E.
and
Jâckle
,
H.
(
1988
).
Differential regulation of the two transcripts from the Drosophila gap gene hunchback
.
EMBO J
.
7
,
2881
2887
.
Scott
,
M. P.
and
Carroll
,
S. B.
(
1987
).
The segmentation and homeotic gene network in early Drosophila development
.
Cell
51
,
689
698
.
Scott
,
M. P.
,
Tamkun
,
J. W.
and
Hartzell
,
G. W.
III
(
1989
).
The structure and function of the homeodomain
.
Biochem. Biophys. Acta Rev. Cane
.
989
,
25
48
.
Scott
,
M. P.
and
Weiner
,
A. J.
(
1984
).
Structural relationships among genes that control development: Sequence homology between the Antennapedia, Ultrabithorax, and fushi tarazu loci of Drosophila
.
Proc. natn. Acad. Sci. U.S.A
.
81
,
4115
4119
.
Scott
,
M. P.
,
Weiner
,
A. J.
,
Polisky
,
B. A.
,
Hazelrigg
,
T. I.
,
Pirrotta
,
V.
,
Scalenghe
,
F.
and
Kaufman
,
T. C.
(
1983
).
The molecular organization of the Antennapedia locus of Drosophila
.
Cell
35
,
763
776
.
Shephard
,
J. C. W.
,
Mcginnis
,
W.
,
Carrasco
,
A. E.
,
Derobertis
,
E. M.
and
Gehring
,
W. J.
(
1984
).
Fly and frog homoeodomains show homologies with yeast mating type regulatory proteins
.
Nature
310
,
70
71
.
Stroeher
,
V. L.
,
Gaiser
,
C.
and
Garber
,
R. L.
(
1988
).
Alternative RNA splicing that is spatially regulated: generation of transcripts from the Antennapedia gene of Drosophila melanogaster with different protein-coding regions
.
Mol. cell. Biol
.
8
,
4143
4154
.
Stroeher
,
V. L.
,
Jorgensen
,
E. M.
and
Garber
,
R. L.
(
1986
).
Multiple transcripts from the Antennapedia gene of Drosophila
.
Mol. cell. Biol
.
6
,
4667
4675
.
Struhl
,
G.
(
1981
).
A homoeotic mutation transforming leg to antenna in Drosophila
.
Nature
292
,
635
638
.
Struhl
,
G.
and
White
,
R. A. H.
(
1985
).
Regulation of the Ultrabithorax gene of Drosophila by other bithorax complex genes
.
Cell
43
,
507
519
.
Sturm
,
R. A.
,
Das
,
G.
and
Herr
,
W.
(
1988
).
The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a horneo box subdomain
.
Genes and Development
2
,
1582
1589
.
Tautz
,
D.
,
Lehmann
,
R.
,
Schnürch
,
H.
,
Schuh
,
R.
,
Seifert
,
E.
,
Kienlin
,
A.
,
Jones
,
K.
and
Jâckle
,
H.
(
1987
).
Finger protein of novel structure encoded by hunchback, a second member of the gap class of Drosophila segmentation genes
.
Nature
327
,
383
389
.
Tautz
,
D.
and
Pfeifle
,
C.
(
1989
).
A non-radioacitve in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback
.
Chromosoma, Berk
98
,
81
85
.
Thali
,
M.
,
Müller
,
M. M.
,
Delorenzi
,
M.
,
Mattias
,
P.
and
Bienz
,
M.
(
1988
).
Drosophila homoeotic genes encode transcriptional activators similar to mammalian OTF-2
.
Nature
336
,
598
601
.
Thireos
,
G.
,
Penn
,
M. D.
and
Greer
,
H.
(
1984
).
5’ untranslated sequences are required for the translational control of a yeast regulatory gene
.
Proc. natn. Acad. Sci. U.S.A
.
81
,
5096
5100
.
Wakimoto
,
B. T.
and
Kaufman
,
T. C.
(
1981
).
Analysis of larval segmentation in lethal genotypes associated with the Antennapedia gene complex in Drosophila melanogaster
.
Devi Biol
.
81
,
51
64
.
Wedeen
,
C.
,
Harding
,
K.
and
Levine
,
M.
(
1986
).
Spatial regulation of Antennapedia and bithorax gene expression by the Polycomb locus in Drosophila
.
Cell
44
,
739
748
.
White
,
R. A. H.
and
Lehmann
,
R.
(
1986
).
A gap gene, hunchback, regulates the spatial expression of Ultrabithorax
.
Cell
47
,
311
321
.
Winslow
,
G. M.
,
Hayashi
,
S.
,
Krasnow
,
M.
,
Hocness
,
D. S.
and
Scott
,
M. P.
(
1989
).
Gene activation by the Antennapedia and fushi tarazu proteins in cultured Drosophila cells
.
Cell
57
,
1017
1030
.
Wirz
,
J.
,
Fessler
,
L. I.
and
Gehring
,
W. J.
(
1986
).
Localization of the Antennapedia protein in Drosophila embryos and imaginai discs
.
EMBO J
.
5
,
3327
3334
.