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Analysis of the ftz upstream element: germ layer-specific enhancers are independently autoregulated.

The Drosophila fushi tarazu (ftz) upstream element is an enhancer-like element that is required for the correct expression of ftz in developing embryos and that directs transcription from a minimal promoter in a ftz-like seven-striped pattern. Using a deletion analysis, we have identified several independent cis-regulatory elements in the upstream element. A distal enhancer directs fusion gene expression in seven stripes primarily in the mesoderm. A more complex proximal enhancer contains a mesodermally active element and a second element with which it interacts to generate seven stripes in the ectoderm. Striped expression directed by each enhancer is ftz-dependent, and each contains binding sites for purified ftz homeo domain. We suggest that ftz protein acts in combination with germ layer-restricted transcription factors directly and positively to regulate the transcription of its own gene.

such cases, cis-acting DNA sequences mediate interactions between trans-acting protein factors and RNA polymerase II or the "general transcription machinery" to activate transcription. Promoter elements, located close to the transcription start site, are required for efficient initiation and positioning of the start site, whereas upstream regulatory elements or enhancers act to increase the rate of transcription from a given promoter. Enhancers are characterized by their abilities to (1) stimulate transcription over large and varying distances, (2) act in an orientation-independent fashion, and (3) stimulate transcription of heterologous promoters (Serfling et al. 1985). For both viral and cellular genes, discrete cisacting regulatory elements have been identified that direct transcription in a cell type-restricted fashion. Although some cell type-specific enhancers have been studied in detail (Atchison 1988), only a small number have been examined in their native cellular environments in transgenic animals (Posakony et al. 1985;Garabedian et al. 1986;Hammer et al. 1987;Pinkert et al.  1987; Johnson et al. 1989;Logan et al. 1989). Much of what we know about enhancer function comes from studies of viral model systems. Analysis of the wellcharacterized viral SV40 enhancers suggests that the basic building blocks of enhancers are short "enhansons" that correspond to the binding sites for transacting factors (Ondek et al. 1988}. Different enhancer motifs may be active in different cell types, suggesting that the availability of cell type-specific transcription factors may control the specificity of enhancer activation {Schirm et al. 1987). Given the complexity of promoter and enhancer elements, it is possible to construct a scenario in which transcriptional specificity is determined by the combinatorial action of general and/or specific trans-acting factors that interact with the same, overlapping, or unique sets of regulatory sequences. This type of model may be particularly informative when applied to developmentally regulated gene expression.
Developmental and genetic studies of Drosophila melanogaster have led to the identification of groups of genes that are required to establish the basic body plan of the fly. These genes include the maternally active coordinate genes and the zygotically active segmentation and homeotic genes {Lewis 1978; Niisslein-Volhard and Wieschavs 1980;Niisslein-Volhard et al. 1987). These genes are expressed in spatially and temporally restricted patterns that are frequently dynamic and rapidly evolving. For example, in the case of the segmentation gene fushi tarazu (ftz), transcripts are first detected throughout the embryo at low levels during nuclear cycle 10 and accumulate to higher levels in a broad band between 15 and 65% egg length by cycle 12. As cellularization begins (early cycle 14), gaps appear in this broad band, generating transiently four and, then, seven stripes that encircle the blastoderm embryo (late cycle 14). At gastrulation and early germ-band extension, ftz transcripts are detected in seven stripes in the primordia of the even-numbered parasegments  in both the ectoderm and underlying mesoderm Martinez-Arias and Lawrence 1985;Weir and Kornberg 1985). Later, ftz is also expressed in a subset of cells in the developing central nervous system and hindgut (Carroll and Scott 1985;Hiromi et al. 1985;Krause et al. 1988).
Analysis of the expression patterns of a number of segmentation and homeotic genes (for review and discussion, see Levine and Wedeen 1985;Gergen et al. 1986;Scott and O'Farrell 1986;Akam 1987;Lawrence 1987;Ingham 1988) and the deleterious effects resulting from their ectopic expression (Struhl 1985;Ish-Horowicz and Pinchin 1987;Schneuwly et al. 1987) suggest that these genes must be expressed in particular domains in time and space to ensure the correct development of the embryo. Thus, understanding how these expression patterns are controlled is essential for understanding embryonic development. The expression patterns of many developmentally regulated genes appear to be controlled primarily at the level of transcription initiation, frequently in response to transcriptional control regions that act over very large distances (Hiromi et al. 1985;Peifer et al. 1987;Bienz et al. 1988; Boulet and Scott 1988;Howard et al. 1988;Goto et al. 1989;Harding et al. 1989}. In the case of the well-characterized ftz gene, a large 5' regulatory region of -6 kb was shown to contain three individual cis-acting regulatory elements: A zebra element directed lacZ fusion gene expression in seven stripes localized primarily in the mesoderm; a neurogenie element was required for expression in neural precursor cells; an upstream element enhanced expression directed by the zebra element, particularly in the ectoderm (for summary, see Fig. 1A; Hiromi et al. 1985). The upstream element, which was active in both orientations relative to and at various distances from the zebra element, alone generated seven ftz-like stripes when linked to a heterologous promoter. This striped expression pattern was ftz-dependent, suggesting that the ftz gene product might regulate the expression of its own gene, directly or indirectly. In addition, there was some indication that the products of some of the gap genes might regulate ftz gene expression via interactions with the upstream element (Hiromi and Gehring 1987).
We have focused on the ftz upstream element as a model to study the cisand trans-acting factors involved in generating discrete patterns of transcription during Drosophila embryogenesis. Here, we present the results of a deletion analysis of the ftz upstream element. We show that it contains several independent regulatory units, including at least two independent enhancers that direct expression of fusion genes in seven ftz-like stripes in a germ layer-restricted fashion. The enhancers are autoregulated independently by the wild-type ftz product and contain multiple binding sites for purified ftz homeo domain. These results suggest that ftz protein is one of the trans-acting factors that, in conjunction with germ layer-restricted transcription factors, acts directly to regulate positively transcription of the ftz gene.

Experimental approach
Previous studies showed that the ftz upstream element, defined by a 2574-bp KpnI-XbaI DNA fragment located between -3.5 and -6.1 kb upstream of the transcription start site (Fig. 1A), contained sequences necessary and sufficient to generate stripes in transformed embryos when linked to a heterologous hsp70 minimal promoter (Hiromi and Gehring 1987). We carried out a deletion analysis of the upstream element. Exonuclease III was used to generate sequential deletions from either end of the element, and restriction endonucleases were used to generate internal deletions (for details, see Experimental procedures). The resulting DNA fragments were assayed for their ability to activate a heterologous minimal promoter in the P-element-containing transformation vector HZ50PL (Hiromi and Gehring 1987). In this vector, the Escherichia coli lacZ gene is used as a reporter gene to monitor fusion gene expression.
Four types of fusion genes were analyzed following insertion into the Drosophila genome by P-element-mediated transformation Spradling and Rubin 1982) and establishment of a number of independent transformant lines: (1) 5' deletion constructs, (2) 3' deletion constructs, (3) internal fragments, and (4) fragments inserted into HZSOPL in reverse orientation (Fig. 1B). Fusion gene expression was assayed by using the chromogenic substrate X-gal to stain wholemount embryos. Because ftz-lacZ fusion gene expression was most readily detectable during germ-band elongation, expression patterns were evaluated at this developmental stage.

Expression of full-length upstream element fusion genes
The expression pattern of a fusion gene containing the -2.6-kb upstream element fused to the minimal hsp70 promoter has been reported previously (UPHZSOH; Hiromi and Gehring 1987;Dearolf et al. 1989). We have analyzed the expression pattern of this fusion gene in more detail. As shown in Figure 2, UPHZSOH was expressed in seven ftz-like stripes, which were readily detectable at the germ-band extension stage. These stripes extended all the way around the outer surface of the embryo and were strongest laterally. The stripes were of unequal intensity. As is the case for ftz RNA and protein, the seventh stripe was the strongest and broadest. In addition, stripes one and six were weaker than stripes two through five. Because f~-galactosidase is extremely stable in transformant embryos, stripes were still detectable in the even-numbered parasegrnents after the formation of Figure 1. ftz upstream element-lacZ fusion genes. (A) The structure of the ftz gene is shown. The indicated 10-kb KpnI fragment complemented the defects of ftz mutants (Hiromi et al. 1985). The transcribed region is indicated by an open box.
The TATA homology at -26 bp (Dearolf et al. 1989;L. Pick and W.J. Gehring, unpubl.), the putative translation start and stop codons, and the 150-bp intron (hatched box) are indicated Laughon and Scott 1984). The -6-kb of 5'-flanking sequence includes three cis-regulatory regions. The zebra element, which contains -600 bp upstream of the transcription start site, directs expression of flz-lacZ fusion genes in seven stripes localized primarily to the mesoderm. In addition, the zebra element appears to contain sequences that mediate interstripe repression to generate the ftz seven-stripe pattern at cellularization (Dearolf et al. 1989}. The neurogenic element, located within the next 2 kb is required for expression in the neural precursor cells in which [tz is normally expressed. The next 1 kb of sequence appears to be dispensable for ftz expression and function, and the remaining -2.6 kb contains the upstream element that enhances expression directed by the zebra element, particularly in the ectoderm (Hiromi and Gehring 1987) fusion genes used in this study are summarized. Fusion genes contain either the full-length or various portions of the ftz upstream element inserted upstream of a minimal promoter in the P-element transformation vector HZ50PL. The minimal promoter and trailer sequences were derived from the Drosophila hsp70 gene (Hiromi and Gehring 1987). The fusion genes are (1.) 5' deletion constructs , (2.) 3' deletion constructs, (3.) internal fragments, and (4.) upstream element fragments inserted in reverse orientation relative to the minimal promoter in HZSOPL. The number of transformant lines examined is indicated in parentheses to the right of each fusion gene. Each deletion end point is indicated, using the numbering system of Harrison and Travers (1988). Position 1 corresponds to the 5' end of the upstream element. Int B (4)
The precise localization of the stripes was investigated further. As reported previously (Dearolf et al. 1989), the seven stripes each extended to the ectodermal cells at the periphery of the embryo. However, in whole-mount embryos, the strong ectodermal staining might obscure the visualization of staining in the underlying meso-derm. Therefore, thin sections of stained UPHZ50H transformant embryos were analyzed. As shown in Figure 3A, B-galactosidase was expressed with roughly equal intensity in the ectoderm and mesoderm in all seven stripes. The internal stain bordered the granular yolk layer and was confined to sharp bands corresponding in width to those in the ectoderm. Thus, the -2.6-kb upstream element contains cis-acting DNA se- quences that direct fusion gene expression in seven stripes in both the ectoderm and the mesoderm.

Expression of 5' deletion constructs
The expression patterns of fusion genes containing the 5' deletion constructs were examined first. Sequential deletions of 289, 822, and 1110 bp from the 5' end of the upstream element (fusion genes 5 ' A -5 ' C ) did not detectably alter the pattern of fusion gene expression {Fig. 4A). Fusion genes 5'D and 5'E, which contain 1072 and 794 bp from the proximal portion of the upstream element, were also expressed in a striped pattern that resembled that directed by the full-length upstream element {Fig. 4B, C). However, these fusion genes were expressed more weakly than longer fusion genes. This weakness was particularly clear on the dorsal and ventral sides of the embryo where the stripes narrow, resulting in a triangular appearance of the stripes when viewed laterally (Fig. 4B, C); and in the first stripe,  which was often barely detectable in 5'D and 5'E transformant embryos (cf. stripe 1 in Fig. 4A with that in B and C). Although the stripes appeared to be narrower, they did extend to the periphery of the embryo and around the entire surface, demonstrating ectodermal localization (Fig. 4D). Examination of sections through these transformant embryos demonstrated that [3-galactosidase was expressed in both the mesoderm and the ectoderm in all seven stripes; the width of the stripes was roughly the same in both germ layers. The relative weakness of these shorter fusion genes was particularly evident in sectioned embryos because all of the stripes were not always readily detectable in each section examined (Fig. 3B). These fusion genes define a proximal element of 794 bp that directs fusion gene expression in seven ftz-like stripes in the mesoderm and ectoderm.

Further analysis of the proximal element
As shown in Figure 4E, a shorter fusion gene, 5'F, conraining the terminal 211 bp from the 3' end of the upstream element, did not express detectable levels of [3galactosidase. This suggested that cis-acting regulatory sequences in the upstream proximal element were located within the 583 bp between positions 1780 and 2363. To test whether or not this 583-bp region contained sequences sufficient for the generation of the ectodermal and mesodermal stripes, a fusion gene containing only this portion of the upstream element was analyzed. As shown in Figures 3C and 4, F and G, fusion gene IntB expression was detectable only in the mesoderm, again in seven ftz-like stripes (the term stripe describes the patch-like mesodermal staining). Thus, se-quences within each of the 211-and 583-bp portions of the proximal element are necessary but not sufficient to generate detectable levels of expression in the ectodermal stripes.
Finally, a fusion gene containing the terminal 531 bp of the upstream element was expressed in seven stripes in the ectoderm and in the mesoderm, as determined by examination of sectioned transformant embryos (data not shown). In addition, because this 531-bp fragment generated a ftz-like seven stripe pattem when inserted into HZ50PL in reverse orientation, the information in the upstream proximal element appears to function in an orientation-independent fashion (Fig. 4H, fusion gene Rev 2043).
We conclude that sequences in the proximal element direct specific expression when linked to a heterologous promoter in an orientation-independent fashion, indicating that the proximal element functions as an enhancer. This proximal enhancer appears to contain two separate regulatory units: a mesodermally restricted element, Prox A, and a second element, Prox B that, when combined with Prox A, directs high levels of expression in the ectodermal stripes (see Fig. 10A, below).

Expression of the 3' deletion constructs
Although the pattem of expression directed by the proximal enhancer was qualitatively similar to that directed by the full-length upstream element, the relative weakness of fusion genes 5'D, 5'E, and Rev 2043 suggested that the proximal element does not contain all of the regulatory information present in the upstream element. Therefore, the expression patterns of fbgalactosidase di-rected by the 3' deletion constructs were examined. Deletion of 527 or 910 bp from the 3' end of the upstream element (fusion genes 3'A and 3'B) resulted in drastically reduced B-galactosidase expression in the ectoderm (Fig. 5A). However, mesodermal staining in seven stripes in the regions in which the full-length UPHZSOH is expressed was retained. A shorter fusion gene, IntA, containing a 1171-bp internal fragment from the distal portion of the upstream element, harbors sequences sufficient to direct f3-galactosidase expression in these seven mesodermal stripes, defining a distal element in the upstream element (Fig. 5B, C). The mesodermal localization of B-galactosidase is clearly visualized i n wholemount embryos and in thin sections of IntA transfor-mant embryos (Fig. 3D). The pattern of expression of these distal element-containing fusion genes resembles that directed by the zebra element alone (Hiromi and Gehring 1987;Dearolf et al. 1989). No B-galactosidase activity was detected in transformant embryos carrying the shorter fusion genes 3'C through 3'G (data not shown). Thus, sequences necessary for expression directed by the distal regulatory element are located between positions 1117 and 1502 of the upstream element. In addition, we believe that sequences within this distal element enhance the expression of proximal enhancercontaining fusion genes because UPHZ50H, 5'A, 5'B, and 5'C, which still contain portions of the distal element, were expressed more strongly than 5'D, 5'E, or Rev 2043, which contain only the proximal enhancer. Figure 5. The distal portion of the upstream element contains a mesodermally restricted regulatory element. Expression of 3' deletion fusion genes was analyzed as described above. (A) 3'A transformant embryo, lateral view. The stripes are graded in intensity: Stripe seven is the strongest; stripe one is the weakest. This gradient-like expression resembles the expression of zebra element-containing fusion genes. In contrast to this, the mesodermal stripes directed by Prox A {see  Hiromi and Gehring (1987) showed previously that insertion of the -2.6-kb upstream element into HZ50PL in reverse orientation (fusion gene UPHZ50T) resulted in fusion gene expression in a single stripe in PS 4, which was readily detectable at the cellular blastoderm stage. Following this phase of expression, UPHZ50T was expressed in the seven stripes characteristic of the forward orientation construct, although strong staining in PS 4 persisted. In this fusion gene, the 5' end of the upstream element was placed -5 0 bp upstream of the transcription start site. When the upstream element was inserted in reverse orientation 154 or 2.5 kb farther away from the transcription start site, this PS 4-specific expression was no longer detected (Y. Hiromi and L. Pick, unpubl.). These results suggested that regulatory sequences at the 5' end of the upstream element might function in a distance-and orientation-dependent manner to direct PS 4-specific expression at the blastoderm stage. To test this possibility, several of the 3' deletion constructs were inserted into HZ50PL in reverse orientation.

Expression of deletion constructs in reverse orientation
As shown in Figure 6A, ~-galactosidase expression directed by fusion gene Rev 2023 was detectable in a strong band in PS 4. This fusion gene also contains the distal regulatory element, which directs f~-galactosidase expression in seven mesodermal stripes that are only weakly detectable at this early developmental stage. These mesodermal stripes increase in intensity as germband elongation proceeds, demonstrating that the upstream distal element functions in an orientation-independent fashion (Fig. 6B). Staining in PS 4 remains very strong at this late stage, overlaying mesodermal stripe 2 and almost obscuring visualization of the seven ftz-like mesodermal stripes.
Fusion genes Rev 3'C, 3'F, and 413, which contain 1117, 763, and 413 bp from the 5' end of the upstream element, do not contain information sufficient to direct fusion gene expression in seven ftz-like stripes. However, as shown for the shortest of these fusion genes in Figure 6C, [3-galactosidase was detectable in the single PS 4 stripe at cellular blastoderm. At later stages, expression in this stripe increased in intensity, but no ftz- overlays mesodermal stripe two. The seven ftz like stripes are clearly detectable at this stage. However, except for stripe seven, these stripes are much weaker than the PS 4-specific stripe.
[C) Rev 413 transformant embryo at the cellular blastoderm stage. A single stripe is detectable in the primordia of PS 4 (arrow). No other stripes are detectable during embryonic development {see also Fig. 7M). (D) Rev 5'B transformant embryo at germ-band extension. The seven-stripe staining pattern is indistinguishable from that of 5'B transformant embryos. Staining anterior to the cephalic furrow was observed for some transformant lines carrying various fusion genes, as noted previously for other ftz-lacZ fusion genes (Hiromi et al. 1985;Dearolf et al. 1989). Sequences in the transformation vector may be responsible for this sporadic expression.
like striped expression was detected (data not shown). As was the case for the forward-orientation fusion genes, expression in the PS 4 stripe became weaker when the distal enhancer was removed, suggesting that this expression can also be enhanced by the distal enhancer (of. Fig. 6A with 6C and 6B with 7M). Thus, sequences in the 413 bp at the 5' end of the upstream element are necessary and sufficient to direct fusion gene expression in a single stripe in PS 4. In addition, this PS 4-specific activator appears to be physically separable from the enhancers that generate the seven ftz-like stripes.
When the PS 4-specific activator and the terminal portion of the distal enhancer are deleted, the remaining portion of the upstream element directs fusion gene expression in seven stripes in an orientation-independent fashion (fusion gene Rev 5'B; Fig. 6D; cf. Fig. 4A). Thus, the pattern-forming elements in the upstream element that are involved in the generation of the seven ftz-like stripes act as enhancers, whereas the PS 4-specific activator functions in an orientation-dependent fashion.

Autoregulation of individual enhancers
The expression of the fusion gene containing the fulllength upstream element (UPHZSOH) was drastically reduced in ftz mutant embryos, indicating that wild-type ftz gene product is required for striped expression directed by the upstream element (Hiromi and Gehring 1987). In hairy (h) mutants, in which the ftz stripes are broadened such that each stripe is about twice its normal width (Carroll and Scott 1986;Howard and Ingham 1986), UPHZ50H expression was correspondingly broadened, presumably in response to the ectopic expression of the endogenous ftz gene (Hiromi and Gehring 1987;Fig. 7A-C). Is this autoregulation a property of the intact, full-length upstream element, that is, is autoregulation of the individual enhancers mediated through a single ftz-responsive region in the upstream element, or are the individual enhancers independently autoregulated?
To address this question, we have examined the ex-pression of fusion genes containing each individual upstream regulatory element in ftz and h mutant embryos, as shown in Figure 7. Expression of fusion genes IntA, 5'E, and IntB (Fig. 7D, G,J) containing the distal enhancer, the proximal enhancer, or Prox A, respectively, was reduced to undetectable levels in ftz mutant embryos (Fig. 7E, H,K). Furthermore, expression of each of these fusion genes was expanded in h mutant embryos (Fig. 7F, I,L). As is the case for the endogenous ftz protein (Lawrence and Johnston 1989), the stripes had a broadened and irregular appearance. Thus, each individual upstream regulatory element appears to be a target for autoregulation by the ftz gene product. Interestingly, the germ layer specificities of the individual fusion genes were retained even under conditions of ectopic ftz expression. For fusion genes IntA and IntB, which are expressed principally in the mesoderm, f~-galactosidase was detected in broadened stripes that retained their mesodermal localization (see Fig. 7F, L).
In contrast to this ftz-dependent seven-stripe expression, the pattern of expression of fusion gene Rev 413 was qualitatively unaffected in either ftz or h mutant embryos ( Fig. 7M-O). Thus, the PS 4-specific activator does not appear to be a target for ftz autoregulation.

Interaction of ftz homeo domain with the upstream element
The genetic experiments presented above demonstrate that wild-type ftz gene product is required for expression of fusion genes in seven stripes. To investigate whether this observed ftz dependence could reflect an indirect requirement for wild-type ftz activity or a direct interaction between ftz product and enhancer DNA, we have used a DNase I protection assay to systematically screen the upstream element for potential ftz protein-binding sites. Preliminary experiments performed with either full-length ftz protein or isolated ftz homeo domain demonstrated that in all cases, identical sites were protected (data not shown; for an example, see Fig. 8B, lanes indicate the single stripe in PS 4. The relative weakness of proximal enhancer-containing fusion genes 5'E and IntB may reflect a decreased responsiveness to ftz protein, as compared with fusion genes that also contain the distal enhancer. In both wild-type embryos and in h mutants, the seven ftz-like stripes are stronger and extend more posteriorly in UPHZ50H transformant embryos than in 5'E transformants and in IntA than in IntB transformants, respectively. Lawrence and his co-workers (1987) and Lawrence and Johnston 11989) observed that by the onset of gastrulation, ftz stripes are asymmetric and graded, the highest levels of expression being in the anterior region of each stripe. We suggest that the full-length upstream element may respond more readily to the lower levels of ftz protein that are present in the posterior portions of the even-numbered parasegments, whereas the proximal enhancer may respond only to the higher levels of protein present in the anterior portion of each parasegment.  (Fig. 8A,  lanes 1-6). As the amount of [tz homeo domain added to the reaction was increased, three lower affinity sites were detected (Fig. 8B). All four binding sites were protected in a similar manner by high concentrations of full-length ftz protein (Fig. 8B, lane 6). Using a DNA fragment containing part of the distal enhancer, several binding sites were also identified ( Fig. 8C and see below}. A summary of the results of the complete analysis is shown in Figure 9. Because different binding sites were protected against DNase I digestion at different concentrations of homeo domain, we have grouped the protected regions into three classes (high, medium, and low affinity; see Fig. 9). Within the 1790 bp analyzed in detail, 20 regions were found to be protected from DNase I digestion (Fig. 9). Most of these regions fall into the medium-affinity class (hatched boxes). Two binding sites . With respect to the regulatory elements identified in transgenic flies, we find that the 794-bp proximal enhancer (positions 1780-2574) contains one high-, seven medium-, and two low-affinity binding sites. Of these sites, three lie within the Prox B portion of the proximal enhancer. The region between these regulatory elements (positions 1502-1780) that does not appear to be required for lacZ fusion gene expression contains one medium-affinity-binding site, one extended medium-affinity site, and one low-affinity site.
The distal enhancer has been more difficult to analyze because the 5'-most 660 bp of this element is extremely AT rich (Harrison and Travers 1988), making DNase I protection analysis of this region virtually impossible.
This region was essentially covered by ftz homeo domain at concentrations used to identify medium-and low-affinity sites, and no individual footprints could be identified. The remaining 710 bp of the distal enhancer has been analyzed systematically, revealing one high-, four medium-, and one low-affinity binding site (Figs. 8C and 9).

The upstream element contains multiple regulatory tmits
The ftz upstream element contains sequences that mediate an autoregulatory feedback loop, which maintains ftz expression in seven stripes in the developing Drosophila embryo. We carried out a deletion analysis of this -2.6-kb element and have shown that it contains a number of separable regulatory elements that are summarized in Figure 10A. A distal regulatory element directs fusion gene expression primarily in the mesoderm and appears to enhance expression of proximal elementcontaining fusion genes. Sequences necessary for expres-  . Several other short (6-8 bp) and generally A-or T-rich regions were not cut by DNase I at a detectable level and could therefore not be assayed for binding. Some ftz-lacZ fusion gene deletion end points are indicated.

AT C G AAG G T CAC C C a~ AAATT CAT C CT O T CTG ~ C T TT C T C TT CA~ T C W AT AG TGAATTT TA6 AJKAC T~'K'T'T'A'4"TT'CATTTJ~T C TAG A 2574 rllll/lll/llllll/]
sion directed by this element were localized to a 385-bp region (positions 1117-15021. A proximal element directs fusion gene expression in seven stripes in the mesoderm and ectoderm. A 794-bp fragment containing the proximal element was further dissected into two elements: Prox A directs expression in seven mesodermal stripes, whereas fusion genes containing only Prox B were not detectably expressed during embryogenesis. However, Prox B is necessary in conjunction with Prox A to direct expression detectable in the ectoderm. Thus, the proximal element identified in this study contains at least two regulatory regions that appear to interact. The resolution of the analysis presented here does not allow us to distinguish between the existence of two completely independent sequence elements--one utilized in the mesoderm and one in the ectoderm--and a more complex situation in which a shared regulatory element (now localized to Prox A) is utilized in both germ layers in combination with regionally restricted regulatory elements that are separable.
Both the distal and proximal elements identified in this study function in an orientation-independent fashion when linked to a heterologous promoter {fusion genes Rev 5'B, Rev 2043, and Rev 2023; data not shown). In addition, the deletion analysis effectively tests the distance dependence of these elements. The 3' deletion series and construct IntA place the distal element progressively closer to the promoter without having any apparent effect on expression. In fusion gene IntB, Prox A is moved -200 bp closer to the promoter without an apparent effect on mesodermal expression, and the distance effect for ectoderm expression of the proximal element is tested by reverse-orientation fusion genes Rev 5'B and Rev 2043. Thus, within the resolution of this analysis, the spacing between the individual regulatory elements and the transcription start site have no effect on expression. Furthermore, each element directs a spatially and temporally restricted pattern of gene expression when linked to a heterologous promoter. These observations indicate that both distal and proximal ele- A model for ftz autoregulation. After general activation of ftz transcription in the embryo, stripes are formed via repression of active transcription in the interstripe regions (Weir and Kornberg 1985;Edgar et al. 1986). Although the initiation of transcription may be mediated by cooperative interactions between the zebra element and the upstream element, interstripe repression appears to be directed by sets of sequences in the zebra element that have been identified by Dearolf et al. (1989). When sufficient levels of ftz protein (f) are synthesized in the stripes, the protein becomes available to interact with sequences in the upstream element, initiating an autoregulatory feedback loop that maintains and increases levels of transcription in the stripes, ftz protein and ectoderm-or mesoderm-specific transcription factors, e and m, respectively, interact with sequences in the proximal and distal enhancers, as indicated. In neural precursor cells, one or more unidentified transcription factor(s) n interacts with sequences in the neurogenic element to direct specific transcription. Although ftz protein is synthesized, it cannot stimulate transcription via its potential binding sites in the upstream element enhancers in the absence of e and m. Autoregulation serves to maintain ftz transcription in the stripes and may also be involved in generating the observed anterior-posterior concentration gradient of ftz within each stripe Lawrence and Johnston 1989). ments function as developmentally regulated enhancers in Drosophila, which have the properties attributed to cell type-specific enhancers [Atchison 1988}.
The identification of two independent regulatory elements that are active in the mesoderm suggests that one of these elements might be redundant in the context of the endogenous ftz gene. However, the observation that fusion genes lacking the distal enhancer are expressed more weakly in both germ layers than those containing the full-length upstream element [ Fig. 4) suggests that the distal enhancer may also be required to increase levels of expression in the ectoderm. In addition, both the significance and complexity of the proximal and distal enhancers are underscored by the observation that the region necessary for activity of the distal enhancer corresponds to the portion of ftz 5'-flanking sequence that is most highly conserved between D. melanogaster and D. hydei and that long stretches of sequences in the proximal enhancer are exactly conserved in D. melanogaster, D. hydei, and D. virilis (Maier 1989; P. Baumgartner and W.J. Gehring, unpubl.).

PS 4-specific activator is functionally separable from the upstream element enhancers
As shown in Figure 6, the 5' 413 bp of the upstream element contains sequences that direct fusion gene expression in a single stripe in the primordia of PS 4 in an orientation-dependent fashion IPS 4 activator, Fig. 10A). We suggest that this PS 4-specific regulatory element is not involved in the regulation of the endogenous ftz pattern, for the following reasons. First, although the single stripe of [3-galactosidase activity overlaps with the region of the second ftz stripe, the stripe is detectable earlier and at higher levels than the upstream element-directed seven ftz-like stripes. Thus, there is a regional overlap but neither a precise temporal nor quantitative correspondence between the patterns. Second, the genetic regulation of this element differs from that of the upstream element enhancers: The PS 4-specific activator is not a target for autoregulation {Fig. 7) and may be more directly regulated by gap genes {Hiromi and Gehring 1987; Y. Hiromi, pers. comm.). Finally, the PS 4-specific activator appears to be physically separate from the upstream element enhancers. Interestingly, in a parallel study, a nuclear scaffold attachment region {SAR) was identified in the ftz upstream element just 3' of the PS 4-specific activator, in a region that is suggested to correspond to the 5' end of the ftz gene (B. Amati, L. Pick, T. LaRoche, and S. Gasser, in prep.).
If the PS 4-specific activator is not part of the ftz gene, what, then, is it.~ As pointed out previously, the PS 4specific expression resembles that of the Antennapedia gene (Antp), although it is not the result of trans-activation by the Antp product (Hiromi and Gehring 1987]. The ftz gene is located in the Antp complex (ANT-C), between the homeotic genes Sex combs reduced (Scr) and Antp (Scott et al. 1983;Kuroiwa et al. 1985). In D. melanogaster, Scr and ftz are transcribed in opposite orientations (Le Motte et al. 1989). In D. hydei the ftz gene, including the upstream element, is highly conserved, and the orientation of ftz with respect to Scr is reversed, that is, the 3' end of ftz lies closer to Scr, presumably placing the 5' end of ftz closer to Antp (D. Maier, pers. comm). If the chromosomal configuration in D. hydei represents the ancestral organization of the ANT-C, then an inversion would have taken place since the divergence of D. melanogaster and D. hydei. An intriguing possibility is that part of an Antp regulatory region that directed Antp expression in PS 4 was included in the inversion. These Antp regulatory sequences would then remain upstream of the ftz gene in D. melanogaster as an evolutionary relic. Such an inversion event would also explain the curious observation that Scr mutations are located on both sides of the ftz gene (Scott et al. 1983;Seeger and Kaufman 1987) and predicts that these mutations would map within the breakpoint of the inversion such that the affected Scr sequences would be located between the ftz and Scr transcription units in D.

Purified ftz homeo domain interacts with the upstream element in vitro
The expression of enhancer-lacZ fusion genes in seven ftz-like stripes is decreased to undetectable levels in ftz mutants, indicating that wild-type ftz product is necessary for fusion gene expression {see Fig. 7 and below); however, these genetic experiments do not address questions regarding the mechanism of ftz action in mediating this effect. The ftz protein contains a homeo domain (McGinnis et al. 1984;Scott and Weiner 1984), binds to specific DNA sequences in vitro [Desplan et al. 1988;Laughon et al. 1988;Miiller et al. 1988), and can stimulate transcription in yeast (Fitzpatrick and Ingles 19891 and in cell culture {Jaynes and O'Farrell 1988;Han et al. 1989;Winslow et al. 1989). These properties of the protein suggest that it could act directly to regulate the transcription of its own gene.
As a first step toward testing this model of direct autoregulation, we asked whether purified ftz protein or ftz homeo domain can interact with enhancer DNA sequences in vitro. We have found that ftz homeo domain interacts with a large number of sites in the upstream Independent ftz enhancers are autoregulated element (Figs. 8 and 9). Both the distal and the proximal enhancers contain a single high-affinity binding site, as well as additional medium-and low-affinity sites. Comparison of all the footprinted regions does not allow us to identify a consensus sequence for all three classes of binding sites. Both of the high-affinity sites contain a GCAATTA sequence element, suggesting that this motif might be partly responsible for the high affinity of these sites. Although some of the medium-affinity sites do contain variations of the GCAATTA motif, others show very little sequence similarity to the high-affinity sites. In the case of the homeo domain-containing Oct-1 protein (also called OTF-1, OBP100, or NFIII; see Sturm and Herr 1988), very dissimilar DNA sequence elements are recognized in vitro, with only slightly different affinities (Baumruker et al. 1988}. The identification of a large number of ftz homeo domain-binding sites in the upstream enhancers was somewhat surprising. Although it is possible that the clustering of binding sites throughout the upstream element has some functional significance, the relatively low selectivity of homeo domain-containing proteins for their DNA target sites suggests that a large number of potential binding sites may be present throughout the entire genome (see also Desplan et al. 1985). We suggest that in vitro DNA-binding studies such as the one presented here identify the total range of sites with which a given homeo domain-containing protein can potentially interact. Mutational analysis of various binding sites should now allow us to ask which of these binding sites are required for ftz responsiveness and to determine whether or not ftz protein interacts directly with these DNA sequences in vivo.

Model for germ layer-restricted upstream element autoregulation
Expression of fusion genes containing either the fulllength upstream element or the independent ftz regulatory elements is dependent on the presence of a wildtype ftz gene in transformant embryos (Fig. 7;Hiromi and Gehring 1987). That this observed autoregulation is a property of the individual enhancers rather than being mediated by a single region within an intact upstream element supports the conclusion from the deletion analysis that the enhancers are independent functional units. Although enhancer-fusion gene expression is decreased to undetectable levels in ftz mutant embryos, it is broadened in h mutant embryos in which the endogenous ftz RNA and protein distribution is expanded. However, even when ftz product is ectopically expressed in h mutants, the germ layer restriction of the regulatory elements appears to be retained. This observation, along with the restricted expression of individual fusion genes, indicates that sequence elements that share the property of being ftz responsive are differentially utilized in the ectoderm and mesoderm. In addition, these DNA sequence elements are apparently unable to respond to ftz protein present in specific neural precursor cells and in the hindgut (Caroll and Scott 1986;Krause et al. 1988), where upstream element-containing fusion genes are Cold Spring Harbor Laboratory Press on July 20, 2018 -Published by genesdev.cshlp.org Downloaded from not detectably expressed (Hiromi and Gehring 1987;L. Pick et al., unpubl.). These observations suggest that although ftz product is required for enhancer activity, it alone is not sufficient to direct enhancer-fusion gene expression. We propose that each enhancer requires the activity of at least one other factor in addition to ftz that determines its specificity.
A simple formulation of this model, as summarized in Figure 10B, is that transcriptional specificity is achieved by the interactions between ftz protein and germ layerspecific factors, which, together, activate transcription via interactions with specific DNA sequences in the distal and proximal enhancers. In ectodermal cells and/or precursors, ftz protein and an ectoderm-specific transcription factor (e) interact with sequences in the proximal enhancer; in mesodermal cells and/or precursors, a mesoderm-specific transcription factor (m} and ftz protein interact with sites in the distal and proximal enhancers, ftz protein may interact with only the single high-affinity binding site in each of the enhancers (in which case mesodermal and ectodermal cells would utilize the same ftz-binding site in the proximal enhancer) or it may interact with several sites, including the medium-or low-affinity binding sites in an additive fashion. However, preliminary results indicate that the single high-affinity site in the proximal enhancer can be deleted without any significant effect on expression (data not shown). In neural and hindgut precursor cells, neither e nor m, nor an equivalent factor allows [tz protein to bind to and/or to activate transcription from the enhancers.
We postulate further that the putative germ layer-specific factors are present throughout each germ layer because ectopic expression of enhancer-fusion genes appears to be limited by the domains of endogenous ftz expression (see Fig. 7). Although we have proposed that the factors limiting the specificity of ftz-dependent expression are other DNA-binding transcription factors, our data are equally consistent with them being soluble factors that alter the specificity of ftz protein-DNA binding or its ability to activate transcription, either by protein-protein interactions with or post-translational modifications of the ftz protein. In the latter case, specific modifications might allow ftz protein to activate transcription via different sites in the upstream element in the mesoderm and in the ectoderm and could inhibit activation in the developing nervous system and hindgut.
The model presented here in which different combinations of trans-acting factors interact to determine transcriptional specificity is consistent with models derived from studies of viral enhancers (for reviews, see introductory section). In the case of the ftz enhancers, the combinatorial interaction of the putative enhancerbinding factors with ftz protein would ensure a regionalspecific restriction of ftz gene expression. In addition, if the putative enhancer-binding factors were necessary to increase the selectivity of ftz DNA binding, it would explain why such a large number of DNA-binding sites have been found by us and others in in vitro experiments using single homeo domain-containing proteins. Such a model would also help to explain how homeo domaincontaining proteins that are expressed in overlapping spatial domains and that bind to overlapping sets of DNA sequences themselves (see Desplan et al. 1988;Hoey and Levine 1988;Laughton et al. 1988;Mfiller et al. 1988), could regulate specific gene expression in a controlled fashion during development. Finally, this model is also supported by experiments carried out with homeo domain-containing proteins in yeast. Mata2 protein has been shown to bind DNA via cooperative interactions with a general transcription factor (Keheler et al. 1988), and Matal protein can modulate the specificity of Mata2 DNA binding, thereby changing its gene regulatory function . The challenge for the future will be to identify the transcription factors that interact with ftz protein.

Construction of lacZ fusion genes
Sequential deletions from either the 5' or 3' end of the ftz upstream element were generated as follows. An XbaI fragment derived from plasmid pKAS-1 (L. Pick, unpubl.), containing the 2574-bp upstream element (Hiromi and Gehring 1987;Harrison and Travers 1988), was inserted in either orientation into the Deletions in both orientations were then generated by BstXI and NotI digestion of pBKmOX-H and pBKmOX-T, followed by exonuclease III/mung bean nuclease treatment, as recommended by the manufacturer (Stratagene Cloning Systems). Protruding ends were filled in using DNA polymerase I large fragment, and the plasmids were religated. Deletion end points were determined by dideoxynucleotide sequencing (Sanger et al. 1977} or by chemical cleavage of isolated DNA fragments {Maxam and Gilbert 1977). Fragments of interest were subsequently released from the vector by digestion with KpnI, gelisolated, and inserted in either orientation into the unique KpnI site of the Carnegie 20-derived vector HZ50PL (Hiromi and Gehrmg 1987). The region cloned into HZ50PL included -80 bp of polylinker sequence between the ftz DNA and the hsp70 basal promoter. Examination of the expression patterns of [3-galactosidase in transformant embryos, carrying the full-length upstream element and several of the deletion derivatives, revealed that expression was significantly weaker than expected (-10-20 times when compared to UPHZ50HI. The full-length upstream element fragment was then reisotated from this fusion gene by use of XbaI and removal of the Bluescript polylinker sequences. Insertion of this fragment into the unique XbaI site of HZSOPL restored quantitative levels of expression. Thus, the use of the large Bluescript polylinker appears to have led to suppression of fusion gene expression. We have not investigated the causes of this phenomenon further; however, in our experience, low levels of 13-galactosidase activity may be Independent ritz enhancers are autoregulated reduced to undetectable levels, leading to misinterpretation of a given pattern (data not shown). We note that repression of expression of fusion genes by vector sequences has also been observed for mouse transgenes (Chada et al. 1985).
To avoid interference by Bluescript polylinker sequences, all 5' deletion fragments were excised from HZSOPL with XbaI (made possible by the 3' XbaI site remaining from the initial cloning into pBKmO and the XbaI site lying 5' to the KpnI site in HZ50PL) and recloned into the XbaI site of HZSOPL. Again, all fusion genes containing XbaI fragments were expressed at higher levels than those containing the corresponding KpnI fragments. The 5' deletion constructs presented here include only those examined after recloning into the XbaI site. Although the 3' deletion constructs did not contain large amounts of the Bluescript polylinker, some new 3' deletion fusion genes were created by using restriction endonucleases. Fragments were excised from the parental plasmid pBKmOX-H with XbaI and an appropriate restriction endonuclease. The XbaI end of each fragment was ligated into the XbaI site of HZ50PL, ends were filled in with the large fragment of DNA polymerase I, and the plasmids were then recircularized with T4 DNA ligase. No apparent difference in expression was seen between 3' deletion fusion genes inserted into the KpnI and XbaI sites.
For plasmids IntA and 5'D, XbaI fragments containing upstream element sequences were isolated from the clones pryFGHR1 or pryFGHRX (kindly provided by Dr. Y. Hiromi) and inserted directly into the XbaI site of HZSOPL. Plasmid IntB was generated by EcoRV digestion and 5'F by HpaI digestion of an isolated XbaI fragment from the plasmid 5'E. Fragments were inserted into the XbaI site of HZ50PL, as described above. All cloning steps were carried out according to standard procedures (Maniatis et al. 1982).

Germ-line transformation and analysis of expression patterns
P-element-mediated transformation and establishment of balanced or homozygous transformant stocks and detection of [3galactosidase activity using the chromogenic substrate 5bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-gal) was as described previously (Hiromi and Gehring 1987). Expression patterns were examined in multiple independent lines for each construct (see Fig. 1). Striped patterns of expression presented here were seen in all lines examined for any given construct. We observed considerable variation in the levels of [3-galactosidase activity between different independent lines transformed with the same construct, although the patterns were qualitatively the same in all lines. As has been seen for other ftz-lacZ fusion genes (Hiromi et al. 1985;Dearolf et al. 1989), staining anterior to the cephalic furrow was observed in some cases. For examination of thin sections, staged embryos were stained for [3-galactosidase activity according to Bellen et al. (1989) or Hiromi and Gehring 11987) and embedded in Tissue-Tek O.C.T. compound (Miles Scientific). Cryostat sections (13 ~m) were prepared as described by .

Analysis of expression in mutant embryos
The mutant alleles used were ftz 9H34 (Jiirgens et al. 1984) or h 5H07 or h IL 79K {Ingham et al. 1985. Mutants {linked to the third chromosome) were crossed to transformant flies carrying a ftz enhancer transgene on the second chromosome. Heterozygous flies for both the mutation and the fusion gene were crossed to each other, and embryos were stained for B-galactosidase activity. Homozygous mutant embryos were identified by characteristic morphological changes observed at later stages of embryogenesis or by double staining with antibodies against engrailed protein {Patel et al. 1989). The analysis of wild-type and h-mutant embryos revealed that the anterior boundaries of even-numbered engrailed stripes correspond to the anterior boundaries of fusion gene stripes (data not shown). As the anterior border of the mature ftz stripes also coincides with the anterior border of engrailed stripes (Lawrence and Johnston 1989), fusion gene expression seems to be confined (in both wild-type and h-mutant embryos) to the region where ftz protein is expressed.

DNase I protection assays
DNA fragments used for footprint analysis were subcloned into a Bluescript M13 + vector, digested with an appropriate restriction enzyme, and either 5'-end-labeled with [~-32P]ATP and T4 polynucleotide kinase or 3'-end-labeled with [~-a2P]dATP and the large fragment of DNA polymerase I (Maniatis et al. 1982). Following cleavage with a second restriction enzyme, DNA fragments were purified after excision from native polyacrylamide gels. Binding reactions (100 ~1) containing 20 mM Tris-HC1 (pH 7.6), 75 mM KC1, 5 I~g BSA, 1 mM DTT, 10% glycerol, a2P-labeled DNA, and varying amounts of purified ftz homeo domain or full-length ftz protein were incubated for 60 min on ice. CaC12, MgC12, and poly[d(I-C}] were added to final concentrations of 2.5 m~, 5 mM, and 10 ~g/ml, respectively, and reaction mixtures were incubated with DNase I (0.5-1.0 ~g/ml) for 8 min on ice. Reactions were stopped by the addition of 100 ~1 of 10 mM Tris-HC1 (pH 7.6), containing 0.4% SDS and 20 mM EDTA. Reaction mixtures were heated at 65~ for 15 rain, and the samples were extracted with equal volumes of phenol/chloroform (1 : 1) and chloroform. Following ethanol precipitation, DNase I cleavage products were separated on 8.3 M urea/6% polyacrylamide gels with G + A reaction cleavage reactions as marker (Maxam and Gilbert 1977]. The full-length ftz protein used in the footprint assays was synthesized in E. coli and purified to near homogeneity (Krause et al. 1988). The ftz homeo domain was synthesized in E. coli BL21 (DE3) lysogen (Studier and Moffat 19861 and purified to homogeneity (M. MUller and W.J. Gehring, unpubl.). Protein concentrations were determined by using the Bio-Rad dye reagent according to the method of Bradford (1976). The purified ftz homeo domain peptide contains two additional amino acids at the amino terminus as a result of the cloning and overexpression procedure and is extended by eight naturally occurring amino acids at the carboxyl terminus, resulting in a 70-aminoacid peptide.