BIOLOGICAL OVERVIEW

The empty spiracles gene is necessary for proper head formation. Organs in the head requiring EMS include antennal sense organs and both dorso-medial and dorso-lateral papillae of the antennomaxillary complex. In these organs ems works in combination with orthodenticle and buttonhead (Cohen, 1990). ems is also involved in brain morphogenesis. The brain is divided into three segments (neuromeres), and ems is required for the development of second and third neuromeres (Hirth, 1995).

To gain further insights into homeotic gene action during CNS development, the role of the homeotic genes was characterized in embryonic brain development of Drosophila. Neuroanatomical techniques were used to map the entire anteroposterior order of homeotic gene expression in the Drosophila CNS. This order is virtually identical in the CNS of Drosophila and mammals. All five genes of the Antennapedia Complex are expressed in specific domains of the developing brain. The labial gene has the smallest spatial expression domain; it is only expressed in the posterior part of the tritocerebral anlage. This contrasts with previous reports that lab is expressed throughout the tritocerebral (intercalary) neuromere. Also studied was the expression of the empty spiracles gene, which in the wild-type brain is expressed in a large domain anterior to lab . In lab loss-of-function mutants, the ems gene is expressed ectopically in the tritocerebral domain in which lab is normally expressed; this ectopic ems expression occurs with 100% penetrance and ranges from 5-7 cells per hemisegment. The expression of pb disappears in the deutocerebrum and tritocerebrum of lab loss-of-function mutants but not in more posterior neuromeres. In contrast, the expression patterns of Dfd and Scr remain unaltered. Thus, in the tritocerebral domain in which lab is normally expressed, two changes in regulatory gene expression occur: activation of ems and inactivation of pb (Hirth, 1998).

Unlike the trunk segments, the anterior head segments of Drosophila are formed in the absence of pair-rule and HOX-cluster gene expression, by the activities of the gap-like genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd). The products of these genes are transcription factors but only Ems has a HOX-like homeodomain. Indeed, ems can confer identity to trunk segments when other HOX-cluster gene activities are absent. In trunk segments of wild-type embryos, however, ems activity is prevented by phenotypic suppression, in which more posterior HOX-cluster genes inactivate the more anterior without affecting transcription or translation. ems is suppressed by all other Hox-cluster genes and so is placed at the bottom of their hierarchy. Misexpression of Ems in the head transforms segment identity in a btd-dependent manner; misexpression of Btd in the trunk causes ems-dependent structures to develop; and Ems and Btd physically interact in vitro. The data indicate that this interaction may allow ems to escape from the bottom of the HOX-cluster gene hierarchy and cause a dominant switch of homeotic prevalence in the anterior-posterior direction (Schock, 2000).

Combined activities of otd, ems and btd generate and specify Drosophila head segments in the absence of pair-rule and homeotic gene activities. btd alone is required for development of the mandibular segment, btd plus ems for the intercalary segment, and btd, ems plus otd for the antennal segment. Misexpression of btd or otd in the prospective head region fails to cause homeotic transformations showing that neither of the two genes carries the proposed homeotic function in head segmentation. To explore the untested homeotic role of ems and to address a possible cooperation with btd, the ems protein (Ems) was misexpressed in the btd domain of otherwise wild-type embryos. Ems expression is achieved by an ems complementary DNA transgene under control of the btd cis-acting promoter region (Schock, 2000).

Ems expression in the btd domain of wild-type embryos causes a second intercalary-like engrailed expression domain in place of the mandibular segment. Furthermore, these embryos develop a duplicate set of intercalary cuticle elements in place of mandibular structures. The same results are observed in response to Ems expression in the anterior third of blastoderm embryos mediated by a Gal4/UAS system. However, misexpression of Otd in the btd domain has no effect on head formation. Thus, among the three head gap-like genes only ems carries both early segmentation and homeotic selector gene function. However, ems misexpression in several head segments causes only the mandibular into intercalary segment transformation. Since the intercalary segment also depends on btd, and ems does not have transforming activity in btd mutant embryos, it is concluded that ems activity is able to specify head segment identity only when acting in concert with btd. Notably, the direction of the ems-dependent transformation is from a posterior into a more anterior segment identity. This is the opposite of the direction of transformation in response to ectopically expressed HOX-cluster genes in the trunk (Schock, 2000).

Btd is a transcriptional activator with in vitro properties that are indistinguishable from those of human Sp1. However, whereas transgene-derived btd activity causes a full rescue of all head segments that are deleted in btd mutant embryos, Sp1 activity can rescue only mandibular segment development. Similarly, expression of the fusion protein VP16^Btdzf , which contains the VP16 transactivator region fused to Btd's zinc finger domain, only rescues mandibular development. Conversely, expression of Btd^Sp1zf, in which the Btd zinc finger domain is replaced by the zinc finger domain of human Sp1, mediates a complete rescue of btd mutant embryos. Thus, Btd must contain specific features outside its zinc finger domain that are needed for intercalary segment development (Schock, 2000).

To identify the Btd region necessary for the ems-dependent intercalary development, it was asked whether Btd can physically interact with Ems in vitro and which parts of Btd are involved. Btd is able to bind [ ³⁵S]methionine-labelled Ems in vitro. This interaction involves the amino-terminal region of the protein. A specific domain could not be found because several parts of the N-terminal region interact with Ems, excluding the zinc finger domain. Sp1, which has the same biochemical features as Btd, does not interact with Ems. The yeast two-hybrid system also shows a direct interaction between Ems and Btd's N-terminal region that does not involve the homeodomain of Ems (Schock, 2000).

The next question to be examined was whether the Btd mutants that interact with Ems are sufficient to allow homeotic Ems activity in vivo. Transgene-dependent rescue experiments were performed in which Btd deletion mutants were expressed in btd mutant embryos. N-Btd, a protein composed of the combined DNA-binding and N-terminal region, rescues all head segments of btd mutant embryos, whereas C-Btd, a protein lacking the N terminus, causes mandibular segment development in all btd mutant embryos but restores only partial intercalary development in rare cases. Furthermore, Btd variants that lack various portions of the N terminus are able to restore intercalary segment development fully, indicating that Btd-dependent intercalary development depends on the parts of its N-terminal region that also allow physical interaction with Ems (Schock, 2000).

To investigate whether the Btd and Ems interaction causes homeotic transformations in other parts of the embryo, use was made of the observation that ems is also expressed in the trunk region of the embryo from stage 9 onward. Lack of ems activity causes no alteration in trunk segments except that the 'filzkörper', a morphologically distinct structure of the last abdominal segment, fails to develop. In the absence of all HOX-cluster gene activities, however, the trunk segments alter identity and develop ems-dependent sclerotic head plates. Their formation can be phenotypically suppressed by the co-expression of any gene of the HOX-cluster including labial, Deformed or Sex combs reduced, all of which are normally expressed and required in the cephalic region of the embryo. Therefore, ems may be a disconnected member of the ancient HOX-cluster, acting at the bottom of the functional HOX-cluster hierarchy (Schock, 2000).

The ems-dependent homeotic transformation in the head region may be because of a requirement of Ems to cooperate with Btd to escape from phenotypic suppression. To test this in the trunk region, Btd was expressed from a heat-shock-inducible transgene at various early embryonic stages. Ectopic Btd expression up to and during blastoderm stage has no effect on trunk segmentation. However, Btd expression during stage 7-9 of embryogenesis, when ems is initially expressed in the prospective trunk region, causes a range of phenotypes. These included the development of sclerotic head plates reminiscent of the ems-dependent structures observed in embryos that lack the HOX-cluster genes (Schock, 2000).

Most Btd misexpressing embryos develop fusions of trunk segments to varying degrees (149 cases out of 218 heat-shocked embryos examined). However, such segment fusions are also observed at a similar frequency in Btd-expressing homozygous ems mutant embryos. Thus, ectopic Btd activity causes metamerization defects independent of ems activity. However, Btd-expressing wild-type embryos also develop sclerotic plates. In a few cases (11 embryos), segmentation is completely abolished, and sclerotic plates are found. Sclerotic plates are never observed in embryos lacking ems activity. Thus, their formation in the trunk region of embryos depends on combined Btd and Ems activities. ems escapes phenotypic suppression by the HOX-cluster genes without Btd affecting the HOX-cluster gene transcription or translation (Schock, 2000).

The results provide evidence that combined Btd and Ems activities specify the intercalary head segment identity. The gnatho-cephalic homeotic genes labial and Deformed are normally expressed in intercalary and mandibular head segments, respectively, and their products cause phenotypic suppression of Ems. Since the intercalary segment development is dependent on the regions of Btd that can associate with Ems in vitro, it is probable that the Ems-Btd interaction releases the phenotypic suppression. Ems can also overcome phenotypic suppression by the HOX-cluster genes in the trunk when co-expressed with ectopic Btd. It is proposed that the interaction with Btd allows Ems to relocate from the bottom to the top of the HOX-cluster gene hierarchy. Ems then functions in an anterior-prevalent manner, that is, in the direction opposite that of other HOX-cluster genes. The unique homeotic feature of ems among the head gap-like genes is therefore consistent with the proposed origin of the gene from the HOX-cluster. By adopting Btd as a partner, Ems could escape phenotypic suppression by gnatho-cephalic HOX gene activities and specify the intercalary head segment identity (Schock, 2000).

Just as ems collaborates with genes in the anterior, so it does in the posterior. Here ems is coexpressed with Abdominal-B. Abdominal B has a direct effect on activation of ems in the posterior spiracular anlagen (filzkörper). EMS alone is not sufficient for filzkörper induction, since overexpression of ems does not compensate for Abd-B deficiency (Jones, 1993).

GENE STRUCTURE

Bases in 5' UTR -299

Exons - two

Bases in 3' UTR - 673

PROTEIN STRUCTURE

Amino Acids - 497

Structural Domains

In addition to a homeodomain, the N-terminal portion of the predicted protein sequence is very proline-rich, whereas the C-terminus has an acidic profile consistent with the role of EMS as a transcriptional activator (Dalton, 1989 and Walldorf, 1992).

date revised: 30 July 2000 

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