Gene name - empty spiracles
Cytological map position - 88A1-2
Function - transcription factor
Keywords - gap gene
Symbol - ems
FlyBase ID: FBgn0000576
Genetic map position - 3-54.0
Classification - homeodomain
Cellular location - nuclear
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 VP16Btdzf , which contains the VP16 transactivator region fused to Btd's zinc finger domain, only rescues mandibular development. Conversely, expression of BtdSp1zf, 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).
In both insects and mammals, second-order olfactory neurons receive input from olfactory receptor neurons and relay olfactory input to higher brain centers. In Drosophila, the wiring specificity of these olfactory projection neurons (PNs) is predetermined by their lineage identity and birth order. However, the genetic programs that control this wiring specificity are not well understood. The cephalic gap gene empty spiracles (ems) encodes a homeodomain transcription factor required for embryonic development of the antennal brain neuromere. ems is expressed postembryonically in the progenitors of the two major olfactory PN lineages. Moreover, ems has cell lineage-specific functions in postembryonic PN development. Thus, in the lateral PN lineage, transient ems expression is essential for development of the correct number of PNs; in ems mutants, the number of PNs in the lineage is dramatically reduced by apoptosis. By contrast, in the anterodorsal PN lineage, transient ems expression is necessary for precise targeting of PN dendrites to appropriate glomeruli; in ems mutants, these PNs fail to innervate correct glomeruli, innervate inappropriate glomeruli, or mistarget dendrites to other brain regions. Furthermore, in the anterodorsal PN lineage, ems controls the expression of the POU-domain transcription factor Acj6 in approximately half of the cells and, in at least one glomerulus, ems function in dendritic targeting is mediated through Acj6. The finding that Drosophila ems, like its murine homologs Emx1/2, is required for the formation of olfactory circuitry implies that conserved genetic programs control olfactory system development in insects and mammals (Lichtneckert, 2008).
The two major groups of olfactory projection neurons, anterodorsal PNs (adPNs) and lateral PNs (lPNs), derive from two brain neuroblasts that generate most of their progeny postembryonically. ems is expressed in these neuroblasts as well as in their GMCs and a subset of their postmitotic neuronal progeny during postembryonic development. Expression data support a model in which ems is persistently expressed in the postembryonic progenitors (neuroblasts and GMCs) of adPN and lPN lineages, remains transiently expressed in their neuronal progeny, and subsequently disappears in these neurons during their differentiation phase and in the adult. This is corroborated by the observation that ems is never coexpressed with the Gal-GH146 driver, which only begins to be expressed in PNs once they differentiate and initiate process outgrowth. The experimental findings that support this model have implications for understanding of ems action in olfactory system development (Lichtneckert, 2008).
During embryogenesis, ems is expressed in a subset of the neuroblasts that give rise to the deutocerebral neuromere. This raises the possibility that ems might also be involved in the development of the small group of olfactory PNs generated in the embryo. Although an unambiguous link between embryonic ems-expressing neuroblasts and the two postembryonic ems-expressing neuroblasts that generate the adPN and lPN lineages is lacking, it is conceivable that ems is required for the development of the entire complement of adPNs and lPNs, embryonic and postembryonic. Indeed, as ems is not only expressed in the embryonic neuroectoderm from which the deutocerebrum derives, but also in the embryonic antennal segment from which the antennal sense organs derive, ems might play a key role in the development of both the peripheral and central olfactory systems (Lichtneckert, 2008).
MARCM-based clonal loss-of-function experiments reveal two lineage-specific mutant phenotypes in adPN versus lPN lineages. In the lPN lineage, ems loss-of-function results in a dramatic cell-autonomous reduction in cell number. A significant contribution to this phenotype is made by cell death. Blocking cell death in mutant clones restores cell number to ~70% of the wild-type number. Although these findings imply that cells in ems mutant lPN lineages die during postembryonic development, it is not known whether cell death occurs at the level of the progenitors or postmitotic neurons. ems is persistently expressed in the neuroblast and GMCs in this lineage and might be required for the survival of these progenitors. Alternatively, since ems is transiently expressed in postmitotic neurons in the PN lineages, this transient neuronal expression might be required for PN survival. Finally, because blocking apoptosis does not always result in a complete rescue of cell number, unknown lineage-specific proliferation defects might also occur (Lichtneckert, 2008).
In the postembryonic adPN lineage, ems loss-of-function does not affect cell number, implying that ems is not required for proliferation or survival of adPNs. Furthermore, adPNs in ems mutant clones have the overall dendritic and axonal features of wild-type PNs, suggesting that ems is not required for general process outgrowth in this lineage. However, ems mutant adPNs do show marked cell-autonomous defects in dendritic targeting: they fail to innervate appropriate glomeruli, ectopically innervate inappropriate glomeruli, or mistarget dendrites. These targeting defects are not random in nature but are limited to a subset of glomeruli. This relative specificity of the mistargeting phenotypes indicates that ems loss-of-function does not simply result in non-specific spillover of adPN dendrites. Moreover, it argues for the existence of other cell-intrinsic determinants that participate in translating adPN lineage information into dendritic targeting specificity (Lichtneckert, 2008).
Previous studies have identified an ensemble of transcription factors that act as intrinsic regulators of dendritic targeting in PNs. For example, the two POU-domain transcription factors Acj6 and Drifter are differentially expressed in adPNs and lPNs, are required for the specific connectivity of these PNs in their lineage, and cause mistargeting when misexpressed in PNs of the alternate lineage. Acj6 and Drifter are expressed in postmitotic Gal4-GH146-positive PNs during their dendritic targeting phase. Hence, the developmental time period in which these transcription factors are expressed coincides with that in which their mutant phenotypes appear. By contrast, the transcription factor-encoding ems gene is expressed in the precursors of PN lineages, but not in Gal4-GH146-positive PNs during their dendritic targeting phase. Thus, ems expression and appearance of the ems mutant dendritic targeting phenotype occur sequentially and do not overlap in developmental time. This suggests that ems acts as an early intrinsic determinant in the adPN lineage to influence cell fate decisions that indirectly result in dendritic targeting later in postembryonic development. Therefore, the mechanism of ems action on dendritic targeting might be mediated by other factors that are themselves regulated by ems and that subsequently affect components of the wiring machinery (Lichtneckert, 2008).
The current findings indicate that Acj6 is one of these factors. Acj6 as downstream mediator of ems action, at least in one class of adPNs. Acj6 expression is lost in approximately half of the ems mutant adPNs. Moreover, the reduced innervation of the VA1lm glomerulus in the ems mutant is significantly rescued by the misexpression of acj6. It is noteworthy that the innervation of the VA1lm glomerulus has been reported to be lost in 63% of acj6 mutant adPN clones and that misexpression of acj6 in these clones rescued innervation to a level similar to that observed in the Acj6 rescue experiments. Given that Acj6 expression is lost in only about half of the ems mutant adPNs, misexpression of Acj6 should not affect the other half of the ems mutant adPNs. Indeed, for the innervation of the VA3 and VM2 glomeruli, it was observes that misexpression of Acj6 in ems mutant adPNs does not result in significant rescue of the projection phenotype, implying that Acj6 expression is ems-independent in the PNs that innervate VA3 and VM2 (Lichtneckert, 2008).
The fact that the other phenotypes observed in this investigation for ems mutant adPN and lPN clones have not been reported in acj6 mutant clones does, however, imply that there are other downstream mediators of ems action in PN development. Similarly, because Acj6 is lost in only about half of the ems mutant adPNs, other upstream regulators of acj6 expression in adPNs are also likely to be present (Lichtneckert, 2008).
Although transient early ems expression is important for appropriate development of the adPN lineage, more prolonged, later expression of ems in the differentiating adPNs can have detrimental effects. Ectopic misexpression of ems in adPNs in differentiating PNs via the Gal4-GH146 driver results in dendritic targeting defects comparable to those caused by ems loss-of-function. Interestingly, ectopic ems misexpression also causes axonal targeting defects in at least one of the adPNs, the DL1 neurons. Since misexpression of ems beyond the time of normal endogenous expression can lead to dual targeting defects (axonal and dendritic), precise temporal regulation of ems expression is likely to be crucial for the correct development of adPNs (Lichtneckert, 2008).
The organization of the insect and mammalian olfactory system is remarkably similar. Olfactory receptor neurons expressing the same receptor project their axons to the same glomeruli in the insect antennal lobe as in the mammalian olfactory bulb. In these glomeruli, the olfactory receptor neurons make specific synaptic connections with the dendrites of second-order olfactory neurons, the PNs in insects and the mitral cells in mammals. Finally, PNs and mitral cells send processed olfactory information to specific regions of higher olfactory centers in the brain (Lichtneckert, 2008).
In both insects and mammals, genes of the ems/Emx family are important for the development of these second-order neurons. In Drosophila, ems is expressed in the two main PN lineages and is required for correct cell number and precise dendritic targeting of these neurons. In the mouse, the two ems gene homologs, Emx1 and Emx2, are expressed in two complementary groups of mitral cells, and the loss of both genes leads to marked defects in the mitral cell layer (Lichtneckert, 2008).
The remarkably similar expression and function of the ems/Emx genes in the development of second-order olfactory neurons in insects and mammals, together with the similarities in expression of these genes in developing olfactory sensory structures in both groups, argue for evolutionarily conserved roles of the ems/Emx genes in olfactory system development. Thus, while the astonishing similarity in anatomical organization of the olfactory system in insects and mammals may be the result of functional convergence, it might also reflect, at least in part, a hitherto unexpected conservation of the molecular genetic mechanisms for olfactory system development in these animals (Lichtneckert, 2008).
Bases in 5' UTR -299
Exons - two
Bases in 3' UTR - 673
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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