even-skipped: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Post-transcriptional regulation | Targets of activity | Protein interactions | Developmental Biology | Effects of Mutation | References

Gene name - even-skipped

Synonyms -

Cytological map position - 46C3-11

Function - transcription factor

Keywords - pair rule gene, germband extension

Symbol - eve

FlyBase ID:FBgn0000606

Genetic map position - 2-[59]

Classification - homeodomain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | UniGene | HomoloGene
Recent literature
Jiang, P., Ludwig, M. Z., Kreitman, M. and Reinitz, J. (2015). Natural variation of the expression pattern of the segmentation gene even-skipped in melanogaster. Dev Biol [Epub ahead of print]. PubMed ID: 26129990
Summary:
The evolution of canalized traits is a central question in evolutionary biology. Natural variation in highly conserved traits can provide clues about their evolutionary potential. This study investigate natural variation in a conserved trait-even-skipped (eve) expression at the cellular blastoderm stage of embryonic development in Drosophila melanogaster. Expression of the pair-rule gene eve was quantitatively measured in three inbred lines derived from a natural population of D. melanogaster. One line showed marked differences in the spacing, amplitude and timing of formation of the characteristic seven-striped pattern over a 50-min period prior to the onset of gastrulation. These changes are larger than those previously reported between D. melanogaster and D. pseudoobscura, two species that are thought to have diverged from a common ancestor over 25 million years ago. This line harbors a rare 448 bp deletion in the first intron of knirps (kni). This finding suggested that reduced Kni levels caused the deviant eve expression, and indeed lower levels of Kni protein were observed at early cycle 14A in L2 compared to the other two lines. A second of the three lines displayed an approximately 20% greater level of expression for all seven eve stripes. The three lines are each viable and fertile, and none display a segmentation defect as adults, suggesting that early-acting variation in eve expression is ameliorated by developmental buffering mechanisms acting later in development. Canalization of the segmentation pathway may reduce the fitness consequences of genetic variation, thus allowing the persistence of mutations with unexpectedly strong gene expression phenotypes.

Holloway, D. M. and Spirov, A. V. (2017). Transcriptional bursting in Drosophila development: Stochastic dynamics of eve stripe 2 expression. PLoS One 12(4): e0176228. PubMed ID: 28437444
Summary:
Anterior-posterior (AP) body segmentation in Drosophila is first seen in the 7-stripe spatial expression patterns of the pair-rule genes, which regulate downstream genes determining specific segment identities. This study developed a stochastic model of the spatial and temporal expression of eve stripe 2 -- binding by transcriptional activators (Bicoid and Hunchback proteins) and repressors (Giant and Kruppel proteins) with all rate parameters constrained by features of the experimental data) -- in order to analyze the noisy experimental time series and test hypotheses for how eve transcription is regulated. Short-time (minute-to-minute) statistics of the data is indicative of eve being transcribed with at least two distinct ON rates, consistent with data on the joint activation of eve by Bicoid and Hunchback. Distinct statistical signatures were predicted for cases in which eve is repressed (e.g. along the edges of the stripe) vs. cases in which activation is reduced (e.g. by mutagenesis of transcription factor binding sites). This approach presents a new way to quantify and analyze time series data during developmental patterning in order to understand regulatory mechanisms and how they propagate noise and impact embryonic robustness.
Crocker, J. and Stern, D. L. (2017). Functional regulatory evolution outside of the minimal even-skipped stripe 2 enhancer. Development 144(17):3095-3101.. PubMed ID: 28760812
Summary:
Transcriptional enhancers are regions of DNA that drive precise patterns of gene expression. While many studies have elucidated how individual enhancers can evolve, most of this work has focused on what are called "minimal" enhancers, the smallest DNA regions that drive expression that approximates an aspect of native gene expression. This study explored how the Drosophila erecta even-skipped (eve) locus has evolved by testing its activity in the divergent D. melanogaster genome. As has been reported previously, it was found that the D. erecta eve stripe 2 enhancer (eveS2) fails to drive appreciable expression in D. melanogaster. However, it was found that a large transgene carrying the entire D. erecta eve locus drives normal eve expression, including in stripe 2. A functional dissection of the region upstream of the D. erecta eveS2 region was performed and multiple Zelda motifs were found that are required for normal expression. These results illustrate how sequences outside of minimal enhancer regions can evolve functionally through mechanisms other than changes in transcription factor binding sites that drive patterning.
Barr, K. A. and Reinitz, J. (2017). A sequence level model of an intact locus predicts the location and function of nonadditive enhancers. PLoS One 12(7): e0180861. PubMed ID: 28715438
Summary:
Metazoan gene expression is controlled through the action of long stretches of noncoding DNA that contain enhancers-shorter sequences responsible for controlling a single aspect of a gene's expression pattern. Models built on thermodynamics have shown how enhancers interpret protein concentration in order to determine specific levels of gene expression, but the emergent regulatory logic of a complete regulatory locus shows qualitative and quantitative differences from isolated enhancers. Such differences may arise from steric competition limiting the quantity of DNA that can simultaneously influence the transcription machinery. This competition was incorporated into a mechanistic model of gene regulation, generated efficient algorithms for this computation, and applied it to the regulation of Drosophila even-skipped (eve). This model finds the location of enhancers and identifies which factors control the boundaries of eve expression. This model predicts a new enhancer that, when assayed in vivo, drives expression in a non-eve pattern. Incorporation of chromatin accessibility eliminates this inconsistency.
Barr, K. A., Martinez, C., Moran, J. R., Kim, A. R., Ramos, A. F. and Reinitz, J. (2017). Synthetic enhancer design by in silico compensatory evolution reveals flexibility and constraint in cis-regulation. BMC Syst Biol 11(1): 116. PubMed ID: 29187214
Summary:
Models that incorporate specific chemical mechanisms have been successful in describing the activity of Drosophila developmental enhancers as a function of underlying transcription factor binding motifs. Despite this, the minimum set of mechanisms required to reconstruct an enhancer from its constituent parts is not known. Synthetic biology offers the potential to test the sufficiency of known mechanisms to describe the activity of enhancers, as well as to uncover constraints on the number, order, and spacing of motifs. Using a functional model and in silico compensatory evolution, putative synthetic even-skipped stripe 2 enhancers with varying degrees of similarity to the natural enhancer. These elements represent the evolutionary trajectories of the natural stripe 2 enhancer towards two synthetic enhancers designed ab initio. In the first trajectory, spatially regulated expression was maintained, even after more than a third of binding sites were lost. In the second, sequences with high similarity to the natural element did not drive expression, but a highly diverged sequence about half the length of the minimal stripe 2 enhancer drove ten times greater expression. Additionally, homotypic clusters of Zelda or Stat92E motifs, but not Bicoid, drove expression in developing embryos. The results show that the gene regulation model explains much of the function of the stripe 2 enhancer. Cases where expression deviated from prediction indicates that undescribed factors likely act to modulate expression. Activation driven Bicoid and Hunchback is highly sensitive to spatial organization of binding motifs. In contrast, Zelda and Stat92E drive expression from simple homotypic clusters, suggesting that activation driven by these factors is less constrained. Collectively, the 40 sequences generated in this work provides a powerful training set for building future models of gene regulation.
Chen, H., Levo, M., Barinov, L., Fujioka, M., Jaynes, J. B. and Gregor, T. (2018). Dynamic interplay between enhancer-promoter topology and gene activity. Nat Genet. PubMed ID: 30038397
Summary:
A long-standing question in gene regulation is how remote enhancers communicate with their target promoters, and specifically how chromatin topology dynamically relates to gene activation. This study combined genome editing and multi-color live imaging to simultaneously visualize physical enhancer-promoter interaction and transcription at the single-cell level in Drosophila embryos. By examining transcriptional activation of a reporter by the endogenous even-skipped enhancers, which are located 150 kb away, three distinct topological conformation states were identified and their transition kinetics were measured. Sustained proximity of the enhancer to its target is required for activation. Transcription in turn affects the three-dimensional topology as it enhances the temporal stability of the proximal conformation and is associated with further spatial compaction. Furthermore, the facilitated long-range activation results in transcriptional competition at the locus, causing corresponding developmental defects. This approach offers quantitative insight into the spatial and temporal determinants of long-range gene regulation and their implications for cellular fates (Chen, 2018).
Lim, B., Fukaya, T., Heist, T. and Levine, M. (2018). Temporal dynamics of pair-rule stripes in living Drosophila embryos. Proc Natl Acad Sci U S A 115(33): 8376-8381. PubMed ID: 30061421
Summary:
Traditional studies of gene regulation in the Drosophila embryo centered primarily on the analysis of fixed tissues. These methods provided considerable insight into the spatial control of gene activity, such as the borders of eve stripe 2, but yielded only limited information about temporal dynamics. The advent of quantitative live-imaging and genome-editing methods permits the detailed examination of the temporal control of endogenous gene activity. This study presents evidence that the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) undergo dynamic shifts in gene expression. Sequential anterior shifting of the stripes along the anterior to posterior axis was observed, with stripe 1 exhibiting movement before stripe 2 and the more posterior stripes. Conversely, posterior stripes shift over greater distances (two or three nuclei) than anterior stripes (one or two nuclei). Shifting of the ftz and eve stripes are slightly offset, with ftz moving faster than eve. This observation is consistent with previous genetic studies, suggesting that eve is epistatic to ftz. The precision of pair-rule temporal dynamics might depend on enhancer-enhancer interactions within the eve locus, since removal of the endogenous eve stripe 1 enhancer via CRISPR/Cas9 genome editing led to precocious and expanded expression of eve stripe 2. These observations raise the possibility of an added layer of complexity in the positional information encoded by the segmentation gene regulatory network.
Vincent, B. J., Staller, M. V., Lopez-Rivera, F., Bragdon, M. D. J., Pym, E. C. G., Biette, K. M., Wunderlich, Z., Harden, T. T., Estrada, J. and DePace, A. H. (2018). Hunchback is counter-repressed to regulate even-skipped stripe 2 expression in Drosophila embryos. PLoS Genet 14(9): e1007644. PubMed ID: 30192762
Summary:
Hunchback is a bifunctional transcription factor that can activate and repress gene expression in Drosophila development. This study investigated the regulatory DNA sequence features that control Hunchback function by perturbing enhancers for one of its target genes, even-skipped (eve). While Hunchback directly represses the eve stripe 3+7 enhancer, in the eve stripe 2+7 enhancer, Hunchback repression is prevented by nearby sequences-this phenomenon is called counter-repression. Evidence was also found that Caudal binding sites are responsible for counter-repression, and that this interaction may be a conserved feature of eve stripe 2 enhancers. These results alter the textbook view of eve stripe 2 regulation wherein Hb is described as a direct activator. Instead, to generate stripe 2, Hunchback repression must be counteracted. How counter-repression may influence eve stripe 2 regulation and evolution is discussed.

BIOLOGICAL OVERVIEW

Even-skipped is a transcriptional repressor of a number of genes, including engrailed (acting indirectly through paired, runt and sloppy paired) (Fujioka, 1996), fushi tarazu, Ultrabithorax and wingless. It fulfills a primary role in segmentation. Its repressive effect on fushi tarazu transciption results in an alternating pattern of FTZ and EVE in the blastoderm. EVE also assures that Wingless is confined to posterior compartments during segmentation. EVE protein forms a symmetrical pattern of seven stripes, subdividing the blastoderm. eve's pair-rule patterning, combined with the activity of other pair-rule genes, assures that even as early as the blastoderm stage, each cell of the fly has a unique identity. Such precision in the determination of cell fate is both startling and mind boggling, taking place as it does within the first three hours of development. This is both exceptionally fast and early.

Each stripe of eve is defined by a combination of maternal factors, transcription activators, and repressors, already patterned in the egg either prior to fertilization or prior to cellularization. The stripe pattern of eve transcription is governed by regional specific enhancers. The stripe two enhancer is the best documented of all Drosophila region specific enhancers, and the work to characterize it is a classic in promoter analysis (Small, 1992).

Each stripe is regulated by a stripe-specific enhancer upstream of the structural gene. For example, in the case of eve stripe 2, a minimal stripe 2 enhancer has been identified between -1.5 and -1.0 kb upstream from the eve start site. It is this site specific enhancer that assures eve transcription in the second pair rule stripe. Enhancers for other stripes have been identified as well.

The repressor Giant forms the anterior border of eve stripe 2. There are three binding sites for this transcription factor in the stripe 2 element. Krüppel, a gap gene expressed in the central portion of the blastoderm, determines the posterior border of the stripe, again by repression. Two of the three Krüppel sites overlap Bicoid activation sites, thus effectively prohibiting activation by Bicoid where Krüppel is expressed. Bicoid and Hunchback, both maternal proteins with anterior/posterior concentration gradients, serve to activate transcription (Small, 1992).

One of EVE's primary functions is regulation of segment polarity through EVE's indirect regulation of engrailed. In odd parasegments, graded expression of eve establishes the en stripes by setting the boundaries of the activator paired and the repressors runt and sloppy paired (Fujioka, 1995). Expression of en in even parasegments results from activation by Fushi tarazu (with Ftz-f1 as a cofactor). Only the most anterior cells of each ftz stripe express en and this restriction is dependent upon odd-skipped and naked.

The existence of regional transcriptional enhancers is perhaps the single most important explanation for regional specific gene transcription in Drosophila development, and therefore the single most important explanation for how regional identity is established during development.

Eve and Ftz regulate a wide array of genes in blastoderm embryos: the selector homeoproteins directly or indirectly regulate most genes in Drosophila

Is it possible to estimate the number of target genes of the homeoproteins Eve and Ftz? Eve and Ftz have been shown to bind with similar specificities to many genes, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Eve and Ftz bind at the highest levels to DNA fragments throughout the length of three probable target genes: eve, ftz and Ubx. However, Eve and Ftz also bind at only two- to ten-fold lower levels to four genes chosen in an attempt to find non targets: Adh, hsp70, rosy and actin 5C, suggesting that Eve and Ftz bind at significant levels to a majority of genes. The expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve (Liang, 1998).

In vitro transcription experiments demonstrate that (1) Eve protein can directly repress the Ubx promoter, and (2) endogenous Eve protein binds to the Ubx gene in embryos. Genetic experiments have shown that eve represses Ubx in stage 11 embryos. However, this effect may be indirect and could be mediated via eve’s effect on engrailed. Consequently, the expression of Ubx was examined in wild-type and eve1.27 embryos at stage 5 -- a time before Engrailed protein is significantly expressed. UBX mRNA is present in four stripes in the posterior half of wild-type stage 5 embryos, with the anterior-most stripe (stripe 1) being the more prominent. In eve1.27 mutants, Ubx expression is derepressed in a region including stripe 1 and stripe 2, reaching the same level of expression as stripe 1. Ubx expression is not significantly affected in the posterior of the embryo, either because Eve binds, but does not regulate Ubx in posterior cells, or because Eve’s function is redundant with that of other transcription factors in these cells. It is also possible that Eve does not bind to Ubx in the posterior of the embryo (though this explanation is considered less likely). Whatever the reason, the early regulation of Ubx supports the evidence that Eve directly represses Ubx (Liang, 1998).

hsp70 is known to be induced uniformly in all cells when Drosophila cells are raised above 25°C. Hence, it initially seemed unlikely that this gene would be regulated by Eve. However, the discovery that Eve crosslinks to hsp70 with only half the strength it crosslinks to Ubx suggested that hsp70 expression should be reexamined. At stages 0-3, Drosophila embryos produce no detectable hsp70 transcripts. In older wild-type embryos, 37 °C heat shocks for more than 1 minute induce hsp70 at relatively uniform levels in most cells, although a weak segmentally repeated pattern of transcripts is observed in embryos after stage 10. In contrast, heat shocks at 37°C for only 15-30 seconds induce hsp70 transcripts in a pair-rule like manner at cellular blastoderm and in segmentally repeated patterns in older embryos. In eve1.27 embryos heat shocked for the same brief period, these patterns are not observed at stage 5 and are much less pronounced in later stages. Thus, Eve does regulate hsp70, and the crosslinking data, together with the fact that this regulation is observed soon after Eve becomes maximally expressed, strongly suggest that this regulation is at least partly direct. To estimate the degree of regulation by Eve, video microscopy was used to measure the changing intensity of stain at stage 5. Quantitation of a number of typical embryos suggests that hsp70 transcript levels vary two fold between the centers of the stripe and inter-stripe regions (Liang, 1998).

Eve crosslinks at the same levels to actin 5C and hsp70: like hsp70, actin 5C was thought to be uniformly expressed throughout most of embryogenesis. However, although maternally derived transcripts are uniformly distributed at stages 0-8, by stages 11 and 12 zygotic actin 5C transcripts are present in a series of different segmentally repeated patterns whose transcript levels vary two- to four-fold between stripe and inter-stripe regions. These patterns do not resemble those of hsp70, suggesting that they are due to specific regulation of actin 5C, and are not due to a general change in transcription of ubiquitously expressed genes. In eve mutant embryos, the initial pattern of actin 5C in the epidermis is not observed, and the later pattern in the mesoderm becomes altered. Since by stage 11 Eve has initiated a complex cascade of regulatory transcription factors and is itself expressed in only a few cells, the effect of Eve on actin 5C at this stage must be indirect. However, it remains plausible that Eve directly regulates actin 5C transcription at stage 5, but that maternal mRNAs obscure this, and that in later development other later expressed selector homeoproteins, such as En and the Hox proteins, directly control actin 5C. Certainly, this analysis of actin 5C adds to the view that more genes are spatially regulated than had been previously thought. The rosy gene is bound by Eve and Ftz with only 1/2 to 1/3 the binding strength of either hsp70 or actin 5C, when bound by Eve and Ftz. At stages 5-6, rosy transcripts are expressed in a broad ventral stripe that in most wild-type embryos show a pair-rule like pattern. The variations in transcript levels are 1.2 to 1.5 fold between the stripe and inter stripe regions. In eve1.27 mutant embryos, these pair-rule like modulations are not observed. Therefore, rosy is downstream of eve at stage 5, and the low levels of Eve binding to this gene may be responsible for this weak regulation. The Adh gene was initially chosen for the in vivo DNA binding studies because Northern blots had indicated that this gene was not expressed at stages 5-9, and thus there was no reason to suspect that this gene might be bound by Eve or Ftz. In situ hybridization confirms that Adh transcripts are not detectable at stage 5, but, interestingly, at stage 14 Adh is expressed in a segmentally repeated pattern that suggests that it is downstream of the Hox genes. In the most simple sense then, the binding of Eve to Adh at stage 5 is probably nonfunctional. However, this binding may indicate that in later development Adh may be a direct target of other selector homeoproteins. When Adh expression is activated, these and other transcription factors may have greater access to this gene (Liang, 1998).

What percentage of genes are downstream of the selector homeoproteins? The above data suggest that the selector homeoproteins may regulate many more genes than initially assumed. To more thoroughly test this idea, the expression patterns of genes selected at random were analyzed. About 200 colonies were randomly picked from three separate plasmid cDNA libraries prepared from the mRNA of either 0-4 hour, 4-8 hour, or 8-12 hour old embryos. In situ hybridizations to whole-mount embryos were then performed using probes prepared from each clone. The DNA sequence of 99 clones from the 8-12 hour library was also determined to identify the genes encoding them. Just after fertilization (stages 0-1), a majority of genes express maternally derived transcripts that are uniformly distributed throughout the embryo. At cellular blastoderm (stages 5-6), maternally derived transcripts can still be detected at reduced levels for most genes, but some genes express zygotically derived transcripts in either pair-rule or other patterns. By stages 10-14, maternal transcripts have largely decayed, and most genes are expressed in either segmentally repeating patterns or in a relatively uniform manner. A subset of genes is more prevalent in the 0-4 hour library and, to a lesser extent, in the 4-8 hour library than in the 8-12 hour library. These highly expressed genes express maternal transcripts that perdure until after stage 8 (5.5 hours) and tend to be uniformly expressed at stages 10-14. Since the 8-12 hour library was prepared from mRNA in which these transcripts no longer predominate, it should give a better estimate of typical gene expression patterns. In support of this, the proportion of genes in the 8-12 hour library that do not have maternal contributions is most similar to that predicted from genetic experiments (i.e. 30%-40%). Also, the estimate of the proportion of zygotic and maternal mRNAs at different stages of development provided by the 8-12 hour library agrees most closely with the results of total RNA labeling experiments. These labeling experiments indicate that 10%-15% of total cytoplasmic poly(A) mRNAs are zygotically derived by stage 5 and that 89% of stage 14 transcripts are zygotic (Liang, 1998).

Taking the 8-12 hour library as most representative of Drosophila genes, a majority of genes whose zygotic transcription can be detected at stage 5 are expressed in pair-rule patterns. These patterns are in a variety of registers relative to one another and to Eve and Ftz, indicating that these patterns are generated by the combinatorial activities of maternal, gap and other pair-rule genes and do not result solely from control by Eve and Ftz. To determine what percent of genes are regulated by Eve and Ftz at stage 5, 11 genes were selected with the most pronounced pair-rule patterns from both the 4-8 hour and 8-12 hour libraries. Of these, the expression of seven clearly differs in eve mutants as compared to wild-type embryos. The expression of all but one of these seven genes also changes in an equally pronounced manner in ftz mutant embryos. The pattern of another gene expressed in pair-rule stripes does not detectably change in either eve or ftz mutant embryos. Of the remaining three genes, their pair-rule patterns are weaker, and it could not be judged if they are regulated by Eve or Ftz. The expression of three genes not expressed in pair-rule patterns was examined. The expression of these genes is not altered in eve or ftz mutant embryos at stage 5. Thus, Eve and Ftz regulate largely the same array of genes at cellular blastoderm. For several reasons it is difficult to give an exact number of genes that are downstream of Eve and Ftz at stage 5: the number of genes assayed is relatively small; there may be possible biases in genes represented in the cDNA library; redundancy or perduring maternal mRNAs may obscure Eve and Ftz’s control of some genes, and weakly patterned genes could not be assayed. However, it is suggested that 25%-50% of genes transcribed at stage 5 are downstream of Eve and Ftz. Assuming that there are 13,000 genes in Drosophila and that 22% of genes are transcribed at this stage, this suggests that about 715-1,430 genes are downstream of both Eve and Ftz at this stage (Liang, 1998).

At stages 10-14, 87% of cDNAs in the 8-12 hour library are likely to be directly or indirectly regulated by Eve, Ftz, Engrailed and all of the Hox proteins. These downstream genes are each expressed in unique, segmentally repeating patterns. Some are expressed at dramatically altered levels between segments. Most vary from segment to segment in the number and position of cells in which they are most prominently expressed. This is not simply because expression follows the distribution of a particular cell type. Between segments, the majority of genes are most highly expressed in differently positioned subsets of the same cell types, indicating that these patterns cannot result solely from the action of cell-type specific transcription factors. Eve, Ftz and Engrailed establish the segmentally repeating structure of the embryo. Therefore, all genes expressed in segmentally repeated patterns by stage 11 should be downstream of these three genes. This has been experimentally confirmed for eve and ftz. The expression of all 14 segmentally expressed genes tested is altered in eve and ftz mutant embryos at stage 11. Equally, the Hox genes establish the differences between segments. Thus, all genes expressed differently in each segment should be downstream of all of the Hox genes. This is indeed the case for the Hox gene Ubx. The expression of all seven segmentally expressed genes tested is regulated by Ubx. These downstream genes can be divided into three classes: genes expressed in strong, moderate or weak segmentally repeated patterns. 33% of cDNAs fall into the strongly repeated class. For this class, staining levels vary five fold or more between cells across a transverse section of a segment along the anterior/posterior axis of the embryo. 24% of clones belong to the moderately regulated class. These genes show two- to five-fold variations in staining across the width of a segment. Finally, the weak segmentally repeated genes vary only 1.2 to 2 fold in staining between cells across a segment. Thus, most downstream genes are expressed in all cells, but each are still subject to specific and precise control by the selector homeoproteins. The more strongly regulated genes include many developmental control genes such as Enhancer of split [E(spl)] , tramtrack, division abnormally delayed (dally), and Dwnt4. A high proportion of the moderate and weakly regulated genes are involved in essential cellular functions such as splicing (e.g. RNA helicases), translation (e.g. met tRNA synthetase), general signal transduction (e.g. G-protein beta13F) and cytoskeletal structure (e.g. alpha tubulin 84B). This raises the question of whether or not modest changes in the expression of essential enzymes and structural proteins are important for morphogenesis. It is argued that they probably are. 11% of the genes picked from the 8-12 hour cDNA library do not appear to be downstream of the selector homeoproteins. Most of these genes are expressed relatively uniformly in all cells. But even these genes show some differences in expression pattern. For example, clone 1.45 (Emp24 - a protein transport gene) is more strongly expressed in the salivary gland. From this analysis, few if any genes are truly uniformly expressed and almost all genes show some distinguishing or specific pattern (Liang, 1998).

Although this analysis suggests that at least 87% of genes are directly or indirectly regulated by the selector homeoproteins, the extent of regulation is not absolute. Around 50% of genes are regulated by five fold or less, and 30% of genes are regulated by two fold or less. Most recessive lethal mutations show little or no obvious mutant phenotype when heterozygous, in comparison to a wild-type copy of the same gene. This could be taken as evidence that two fold changes in gene expression are not significant. However, this assumption is not valid. Two to three fold changes in the levels of multiple proteins involved in the same process generally have important effects on cell physiology. The metabolic flux through most pathways is not controlled at a single rate limiting step, as early theories assumed. Instead, the control of flux is generally shared by many enzymes in a pathway. For this reason, large increases in flux require the activities of a number of enzymes to be raised, and cannot be accomplished by increasing the level of just one protein. In general, moderate changes in the activities of multiple enzymes in a pathway will alter flux more than a large change in the activity of a single enzyme. This point is illustrated by the obese mouse. Here, 1.5- to 3-fold increases in the activities of eight glycolytic and lipogenic enzymes lead to a profound change in the physiology of the mouse. Genetic experiments also suggest that small changes in gene expression are significant. For example, hypomorphic mutations are often enhanced by lowering the dose of another gene in the same pathway by half. Similarly, although there are only 73 known haplo-insufficient loci in Drosophila, and only a few of these are haplo-lethals, heterozygotes for deficiencies of 3% or more of the genome are lethal for almost all regions of the genome. Thus, to determine the significance of a change in a gene’s expression, it is essential to consider changes that may also have occurred in the levels of other proteins. One of the processes controlled by the selector homeoproteins is cell size. A two fold change in cell volume should require modest changes in the expression of most cytoskeletal proteins, membrane proteins, enzymes etc. It ought not to require a change in the levels of chromatin binding proteins. Different cell types may have different requirements for which gene’s expression must be altered during changes in cell size. Selector homeoproteins also control the number of cell divisions, the orientation of cell divisions, cell shape, cell affinities, differentiation, and cell movement. Thus, it seems entirely reasonable that changing morphology may require the coordinated, differential regulation of a large percentage of genes, often to only moderate extents (Liang, 1998 and references).

The Drosophila wing hearts originate from pericardial cells and are essential for wing maturation

In addition to the heart proper, insects possess wing hearts in the thorax to ensure regular hemolymph flow through the narrow wings. In Drosophila, the wing hearts consist of two bilateral muscular pumps of unknown origin. This paper presents the first developmental study on these organs and reports that the wing hearts originate from eight embryonic progenitor cells arising in two pairs in parasegments 4 and 5. These progenitors represent a so far undescribed subset of the Even-skipped positive pericardial cells (EPC) and are characterized by the early loss of tinman expression in contrast to the continuously Tinman positive classical EPCs. Ectopic expression of Tinman in the wing heart progenitors omits organ formation, indicating a crucial role for Tinman during progenitor specification. The subsequent postembryonic development is a highly dynamic process, which includes proliferation and two relocation events. Adults lacking wing hearts display a severe wing phenotype and are unable to fly. The phenotype is caused by omitted clearance of the epidermal cells from the wings during maturation, which inhibits the formation of a flexible wing blade. This indicates that wing hearts are required for proper wing morphogenesis and functionality (Tögel, 2008).

Unlike in vertebrates, where an elaborate closed blood vessel system extends throughout the whole body, insects possess only one vessel, the tubular heart, in their otherwise open circulatory system. Once the hemolymph has left the heart, it moves freely between the internal organs and can not be directed into narrow body appendages such as antennae, legs or wings. To ensure sufficient hemolymph supply of these appendages additional circulatory organs evolved (Pass, 2000; Pass, 2006). In Drosophila, circulation in the wings is maintained by the so-called wing hearts (Krenn, 1995), a pair of autonomous muscular pumps located bilaterally in the scutellum, the dorsal elevation of the second thoracic segment. Due to this location, they are also referred to as scutellar pulsatile organs. Although known for many years, no developmental studies on the origin or morphogenesis of these organs have been performed. Probably, this was due to the lack of available methods to track their differentiation. However, studies on the origin of the thoracic somatic muscles in Drosophila and comparative anatomical investigations in insects suggested that the wing hearts originate from the cardiac mesoderm or from the heart itself (Tögel, 2008).

A previous study identified an enhancer region of the Drosophila hand gene that is able to drive reporter gene activity in the wing hearts (Sellin, 2006). In the present work, this reporter was used to identify the embryonic anlagen of the wing hearts and to elucidate the dynamics of their postembryonic development with in vivo time lapse imaging. It was found that the anlagen of the Drosophila wing hearts indeed derive from the cardiac mesoderm but, astonishingly, not from the muscular cardioblast lineage. Instead, they represent a so far undescribed subpopulation of the well-known Even-skipped (Eve) positive pericardial cells (EPCs) (Tögel, 2008).

In addition to their unknown origin, little is known about the contribution of wing hearts to wing morphogenesis and functionality. After eclosion, wings are unfolded by a sudden influx of hemolymph and subsequently undergo maturation. During this process, the epidermal cells that until then bonded the dorsal and ventral wing surfaces enter programmed cell death, delaminate from the cuticle, and disappear into the thorax (Kimura, 2004). Subsequently, the cuticles of the intervein regions become tightly bonded to form a flexible wing blade, while the cuticles of the vein regions form tubes, lined by living cells, through which hemolymph circulates in mature adult insects. Measurements of hemolymph flow in adult butterflies showed that wing hearts function as suction pumps that draw hemolymph out of the wings starting shortly after wing unfolding. Whether wing hearts might play a role in wing maturation was tested by generating flies lacking wing hearts. The findings demonstrate that the delaminated epidermal cells are removed from the wings by the hemolymph flow generated by the wing hearts. Loss of wing heart function leads to remains of epidermal cells resting between the unbonded dorsal and ventral wing surfaces which results in malformation of the wing blade and flightlessness. It is concluded that wing hearts are essential for wing maturation and, thus, for acquiring flight ability in Drosophila (Tögel, 2008).

A hand-C-GFP reporter was generated (Sellin, 2006) that reflects the described hand expression pattern and was found to be active in wing hearts. To confirm that the hand-C-GFP reporter is expressed in all cells of mature wing hearts, their morphology was examined based on the signal from the reporter in conjunction with histological sections. In the adult fly, wing hearts are located at the lateral angles of the scutellum, which are joined to the posterior wing veins by cuticular tubes. Each organ is curved in anterior–posterior direction as well as dorso-ventrally. It consists of about 7-8 horizontally arranged rows of prominent muscle cells, which are attached at their proximal side to a thin layer of cells that has a greater dorsal extension than the muscle cells. Both cell types are labeled by the reporter. The fine acellular strands that hold the wing hearts to the adjacent epidermal cells were not observed to be marked by the reporter. Movies are provided to demonstrate the location and the beating of wing hearts (Tögel, 2008).

The hand-C-GFP reporter was tested for expression in earlier stages of wing heart development and it was found to be active throughout the entire organogenesis. This enabled identification of the embryonic anlagen of the wing hearts, which consist of eight progenitor cells located dorsally and anterior to the heart, in two pairs in the second and third thoracic segment from stage 16/17 onward. The progenitors exhibit a flattened triangular shape and are interconnected by thin cytoplasmic extensions. In addition, the second and the fourth pair of the progenitors are closely associated with the dorsal tracheal branches at their interconnection in the second and third thoracic segments. The characteristic pairwise arrangement and the connection to the tracheae are retained during the subsequent three larval stages. Proliferation starts at about the transition from the second to the third larval instar, leading to eight clusters of cells that remain arranged in four pairs in the anterior region until 1h after puparium formation (APF). Between 1 and 10h APF, the cell number increases significantly and the anterior three pairs of cell clusters are retracted to join the last pair of clusters, eventually forming one large median cluster. Between 13 and 50h APF, the single large cluster splits along its anterior-posterior axis into two groups of cells that migrate laterally in the forming scutellum, thereby adopting the characteristic arched appearance of the adult wing hearts. During this process some of the cells on either side form the underlying thin layer while the remaining cells arrange in horizontal rows along that layer. First contractions of the mature organs were observed at about 45-50h APF (Tögel, 2008).

The expression of the bHLH transcription factor Hand in the wing heart progenitors, which serves as a general marker for all classes of heart cells in Drosophila, prompted a to screen for the expression of genes known to be active in cardiac lineages. Analysis of Even-skipped (Eve) expression revealed that the embryonic wing heart progenitors arise through the same lineage as the well described Eve expressing pericardial cells (EPCs). At stage 10 in embryogenesis, 12 Eve clusters are present on either side of the embryo, located in parasegments (PS) 2 to 12. Each cluster gives rise to a pair of EPCs, except for the most posterior cluster in PS 14, which generates only one EPC. During subsequent development, the first and the second pair of EPCs, located in parasegment 2 and 3, turn toward the midline of the embryo to accompany the tip of the heart, which later bends ventrally into the embryo. The third and the fourth pair of EPCs in PS 4 and 5 are shifted anteriorly in relation to the heart. This step is not based on migration but on the remodeling of the embryo during head involution, since the cells remain in their PS close to the likewise Eve positive anlagen of the DA1 muscle. The EPCs in PS 4 and 5 subsequently differentiate into the later wing heart progenitors, while all others become the classical EPCs and accompany the heart in a loosely associated fashion. At least from PS 4 to 12, all pairs of Eve positive cells (wing heart progenitors and classical EPCs) are interconnected by cytoplasmic extensions forming a rope ladder-like strand above the heart after dorsal closure at stage 16/17. This mode of contact between the cells persists in the wing heart progenitors in postembryonic stages and might be essential for proper relocation in the prepupae (Tögel, 2008).

Although the Drosophila wing hearts have been known for many years, their origin and development have remained unknown. This study provides the first developmental approach on these organs using in vivo time lapse imaging as well as genetic and immunohistochemical methods. It was found that the wing hearts develop from embryonic anlagen that consist of eight progenitor cells located anterior to the heart. Analysis of gene expression in these progenitors confirmed the hypothesis that the wing hearts originate from the cardiac mesoderm, but not from the contractile cardioblast lineage, as has been suggested based on anatomical data. Surprisingly, the embryonic anlagen derive from a particular subset of the well-known EPCs. EPCs arise in pairs in PS 2 to 12 from the dorsal progenitor P2, which divides asymmetrically into the founder of the dorsal oblique muscle 2 and the founder of the EPCs in a numb-dependent lineage decision. Additionally, a single EPC arises in PS 14. The subsequent differentiation of the founders into EPCs requires the activity of the transcription factors Zfh1 and Eve. This study shows that the EPCs located in PS 4 and 5 are relocated in relation to the heart during head involution at stage 14/15 of embryogenesis and subsequently differentiate into the wing heart progenitors. Until this step, no difference to the EPCs in the anterior and posterior PS could be detected. Like the classical EPCs, which remain close to the heart, the EPCs that give rise to the wing heart progenitors depend on factors involved in asymmetric cell division, e.g. Insc or Numb, and fail to differentiate in embryos mutant for zfh1 as well as in animals lacking mesodermal Eve. Loss of tinman expression is the only event that could be identified that discriminates between a classical EPC fate and the specification of wing heart progenitors. Consistently, ectopic expression of Tinman in the wing heart progenitors effectively represses their specification, probably by committing them to a classical EPC fate, indicating that Tinman plays a crucial role in the involved regulatory pathway (Tögel, 2008).

So far, the biological role of pericardial cells (PCs), and EPCs in particular, is not well understood. In the embryo, three populations of PCs arise in each segment, which are characterized by the expression of different combinations of genes (Odd positive PCs, Eve positive PCs, and Tinman positive PCs). During postembryonic stages, the number of PCs decreases, raising the question which population contributes to the final set of PCs in the adult and whether all PCs have the same function throughout development. Recent studies have shown that postembryonic PCs express Odd and Eve, a combination which is not observed in the embryo, and are dispensable for cardiac function. Genetic ablation of all larval PCs had no effect on heart rate, but increased sensitivity to toxic stress. In contrast, the specification of the correct number of embryonic PCs is crucial for normal heart function. Loss of mesodermal Eve during embryogenesis results in fewer larval pericardial cells, which causes a reduction in heart rate and lifespan. Conversely, hyperplasia of embryonic PCs has no effect on heart rate but causes decreased cardiac output. This was explained by an excess of Pericardin secreted by the PCs into the extracellular matrix enveloping the heart (Johnson, 2007). Taken together, embryonic PCs seem to influence cardiac development by e.g., secreting substances whereas postembryonic PCs function as nephrocytes. However, in this study, functional data is provided on a subset of embryonic EPCs, which differentiate into adult progenitors giving rise to a myogenic lineage. This represents a completely new function of PCs, raising the question whether EPCs might in general have myogenic potential and rather represent a population of adult progenitors, than PCs in a functional sense (Tögel, 2008).

The organogenesis of the wing hearts is a highly dynamic process, which includes distinct cellular interactions. At first, adjacent EPCs (including the wing heart progenitors) on either side of the embryo establish contact via cytoplasmic extensions. After dorsal closure of the embryo, interconnections are also formed between opposing EPCs resulting in a rope ladder-like strand above the heart. These interconnections are assumed to be needed to retain contact between the wing heart progenitors during the subsequent development. During larval stages, some of the wing heart progenitors establish a second contact to specific tracheal branches and proliferation starts. In the prepupa, a relocation event joins all wing heart progenitors in one large cluster. During this step, the progenitors are probably passively relocated in conjunction with the tracheal branches to which they are connected. Finally, the wing heart progenitors initiate active migration and form the mature wing hearts in the pupa. Considering the complexity of their development, it is proposed that wing hearts provide an ideal model for studying organogenesis on several different levels such as signaling, cell polarity, or path finding (Tögel, 2008).

Elimination of the embryonic progenitors by ectopic expression of tinman or by laser ablation causes the loss of wing hearts, which results in a specific wing phenotype in conjunction with flightlessness. In the identified phenotype, the delaminated epidermal cells are not cleared from the wings during wing maturation and bonding of the dorsal and ventral wing surfaces is omitted. Recently, it was reported that the epidermal cells transform into mobile fibroblasts and actively migrate out of the wings. However, in in vivo time-lapse studies migration of epidermal cells could not be observed during wing clearance. Conversely, their movements correlated with the periods of wing heart beating, indicating that they are passively transported by the hemolymph flow. One-sided ablation of mature wing hearts in pupae, confirms that wing hearts play a crucial physiological role in wing maturation, since the wing phenotype occurs only on the treated side, but in the same genetic background. In contrast, mutations in genes coding for proteins involved in cell adhesion, e.g. integrins, or in adhesion to the extra cellular matrix, cause a blistered wing phenotype. In the latter phenotype, the epidermal cells of the immature wings are not attached to their opposing cells or to the cuticle and the wing surfaces are separated during unfolding by the sudden influx of hemolymph. In contrast, in animals lacking wing hearts the wings resemble those of the wild-type shortly after unfolding. The epidermal cells also delaminate later from the cuticle, as indicated by their disarrayed pattern, but are not removed from the wings due to the missing hemolymph circulation and probably impede spatially the bonding of the dorsal and the ventral cuticle. Thus, the wings remain in their immature state and do not acquire aerodynamic properties, which accounts for the flightlessness. It is concluded that wing hearts are crucial for establishing proper wing morphology and functionality in Drosophila (Tögel, 2008).

Wing hearts occur in all winged insects, but differ considerably in their morphology. However, their function is highly conserved, since they all function as suction pumps that draw hemolymph from the wings. In the basal condition, the heart itself is directly connected to the scutellum and constitutes the pump. This connection was lost several times during evolution and other muscles, e.g. the separate wing hearts in Drosophila, were recruited to retain the function indicating a high selection pressure on wing circulation. It is suggested that this is due to the crucial role of wing hearts during wing maturation. Since proper wing morphogenesis is essential for flight ability, insect flight might not have been possible before the evolution of wing hearts (Tögel, 2008).


GENE STRUCTURE

cDNA clone length - 1450

Bases in 5' UTR -93

Exons - two

Bases in 3' UTR - 165


PROTEIN STRUCTURE

Amino Acids - 376

Structural Domains

The EVE homeodomain shares only 50% homology with Fushi tarazu, Engrailed and Antennapedia (Frasch, 1987).

even-skipped is a homeobox gene important in controlling segment patterning in the embryonic fruit fly. Its homeobox encodes a DNA binding domain which binds with similar affinities to two DNA consensus sequences, one AT-rich (ATTAAATTC), the other GC-rich. A crystallographic analysis of the Even-skipped homeodomain complexed to an AT-rich oligonucleotide at 2.0 A resolution is described. The structure reveals a novel arrangement of two homeodomains bound to one 10 bp DNA sequence in a tandem fashion. This arrangement suggests a mechanism for the homeoproteins' regulatory specificity. In addition, the functionally important residue Gln50 is observed in multiple conformations making direct and water-mediated hydrogen bonds with the DNA bases (Hirsch, 1995).

The minimal 57-residue repression domain is protein rich and contains a high perentage of hydrophobic amino acids (Han, 1993).


Interactive Fly, Drosophila even-skipped: Evolutionary Homologs | Transcriptional regulation | Post-transcriptional regulation | Targets of activity | Protein interactions | Developmental Biology | Effects of Mutation | References

date revised: 20 November 98

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