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 eve^1.27 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 eve^1.27 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 eve^1.27 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 eve^1.27 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).

Determinants of chromosome architecture: Insulator pairing in cis and in trans

To elucidate the principles governing insulator architectural functions, this study used two insulators, Homie and Nhomie, that flank the Drosophila even-skipped locus. It was shown that homologous insulator interactions in trans, between Homie on one homolog and Homie on the other, or between Nhomie on one homolog and Nhomie on the other, mediate transvection. Critically, these homologous insulator:insulator interactions are orientation-dependent. Consistent with a role in the alignment and pairing of homologs, self-pairing in trans is head-to-head. Head-to-head self-interactions in cis have been reported for other fly insulators, suggesting that this is a general principle of self-pairing. Homie and Nhomie not only pair with themselves, but with each other. Heterologous Homie-Nhomie interactions occur in cis, and they serve to delimit a looped chromosomal domain that contains the even skipped transcription unit and its associated enhancers. The topology of this loop is defined by the heterologous pairing properties of Homie and Nhomie. Instead of being head-to-head, which would generate a circular loop, Homie-Nhomie pairing is head-to-tail. Head-to-tail pairing in cis generates a stem-loop, a configuration much like that observed in classical lampbrush chromosomes. These pairing principles provide a mechanistic underpinning for the observed topologies within and between chromosomes (Fujioka, 2016).

The highly regular and reproducible physical organization of chromosomes in multicellular eukaryotes was recognized a century ago in cytological studies on the lampbrush chromosomes that are found in oocytes arrested at the diplotene phase of meiosis I. At this stage, homologous chromosomes are paired. The two homologs display a similar and reproducible architecture. It consists of a series of loops emanating from the main axis, that are arranged in pairs, one from each homolog. In between the loops are regions of more compacted chromatin. A similar physical organization is evident in insect polytene chromosomes. As with lampbrush chromosomes, the paired homologs are aligned in precise register. However, instead of one copy of each homolog, there are hundreds. While loops are not readily visible, each polytene segment has a unique pattern of bands and interbands that depends upon the underlying DNA sequence and chromosome structure (Fujioka, 2016).

Subsequent studies have shown that the key features of chromosome architecture evident in lampbrush and polytene chromosomes are also found in diploid somatic cells. One of these is the subdivision of the chromatin fiber into a series of loop domains. There are now many lines of evidence indicating that looping is a characteristic architectural feature. Biochemical evidence comes from chromosome conformation capture (3C) experiments, which show that distant sites come into contact with each other in a consistent pattern of topologically associating domains (TADs). While the first studies in mammals suggested that TADs have an average length of 1 Mb, subsequent experiments showed that the average is only about 180 kb. In flies, TADs are smaller, between 10-100 kb (Sexton, 2012; Hou, 2012). Neighboring TADs are separated from each other by boundaries that constrain both physical and regulatory interactions. In mammals and also in flies, these boundaries typically correspond to sequences bound by insulator proteins like CTCF (Fujioka, 2016).

That TAD boundaries correspond to insulators is consistent with the known properties of these elements. Insulators subdivide the chromosome into functionally autonomous regulatory domains. When interposed between enhancers or silencers and target promoters, insulators block regulatory interactions. They also have an architectural function in that they can bring distant chromosomal sequences together, and in the proper configuration can promote rather than restrict regulatory interactions. Moreover, insulators are known to mediate contacts between distant sequences (loop formation), and these physical contacts depend upon specific interactions between proteins bound to the insulators (Fujioka, 2016).

The notion that insulators are responsible for subdividing eukaryotic chromosomes into a series of looped domains raises questions about the rules governing loop formation in cis. One of these is the basis for partner choice. Is choice based simply on proximity, or is there an intrinsic partner preference? A second concerns the topology of the loop formed by interacting partners in cis. Do the partners interact to form a stem-loop-like structure, or does the interaction generate a circular loop ('circle-loop')? The answer to this question will depend upon whether there is an orientation dependence to the interactions between two heterologous insulators. In flies, homologs are typically paired in somatic cells, not just in cells that are polyploid. This means that the loop domains in each homolog must be aligned in precise register along their entire length. A plausible hypothesis is that both alignment and homolog pairing are mediated by insulator interactions in trans. If this is case, there are similar questions about the rules that govern trans interactions. Is there a partner preference in the interactions that mediate homolog pairing? Is there an orientation dependence, and if so, what is the topological outcome of the looped domains generated by insulator interactions in paired chromosomes in cis and in trans? (Fujioka, 2016).

This study has used insulators from the even skipped (eve) locus to address the questions posed above about the architecture of eukaryotic chromosomes. The eve domain spans 16 kb and is bordered upstream by the Nhomie (Neighbor of Homie) insulator and downstream by Homie (Homing insulator at eve). eve encodes a homeodomain transcription factor that is required initially for segmentation, and subsequently in the development of the CNS, muscles, and anal plate. It has a complex set of enhancers that activate expression at different stages and tissueS, and a Polycomb response element (PRE) that silences the gene in cells where it isn't needed. In early embryos, the stripe enhancers upstream (3+7, 2, late stripes) and downstream (4+6, 1, and 5) of the eve gene activate transcription in a pair-rule pattern. Later in development, around the time that germband retraction commences, mesodermal (Me) and neuronal (CNS) enhancers turn on eve expression in a subset of cells in each of these tissues. These late enhancers continue to function once germband retraction is complete, while another enhancer (APR) induces transcription in the presumptive anal plate. Located just upstream of eve is CG12134, while the TER94 gene is downstream. Unlike eve, both of these genes are ubiquitously expressed throughout much of embryogenesis (Fujioka, 2016).

The importance of insulators in organizing eukaryotic chromosomes has been recognized since their discovery in the 1980's. However, the principles underlying their architectural and genetic functions have not been fully elucidated. With this goal in mind, this study asked how these elements shape two critical architectural features of chromosomes. The first is homolog pairing. Homologs pair in flies from the blastoderm stage onward, and the consequent trans-interactions are important for proper gene regulation. The phenomenon of homolog pairing is not unique to Drosophila. Homologs are paired in lampbrush chromosomes of invertebrate and vertebrate oocytes. The second is the looped domain organization. Although there is now compelling evidence that insulators subdivide chromosomes into topologically independent looped domains (and that these domains play a central role in gene regulation), the topology of the loops is unknown. Moreover, while the loops must emanate from the main axis of the chromosome, the relationships between the loops, the insulators that delimit them, and the main chromosomal axis are not understood. As homolog pairing is more straightforward and the likely mechanism better documented, it is considered first (Fujioka, 2016).

Homolog pairing requires mechanisms for aligning homologs in precise register, and maintaining their stable association. While many schemes are imaginable, the simplest utilizes elements distributed along each homolog that have self-interaction specificity. Such a mechanism would be consistent with the persistence of local pairing and transvection in chromosomal rearrangements. It would also fit with studies on the pairing process. Self-association of pairing elements would locally align sequences in register, and ultimately link homologs together along their entire length. In this mechanism, self-association must be specific and also directional, namely head-to-head. This avoids the introduction of unresolvable loops and maximizes pairing for transvection (Fujioka, 2016).

In Drosophila, the homing of P-element transgenes, in which normally random insertion becomes targeted, suggested the ability of genomic elements to self-interact. Such a homing activity was found in the engrailed locus for a region that includes two PREs, and later studies showed that some insulators and a promoter region also possess homing activity. The self-interaction implied by homing suggests that these elements may facilitate homolog pairing. However, in contrast to PREs and promoters, insulators have consistently been found to engage in specific self-interactions. Thus, among the known elements in the fly genome, insulators are the best candidates to align homologs in register and maintain pairing. Moreover, genome-wide chromatin immunoprecipitation experiments (ChIPs) show that insulators are distributed at appropriate intervals along each chromosome (Fujioka, 2016).

A role in homolog pairing was first suggested by the discovery that the su(Hw) and Mcp insulators each can mediate regulatory interactions between transgenes inserted at distant sites. The Fab-7 insulator can also mediate long-range regulatory effects. Further evidence that self-association is characteristic of fly insulators came from insulator bypass experiments. These experiments showed that bypass is observed when an insulator is paired with itself, while heterologous combinations are less effective or don't give bypass. Moreover, self-pairing is, with few exceptions, head-to-head (Fujioka, 2016).

That insulators mediate homolog pairing through specific self-interactions is further supported by the current studies. Using a classical transvection assay, this study found that Homie-Homie and Nhomie-Nhomie combinations stimulate trans-regulatory interactions between enhancers on one homolog and a reporter on the other. Moreover, the parameters that favor transvection dovetail with those expected for a pairing mechanism based on insulator self-interactions in trans. First, the two insulators must be in the same orientation. When they are in opposite orientations, transvection is not enhanced (or enhancement is much weaker). Second, the enhancers and reporter must be located on the same side (centromere proximal or distal) of the insulators. In addition to transvection, Homie and Nhomie also engage in highly specific and directional distant regulatory interactions (Fujioka, 2016).

While there is compelling evidence that insulator self-interactions are responsible for homolog pairing, many issues remained unresolved. Perhaps the most important is the nature of the code used for self-recognition and orientation. The best hint comes from bypass experiments using multimerized binding sites for Su(Hw), dCTCF, or Zw5. Homologous multimer combinations give bypass, while heterologous combinations do not. However, bypass is observed for composite multimers when they are inserted in opposite orientations (e.g., Su(Hw) dCTCF >-< dCTCF Su(Hw)), but not the same orientation (e.g., Su(Hw) dCTCF >> Su(Hw) dCTCF). These findings argue that the identity and order of proteins bound to the insulator determine its self-association properties (Fujioka, 2016).

The first direct evidence that insulators generate loops came from 3C experiments on the mouse β-globin and the fly 87A7 heat shock loci. These studies suggested that physical interactions between adjacent insulators in cis could subdivide chromosomes into looped domains. Subsequent work has confirmed this conclusion (Rao, 2014). However, while these experiments demonstrate that cis insulator interactions generate loops, they provided no information about the topology of these loops, or how they are arranged (Fujioka, 2016).

Cis interactions could, a priori, be either head-to-head like self-association in trans, or head-to-tail. The consequences are quite different. Head-to-head interactions generate a circle-loop, while head-to-tail interactions generate a stem-loop. If heterologous insulators interact with only one specific partner, the circle-loop or the stem-loop will be linked to neighboring circles or stem-loops by loops without anchors. These unanchored loops would correspond to the main axis of the chromosome, and the circle-loops or stem-loops would then protrude from the main axis in a random orientation and at distances determined by the length and compaction of the unanchored loops (Fujioka, 2016).

On the other hand, if insulators in a chromosomal segment are able to interact with both of their neighbors, then the main axis of the chromosome in this region would be defined by the insulators. Quite different structures are predicted for head-to-head and head-to-tail interactions. Head-to-head would give a series of variably sized circle-loops linked together at their base by an array of interacting insulators. The base would correspond to the main axis of the chromosome, and each circle-loop would extend from one side of the main axis to the other. If the direction of coiling were always the same, this would give a structure resembling a helix anchored to a rod. If the direction of coiling were random, the structure would be more complicated and variable, since neighboring circle-loops could extend out from the main axis in either the same or the opposite direction (not illustrated). The loop-axis relationship would be more regular for head-to-tail insulator pairing in cis. Adjacent stem-loops would extend out from the main axis in opposite directions much like the lampbrush chromosomes formed when haploid sperm heads are injected into amphibian oocytes. This stem-loop organization would also fit with the radial loop model proposed by Laemmli and others for the first level of folding of metaphase chromosomes (Fujioka, 2016).

Since the current experiments show that Homie-Nhomie association is head-to-tail, the topology of the eve locus in vivo is a stem-loop, not a circle-loop. This finding raises a number of questions. Perhaps the most important is whether head-to-tail interactions are the rule rather than the exception. While the orientation dependence of homologous interactions has been extensively investigated, there have been no systematic studies on interactions between neighboring insulators. However, there are reasons to think that cis interactions are more likely head-to-tail than head-to-head. One is homolog pairing. As mentioned above, the circle-loops formed by head-to-head interactions can coil in either direction, either left-handed or right-handed. If coiling were random, then about half of the circle-loops on each homolog would be coiled in opposite directions. In this case, head-to-head pairing of homologous insulators in each homolog would generate a structure in which the circle-loops would point in opposite directions. This topology would not be compatible with transvection. Coiling of the circle-loops in the same direction on both homologs would permit interdigitation of one circle-loop inside the other; however, the chromatin fiber from the inside circle-loop would need to cross in on one side and out on the other. If the main axis of the chromosome in the paired region is defined by a series of interacting insulators in cis, then generating a topology permissive for transvection (not illustrated) would require coiling of successive homologous circle-loops on each homolog in the same direction, one inside the other (Fujioka, 2016).

These topological issues aren't encountered when heterologous insulator interactions in cis are head-to-tail. Head-to-head pairing of homologous insulators in trans would bring regulatory elements and genes in the two homologous stem-loops into close proximity. Alignment of the two homologs is straightforward whether or not the main axis of the chromosome is defined by a series of interacting insulators. Alternating loops extending upwards and downwards from the main axis of the chromosome would be directly aligned when homologous insulators pair head-to-head in trans (Fujioka, 2016).

While the requirements for aligning and pairing homologs would appear to favor stem-loops between heterologous insulators in cis in flies, homolog pairing does not occur in vertebrates except in specialized cell types. This could mean that circle-loops formed by cis interactions between heterologous insulators are permissible in vertebrate chromosomes. However, even in organisms in which homolog pairing doesn't occur in somatic cells, it seems possible that cis-pairing interactions more commonly generate stem-loops than circle-loops (see Chromosome architecture: pairing head-to-head and head-to-tail in cis). First, following DNA replication and before mitosis (during the S and G2 phases of the cell cycle), sister chromatids are aligned. Maintaining this alignment may facilitate epigenetic mechanisms that template chromatin structures from one cellular generation to the next, such as the copying of histone modifications onto both daughter chromosomes. The simpler topology of stem-loops could facilitate this sister chromatid pairing, as well as their separation during mitosis. Second, recent studies on the relationship between loop domains and CTCF insulators showed that in more than 90% of the cases, the CTCF binding sites on opposite ends of a loop are in opposite orientation. Thus, assuming that the orientation of pairing is such that the CTCF sites are aligned in parallel to form the loop, pairing between CTCF insulators at the ends of the loop would generate stem-loops rather than circle-loops. If insulators form the main axis of the chromosome, there is an additional explanation for such a bias. Head-to-head pairing in cis could generate a series of circular loops that extend out from the same side of the main axis. This configuration would be favorable for crosstalk between regulatory elements and genes in adjacent loops. By contrast, head-to-tail pairing, where adjacent stem-loops extend out in opposite directions, would disfavor crosstalk, helping to explain how insulators block enhancer-promoter communication between adjacent loops (Fujioka, 2016).

Multimodal transcriptional control of pattern formation in embryonic development

Predicting how interactions between transcription factors and regulatory DNA sequence dictate rates of transcription and, ultimately, drive developmental outcomes remains an open challenge in physical biology. Using stripe 2 of the even-skipped gene in Drosophila embryos as a case study, this study dissected the regulatory forces underpinning a key step along the developmental decision-making cascade: the generation of cytoplasmic mRNA patterns via the control of transcription in individual cells. Using live imaging and computational approaches, it was found that the transcriptional burst frequency is modulated across the stripe to control the mRNA production rate. However, it was discovered that bursting alone cannot quantitatively recapitulate the formation of the stripe and that control of the window of time over which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical modeling revealed that these regulatory strategies (bursting and the time window) respond in different ways to input transcription factor concentrations, suggesting that the stripe is shaped by the interplay of 2 distinct underlying molecular processes (Lammers, 2019).

This work investigated how single-cell transcriptional activity leads to the formation of stripe 2 of the widely studied even-skipped (eve) gene in the developing fruit fly embryo. Previous work has established that the stripe is formed through the interplay of transcriptional activators and repressors. In addition, recent studies have indicated that the eve stripe mRNA profiles are graded and highly reproducible, suggesting that the detailed cytoplasmic distribution of mRNA that makes these stripes is key to the transmission of spatial information along the gene regulatory network that drives Drosophila development and reinforcing the need to develop models of gene regulation capable of connecting quantitative variations in input transcription factor patterns to graded output rates of transcription. To do this, live imaging was combined with theoretical modeling to study transcription at the single-cell level in real time, seeking a quantitative connection between the spatiotemporal variations in input transcription factor concentrations, the control of eve transcription, and the formation of cytoplasmic patterns of mRNA (Lammers, 2019).

All three regulatory strategies outlined in an accompanying figure (see Multiple modes of pattern formation by single-cell transcriptional activity) quantitatively contribute to the formation of eve stripe 2. First, a smaller fraction of nuclei become active and engage in transcription in the periphery of the stripe than in the center, although this regulation of the fraction of active nuclei makes only a minor contribution to stripe formation. Second, consistent with previous studies, the rate of mRNA production is significantly elevated in the center of the stripe. Strikingly, however, it was discovered that this analog control of the transcription rate is insufficient to quantitatively recapitulate the cytoplasmic mRNA stripe pattern. In addition to the control of the rate of mRNA production among nuclei, a pronounced regulation of the window of time during which eve loci were engaged in transcription across the stripe was observed, with those in the stripe center expressing for approximately 3 times longer than those in the flanks. While it is widely appreciated that genes are transcriptionally competent for limited windows of time during development, this study found that-in the case of eve stripe 2-this binary transcriptionally engaged/disengaged logic is not merely a necessary precondition for pattern formation-it is the primary driver thereof. Thus, it is concluded that the regulation of eve stripe 2 is multimodal in nature, with contributions from three distinct regulatory strategies. Nonetheless, stripe formation can be quantitatively explained almost entirely through the interplay between 2 distinct control strategies: binary control of the duration of transcriptional engagement and control of the mean rate of transcription (Lammers, 2019).

Building upon this result, computational approaches were developed to uncover the mechanistic underpinning of each regulatory strategy. A compound-state hidden Markov model (cpHMM) was used to uncover variations in transcriptional bursting dynamics in individual nuclei across space and time. Consistent with previous results, transcription factors control the rate of transcription by altering the frequency of transcriptional bursts. In addition, logistic regressions to correlate eve stripe 2 transcriptional dynamics with changes in input transcription factor concentrations. This analysis revealed that the transcriptional time window adheres to different regulatory logic than transcriptional bursting: While repressor levels alone were sufficient to explain the early silencing of nuclei in the anterior and posterior stripe flanks, the control of bursting among transcriptionally engaged nuclei depends upon the input concentrations of both activators and repressors. Thus, these findings point to the presence of two distinct regulatory mechanisms that control transcription and gene expression patterns in early development, showcasing the potential for theoretical modeling and biological numeracy to yield additional biological insights when coupled with precise and quantitative experimental observation (Lammers, 2019).

The role of Even-skipped in Drosophila larval somatosensory circuit assembly

Proper somatosensory circuit assembly is critical for processing somatosensory stimuli and for responding accordingly. In comparison to other sensory circuits (e.g., olfactory and visual), somatosensory circuits have unique anatomy and function. However, understanding of somatosensory circuit development lags far behind that of other sensory systems. For example, there are few identified transcription factors required for integration of interneurons into functional somatosensory circuits. This study examined one type of somatosensory interneuron, Even-skipped expressing Laterally placed interneurons (ELs) of the Drosophila larval nerve cord. Even-skipped (Eve) is a highly conserved, homeodomain transcription factor known to play a role in cell fate specification and neuronal axon guidance. Because marker genes are often functionally important in the cell types they define, this study deleted eve specifically from EL interneurons. On the cell biological level, using single neuron labeling, this study found eve plays several previously undescribed roles in refinement of neuron morphogenesis. Eve suppresses aberrant neurite branching, promotes axon elongation, and regulates dorsal-ventral dendrite position. On the circuit level, using optogenetics, calcium imaging, and behavioral analysis, it was found that eve is required in EL interneurons for the normal encoding of somatosensory stimuli and for normal mapping of outputs to behavior. It is concluded that eve coordinately regulates multiple aspects of EL interneuron morphogenesis and is critically required to properly integrate EL interneurons into somatosensory circuits. These data shed light on the genetic regulation of somatosensory circuit assembly (Marshall, 2022).

This study shows that eve expression is required for positioning EL interneuron neurites in all three axes (i.e., medial-lateral, anterior-posterior, and dorsal-ventral). In Drosophila, each axis is patterned by a separate ligand/receptor signaling system. However, how individual interneurons read and interpret each signal is not well understood. The data suggest eve is important for ELs to simultaneously read and/or interpret multiple ligand gradients simultaneously (Marshall, 2022).

Generally, eve is considered a cell fate determinant. For example, in mouse V0v interneurons, evx1 represses expression of en1, a marker of V1 interneuron identities. In V0v interneurons that lack evx1, en1 expression is derepressed and take on V1-like axonal projections. Similar fate changes are seen in Drosophila and C. elegans motor neurons when eve is disrupted. The current data are more consistent with the idea that eve plays a role in the refinement of EL morphogenesis. In support for the morphogenetic refinement model is, first, in wild-type, there are no neurons with morphology that matches the morphology of Eve^- ELs, as would be expected by a cell fate switching model. Second, there are no obvious large-scale changes in gene expression, which are typically associated with cell fate changes. Third, eve expression in ELs is not playing a role in initial morphogenesis (Marshall, 2022).

Both Eve^- and Eve⁺ ELs cross the midline at embryonic stage 15. Thus, eve expression is either dispensable for initial morphogenesis, or in EL eve mutants there is an undetectable pulse of early eve expression in ELs. But, no Eve protein expression was found in ELs in EL eve mutants at any stage of development. In later stage embryos and larvae, morphologic defects were observed in Eve^- ELs. This raises the possibility that, in general, eve genes may play a later role in morphogenesis. This is consistent with the observation that, in mouse V0v interneurons, there is early evx1 expression and later evx2 expression. However, the later role of evx2 is unknown (Marshall, 2022).

In general, eve genes are known to regulate axon morphogenesis. This study shows that late-born Eve^- ELs have axonal defects. Notably, the role of eve in dendrite morphogenesis is extremely poorly characterized. The distinction between dendrite and axon is important because these two compartments carry out different functions. Further, in Drosophila, interneuron axons and dendrites are structurally different. Dendrites are often highly branched, and lack mitochondria and postsynaptic machinery. Whereas, axon terminals (boutons) are full of mitochondria, pools of synaptic vesicles, microtubules, and vesicle release sites, each part of the arbor (axon or dendrite) can be independently controlled by different transcription factors. For example, in Drosophila sensory neurons, the transcription factors Knot and Cut specifically regulate dendrite morphogenesis, but not axonal morphology. Thus, in Drosophila, axon and dendrite morphology can be controlled as independent modules. This study has shown that in addition to regulating axon morphology, eve regulates dorsal-ventral dendrite positioning. eve expression is also required for dendrite morphogenesis in RP motor neurons. Taken together, these data show that eve coordinately regulates multiple aspects of neuronal morphogenesis, and that coordinate control may be a widely-occurring role for neuronal eve (Marshall, 2022).

Neuronal circuits are functional units of the nervous system. Sensorimotor circuits, specifically, transform somatosensory stimuli into motor output. Therefore, functional assays are required for the study of somatosensory circuit assembly. However, because the circuit context of individual interneurons is not well characterized, often researchers rely on anatomic assays to infer changes at the circuit level. One reason an anatomic approach can be flawed is the existence of compensatory mechanisms that allow for relatively normal circuit wiring despite changes in neuron morphology. This study links defects in neuronal morphology to changes in circuit function, thereby explicitly demonstrating the role of eve expression in somatosensory circuit assembly (Marshall, 2022).

It was shown that eve is required for somatosensory stimulus encoding by ELs. Based on known connectivity of ELs with other neurons, it is inferred that in ELs, eve is required for the formation of at least four types of functional input synapses: those from vibration (chordotonal) sensory neurons to early-born ELs, from vibration-sensitive interneurons (Basins) to early-born ELs, from proprioceptive sensory neurons to late-born ELs, and from proprioceptive-sensitive interneurons (Jaams) to late-born ELs. The likely cell biological underpinning, at least for late-born ELs, is that axons from input sensory neurons are not in close enough proximity to make synaptic contact with Eve^- ELs. Because of technical limitations, dendrite morphology of early-born ELs could not be visualized (Marshall, 2022).

In the Drosophila nerve cord, there is unidirectional compensatory growth from interneurons to genetically misplaced sensory neurons. Thus, Drosophila sensory neuron-to-interneuron wiring can be robust to morphologic alterations to circuit components. The observation that sensory neurons do not grow to reach mispositioned Eve^- EL dendrites raises two possibilities: (1) in this system, compensatory growth is unidirectional (i.e., interneurons grow to misplaced sensory neurons, but not vice versa); and (2) alternatively, compensatory growth is bidirectional, however, eve expression is required for this process. Future experiments will be needed to distinguish between these models (Marshall, 2022).

The data show Eve^- EL output synapses are functional, but remapped. Spontaneously-occurring crawling behavior is disrupted in EL eve mutants, and that this disruption is significantly worse than in larvae which lack EL neurons altogether. This could be explained by requirement for ELs during early circuit development (e.g., acting as a scaffold for normal axonal pathfinding for other neurons). Alternatively, mature Eve^- ELs could exert a dominant negative effect at the level of circuit function. The latter idea is favored because it is consistent with optogenetic experiments, and anatomic data. In controls, EL output synapses are excluded from many zones of the neuropile including the dorsal lateral zone, which houses the dendrites of dorsally-projecting motor neurons. However, Eve^- ELs are likely to form output synapses in this region. This specific re-distribution of output synapses is notable because it raises the possibility that Eve^- ELs output synapses (ELs are excitatory) could be directly re-mapped to dorsal motor neurons. Such a re-mapping could explain the novel behavioral phenotype, dorsal body bending phenotype seen on optogenetic activation of Eve^- ELs. Regardless of the exact anatomic changes, the data show that output synapses of Eve^- ELs are functional, but are functionally re-mapped to new output circuits (Marshall, 2022).

In conclusion, this study has provided an updated understanding of the role of eve expression in neurons. The data provide understanding of the role of neuronal eve at the levels of circuit physiology and animal behavior. Further they provide insight into the genetic logic of somatosensory circuit assembly, demonstrating that multiple terminal neuronal features can be coordinately regulated by the activity of a single postmitotic transcription factor. Finally, the data raises new questions about the role of eve expression in other neuron types and enable future experimental inquiry into somatosensory circuit assembly in Drosophila (Marshall, 2022).

GENE STRUCTURE

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).

date revised: 22 May 2023

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