fushi tarazu

Targets of Activity

In the even-numbered parasegments of the Drosophila embryo, expression of the fushi tarazu gene is necessary for transcription of engrailed (en). Yet of those cells expressing ftz+ in a stripe, only the anteriormost come to express en. One explanation is that the level of ftz+ might be graded across the stripe: in order to express en, it would be sufficient for cells to exceed a threshold concentration of Ftz protein. Photographs and microspectrophotometry were used to measure differences in Ftz antigen concentration; no gradient within the Ftz stripe is observed. Rather, the stripe appears to contain cells with similar amounts of antigen plus a few weakly staining cells that are usually at the posterior edge. Further, varying the amount of Ftz protein about fourfold has no effect on en expression. Finally, embryos lacking the even-skipped gene have normal levels of Ftz but do not express en. This result may suggest that wild-type levels of Ftz are necessary but not sufficient to activate en expression. Alternatively, in the absence of Eve, repression by the pair-rule gene odd-skipped (odd) may block the ability of Ftz to activate even-numbered en stripes. This idea is supported by the observation that the Ftz-dependent en stripes are expressed in eve/odd double mutants. These observations appear to rule out the threshold hypothesis (Lawrence, 1998).

Recent advances have shed new light on how the Q50 homeoproteins act in Drosophila. Q50 homeoproteins all contain a glutamine residue at position 50 of the homeodomain. These transcription factors, encoded by the segmentation genes even-skipped, fushi-tarazu and engrailed, have remarkably similar and promiscuous DNA-binding specificities in vitro, yet they each specify distinct developmental fates in vivo. One current model suggests that because the Q50 homeoproteins have distinct biological functions, they must each regulate different target genes. According to this 'co-selective binding' model, significant binding of Q50 homeoproteins to functional DNA elements in vivo would be dependent upon cooperative interactions with other transcription factors (cofactors). If the Q50 homeoproteins each interact differently with cofactors, they could be selectively targeted to unique, limited subsets of their in vitro recognition sites and thus control different genes. Thus cofactors would selectively target different Q50 homeoproteins to bind to different DNA sites. However, a variety of experiments question this model. Molecular and genetic experiments suggest that the Q50 homeoproteins do not regulate very distinct sets of genes. Instead, they mostly control the expression of a large number of shared targets. The distinct morphogenic properties of the various Q50 homeoproteins may result principally from the different manners in which they either activate or repress these common targets. Further, in vivo binding studies indicate that at least two Q50 homeoproteins, Eve and Ftz, have very broad and similar DNA-binding specificities in embryos, a result that is inconsistent with the 'co-selective binding' model. Based on these and other data, it is suggested that Q50 homeoproteins bind many of their recognition sites without the aid of cofactors. In this 'widespread binding' model, cofactors act mainly by helping to distinguish the way in which homeoproteins regulate targets to which they are already bound (Biggin, 1997).

The exact positioning of neuroblasts in the neuroectodermal region that gives rise to the CNS is regulated by a combination of pair-rule genes. Proneural achaete-scute complex genes are controlled by combinations of axis-patterning genes through a common intergenic control region. Specifically, in fushi tarazu mutants, the fourth row of achaete clusters is removed in each segment (Skeath, 1992).

fushi tarazu, and especially engrailed, appear to act as transcriptional activating factors of abdominal-A. abd-A is normally expressed in parasegments 7 to 13. The initial distribution of the product is approximately uniform within this domain, but the subsequent elaboration of the expression pattern results in differences between, as well as within, parasegments. The establishment of the original abd-A expression domain is independent of any of these genes, but most of them are required for the subsequent elaboration of abd-A expression within the domain (Macias, 1994).

Within an engrailed enhancer, adjacent and conserved binding sites for the Fushi tarazu protein and a cofactor are each necessary, and together sufficient, for transcriptional activation. Footprinting shows that the cofactor site can be bound specifically by Ftz-F1, a member of the nuclear receptor superfamily. Ftz-F1 and the Fushi tarazu homeodomain bind the sites with 4- to 8-fold cooperativity, suggesting that direct contact between the two proteins may contribute to target recognition. Even parasegmental reporter expression is dependent on Fushi tarazu and maternal Ftz-F1, suggesting that these two proteins are indeed the factors that act upon the two sites in embryos. The two adjacent binding sites are also required for continued activity of the engrailed enhancer after Fushi tarazu protein is no longer detectable, including the period when both engrailed and the enhancer become dependent upon wingless. A separate negative regulatory element exists that apparently responds to odd-skipped (Florence, 1997).

Fushi tarazu and Even-skipped act through particular key control regions of the homeotic gene Ultrabithorax to generate ftz- or eve-like stripe patterns. FTZ protein acts directly as a transcriptional activator of Ubx. Its activity outside the Ubx expression domain is suppressed by Hunchback, a repressor of Ubx. Some DNA binding sites for FTZ protein are adjacent to binding sites for HB protein, while others overlap the HB sites. FTZ protein competes with HB protein for DNA binding and/or for transcriptional activation. This competition mechanism results in a sharp anterior expression boundary. Direct activation of homeotic gene control regions by FTZ (or EVE) protein may be a regulatory step which is generally used to align expression of homeotic genes with parasegmental boundaries (Muller, 1992).

The 18 wheeler stripes require pair rule gene function for their establishment and later become dependent upon segment-polarity gene function for their maintenance. The establishment of the first even-numbered stripes of 18w depends on the function of the pair-rule gene fushi tarazu; the appearance of odd-numbered stripes depends on the function of even skipped. In wingless and hedgehog mutants, 18w expression rapidly declines as the germband reaches full extension, with the exception of small regions including a subset of neuroblasts along the midline. In engrailed mutant embryos, although general loss of expression is consistently observed, the loss of expression is not as striking as in wg and hh mutants. In naked and patched mutant embryos, the 18w stripes expand to about twice their normal width with occasional broadening in naked mutants such that the space between stripes is obliterated (Chiang, 1995).

To understand the nature of the regulatory signals impinging on the second promoter of the Antennapedia gene (Antp P2), analysis of its expression in mutants and in inhibitory drug injected embryos has been carried out. Products of the zygotically-active segmentation genes ftz, hb, Kr, gt and kni act as activators or repressors of Antp P2 in a combinatorial fashion. The timing of these events, and their positive versus negative nature, is critical for generating the expression patterns normal for Antp (Riley, 1991).

Tenascin major, the extracellular protein related to vertebrate Tenascin, is under the control of fushi tarazu and even-skipped(Baumgartner, 1994).

The Drosophila melanogaster gene teashirt (tsh) is essential for normal segment identity in the embryonic thorax and abdomen. Null alleles of tsh reduce the size of all trunk segments. During the cellular blastoderm stage, tsh is expressed in a broad central domain that expands into parasegments 3 to 13. A deletion 3' to the tsh transcription unit causes the loss of tsh early expression in the even-numbered parasegments, and the corresponding larval cuticular patterns are disrupted. tsh function in the odd-numbered parasegments in these mutants is normal by both criteria. The in vivo activities of genomic fragments from the deleted region were tested in transgenic embryos. A 2.0 kb enhancer from the 3' region (thousands of base pairs from the tsh 3' terminus) acts mainly in the even-numbered parasegments and is dependent on fushi tarazu (ftz) activity. Ftz protein binds in vitro to four distinct sequences in a 220 bp sub-fragment; these and neighboring sequences are conserved in the equivalent enhancer isolated from Drosophila virilis. Tsh protein produced under the control of the 220 bp enhancer partially rescues a null tsh mutation, with its strongest effect in the even-numbered parasegments. Mutation of the Ftz binding sites partially abrogates the capacity for rescue. These results suggest a composite mechanism for the regulation of tsh, with different activators such as ftz contributing to the overall pattern of expression of this key regulator (Core, 1997).

Expression of a serotonin receptor (5-HT2) occurs as early as 3 hr into Drosophila embryogenesis. This early embryonic expression is surprisingly well organized in a seven-stripe pattern that appears at the cellular blastoderm stage. In addition, this pattern is in phase with that of the even-parasegment-expressed pair-rule gene fushi-tarazu and is similarly modified by mutations affecting segmentation genes. Simultaneously with this pair-rule expression, the complete machinery of serotonin synthesis is present and leads to a peak of ligand concomitant with a peak of 5-HT2-specific receptor sites in blastoderm embryos (Colas, 1995).

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 early expression of the Drosophila segment polarity gene gooseberry is under the control of the pair-rule genes. A 514-bp enhancer, -5.3 to -4.8 kb interval (called fragment IV), has been identified that reproduces the early gsb expression pattern in transgenic flies. The transcription factor Paired (Prd) is the main activator of this enhancer in all parasegments of the embryo. It binds to paired-and homeodomain-binding sites, which are segregated on the enhancer. Using site-directed mutagenesis, sites critical for Prd activity have been identified. Negative regulation of this enhancer is mediated by the Even-skipped protein (Eve) in the odd-numbered parasegments and by the combination of Fushi-tarazu (Ftz) and Odd-skipped proteins in the even-numbered parasegments. The organization of the Prd-binding sites, as well as the necessity for intact DNA binding sites for both the paired- and homeodomain-binding sites, suggests a molecular model whereby the two DNA-binding domains of the Prd protein cooperate in transcriptional activation of gsb. This positive activity appears to be in competition with Eve and Ftz on Prd homeodomain-binding sites (Bouchard, 2000).

The establishment of the posterior border of gsb in the even-numbered parasegments requires an efficient mechanism of repression, since Prd is present throughout all all even-numbered parasegments at the time of gsb initiation. The expression of transgenic line IV-LacZ is derepressed in ftz and odd mutant embryos. Moreover, Prd activity is directly competed by Ftz and Odd in tissue cultured cells. These data identify Ftz and Odd proteins as being responsible for the establishment of gsb expression borders in the even-numbered parasegments (Bouchard, 2000).

In the genetic analysis of fragment IV, it was observed that neither ftz nor odd mutant embryos show a complete derepression in the even-numbered parasegments. An odd embryo shows an anterior widening of the odd-numbered gsb stripes, suggesting a more important role for Odd in the region of low Ftz concentration. This result also indicates that Ftz is a potent repressor in the embryo since it is still able to partially repress gsb, even though Prd levels remain high in the central part of the even-numbered parasegments in an odd embryo, as opposed to its gradual repression in this region in a wild-type background. In a ftz embryo, a posterior widening of two to three cells in the even-numbered stripes is observed. This limited expansion can be explained by the action of Odd in the posteriormost portion of the parasegment combined with the fact that Prd is fading exclusively in this region in a ftz mutant embryo. The true repressor effect of Odd on fragment IV is possibly masked in these genetic experiments by the fact that, in an odd mutant embryo, Ftz is not properly repressed in the posterior portion of the parasegment. In such an embryo, Ftz is thus compensating for the absence of Odd. At the molecular level, the mechanism of action of Odd is unclear. It is possible that Odd binds directly to fragment IV via its zinc-finger domain, but this interaction would have been missed due to insufficient binding activity in vitro. Alternatively, Odd could bind Prd via protein-protein interaction and thereby interfere with its transactivation properties (Bouchard, 2000).

Homeodomain proteins are DNA-binding transcription factors that control major developmental patterning events. Although DNA binding is mediated by the homeodomain, interactions with other transcription factors play an unusually important role in the selection and regulation of target genes. A major question in the field is whether these cofactor interactions select target genes by modulating DNA binding site specificity (selective binding model), transcriptional activity (activity regulation model) or both. A related issue is whether the number of target genes bound and regulated is a small or large percentage of genes in the genome. These issues have been addressed using a chimeric protein that contains the strong activation domain of the viral VP16 protein fused to the Drosophila homeodomain-containing protein Fushi tarazu. Genes previously thought not to be direct targets of Ftz remain unaffected by FtzVP16. Addition of the VP16 activation domain to Ftz does, however, allow it to regulate previously identified target genes at times and in regions that Ftz alone cannot. It also changes Ftz into an activator of two genes that it normally represses. Taken together, the results suggest that Ftz binds and regulates a relatively limited number of target genes, and that cofactors affect target gene specificity primarily by controlling binding site selection (Nasiadka, 2000).

Activity regulation plays an important role in Ftz function, but this role is mainly to refine the temporal and spatial windows of target gene regulation and to modulate levels of expression. This conclusion is supported by the following observations. Five of the genes tested (ftz, odd, slp, en and wg) could be activated ectopically by FtzVP16 in regions and at times that Ftz could not induce a response. This shows that Ftz has the ability to bind to these promoters, but that it must be bound in an inactive state. For Ftz to function in these cells, it probably requires the addition of requisite cofactors, the removal of repressors or both. For the five genes listed above, the VP16 activation domain is able to overcome some of these limitations. The regulation by Ftz of en is a good example of this type of temporal and spatial refinement in activity. Results with FtzVP16 show that Ftz can bind to the en promoter during the time that ftz autoregulation and odd activation are well under way. However, the ability of Ftz to activate en is normally delayed until cellularization is completed (approx. 45 minutes). This delay may be necessary to allow other en regulators to resolve into the complex patterns of expression that are required for en to initiate in 14 narrow stripes. Like most homeodomain proteins, Ftz has the ability to function as both a transcriptional activator and repressor. This dual capacity suggests a requirement for distinct activity-regulating cofactors. However, differential activity can also be achieved, at least in part, by binding to different sites on different genes. For example, the response elements required for repression of the Distalless gene by Ubx and activation by Dfd are different. This also appears to be the case for activation of the dpp gene by Ubx and its repression by Abd-A. The different cofactors that help recruit the three proteins to these sites may also be partly responsible for their differences in transcriptional activity. For example, Exd is thought to generally work as a coactivator, acting in part to alter Hox protein conformation. Other factors bound in the vicinity of these sites are also likely to play a major role in activity regulation (Nasiadka, 2000).

In addition to showing that positively acting cofactors are important for Ftz specificity, these data implicate the actions of powerful negative regulators that limit the gene's temporal and spatial domains of activity. The strength and diversity of these negative regulators were emphasized by their ability to suppress the actions of the fused VP16 activation domain despite its previously reported reputation of strength and autonomy. It may be the low DNA binding specificity of the homeodomain that has necessitated this need for diverse mechanisms of repression, since low DNA specificity provides the potential to regulate a large number of inappropriate target genes. Indeed, a rapidly growing number of homeodomain proteins have been shown to be capable of functioning as oncogenes or proto-oncogenes, and oncogenicity can be conferred by fusions to other transcriptional activators. Further studies will be required to identify many of the cofactors and inhibitors that modulate Ftz activity and to determine how they do so (Nasiadka, 2000).

Target selectivity of Bicoid and Ftz

Described here are experiments to compare the activities of two Drosophila homeodomain proteins, Bicoid (Bcd) and an altered-specificity mutant of Fushi tarazu, Ftz(Q50K). Although the homeodomains of these proteins share a virtually indistinguishable ability to recognize a consensus Bcd site, only Bcd can activate transcription from natural enhancer elements when assayed in both yeast and Drosophila Schneider S2 cells. Analysis of chimeric proteins suggests that both the homeodomain of Bcd and sequences outside the homeodomain contribute to its ability to recognize natural enhancer elements. Unlike the Bcd homeodomain, the Ftz(Q50K) homeodomain fails to recognize nonconsensus sites found in natural enhancer elements. The defect of a chimeric protein containing the homeodomain of Ftz(Q50K) in place of that of Bcd can be preferentially restored by converting the nonconsensus sites in natural enhancer elements to consensus sites. These experiments suggest that the biological specificity of Bcd is determined by combinatorial contributions of two important mechanisms: the nonconsensus site recognition function conferred by the homeodomain and the cooperativity function conferred primarily by sequences outside the homeodomain. A systematic comparison of different assay methods and enhancer elements further suggests a fluid nature of the requirements for these two Bcd functions in target selection (Zhao, 2000).

The two K50 homeodomain proteins, Bcd and Ftz(Q50K), which have similar affinities to a consensus TAATCC site, exhibit distinct abilities in mediating transcriptional activation from natural enhancer elements. This observation exemplifies a puzzle underlying target selection by homeodomain proteins: why do homeodomain proteins behave differently in vivo while sharing similar or identical DNA binding specificities? It is suggested that the recognition of nonconsensus sites represents an essential biochemical function that helps define biological specificity. This idea is supported by experiments demonstrating that the activity of LexA-Bcd-Ftz(Q50K)HD, which contains the Ftz(Q50K) homeodomain and fails to bind to nonconsensus sites, can be preferentially restored by converting the natural nonconsensus sites to consensus sites. Nonconsensus sites are also found in the hb enhancer elements from other fly species. Previous studies have shown that efficient activation by homeodomain proteins requires a minimal number of recognition sites, reflecting their intrinsically weak properties. Thus, nonconsensus sites found in natural enhancers, depending on their architectures (e.g., number and type of sites), are expected to either merely modulate transcription levels or act as specificity-defining elements (Zhao, 2000).

Because of their critical role in mediating Bcd function, it is important to understand how nonconsensus sites are recognized by the Bcd homeodomain. Chemical-footprint experiments with the consensus site A1 and the nonconsensus site X1 suggest that the Bcd homeodomain can establish different sets of contacts with different recognition sequences. The experiments further suggest that Arg 54 of the Bcd homeodomain makes a base-specific contact with the fourth-position guanine (i.e.,TAAGCT, shown underlined) unique to X1. In the Ftz(Q50K) homeodomain, the 54th position contains methionine. However, an arginine residue artificially introduced in the 54th position of Ftz(Q50K) fails to confer an X1 recognition ability on the protein. It is suggested that both the homeodomain framework and specific residues play important roles in nonconsensus-site recognition. In this context, it is interesting to note that complexes containing Ftz(Q50K) and Bcd homeodomains exhibit slightly different mobilities in electrophoresis. The analysis of several other natural K50 homeodomains further reveals that the ability to recognize all tested nonconsensus sites is unique to the Bcd homeodomain. It is proposed that the nonconsensus site recognition function of the Bcd homeodomain is a noncoincidental property that defines a unique biological specificity for Bcd (Zhao, 2000).

The present study also further underscores the importance of protein-protein interaction between Bcd molecules in natural-target selection. Such a protein interaction function, which is conferred by Bcd sequences outside its homeodomain, is responsible primarily for its cooperative DNA binding activity. Interestingly, the hb and kni enhancer elements used in this study exhibit different requirements for the protein interaction function. In particular, Ftz-BcdHD, which contains the Bcd homeodomain in the framework of Ftz, can efficiently activate transcription from the hb enhancer element while it is virtually inactive on the kni enhancer element. It is proposed that a residual cooperativity function conferred by the Bcd homeodomain, while insufficient on the kni enhancer element, contributes to the chimeric protein's ability to recognize the hb enhancer element. It is noted that the hb and kni enhancer elements have architectural differences in both Bcd site composition and alignments. The hb enhancer element contains three dispersed perfect TAATCC consensus sites, in addition to at least three centrally located, tightly linked nonconsensus sites. In contrast, the kni enhancer element contains symmetrically arranged and tightly linked sites that do not match the TAATCC consensus. Exactly how these architectural features determine the different requirements for Bcd functions remains to be determined (Zhao, 2000).

The results suggest that both the cooperativity and nonconsensus site recognition functions of Bcd contribute combinatorially to target selection. Interestingly, the degree of reliance on these two functions can be influenced not only by enhancer architecture but also by the host factor(s). In particular, Bcd-Ftz(Q50K)HD-VP16 can activate transcription from the kni enhancer elements in Schneider cells but not in yeast. This difference is unlikely to be due to the reporter gene status, because this protein fails to activate the kni-lacZ reporter gene in yeast regardless of whether it is integrated or carried on a replicating plasmid. It is possible that a factor(s) present in Schneider cells but absent from yeast can influence the activity of this derivative on the kni enhancer element (but not on the hb enhancer element). Although a cofactor for Bcd has also been proposed previously, its identity remains elusive; interestingly, a recent study suggests that Bcd activity can be potentiated modestly by the Drosophila protein Chip. A systematic comparison of different assay systems also reveals that, in many instances, dependence on Bcd functions is reduced on reporter genes carried on plasmids, presumably because they are more accessible to activators than are integrated reporters. For example, Ftz-BcdHD-VP16 shows a higher relative activity on plasmid reporters containing the hb, kni, and kni(6A) enhancer elements than on integrated reporters. Similarly, Bcd-Ftz(Q50K)HD-VP16 has a higher relative activity on hb(6A)-lacZ and kni(6A)-lacZ plasmid reporters than on the integrated reporters. Together, these results illustrate a fluid nature of the requirements for Bcd functions in target selection, a process reflective of an efficient interaction between the activator and specific enhancers in physiological environments (Zhao, 2000).

Extensive studies of Q50 homeodomain proteins have produced two contrasting models to explain how their biological specificities are achieved. Both models center on the existence of cofactors, but the roles of these cofactors differ. The first model, referred to as the coselector model, suggests that cofactors selectively interact with different homeodomain proteins to enhance their DNA binding specificities. The second model, referred to as the widespread-binding model, proposes that, although most Q50 homeodomain proteins recognize similar or identical targets in vivo, cofactors can modulate the regulatory activities of these DNA-bound proteins. The latter model is supported by in vivo cross-linking experiments and a recent finding that a Ubx derivative with a strong activation function gains a novel biological specificity. Although the present studies focus on the K50 homeodomain protein Bcd, nonconsensus site recognition most likely also plays an important role, to various extents, in target selection by all homeodomain proteins (Zhao, 2000 and references therein).

Specification of motoneuron fate in Drosophila: Integration of positive and negative transcription factor inputs by a minimal eve enhancer: Hkb activates eve

The mechanisms that generate neuronal diversity within the Drosophila central nervous system (CNS), and in particular in the development of a single identified motoneuron called RP2, are of great interest. Expression of the homeodomain transcription factor Even-skipped (Eve) is required for RP2 to establish proper connectivity with its muscle target. The mechanisms by which eve is specifically expressed within the RP2 motoneuron lineage have been examined. Within the NB4-2 lineage, expression of eve first occurs in the precursor of RP2, called GMC4-2a. A small 500 base pair eve enhancer has been identified that mediates eve expression in GMC4-2a. Four different transcription factors (Prospero, Huckebein, Fushi tarazu, and Pdm1) are all expressed in GMC4-2a, and are required to activate eve via this minimal enhancer; one transcription factor (Klumpfuss) represses eve expression via this element. All four positively acting transcription factors act independently, regulating eve but not each other. Thus, the eve enhancer integrates multiple positive and negative transcription factor inputs to restrict eve expression to a single precursor cell (GMC4- 2a) and its RP2 motoneuron progeny (McDonald, 2003).

GMC4-2a forms at stage 9, becomes Eve⁺ at stage 11, and generates the Eve⁺ RP2/sib neurons at late stage 11. The second-born Eve-negative GMC4-2b forms at stage 10, and generates an unknown pair of neurons. The first transcription factors detected in GMC4-2a are Pros and Hkb, due to inheritance of the proteins from the neuroblast. The next transcription factors detected in GMC4-2a are Ftz and Pdm1. Ftz is first detected at stage 10, and Pdm1 is first detected at stage 11. The de novo expression of Pdm1 is distinct from its inheritance in GMCs produced by Pdm⁺ neuroblasts during the assignment of temporal identity. The last protein to be detected is Eve, which appears only at late stage 11. Pros, Hkb, Ftz, and Pdm1 are each expressed transiently in the RP2/sib neurons at stage 12, but by stage 16 none of these proteins is detectable in the mature RP2 neuron. It is concluded that there is a temporal sequence of transcription factor expression in GMC4-2a: first Pros and Hkb, then Ftz, then Pdm1, and that Eve is detected only after all of these proteins are present (McDonald, 2003).

GMC4-2b forms at late stage 10, never expresses Eve, and generates two unknown Eve-negative neurons. Three transcription factors that positively regulate eve expression are detected in GMC4-2b: Pros, Ftz, and Hkb. The pattern of Pdm1 expression is too complex to score at the time GMC4-2b is born. The negative regulator Klu is detected in GMC4-2b but not GMC4-2a. It is concluded that GMC4-2b expresses at least three of the four positively acting transcription factors that are required to activate eve (Pros, Ftz, Hkb), and at least one negative regulator of eve expression (Klu). The absence of eve expression is likely due to the presence of Klu, rather than the absence of a positive regulator, because klu mutants can activate eve transcription in GMC4-2b (McDonald, 2003).

The sequential expression of Pros, Hkb, Ftz, Pdm1, and Eve in GMC4-2a raises the possibility that these four transcription factors act in a linear pathway to regulate eve expression. If so, then a mutant in an early-acting gene should lead to loss of expression of all later-acting genes in the pathway. Alternatively, the four transcription factors could all act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present. In this case, mutants in one gene should have no effect on any other gene except eve. To distinguish between these two models, pros, hkb, ftz, and pdm1 mutants were examined for expression of all four transcription factors and eve. Pdm1 is detected in GMC4-2a in all mutant genotypes: Ftz is detected in GMC4-2a in all mutant genotypes: pros, hkb, and pdm1, and Hkb is detected in GMC4-2a in all mutant genotypes. Finally, Pros is observed in GMC4-2a in all mutant genotypes, as expected because Pros is transcribed and translated in neuroblasts and is asymmetrically partitioned into each GMC. Taken together, these data support the model that all four transcription factors act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present (McDonald, 2003).

To test the model that Pros, Hkb, Ftz, and Pdm1 transcription factors directly regulate eve expression, the eve cis-regulatory DNA that confers regulated expression in the NB4-2 lineage was identified. Eve is expressed in a subset of neurons in the embryonic CNS, including the aCC/pCC neurons derived from NB1-1, the U1-5 neurons derived from NB7-1, the EL neurons derived from NB 3-3, and the RP2/sib neurons derived from NB4-2. An eve cis-regulatory element [R79R92; from ~7.9 and ~9.2 kilobase pair (kb) on the eve genomic map] has been defined that accurately directs lacZ expression to the Eve⁺ cells within two NB lineages: GMC4-2a and its RP2 progeny and GMC1-1a and its aCC/pCC progeny. The properties of this element are examined in this study in detail. When the R79R92 eve element was truncated to ~7.9 to ~8.6 kb (R79N86), lacZ expression in RP2 and aCC was normal, whereas expression in the pCC neuron was reduced. Truncation of the eve element to ~7.9 to ~8.4 kb (R79S84) almost completely abolished expression of lacZ in pCC, although occasionally expression in pCC was observed at low levels, whereas expression in RP2 and aCC remained high. Further truncation of the left end point to ~8.0 kb (S80S84) resulted in a reduction of expression in both aCC and RP2. Addition of the region ~8.4 to ~8.6 kb to this fragment (S80N86) increased the level of expression. However, because the region ~8.4 to ~9.2 kb (S84R92) did not show any ability to activate lacZ, the region ~8.4 to ~8.6 kb is apparently insufficient on its own to direct expression, and thus serves an auxiliary function. The removal of ~8.2 to ~8.4 kb from P80N86 abolished expression (SNdeltaSC). Together with the fact that each of the fragments ~7.9 to ~8.2 kb (S79C82) and ~8.2 to ~9.2 kb (C82R92) failed to activate lacZ, this indicates that both of the regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are necessary to direct expression, and that neither alone is sufficient. Consistent with this, two tandem copies of ~8.2 to ~8.4 kb failed to activate lacZ (C82S84x2), suggesting that the two regions may provide qualitatively different activities. In summary, the critical eve cis-regulatory element for the GMC4-2a and RP2 lies in a 0.5 kb fragment of genomic DNA between ~7.9 and ~8.4 kb (McDonald, 2003).

Do the genes that activate or repress eve expression in the NB4-2 lineage work through the minimal 500 bp RP2/aCC eve enhancer? Expression of R79S84-lacZ was assayed in pros, ftz, hkb, pdm1, and klu mutant embryos, and whether it was regulated identically to the endogenous eve gene was tested. ftz, pdm1, and hkb mutant embryos show loss of R79S84-lacZ in the RP2 neuron but not the aCC neuron, identical to the pattern of endogenous eve expression in these mutants. pros mutants show loss of eve-lacZ in both RP2 and aCC, identical to the pattern of endogenous eve expression in pros mutants. In embryos lacking klu, R79S84-lacZ is expressed in two cells at the RP2 position, whereas expression in aCC is normal; this matches the pattern of endogenous eve expression in klu mutant embryos. It is concluded that the R79S84 minimal eve cis-regulatory element precisely reproduces the pattern of endogenous eve expression within the NB4-2 lineage, and that transcription factors regulating eve in GMC4-2a can act through this enhancer to activate or repress eve expression (McDonald, 2003).

Expression of eve is not detected in GMC4-2b in wild-type embryos, but mutations in the klu gene result in ectopic expression of eve in GMC4-2b. Klu contains four predicted zinc fingers, one of which is highly homologous to the WT1 zinc finger domain. The consensus binding site for the WT1 zinc finger transcription factor is a ten nucleotide sequence, 5'-(C/G/T)CGTGGG( A/T)(G/T)(T/G)-3', with variable nucleotides shown in parentheses. It was reasoned that if Klu directly binds to the eve enhancer to repress expression in GMC4-2b, one or more WT1 consensus binding sites should be found in the minimal eve enhancer R79S84. Three conserved putative Klu-binding sites were found in the R79S84 sequence: site 1, GGGTGGGGAG at nucleotides ~8066 to ~8075; site 2, GCGTGGGTGA at nucleotides ~8090 to ~8099; and site 3, TCGCCCACCA at ~8262 to ~8271. Based on the fact that altering the C2, G3, G5, G6, and G7 to T or T4 to A in the WT1-consensus binding site abolished WT1 binding, nucleotide substitutions were made in the three putative Klu-binding sites. In sites 1 and 2, As were substituted for T4, G6, and G7. In site 3, which is a reversed binding site, Ts were substituted for C4, C6, and A7. These substitutions were made at all three sites; transgenic lines were constructed expressing the mutant enhancer driving lacZ (eveK123-lacZ), and the pattern of lacZ expression was examined in the CNS of wild-type embryos and embryos misexpressing Klu protein in the NB4-2 lineage (McDonald, 2003).

In wild-type embryos, the eveK123-lacZ transgene is expressed in the aCC and RP2 neurons, similar to the wild-type (R79S84) eve-lacZ transgene. However, in one or two hemisegments per embryo, an extra cell expressing eveK123-lacZ adjacent to the RP2 neuron was observed. This phenotype is very similar to wild-type (R79S84) eve-lacZ expression in klu mutant embryos, although slightly less penetrant. It is concluded that the eveK123-lacZ transgene mimics the klu mutant phenotype, and it is proposed that Klu represses eve expression via direct binding to one or more of these sites (McDonald, 2003).

To further test this hypothesis, gain of function experiments were used to test whether ectopic Klu in GMC4-2a can repress eve-lacZ expression via these sites. Expression of a wild-type (R79S84) eve-lacZ transgene was compared with a transgene containing three mutated Klu consensus binding sites (eveK123-lacZ) in embryos where Scabrous-Gal4 (Sca-Gal4) drives ectopic expression of UAS-klu in all neuroblast lineages. The wild-type (R79S84) eve-lacZ expression is partially repressed by ectopic Klu expression, but the eveK123-lacZ transgene with mutated Klu sites is repressed to a lesser extent. This difference in repression is only observed when the levels of transgene expression are lowered by raising the embryos at 18°C; when the transgenes are more strongly expressed (by raising the embryos at 23°C) no detectable repression was observed. Taken together, Klu loss of function and misexpression studies indicate that Klu acts partly, but not completely, through three predicted Klu-binding sites to repress eve expression in the NB4-2 lineage (McDonald, 2003).

In summary, hkb, ftz, pdm1, and pros are independently required to activate eve expression in GMC4-2a. This suggests that the eve enhancer is capable of integrating the input of all four of these transcription factors to activate transcription. Hb and Ind are also necessary for eve expression in GMC4-2a, but it is not known if they act directly on the eve element or via one of the four transcription factors described in this study. Putative binding sites were found for each of the positively acting transcription factors within the minimal eve element, but mutation of these sites had no effect on expression of the eve-lacZ transgene in embryos (M. Fujioka, J.A. McDonald, and C.Q. Doe, unpublished results reported in McDonald, 2003). It remains to be determined whether Pros, Hkb, Ftz, or Pdm1 activate eve transcription via direct binding to the minimal eve element, or indirectly by activating or facilitating the binding of other transcriptional activators (McDonald, 2003).

Based on functional dissection of the RP2/aCC/pCC eve element, it seems to be composed of three parts. The regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are each necessary to direct the expression pattern (together they comprise the minimal element for expression in RP2 and aCC), while the region ~8.4 to ~8.6 kb enhances the level of expression. Expression in the pCC neuron is further enhanced by the region extending to ~9.2 kb. The two regions within the minimal element seem to be regulated by different factors, because two copies of ~8.2 to ~8.4 kb (increasing the number of activator binding sites within this region by twofold) could not substitute for the function of the region ~7.9 to ~8.2 kb. This is consistent with the fact that at least four factors are independently required to activate eve in RP2 neurons. How does Klu repress eve expression in GMC4-2b? Negative regulation of eve expression by Klu is due to direct binding to the eve minimal element. (1) It is shown that klu mutants exhibit similar derepression of the eve minimal element transgene and the endogenous eve gene in the NB4-2 lineage; (2) three consensus binding sites are detected for Klu in the eve minimal element (comparison of Drosophila virilis and Drosophila melanogaster shows that the three identified sites are highly conserved); (3) mutation of these sites results in ectopic expression of eve-lacZ in the NB4-2 lineage in wild-type, and (4) mutation of these sites impairs repression of eve-lacZ by ectopic Klu in the NB4-2 lineage. The predicted Klu binding sites (K123) are probably only a subset of relevant Klu binding sites, however, because mutation of the sites gives only partially penetrant phenotypes (McDonald, 2003).

Surprisingly, it was not possible to separate the GMC4-2a/ RP2 element from the GMC1-1a/aCC/pCC element. In both NB 1-1 and NB 4-2 lineages, eve is expressed in the first-born GMC and its neuronal progeny. Both first-born GMCs share expression of several transcription factors, including Pros and Ftz. However, many other transcription factors are differentially expressed, such as the GMC1-1a specific expression of Vnd and Odd-skipped, and the GMC4-2a specific expression of Hkb, Pdm1, and Ind. It is possible that one or more commonly expressed transcription factors are required for expression of eve in both GMC1-1a and GMC4-2a, such as Pros, and this is why the elements cannot be subdivided (McDonald, 2003).

lilliputian, the sole Drosophila member of the FMR2/AF4 (Fragile X Mental Retardation/Acute Lymphoblastic Leukemia) family of transcription factors, is widely expressed with roles in segmentation, cellularization, and gastrulation during early embryogenesis with additional distinct roles at later stages of embryonic and postembryonic development. This study identified lilli in a genetic screen based on the suppression of a lethal phenotype that is associated with ectopic expression of the transcription factor encoded by the segmentation gene runt in the blastoderm embryo. In contrast to other factors identified by this screen, lilli appears to have no role in mediating either the establishment or maintenance of engrailed (en) repression by Runt. Instead, it was found that Lilli plays a critical role in the Runt-dependent activation of the pair-rule segmentation gene fushi–tarazu (ftz). The requirement for lilli is distinct from and temporally precedes the Runt-dependent activation of ftz that is mediated by the orphan nuclear receptor protein Ftz-F1. A role is described for lilli in the activation of Sex-lethal (Sxl), an early target of Runt in the sex determination pathway. However, lilli is not required for all targets that are activated by Runt and appears to have no role in activation of sloppy paired (slp1). Based on these results it is suggested that Lilli plays an architectural role in facilitating transcriptional activation that depends both on the target gene and the developmental context (Vanderzwan-Butler, 2006).

This study uncovered Lilli's role in Runt-dependent transcriptional regulation based on the identification of lilli mutations as dose-dependent suppressors of the lethality produced by threshold levels of NGT-driven Runt expression. In contrast to all of the other Runt-interacting genes and deficiency intervals identified as suppressors in this genetic screen, a reduction in maternal lilli dosage has no effect on either the establishment or maintenance of Runt-dependent en repression. The target of Runt that provides the most dramatic and clearest evidence for a functional interaction between runt and lilli is the pair-rule gene ftz. It is notable that the ftz expression is not discernibly altered by the relatively low levels of NGT-driven Runt used in the genetic screen. This raises a question regarding the basis for lilli acting as a dose-dependent suppressor of the lethality associated with ectopic Runt expression. One explanation is that there are subtle changes in ftz expression at the threshold levels of NGT-driven Runt used in the viability assays that contribute to lethality. A second possibility is that there are other targets of Runt and Lilli that contribute to the lethality associated with ectopic Runt expression. Although Sxl would seem to be one obvious candidate for such a target, the developmental window for Sxl activation occurs prior to the stage during which the NGT-drivers are useful for manipulating gene expression. Indeed, it has not been possible to detect activation of Sxl by NGT-driven Runt, even at levels that are tenfold higher than the levels used in the genetic screen. Finally, it is possible that the effect on viability is in part due to a non-specific effect of Lilli on GAL4-dependent activation of Runt. There is some evidence for non-specificity, especially with Df(2L)C144; however, there is also a clear suppression of lethality with other lilli alleles that do not show a comparable reduction in NGT-driven UAS-lacZ expression. Thus it seems likely that the identification of lilli is due to a combination of specific and non-specific effects on the lethality produced by NGT-driven Runt expression. If this interpretation is correct, then it also seems likely that other deficiency intervals that were eliminated from further consideration due to apparent non-specific effects may in fact have specific and interesting effects that would be revealed by more directly assaying the effects of these mutations on the responses of different targets to NGT-driven Runt expression (Vanderzwan-Butler, 2006).

These observations confirm and extend findings regarding a role for Lilli in the transcriptional activation of the pair-rule gene ftz (Tang, 2001). Lilli does not appear to have any role in regulating other pair-rule genes, and the effects of eliminating maternal Lilli on segment-polarity gene expression have been interpreted as an indirect effect due to the loss of Ftz (Tang, 2001). Thus ftz appears to stand out as the sole gene in the segmentation pathway that shares a requirement for both Lilli and Runt. The previous work from Tang did identify two other targets for Lilli in the early Drosophila embryo, serendipity-α (sry-α) and huckebein (hkb). There is no evidence that either of these genes is regulated by Runt. Thus, just as there are targets of Runt in the segmentation pathway whose regulation is Lilli-independent, there are also targets of Lilli that do not involve interactions with Runt (Vanderzwan-Butler, 2006).

This work adds Sxl as an additional candidate target for Lilli. The elimination of maternal Lilli interferes with the activation of the Sxl_PE:lacZ reporter gene in all somatic cells of the female embryo. This global effect stands in contrast to the more localized role of Runt which is only required for Sxl activation in cells within the pre-segmental region of female embryos. The failure in Sxl activation observed in the absence of maternal Lilli is similar to the phenotype of embryos that are mutant for either sisA or sisB. Indeed the possibility is considered that the primary defect in lilli germline clone embryos was the failure to activate either of these two X-chromosome linked numerators. Expression of sisA was found to be normal in lilli germline clone embryos (VanderZwan, 2003). The low level of sisB expression in wild-type embryos made it difficult to unambiguously determine whether lilli was important for sisB activation. To further investigate the role of Lilli in Sxl activation the expression of the Sxl_GOF:lacZ reporter gene was examined. Both Runt and SisB contribute to the ectopic expression of this reporter in male embryos. The elimination of maternal Lilli has a more severe effect on the expression of the Sxl_GOF:lacZ reporter than is observed in embryos that are mutant for either runt or sisB (VanderZwan, 2003). The most straightforward interpretation of these results is that Lilli is directly involved in the transcriptional activation of the early embryonic Sxl promoter, in this case in cooperation with the four different X-linked factors that are responsible for the sex-specific expression of Sxl in female embryos (Vanderzwan-Butler, 2006).

The inclusion of Sxl gives four putative direct targets of Lilli in the Drosophila embryo. These four genes, Sxl, ftz, sry-α and hkb are normally activated at very early stages, and in all four cases this activation is reduced, if not abrogated, in the absence of maternally provided lilli. The notion that Lilli functions primarily in activation is consistent with observations on the properties of the mammalian homologs FMR2 and LAF4 (Hillman, 2001). However, early activation is clearly not the sole identifying characteristic of Lilli's targets. Indeed, for three of these targets, there are other genes in the same developmental pathway that are activated at the same time that do not require Lilli. In the cellularization pathway, Lilli is required for expression of sry-α but has no role in the activation of bottleneck (bnk) or nullo (Tang, 2001). In the segmentation pathway, the gap gene hkb is expressed in the anterior and posterior poles in response to signaling by the terminal pathway. Elimination of maternal Lilli greatly reduces hkb expression, but has no obvious effect on tailless (tll), another gap gene that is activated at the same stage in response to the terminal signaling pathway (Tang, 2001). Finally, the requirement for maternally provided Lilli that is observed for ftz is not shared with the pair-rule segmentation genes eve, hairy and runt (Tang, 2001). This last observation is perhaps most important as the wealth of information that exists on pair-rule gene regulation provides a useful framework for further considering the potential attributes of Lilli-dependent targets (Vanderzwan-Butler, 2006).

Elimination of maternal Lilli reduces, but does not eliminate ftz expression. The reduced expression that remains is similar to what is obtained in embryos that lack Runt, and is presumed to be in response to other activating factors. The complications presented by these other factors are bypassed in experiments in which Runt is over-expressed, either by heat-shock or by NGT-driven expression. Indeed, the inability of ftz to respond to ectopic Runt in the absence of maternal Lilli provides very compelling evidence for the importance of Lilli in ftz activation (Vanderzwan-Butler, 2006).

Lilli is acutely required for mediating Runt-dependent activation of ftz during the blastoderm stage, and this requirement precedes the temporal requirements for Ftz-F1. Ftz-F1 was initially identified as a factor that interacts with sequences within the ftz 'zebra element', a 669-bp, promoter proximal element that drives early expression in response to gap and pair-rule gene transcription factors. However, subsequent studies revealed that Ftz-F1 plays a more significant role in mediating Ftz-dependent auto-regulation by the so-called 'upstream element' during the early stages of germ-band extension. The earlier requirement for Lilli strongly suggests it contributes to the early 'zebra element'-dependent activation of ftz in response to activating inputs from Runt (Vanderzwan-Butler, 2006).

What is the role of Lilli in mediating Runt-dependent activation? Directed yeast two-hybrid assays fail to detect direct interactions between the full length Runt and Lilli proteins. This observation suggests that other factors contribute to the functional interactions described above. A notable conserved feature that Lilli shares with its mammalian homologs is an HMG-box. This structural DNA-binding motif interacts with the minor groove of DNA and modulates DNA structure by bending. These properties have been interpreted to reflect an architectural role for HMG-box proteins in facilitating the formation of higher order chromatin structures that contribute to the regulation of gene expression. It seems likely that chromatin architecture is important for ftz zebra element function. Although the 'zebra element' was one of the very first cis-regulatory elements in the segmentation pathway to be described, there is not yet a clear understanding of the rules that govern its activity. This is in contrast to the relatively simple combinatorial rules that have been elucidated for several of the stripe-specific elements of the pair-rule genes eve, hairy, and runt. It is proposed that the difficulty in identifying discrete regulatory modules within the zebra element reflects the importance of chromatin architecture in conferring high-fidelity regulation of the zebra element in response to inputs from Runt and other gap and pair-rule transcription factors (Vanderzwan-Butler, 2006).

It is interesting to note that the cis-regulatory element responsible for the initial activation of Sxl shares several properties with the ftz zebra element. The minimal DNA element necessary to faithfully recapitulate the strong, early sex-specific activation of the Sxl promoter is 1.7 kb in size. As found for the ftz zebra element, smaller reporter gene constructs do not function properly, although sub-elements that confer sex-specific regulation, and augment this activation have been identified. The on/off regulation of Sxl is in response to a twofold difference in the activity of four different DNA-binding transcription factors. It is easy to imagine that chromatin architecture may be critical in sensing this twofold difference in a robust and reproducible manner. There is one further similarity shared by Sxl and ftz that is intriguing. The initial Lilli-dependent activation of both genes is followed by a second phase of gene expression that involves distinct cis-regulatory components. In the case of ftz, the switch is from regulation by the zebra element to regulation by the upstream element, whereas for Sxl the switch is from expression at the Sxl_Pe promoter to expression at Sxl_M a different promoter that is activated in all somatic cells of both male and female embryos . Perhaps the unique requirements for Lilli reflect architecture-dependent regulatory elements that retain the ability to be rapidly re-organized in a developmentally dynamic manner. Further studies on the mechanisms by which Lilli participates in the activation of ftz and Sxl in the early Drosophila embryo should provide new insights on the role of chromatin architecture in developmentally regulated gene expression (Vanderzwan-Butler, 2006).

Non-additive interactions involving two distinct elements mediate sloppy-paired regulation by pair-rule transcription factors

The relatively simple combinatorial rules responsible for establishing the initial metameric expression of sloppy-paired-1 (slp1) in the Drosophila blastoderm embryo make this system an attractive model for investigating the mechanism of regulation by pair rule transcription factors. This investigation of slp1 cis-regulatory architecture identifies two distinct elements, a proximal early stripe element (PESE) and a distal early stripe element (DESE) located from −3.1 kb to −2.5 kb and from −8.1 kb to −7.1 kb upstream of the slp1 promoter, respectively, that mediate this early regulation. The proximal element expresses only even-numbered stripes and mediates repression by Even-skipped (Eve) as well as by the combination of Runt and Fushi-tarazu (Ftz). A 272 basepair sub-element of PESE retains Eve-dependent repression, but is expressed throughout the even-numbered parasegments due to the loss of repression by Runt and Ftz. In contrast, the distal element expresses both odd and even-numbered stripes and also drives inappropriate expression in the anterior half of the odd-numbered parasegments due to an inability to respond to repression by Eve. Importantly, a composite reporter gene containing both early stripe elements recapitulates pair-rule gene-dependent regulation in a manner beyond what is expected from combining their individual patterns. These results indicate interactions involving distinct cis-elements contribute to the proper integration of pair-rule regulatory information. A model fully accounting for these results proposes that metameric slp1 expression is achieved through the Runt-dependent regulation of interactions between these two pair-rule response elements and the slp1 promoter (Prazak, 2010).

This work identifies two distinct CRMs from the slp1 gene that drive early expression in response to pair-rule gene regulation. The observation that a composite reporter gene containing both elements faithfully emulates the initial metameric expression of slp1 in wild-type embryos as well as the response to manipulations in pair-rule activity strongly suggests these two CRMs together account for most of the early regulation of slp1 in response to pair-rule transcription factors. This view is supported by recent ChIP/chip results from the Berkeley Drosophila Transcription Network Project indicating there are two major regions of association for both Runt and Ftz in the 20 kb of DNA flanking the slp1 transcription unit, a distal region spanning from ~8.4 to ~6.7 kb and a proximal region spanning from ~3.7 to ~2.2 kb upstream of the transcription start site. These two intervals correspond extremely well to the minimal intervals defined by the functional analysis of ~8.1 to ~7.1 and ~3.1 to ~2.5 kb, respectively. Although this genome-wide analysis did not include results for either Eve or Opa it is interesting to note that Paired (Prd) and Hairy, the two other pair-rule transcription factors included in this study also show association with these same two regions. This observation further suggests Prd and Hairy may also participate in slp1 regulation, although genetic experiments do not provide any evidence indicating that either of these factors play important direct roles in regulating the early slp1 stripes (Prazak, 2010).

A most conspicuous finding from the work presented in this study is that pair-rule dependent regulation of slp1 transcription involves non-additive interactions between two distinct upstream CRMs. The ability of composite reporters containing both the DESE and PESE enhancers to mimic expression of the endogenous gene cannot be explained solely by the independent regulatory capabilities of the two elements as a simple addition of the two patterns will include inappropriate DESE-driven expression in anterior even-numbered parasegments. This non-additive interaction potentially conflicts with the generally accepted paradigm for the modular and independent action of distinct CRMs, a point that will be discussed further below (Prazak, 2010).

Although the early stripe elements need to be combined in order to fully recapitulate pair-rule regulation, studies on the independent elements provide new insights on the pair-rule to segment polarity gene transition. The homeodomain proteins Eve and Ftz both participate in slp1 repression. Several lines of evidence indicate differences in the cis-regulatory requirements for repression by these two structurally related transcription factors. DESE is insensitive to repression by Eve, but is capable of mediating repression by Ftz. The exact opposite specificity is demonstrated by the PESE:C1+ element, which is repressed by Eve but not by Ftz. The DNA-binding specificities of Eve and Ftz are similar both in vitro and in vivo and their specificity of action is thought to involve co-factor interactions that dictate the manner in which they regulate different targets. An established cofactor for Ftz is the orphan nuclear receptor protein Ftz-F1. Indeed, elimination of maternally provided Ftz-F1 results in alterations in slp1 expression that are identical to those seen in ftz mutants. The Ftz-dependent repression of slp1 also requires Runt, making this a second prospective co-factor for this activity of Ftz. Further studies on slp1 regulation should provide valuable information on the mechanisms that underlie repression by the Eve and Ftz proteins (Prazak, 2010).

These studies on the independent DESE and PESE reporters also provide information on the properties of the unidentified factor(s) that are responsible for slp1 activation in posterior even-numbered parasegments. In the case of PESE, expression of the minimal slp1[PESE:C1+]lacZ^att reporter throughout the entire pre-segmental region of eve mutant embryos indicates that a factor(s) capable of activating this element is present in all cells within this region of the embryo. DESE also drives expression of even-numbered stripes, but interestingly fails to generate stripe 0. This difference between DESE and PESE suggests there are differences in the factors responsible for activating these two elements in even-numbered parasegments. DESE also generates the odd-numbered stripes, an aspect of slp1 expression that is normally driven by the combination of Runt and Opa. Runt is normally expressed in the posterior half of only the odd parasegments and not in the posterior half of even-numbered parasegments. However, the observation that transient elimination of Runt does not abrogate DESE-driven expression in odd parasegments suggests Opa may be capable of activation in the absence of Runt. This same proposal could account for the ectopic DESE-driven expression in the anterior half of the odd parasegments as Opa is uniformly expressed in all cells within the pre-segmental region that are posterior to the cephalic furrow. The observation that Opa expression is lost anterior to the head-fold in late blastoderm stage embryos may further account for the failure of DESE to generate stripe 0. Although the only Opa-expressing cells that do not activate the DESE-lacZ reporters are those that express the combination of Runt and Ftz, there are differences in the level of expression in different cells. The increased expression in posterior versus anterior odd-numbered parasegments may reflect a contribution from Runt in potentiating DESE-driven expression (Prazak, 2010).

A central issue raised by the results is to understand how interactions involving two distinct CRMs can account for their ability to faithfully recapitulate the regulation of slp1 in response to the pair-rule transcription factors. A major discrepancy between the expression of the composite [DESE+PESE] reporter and the pattern expected from the independent action of the separate CRMs is repression of DESE-driven expression in anterior odd parasegments. One potentially trivial explanation for inappropriate expression of the DESE-lacZ reporters in these cells is close juxtaposition of binding sites for DESE-interacting activators with the basal promoter region. However, the observations that this inappropriate expression is seen for DESE-lacZ reporters that have slp1 basal promoter segments that extend anywhere from ~71 bp to ~1.8 kb upstream of the transcription start site indicates it is not due to short range interactions between DESE-bound activators and the promoter region (Prazak, 2010).

A second potential explanation is that repression in these cells involves interactions that allow the Eve-sensitivity of PESE to be transmitted to DESE. In this version of the model, Eve-interacting PESE is acting as an insulator element that prevents DESE from communicating with the slp1 promoter by blocking propagation of signals that track along the chromosome. It is also possible to imagine insulator models involving looping, such as sequestration of DESE by Eve-interacting PESE, thus preventing DESE-dependent activation at the promoter. However, in both of these insulator models ectopic Eve expression would be expected to block expression of both the odd- and even-numbered stripes, an effect that is not observed for slp1 or for the composite [DESE+PESE] reporter (Prazak, 2010).

A second discrepancy between the single elements and the composite [DESE+PESE] reporter is the Runt-independent activation by DESE in odd parasegments. This observation strongly suggests that Runt’s role in activating the composite reporter involves enabling DESE-dependent activation. This could be due to Runt-dependent antagonism of the insulator-like activities of PESE depicted in. However, there is a perhaps more straightforward explanation that does not invoke Runt-dependent regulation of PESE insulator activity, but that instead involves regulating competition between the two upstream enhancers and the slp1 promoter. In this model it is proposed that Runt plays a role in switching the promoter from interacting with PESE to interacting with the further upstream DESE. This proposed promoter-targeting role of Runt is bypassed in the DESE-lacZ reporter due to the lack of competition from PESE, thus accounting for the expression of this reporter in all cells within the segmented region of the embryo except for those that express both Runt and Ftz. Importantly, this model fully accounts for expression of both slp1 and the composite [DESE+PESE] reporters in both wild-type and mutant embryos. All expression in anterior odd-parasegments of wild-type embryos is restricted to PESE-dependent regulation as these cells do not express Runt and thus will not reveal the activating potential of DESE. The role that Eve normally plays in repressing both slp1 and the [DESE+PESE] reporter in anterior odd parasegments is also clearly accounted for by Eve’s activity as a repressor of PESE. Further work is needed to determine whether an insulator or enhancer competition model more readily accounts for the non-additive interaction between these two cis-elements, but in either event the regulatory output would appear to be due to functional attributes of the Runt transcription factor (Prazak, 2010).

There are two aspects of the current results that have widespread implications for studies on cis-regulatory DNA elements and their role in developmentally regulated gene expression. The first point is that cis-elements (such as DESE) that have broad activities when tested as autonomous single elements can have more restricted roles in regulating gene expression in the context of their normal chromosomal environment. A second, and perhaps even more crucial point is that transcription factors (such as Runt) that are not essential for activation by a single autonomous enhancer, even when tested in a physiologically relevant context, may have critical roles in enabling the activity of this enhancer in a developmental setting. Further studies on the functional interplay between the slp1 early stripe elements during Drosophila segmentation should provide insights on a phenomenon of potentially far-reaching importance in understanding the developmental regulation of gene expression (Prazak, 2010).

A targeted genetic screen identifies crucial players in the specification of the Drosophila abdominal Capaergic neurons

The central nervous system contains a wide variety of neuronal subclasses generated by neural progenitors. The achievement of a unique neural fate is the consequence of a sequence of early and increasingly restricted regulatory events, which culminates in the expression of a specific genetic combinatorial code that confers individual characteristics to the differentiated cell. How the earlier regulatory events influence post-mitotic cell fate decisions is beginning to be understood in the Drosophila NB 5-6 lineage. However, it remains unknown to what extent these events operate in other lineages. To better understand this issue, a very highly specific marker was used that identifies a small subset of abdominal cells expressing the Drosophila neuropeptide Capa: the ABCA neurons. The data support the birth of the ABCA neurons from NB 5-3 in a cas temporal window in the abdominal segments A2-A4. Moreover, it was shown that the ABCA neuron has an ABCA-sibling cell which dies by apoptosis. Surprisingly, both cells are also generated in the abdominal segments A5-A7, although they undergo apoptosis before expressing Capa. In addition, a targeted genetic screen was performed to identify players involved in ABCA specification. It was found that the ABCA fate requires zfh2, grain, Grunge and hedgehog genes. Finally, it was shown that the NB 5-3 generates other subtype of Capa-expressing cells (SECAs) in the third subesophageal segment, which are born during a pdm/cas temporal window, and have different genetic requirements for their specification (Gabilondo, 2011).

The findings strongly suggest that the Capaergic abdominal ABCA neuron arises from NB 5-3. This conclusion is based on the expression in ABCA cells of gsb, wg and unpg, and the absence of the markers lbe(K) and hkb. However, even though gsb expression is known to be maintained specifically in the lineage of rows 5 and 6 NBs, whether expression of the other genetic markers used to identify NBs at stage 11 changes late in embryogenesis remains unanswered. Nonetheless, the specific combination of NB markers found in ABCA cells and their position in the hemineuromere are consistent with their birth from NB 5-3. Previous accounts showed that this NB gives rise to a lineage of 9–15 cells. Additionally, observations derived from studies in which PCD was blocked showed that NB 5-3 can potentially produce a large lineage (ranging from 19 to 27 cells), suggesting that it could generate 13 or 14 GMCs. The lack of a NB 5-3 specific-lineage marker prevented resolution of its complete lineage, and thus determining the birth order of the ABCA cell (Gabilondo, 2011).

Recent findings on the NB 5-6 and NB 5-5 demonstrate that cas and grh act together as critical temporal genes to specify peptidergic cell fates at the end of these lineages. cas mutants lack ABCA cells and Cas is expressed in these cells, while the normal pattern of ABCA cells is found in grh mutants, and Grh is not present in ABCA neurons. These data strongly support the birth of ABCA cells in a cas-only temporal window. This is different from the subesophageal Capaergic SECA cells, which while also arising from NB 5-3, show a reduction in cell number in both pdm and cas mutants, demonstrating birth at a mixed pdm/cas temporal window. Previous studies in other lineages have shown that when a temporal gene is mis-expressed, all progeny cells posterior to that temporal window can be transformed to the specific fate born at that particular temporal window. However, cas mis-expression failed at inducing ectopic ABCA cells, suggesting that cas in necessary but not sufficient to specify the ABCA fate (Gabilondo, 2011).

Programmed cell death (PCD) is a basic process in normal development. The results suggest that the ABCA and its sibling are equivalent cells committed to achieve the ABCA fate. First, it was shown that the ABCA-sibling cell dies by apoptosis, but produces an ABCA-like Capaergic neuron if PCD is inhibited. Second, when PCD is blocked, NB 5-3 also produces a GMC generating two ABCA-like Capaergic cells in the A5–A7 segments. These data indicate that a segment-specific mechanism prevents death of the ABCA cells in A2–A4 neuromeres. Segment specific cell death has been previously reported for the NB 5-3 lineage, and detailed studies on segment-specific apoptosis of other lineages have shown that this process is under homeotic control. In addition, the results show a different timing in the PCD undergone by the ABCA sibling and the ABCA cells born in A5–A7. This interpretation is based on the differential effect of p35 expression when cas-Gal4 or elav-Gal4 drivers were used. Although elav-Gal4 is transiently express in NBs and GMCs, robust and maintained driver expression commences in differentiating neurons. In contrast, cas expression starts in the NB and is maintained in the GMC and neuronal progeny. Therefore, the finding that death of the ABCA-sibling cell can be prevented by directing p35 with cas-Gal4, but not with elav-Gal4, suggests that the death of the ABCA sibling occurs earlier in development than the death undergone by the ABCA cells in A5–A7 segments (Gabilondo, 2011).

In the ABLK/LK peptidergic fate (derived from the NB 5-5), activation of Notch (N) signaling in the peptidergic cell prevents its death, while its sibling, N^OFF cell undergoes apoptosis. On the contrary, in the EW3/Crz peptidergic fate (derived from the NB 7-3), silencing of N signaling is essential for the neuron survival, and therefore for it proper specification. The current results are in accordance with the last scenario, in which the ABCA cell is N^OFF, and it sibling, which undergoes apoptosis, is N^ON. Therefore, Notch signaling must be switch off for the proper specification of the ABCA neuron (Gabilondo, 2011).

To search for genes involved in specification of the ABCA neural fate, a reduced set of mutants was screened of genes that are expressed in the embryonic CNS at stage 11, a time at which distinctly defined sublineages are being generated from all active NBs. Even though this method will certainly overlook important genes, the results reveal that it is in fact a very effective way to find genes involved in specification of a particular neural fate. Indeed, the ratio of success has been very satisfactory: 33.3% of the genes analyzed display a significant phenotype. Moreover, the set of identified genes could be further expanded by, for example, searching in interactome databases, and performing the subsequent screen on those putative interactors (Gabilondo, 2011).

It is assumed that the specification of a concrete cellular fate requires the combination of several transcription factors, namely a genetic combinatorial code. Recently, a detailed combinatorial code has been reported for three neuropeptidergic fates: ap4/FMRFa, ap1/Nplp1 and ABLK/Lk. However, very little is known about the specification of the rest of the 30 peptidergical fates. This study has identified several genes involved in the specification of the ABCA fate, which fit into three categories. First, genes were found whose loss-of-function produces a relevant increase of the number of ABCA cells. Most remarkable are the klu and rn phenotypes, which consist of duplications of the ABCA cells. These phenotypes suggest that these two transcription factors repress the ABCA fate in other neural cells (or/and NBs/GMCs). Interestingly, the normal phenotype of nab mutants indicates that, contrary to its mode of action in the wing, Rn does not work with the transcription cofactor Nab in this context (Gabilondo, 2011).

Second, genes were found whose loss-of-function produces a significant decrease of the number of ABCA neurons. In this category, the zhf2, ftz and grain phenotypes stand out. The effects of mutations on ftz are in agreement with its early role in segmentation: ftz is a pair-rule segmentation gene that defines even-numbered parasegments in the early embryo, and absence of ABCA cells was found in the A3 segment in ftz mutants. However, zfh2 and grain seem to be part of the specific combinatorial code of the ABCA cells. The Drosophila GATA transcription factor Grain has been reported to be involved in the specification of other cell fates, such as the aCC motoneuron fate. Based on its expression, the zinc finger homeodomain protein zfh2 has been proposed to mediate specification of the serotoninergic fate, but this has not been further demonstrated. Interestingly, during wing formation, zfh2 is required for establishing proximo-distal domains in the wing disc, and it does so partly by repressing gene activation by Rn. The opposite phenotypes that was observed in rn and zfh2 mutants suggest that similar interactions occur during ABCA specification. Analyses aimed to test this hypothesis are currently being performed (Gabilondo, 2011).

Third, two genes were found whose loss-of-function abolishes the ABCA fate: Grunge and hh. Grunge encodes a member of the Atrophin family of transcriptional co-repressors that plays multiple roles during Drosophila development. Taken together, studies from C. elegans to mammals suggest that Atrophin proteins function as transcriptional co-repressors that shuttle between nucleus and cytoplasm to transduce extracellular signals, and that they are part of a complex gene regulatory network that governs cell fate in various developmental contexts. Similarly, Hh is an extracellular signaling molecule essential for the proper patterning and development of tissues in metazoan organisms. It is noteworthy that two genes implicated in extracellular signaling pathways, Grunge and hh, are absolutely required for ABCA fate. Further studies will be needed to identify at which step/s they exert their actions, and to unravel possible interactions between them and with other players of the combinatorial code for ABCA specification (Gabilondo, 2011).

Stripy Ftz target genes are coordinately regulated by Ftz-F1

During development, cascades of regulatory genes act in a hierarchical fashion to subdivide the embryo into increasingly specified body regions. This has been best characterized in Drosophila, where genes encoding regulatory transcription factors form a network to direct the development of the basic segmented body plan. The pair-rule genes are pivotal in this process as they are responsible for the first subdivision of the embryo into repeated metameric units. The Drosophila pair-rule gene fushi tarazu (ftz) is a derived Hox gene expressed in and required for the development of alternate parasegments. Previous studies suggested that Ftz achieves its distinct regulatory specificity as a segmentation protein by interacting with a ubiquitously expressed cofactor, the nuclear receptor Ftz-F1. However, the downstream target genes regulated by Ftz and other pair-rule genes to direct segment formation are not known. In this study, candidate Ftz targets were selected by virtue of their early expression in Ftz-like stripes. This identified two new Ftz target genes, drumstick (drm) and no ocelli (noc), and confirmed that Ftz regulates a serotonin receptor (5-HT2). These are the earliest Ftz targets identified to date and all are coordinately regulated by Ftz-F1. Engrailed (En), the best-characterized Ftz/Ftz-F1 downstream target, is not an intermediate in regulation. The drm genomic region harbors two separate seven-stripe enhancers, identified by virtue of predicted Ftz-F1 binding sites, and these sites are necessary for stripe expression in vivo. It is proposed that pair-rule genes, exemplified by Ftz/Ftz-F1, promote segmentation by acting at different hierarchical levels, regulating first, other segmentation genes; second, other regulatory genes that in turn control specific cellular processes such as tissue differentiation; and, third, 'segmentation realizator genes' that are directly involved in morphogenesis (Hou, 2009).

This study identified Ftz targets based on a search for genes expressed in striped patterns in the early Drosophila embryo. Each of these Ftz-dependent genes is also regulated by Ftz-F1, an orphan nuclear receptor previously shown to interact with Ftz in vitro and in vivo. Unlike Ftz, which is expressed in a striped pattern in the Drosophila blastoderm, Ftz-F1 is expressed ubiquitously, in all somatic cells at the blastoderm stage. The finding in this study that all three additional Ftz-dependent genes, identified by virtue of their striped expression patterns, require Ftz-F1 for expression in stripes lends support to the model that interaction with Ftz-F1 is the key to Ftz functional specificity as a segmentation protein. The three genes characterized in this study, 5-HT2, noc and drm, are the earliest identified downstream targets of Ftz. Expression in stripes was observed at the cellular blastoderm stage when Ftz-F1 is highly expressed throughout the embryo and the seven Ftz stripes are at their peak levels. These early target gene stripes were lost in ftz and also in ftz-f1 mutants. In addition, ectopic expression was observed at early stages when Ftz was ectopically expressed throughout the embryo. En, long thought to be a major mediator of Ftz function in segmentation, is expressed later than these target genes, and it was verified that En is not required for the Ftz-dependent stripe expression of noc or drm. These findings suggest that Ftz and Ftz-F1 directly regulate expression of these three target genes. This new study brings to seven the targets of Ftz that appear to be directly co-regulated by Ftz and Ftz-F1: ftz itself, en, apt, Dsulf1, 5HT-2, noc and drm. For each gene, multiple potential Ftz-F1 binding sites were found within a 15-20 kb genomic region. In all cases, multiple potential Ftz binding sites surround the Ftz-F1 binding sites that could mediate cooperative interactions between Ftz and Ftz-F1. Many of these sites have been maintained during evolution and are present in distant Drosophila species. Other Ftz targets, such as Ubx, prd, odd and tsh are also likely to be co-regulated by Ftz-F1 (Hou, 2009).

The seven Ftz/Ftz-F1 target genes identified to date play diverse roles in segmentation and act at different levels of the embryonic hierarchy. First, Ftz acts in a cross-regulatory fashion to modulate expression of other pair-rule genes: it interacts with Ftz-F1 in autoregulation and also has been shown to regulate the pair-rule genes prd, odd and slp. Second, Ftz/Ftz-F1 directly regulate components of the segment polarity system: first, they activate en expression in alternate stripes, and, second, they regulate Dsulf1, thought to modulate Wg activity. Ftz has also been shown to repress wg expression. Ftz/Ftz-F1 thus indirectly control compartment border formation, via regulation of En and Wg. Third, Ftz/Ftz-F1 regulate transcription factors that in turn control the differentiation of specific cell types: apt, noc, drm. drm encodes an odd-skipped family zinc finger transcription factor that it is required for patterning the dorsal epidermis, thus regulating the differentiation of specific cell types. noc plays a role in trachael morphogenesis with mutants displaying defects in branch migration and expanded expression of trachael-specific genes. Similarly, apt is involved in this process as a regulator of the migration of trachael precursor cells. Finally, Ftz/Ftz-F1 regulate a target gene more directly involved in morphogenesis, 5HT-2. 5-HT2 encodes a serotonin receptor that demonstrates specific ligand binding in transfected cells and in Drosophila embryo extracts. Phenotypic analysis suggested a role for 5-HT2 and other genes involved in serotonin biosynthesis in morphogenetic movements during gastrulation: deficiency embryos lacking 5HT-2 displayed delayed and incomplete movements during germband extension accompanied by mislocalization of Armadillo protein, suggestive of abnormalities in adherens junctions. It will be of interest in the future to determine whether other pair-rule genes direct expression of additional cell surface proteins that coordinate these processes (Hou, 2009).

This study has identified enhancers of drm by combining bioinformatics with enhancer-reporter gene expression analysis in vivo. Fragments chosen for the in vivo analysis contained one or more matche(s) to a Ftz-F1 binding site. Three of the four fragments directed expression in drm-like patterns in vivo. The upstream fragment, drm1, harbors a late stage enhancer that directs segmental expression of drm. drm2 directed expression in seven strong stripes. drm34 harbors enhancers for the region-specific expression of drm in the proventriculus and hindgut, expression that is important for the development of the fore- and hindgut, as well as an early 7-stripe enhancer. Whether any of these enhancers also direct expression in the leg imaginal discs was not investigated. Two of the fragments, drm2 and drm34, directed expression in 7-stripe patterns. Surprisingly, for each of them, the set of seven stripes is in register with Ftz, suggesting that both regulate expression of the drm-primary stripes. Although unexpected, this phenomenon has been observed in other cases where it was suggested that enhancers directing the same or similar expression patterns function as shadow enhancers to enhance the precision of expression patterns and facilitate the rapid evolution of cis-regulatory sequences. Point mutations of either or both of the predicted Ftz-F1 binding sites in the drm34 Early 7-Stripe Enhancer abolished expression of lacZ fusion genes. Stripe expression was decreased but not completely abolished by mutation of the single predicted Ftz-F1 binding site in the drm2 7-Stripe Enhancer, suggesting additional inputs into regulation of the drm primary stripes by this enhancer. Together, these results suggest that Ftz-F1 activates expression in the primary drm stripes via the drm34 Early 7-Stripe Enhancer. It is speculated that following this initial activation, autoregulation by Drm may augment Ftz-F1 activation of stripes via the drm2 7-Stripe Enhancer to raise levels of transcription in drm primary stripes (Hou, 2009).

Drosophila ftz is a typical pair-rule gene: ftz mutant embryos die lacking even-numbered body segments. How this wild type function of ftz, and other pair-rule genes, is executed is not yet known. As for other segmentation mutants, the pair-rule mutant phenotype results from cell death. However, this cell death appears to be an indirect effect. Similarly, pair-rule genes regulate segment border formation indirectly, via activation of the segment polarity genes such as en and wg. In addition to this, segment-polarity-independent roles for the pair-rule genes in morphogenesis have been revealed by careful studies from the Wieschaus lab. For example, it was found that cell intercalation and germ band extension are regulated by the pair-rule genes, independent of segment polarity genes. Similarly, cellular studies defined two subtle morphogenetic processes that occur before gastrulation - one, controlled by the pair-rule gene paired. More recently, studies have shown that the planar polarity and organization of intercalating cells during germ band extension are controlled by the striped expression patterns of eve and runt and that the longitudinal division of cells during germ band extension is controlled by eve. These studies are suggestive of direct roles for the pair-rule system in cell shape changes and rearrangements during germ band extension. Together, these studies support the notion that combinatorial expression of early patterning genes assigns unique identities in the blastoderm at a single cell level. This study has shown that the pair-rule gene ftz regulates target genes prior to and independently of En. These findings support the model that the stripes of pair-rule genes play active roles in patterning the embryo rather than serving solely as intermediary patterns whose function is to produce the segmental stripes of segment polarity genes. One role for these pair-rule stripes may be to establish differential adhesiveness to groups of cells in the blastoderm embryo. Future work identifying additional pair-rule targets will be required to explain the fundamental biological roles of pair-rule patterning and to understand how the assignment of positional identities by pair-rule genes, prior to morphogenesis, translates into the development and differentiation of body segments (Hou, 2009).

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