Sex combs reduced

Targets of Activity

Dfd is repressed, both transcriptionally and at the phenotypic level, by Scr, Antp, Ubx and Abd-B, if their products are supplied in sufficient quantity (Gonzalez-Reyes, 1992).

Expression of Creb-A in salivary glands depends on Sex combs reduced, the master regulator of salivary gland fate, since Scr mutants do not express CrebA in salivary glands and embryos expressing Scr in new places also express CrebA in new places. Activation is blocked by the trunk gene, teashirt and the posterior homeotic gene Abdominal-B. As with two other salivary gland genes, forkhead and trachealess, activation of CrebA in the salivary gland by Scr is blocked by ectodermal dpp (Andrew, 1997).

Based on its pattern of expression, eyegone is thought to play a role in salivary gland organogenesis. Salivary gland primordium (SGP) development responds to positional information. On the anteroposterior axis, Sex combs reduced (Scr) specifies parasegment 2. In Scr minus embryos, no salivary glands are formed and eyg expression is lost, except for a small patch of cells present at the PS1/PS2 border. In a teashirt minus mutation, Scr is expanded to both PS2 and PS3 and results in enlarged SGPs. The SGP expression of eyg is duplicated in PS3, although its appearance and fading are delayed slightly. Along the dorsoventral axis, the SGP is restricted dorsally by decapentaplegic (dpp), and ventrally by the spitz group of genes. In dpp minus embryos, eyg expression expands dorsally to form a ring that is interrupted ventrally. In several spitz-group mutant embryos, such as single minded (sim), the SGPs from each side move ventrally, and eyg expression expands ventrally. Expression in the trunk is also disordered, which may be a secondary effect of the disruption of the mesoderm (Jun, 1998).

Salivary eyegone expression is regulated positively by Sex combs reduced and trachealess (trh) but is regulated negatively by forkhead. Scr, the homeotic gene responsible for patterning parasegment 2, is responsible for the activation of every salivary gene that has been tested. As expected, eyg is not expressed in the salivary primordium of Scr-mutant embryos (Jones, 1998).

Sulfation is a widespread modification for many biological active molecules. This modification has been implicated in an increasing number of biological processes such as the clotting of blood, the formation of connective tissue, the metabolism of drugs and toxins and the action of growth factors and hormones. Sulfation involves the transfer of a sulfuryl group from a sulfate donor, including protein, glycolipids, glycosaminoglycans and steroids. PAPS synthetase is a bifunctional enzyme containing both ATP sulfurylase and APS kinase activities required for the biosynthesis of PAPS, the sulfate donor in sulfation reactions. The PAPS synthetase is the first gene implicated in the sulfation pathway to be described in Drosophila; its specificity of expression in embryos is described. DDmPAPSS is a novel salivary gland marker. Expression in the salivary glands is regulated by Sex combs reduced. At the end of embryogenesis, expression of DmPAPSS is also observed at the entry and exit of the gut and the posterior spiracles. The pattern of expression of the DmPAPSS gene might reflect a major role for sulfation in mucus biosynthesis at the end of Drosophila embryogenesis (Jullien, 1997).

The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. Several of the genes necessary to generate this embryonic pattern of expression have been identified. These genes can be roughly split into three categories based on their time of action during development. (1) Prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. (2) The initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). cnc and tsh act as as negative regulators of pb expression in the mandible and first thoracic segments, respectively. (3) Maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct (Rusch, 2000).

The early phase reflects a requirement for gap gene function for normal expression of pb to occur during later stages. Specifically, btd, gt, and hb have been identified as being required for proper gnathal expression of pb. The function of the head gap gene btd has been shown to be required only during early stages of embryogenesis. The expression patterns of gt and hb are such that they are no longer expressed in the labial segment at the time when pb expression begins. This is taken as a strong indication that the gap genes influence pb indirectly. Consistent with this hypothesis, no gt or hb binding sites could be detected in the regulatory elements of the pb reporter. In the case of hb, the role that various trans-acting factors might play in mediating loss of pb expression in the labial segment was investigated. Expression of Scr, the positive regulator of pb in the labial segment, is not eliminated. Further, repression of pb is not attributable to expansion of tsh expression. One possibility is that another negative regulator is being expressed such that Scr can no longer activate pb. Given the negative regulatory interactions that occur between the gap genes, it is possible that one of the other gap genes might be misexpressed and downregulate pb. However, it may be misexpression of cnc or some other gene that has yet to be identified. Alternatively, the 'hit-and-run' hypothesis, proposed to explain the long-term repression of Ultrabithorax (Ubx) by hb, may describe how transient expression of the gap genes is required very early in development to permit later expression of pb. In this hypothesis, heritable changes in chromatin structure, mediated by the PcG genes, are invoked to explain how hb regulates Ubx long after hb expression has ceased. In the case of pb regulation, one or more of these gap genes may be required to alter chromatin structure in and around the pb locus, thereby allowing the various trans-acting factors access to the pb cis-acting regulatory elements (Rusch, 2000).

During the middle phase, the initial expression pattern of pb is set by a variety of trans-acting factors. Focus was placed on the identification of those factors that determine the ectodermal pattern of pb expression along the A/P axis of the embryo. The Hox genes Dfd and Scr act as positive regulators of pb and Dfd can bind to pb regulatory elements in vitro. It is thought likely that Scr also regulates pb directly based on the similarity with which mutations in Dfd and Scr affect expression of pb and the pb reporter. In addition to the Hox genes, the region-specific homeotics cnc and tsh have been identified as negative regulators of pb and serve to restrict pb expression to the gnathos. It is not clear whether these genes regulate pb directly, though in the case of tsh the sequence TGGAAAAGT has been identified in the 500-bp regulatory fragment used in the pb reporter; this sequence is very similar to the identified tsh binding site. While this regulatory paradigm does not completely describe the regulation of the endogenous gene, based on the presence of pb residual expression, it is sufficient to explain the behavior of the 500-bp pb reporter. This mechanism of regulation places pb downstream of the Hox genes and is the first instance in Drosophila where one Hox gene is positively and directly regulated by another, a distinction previously accorded only to vertebrate Hox genes. Studies by others have suggested that wg may be mediating the nonautonomous residual expression of pb that is uncovered by mutations in Dfd and/or Scr. With the exception that wg has the strongest effect on pb expression of the segment polarity genes tested, the results shed little light on the mechanism that underlies this phenomenon. However, non-cell autonomous signaling has been implicated to explain regulation of ectodermal pb function by mesodermal expression of Scr; perhaps the residual expression in the embryo is an example of this pathway. Further experiments, including identification of an enhancer that mediates this residual expression, are needed (Rusch, 2000).

Cross-regulation of Homeotic Complex (Hox) genes by ectopic Hox proteins during the embryonic development of Drosophila was examined using Gal4 directed transcriptional regulation. The expression patterns of the endogenous Hox genes were analyzed to identify cross-regulation while ectopic expression patterns and timing were altered using different Gal4 drivers. Evidence is provided for tissue specific interactions between various Hox genes and the induction of endodermal labial (lab) by ectopically expressed Ultrabithorax outside the visceral mesoderm (VMS). Similarly, activation and repression of Hox genes in the VMS from outside tissues seems to be mediated by decapentaplegic gene activation. Additionally, it has been found that proboscipedia (pb) is activated in the epidermis by ectopically driven Sex combs reduced (Scr) and Deformed (Dfd); however, mesodermal pb expression is repressed by ectopic Scr in this tissue. Mutant analyses demonstrate that Scr and Dfd regulate pb in their normal domains of expression during embryogenesis. Ectopic Ultrabithorax and Abdominal-A repress only lab and Scr in the central nervous system (CNS) in a timing dependent manner; otherwise, overlapping expression in the CNS in tolerated. A summary of Hox gene cross-regulation by ectopically driven Hox proteins is tabulated for embryogenesis (Miller, 2001a).

The pb gene is normally expressed during embryogenesis but mutants have no apparent embryonic phenotype. However, ectopic Pb protein in the embryo does produce homeotic transformations in embryos. These observations suggest that the regulation of pb expression during embryogenesis may be important for proper development. Both Scr and Dfd are necessary for establishing the proper expression patterns of pb during embryogenesis. Although ectopic Scr and Dfd function equivalently to activate pb in the antennal segment epidermis, they have an opposite effect on native pb expression in the mandibular mesoderm (Mn). Ectopic Dfd accumulation has no significant effect on pb expression in the Mn, which is part of the Dfd expression domain. However, ectopic expression of Scr by the prd and 69B drivers represses pb expression in the Mn, demonstrating the opposite tissue specific regulation of pb by Scr (Miller, 2001a).

Genetic analyses demonstrate native regulatory interactions between pb, Dfd and Scr during embryogenesis. The reduction of pb expression in Dfd and Scr mutant backgrounds shows that normal pb expression is dependent on these genes. Dfd is required in the mandibular mesoderm (Mn) and anterior maxillary (Mx) segments (Miller, 2001a).

Similarly, Scr is necessary in the posterior Mx and Labial (Lb) segments. The functional significance of these regulatory interactions is debatable due to the lack of mutant embryonic phenotypes in pb nulls; however, the evolutionary implications are perhaps more interesting. Positive cross-regulation between Hox genes has not been previously demonstrated in Drosophila except through signal transduction. Nevertheless, vertebrate enhancers that are responsible for direct positive cross-regulation exhibit similar activity when tested in Drosophila suggesting that these differences are probably due to evolutionary changes at cis-regulatory elements. Since there is no clear mutant pb embryonic phenotype, these cis-regulatory elements apparently direct positive Hox regulatory interactions between Scr, Dfd and pb may be atavistic and non-functional in derived insects such as Drosophila. However, these cis-regulatory elements may also be necessary for proper Hox gene expression later in development (Miller, 2001a).

Homeosis and Homeotic Complex (Hox) regulatory hierarchies have been evaluated in the somatic and visceral mesoderm. Both Hox control of signal transduction and cell autonomous regulation are critical for establishing normal Hox expression patterns and the specification of segmental identity and morphology. Novel regulatory interactions have been identified associated with the segmental register shift in Hox expression domains between the epidermis/somatic mesoderm and visceral mesoderm. A proposed mechanism for the gap between the expression domains of Sex combs reduced (Scr) and Antennapedia (Antp) in the visceral mesoderm is provided. Previously, Hox gene interactions have been shown to occur on multiple levels: direct cross-regulation, competition for binding sites at downstream targets and through indirect feedback involving signal transduction. Extrinsic specification of cell fate by signaling can be overridden by Hox protein expression in mesodermal cells and the term autonomic dominance is proposed for this phenomenon. The endoderm was used to monitor target gene regulation by the Hox proteins (specifically wg, dpp and lab) through signal transduction (Miller, 2001b).

The redundancy of ectopic Scr and Dfd in transforming the mesothoracic muscle pattern to resemble the prothorax indicates that these proteins may regulate similar target genes in this developmental pathway. This may be a reflection of their strong homologies resulting in similar binding specificities. Transformations caused by ectopic Scr, Pb and Dfd in the thorax may also be due, in part, to their ability to compete with the Teashirt protein for targets. Additionally, the strong Dfd autoregulatory mechanism may be responsible for its more potent transformation of the thorax (Miller, 2001b).

The co-linear relationship to homeotic penetrance in the thoracic sm by ectopic Hox expression may be related to their relative homologies. For example, the frequency of transformation by these ectopic Hox proteins is representative of their chromosomal organization; namely (Lab<Pb<Dfd<Scr<Antp<Ubx). This ordering is similar to their respective homologies; this ordering is also a reflection of their dependence on Exd as a co-factor. Interestingly, this same order of penetrance is represented in reverse, when these Hox proteins are used to rescue lab mutant phenotypes in the embryonic CNS (i.e. Lab>Pb>Scr>Antp>Ubx>abdA>Abd-B). In summary, segmental identity was followed in ectopic Hox protein expression experiments by monitoring ventral muscle development. It was found that nearly all the homeotic proteins are capable of transforming the T2 segment in a pattern that is not predicted by previously described hierarchies such as posterior dominance and phenotypic suppression. Since the T2 segment musculature is at least in part inductively regulated by Hox gene expression in ectodermal tissue, 'autonomic dominance' is proposed as an additional component of the Hox regulatory hierarchy to explain this phenomena; namely, the ability of Hox encoded proteins to cell autonomously override an exogenous signal. A better understanding of signal transduction between germ layers is needed in order to determine the mechanism of autonomic dominance (Miller, 2001b).

During an investigation of Hox cross-regulation in the midgut visceral mesoderm it was demonstrated that both Antp and Ubx are responsible for the proper maintenance of the posterior boundary of Scr expression in ps4. It is proposed that Ubx represses Scr at this position extrinsically from nearby tissues. The segmental register shift in Hox expression domains found between the epidermis/somatic mesoderm/CNS and visceral mesoderm juxtaposes Ubx expression (ps5) to a position where it can influence Scr expression in the visceral mesoderm (ps4). Since Ubx activates dpp, which represses Scr in the visceral mesoderm, it seems reasonable to conclude that the interaction seen involves the action of dpp. Hox cross-regulation studies demonstrate that ectodermal Gal4 drivers producing ectopic Ubx repress Scr in the visceral mesoderm while stimulating dpp-LacZ expression. Ubx expression in the somatic mesoderm, which is between the epidermis and visceral mesoderm, may be the tissue that actually contributes the signaling influence demonstrated in this interaction. However, the responder only contains the visceral mesoderm regulatory elements and does not demonstrate that dpp gene activation is the signaling source in these outer tissues. Interestingly, ectopic expression of Abd-A outside the visceral mesoderm also demonstrates a posterior expansion of Scr expression in the visceral mesoderm, presumably since it represses Ubx there. Similarly, Antp repression of Scr in ps5 of the visceral mesoderm appears to be through signaling. Scr and Antp expression does not entirely fill the gap when Ubx expression is removed. Additionally, in Antp null mutants, Scr accumulation is seen in cells that normally express Antp in the presence of normal Ubx expression. By counting Scr expressing cells in the visceral mesoderm, it was found that Antp represses Scr in this tissue, contrary to previous reports. Interestingly, ectopic Antp in ectodermal tissues has no effect on ectodermal Scr expression. Thus, both Ubx and Antp contribute to define the Scr domain at its posterior visceral mesoderm boundary apparently through signal transduction (Miller, 2001b).

The segmental register shift in Hox gene expression domains between developing germ layers is well documented and appears to be a result of midgut morphogenesis. However, functional consequences of that shift have not been previously demonstrated. The regulation of Scr in the visceral mesoderm by Ubx suggests that the segmental register shift is functional and through signal transduction serves to specify the observed segmental register of Hox gene expression. In addition to Hox cross regulation and feedback loops, combined signal transduction pathways contribute to development and morphogenesis of the gastric caecae and midgut constrictions. However, a more thorough time dependent analysis of Hox expression and signaling domains in the embryo is necessary for a complete understanding of how these processes conspire to regulate these specific aspects of gut ontogeny (Miller, 2001b).

Scr inductively stimulates growth of the gastric caecae in the visceral mesoderm but seems to block it cell autonomously. Evidence was found for ectopic gastric caecae primordia in ps7 of the visceral mesoderm associated with ectopic Scr. Interestingly, the presumptive gastric caecae primordia at ps7 is generated in the same signaling environment as the native gastric caecae in ps3. During normal gastric caecae development at ps3, wg expression is observed at ps2 and dpp expression is observed at ps3. In ectopic Scr embryos polyps (ectopic gastric caecae?) are found at ps7. The signaling environment for this region of the midgut has dpp at ps7 and wg at ps8. Ectopic expression of Scr alters neither wg expression in ps8 nor dpp expression at ps7. Ectopic Hox expression (including Scr) suppresses complete gastric caecae development at ps3. This suggests that the signaling environment at ps3 may be responsible for development of the gastric caecae while Hox proteins block this morphogenesis cell autonomously (autonomic dominance). A complete map of signal transduction gene expression and Hox influences is critical for understanding this morphogenic process since other signaling agonists are likely also involved (Miller, 2001b).

Ectopic Hox protein expression in the mesoderm can induce lab, lab-LacZ and dpp-LacZ expression in the midgut. Typically, the anterior ectopic endodermal lab expression parallels the observed expression pattern in the visceral mesoderm. The lack of ectopic lab expression posterior to ps7 is probably due to the unaltered high levels of wg expression, that repress lab. Normal lab induction in the endoderm requires wg, dpp and vein; however, dFos dependent (wg independent) lab transcription can be accomplished with high Dpp levels. Typically, lab induction by dpp, wg and vein is coordinated by sgg (GSK3) which may be responsible for the ps4 gap in lab, lab-LacZ, and expression patterns seen in experiments involving ectopic Antp visceral mesoderm expression. Moreover, the lack of expanded lab-LacZ expression (unlike native lab) by ectopic Antp indicates the existence of presently undefined cis-regulatory elements at the lab locus that are not contained in genomic fragments of the identified enhancers. Antp protein may be regulating other influential signaling pathways while the corresponding cis-acting elements are not located in the genomic lab enhancers tested. Antp expression is functionally linked to another TGF-beta agonist 60A (glass bottom boat), as well as the Wnt pathway agonist DWnt4 (Miller, 2001b).

It is concluded that Hox gene interactions in the mesoderm are not always consistent with previous governing hierarchies: posterior dominance and phenotypic suppression. In the visceral mesoderm it is found that posterior dominance (Hox direct cross-regulation) seems legitimate but may be mediated by signal transduction. Phenotypic suppression is violated by morphological changes and target gene regulation. In the somatic mesoderm, more anterior Hox genes alter the identity of the ventral T2 segment, but this tissue is largely extrinsically regulated in the absence of direct Hox expression. In light of this result, the notion of autonomic dominance is proposed: Hox genes cell-autonomously dominate tissues regulated by signal transduction (Miller, 2001b).

The predominant paradigm depends on whether cells are extrinsically or autonomously specified by Hox gene expression. It is argued that non-typical homeosis caused by ectopically expressed Hox proteins (i.e. not following the dictates of posterior prevalence) can be taken to indicate inductively specified tissues and hence, confer autonomic dominance. Interestingly, ectopic expression of the Hox proteins also exhibit non-typical homeosis in the chordotonal organs of the PNS and the thoracic cuticle, suggesting that inductive specification and autonomic dominance may not be restricted to the mesoderm. However, Hox regulatory hierarchies seem to be of limited value in other tissues as well. The mechanism responsible for autonomic dominance has not been determined in this study; only the correlation between autonomous Hox dominance over inductively specified tissue. Signal transduction pathway cross-talk could be the predominant cause of autonomic dominance phenotypes (homeosis) due to Hox regulation of signaling agonists. These agonists could then contribute to the signaling environment to alter the tissue, since these morphogens are potent factors in differentiation. Meanwhile, Hox genes cross-regulate each other cell autonomously and in nearby tissues through signal transduction. This occurs in a tissue specific manner that likely depends on both the signaling environment, transcriptional co-factors, and perhaps any of an estimated 100 target genes for a given Hox protein. The signaling environment of any given tissue is dictated primarily by Hox genes, which is critical for maintenance of Hox expression domains and subsequent differentiation, determination and morphogenesis. This complex set of intrinsic and extrinsic Hox controls are likely responsible for the means by which Hox genes were genetically identified for their abilities to dominate segmental identities as homeotic selector genes (Miller, 2001b).

In the early Drosophila embryo, a system of coordinates is laid down by segmentation genes and dorsoventral patterning genes. Subsequently, these coordinates must be interpreted to define particular tissues and organs. To begin understanding this process for a single organ, a study has been carried out of how one of the first salivary gland genes, fork head (fkh), is turned on in the primordium of this organ, the salivary placode. A placode-specific fkh enhancer was identified 10 kb from the coding sequence. Dissection of this enhancer shows that the apparently homogeneous placode is actually composed of at least four overlapping domains. These domains appear to be developmentally important because they predict the order of salivary invagination, are evolutionarily conserved, and are regulated by patterning genes that are important for salivary development. Three dorsoventral domains are defined by Egf receptor (Egfr) signaling, while stripes located at the anterior and posterior edges of the placode depend on wingless signaling. Further analysis has identified sites in the enhancer that respond either positively to the primary activator of salivary gland genes, Sex combs reduced (Scr), or negatively to Egfr signaling. These results show that fkh integrates spatial pattern directly, without reference to other early salivary gland genes. In addition, a binding site for Fkh protein was identified that appears to act in fkh autoregulation, keeping the gene active after Scr has disappeared from the placode. This autoregulation may explain how the salivary gland maintains its identity after the organ is established. Although the fkh enhancer integrates information needed to define the salivary placode, and although fkh mutants have the most extreme effects on salivary gland development thus far described, it is argued that fkh is not a selector gene for salivary gland development and that there is no master, salivary gland selector gene. Instead, several genes independently sense spatial information and cooperate to define the salivary placode (Zhou, 2001).

In the 219-308 interval of a 1-kb promoter subfragment, bases 242-277 is strongly conserved in the D. virlilis enhancer, and it has been designated as the 250 region for further analysis. To test the activity of this 36-bp region, three copies of 250 were placed upstream of the minimal heat shock promoter and lacZ. Multiple transformed lines containing this construct were obtained, and all showed ß-galactosidase expression throughout the ectoderm of PS2, as well as in a few cells in each trunk segment and patches in the head. These results suggest that, in its normal context, 250 activates fkh expression in the salivary placode, perhaps by binding the homeotic regulator Scr. Within the 250 element, there is one region with two overlapping sequences of ATTAAT that are similar to homeodomain core binding sites. To test whether this consensus site is important for PS2 expression, the two contiguous As were changed to Gs. These changes should destroy homeodomain protein binding, but have minimal effect on the surrounding regions. Staining of transformants carrying this 250-GG construct is consistent with the direct involvement of Scr in the enhancer activity of the 250 element (Zhou, 2001).

Sex combs reduced and Ultrabithorax establish differential expression of the proneural gene achaete by modifying expression of the achaete prepattern regulator Delta in Drosophila legs

Many studies have shown that morphological diversity among homologous animal structures is generated by the homeotic (Hox) genes. However, the mechanisms through which Hox genes specify particular morphological features are not fully understood. This issue was addressed by investigating how diverse sensory organ patterns are formed among the legs of the Drosophila adult. The Drosophila adult has one pair of legs on each of its three thoracic segments (the T1-T3 segments). Although homologous, legs from different segments have distinct morphological features. Focus was placed is on the formation of diverse patterns of small mechanosensory bristles or microchaetae (mCs) among the legs. On T2 legs, the mCs are organized into a series of longitudinal rows (L-rows) precisely positioned along the leg circumference. The L-rows are observed on all three pairs of legs, but additional and novel pattern elements are found on T1 and T3 legs. For example, at specific positions on T1 and T3 legs, some mCs are organized into transverse rows (T-rows). The T-rows on T1 and T3 legs are established as a result of Hox gene modulation of the pathway for patterning the L-row mC bristles. The findings suggest that the Hox genes, Sex combs reduced (Scr) and Ultrabithorax (Ubx), establish differential expression of the proneural gene achaete (ac) by modifying expression of the ac prepattern regulator, Delta (Dl), in T1 and T3 legs, respectively. This study identifies Dl as a potential link between Hox genes and the sensory organ patterning hierarchy, providing insight into the connection between Hox gene function and the formation of specific morphological features (Shroff, 2007).

It is proposed that T-rows are formed on T1 and T3 legs in response to Scr or Ubx alteration of the L-row prepattern via repression of Dl expression. Dl is expressed in narrow longitudinal stripes that correspond to the L-row primordia. Dl-expressing cells in the L-row primordia signal to adjacent cells to activate N signaling and repress ac expression in the hairy-OFF interstripes and in one hairy-ON interstripe, between the L-row 1 and 8 proneural fields. It is suggested that in T1 and T3 legs, reduction of Dl expression in cells with high-levels of Scr or Ubx establishes a zone where there is no repressive influence on ac expression, resulting in expression of Ac in broad domains from which the T-row precursors will be selected. Cells in the center of T-row primordia are presumably out of range of the Dl signaling that takes place at the interface of Dl-expressing and Dl-non-expressing cells. The anterior and posterior boundaries of Ac expression in the T-row primordia of T1 prepupal legs are likely established by Dl/N signaling. In T3 legs, in contrast, it appears that Hairy rather than Dl/N signaling establishes the boundaries of ac on either side of the T-row primordia. Reduced Dl expression in the T-row primordia of T3 legs, however, is likely required to establish a broader domain of Ac expression than would be observed in the corresponding domain of T2 legs (Shroff, 2007).

A key feature of the model for mC patterning is that position-specific expression of ac expression in the mC proneural fields is established mainly by repression and that differential mC patterns are generated by altering expression or function of the repressive factors, Hairy and/or Dl. It is suggested that altered Dl expression is required in order to reduce N signaling, which allows proneural gene expression within the T-row primordia. An alternative hypothesis is that Dl function in during leg mC development is limited to selection of SOPs via lateral inhibition and that regulation of Dl by Scr/Ubx alters lateral inhibition within the T-row proneural fields. However, the hypothesis that Scr/Ubx regulation of Dl alters the proneural prepattern is supported by several observations. A prepattern function for Dl in mC patterning has been previously demonstrated in the notum. Similarly, it has been observed that in prepupal legs with reduced Dl function, proneural Ac expression expands along the leg circumference and is excluded only from Hairy-expressing cells. Furthermore, in prepupal legs, proneural Ac expression fills the center of large clones lacking Dl function. It is also observed that N signaling is activated only in narrow stripes on either side, but not within the T-row proneural fields. The genetic observations are substantiated by analysis of an enhancer that directs ac expression in both the L-row and T-row proneural fields. This enhancer consists of an activation element that directs uniform expression of ac along the leg circumference and two associated repression elements, one that is N-responsive and another that is Hairy-responsive. This is consistent with genetic studies suggesting that in the absence of repressive influences from Hairy and Dl, proneural ac expression would be uniformly along the leg circumference. Combined, these observations suggest that the mC patterning pathway is modified upstream of proneural gene expression by establishment of differential Dl expression in legs from different thoracic segments (Shroff, 2007).

The finding that Dl expression is down-regulated in the T-row primordia, does not necessarily imply that Dl expression is incompatible with mC formation. That this is not the case is suggested by the observation that Dl is expressed in the L-row primordia. Previous studies in a number of tissues have shown that high-level N-ligand expression renders cells non-responsive to N signaling. Hence, it appears that Dl/N signaling at the boundary of Dl-expressing and Dl-non-expressing cells, not Dl expression per se, is incompatible with mC development (Shroff, 2007).

Many studies have made clear the importance of establishing spatially defined proneural gene expression, largely via transcriptional regulation, for patterning of both the vertebrate and invertebrate nervous system. For example, it has been shown that ectopic proneural gene expression causes disruption of the sense organ pattern in adults. In the leg, compromised hairy function results in ectopic proneural ac expression and disorganization of the adult mC pattern, including formation of extranumerary mCs. However, other studies have implicated post-transcriptional regulation of proneural gene function in neural patterning. This was suggested by a study that showed that generalized and transient sc expression in a background devoid of ac and sc function results in an almost normal sense organ pattern in adult flies. Studies in the notum have provided an explanation for this observation by identification of the Extra macrochaetae (Emc) protein as a post-transcriptional regulator of proneural gene function. Emc, an HLH protein that lacks a basic DNA binding domain, binds proneural bHLH proteins, such as Ac, and inhibits their activity. In the notum, emc is expressed in a complex pattern that partially overlaps proneural gene expression, and it appears that SOPs are selected from cells with the lowest levels of Emc. This would suggest that on the notum, competence to acquire a neural fate depends on the balance of proneural protein to Emc levels. It is probable that similar mechanisms function in leg mC patterning as well, since largely normal sense organ patterns are found in legs ubiquitously expressing Sc. These observations indicate that sense organ patterning is a complex process that involves regulation of both proneural gene expression and function. Hence, it would be of interest to assess the relative contribution of post-transcriptional regulation of proneural gene function on leg mC patterning (Shroff, 2007).

It is proposed that T-row mCs are selected from domains of up-regulated Scr or Ubx expression and that one essential function for Scr and Ubx in T-row development is repression of Dl expression. This proposal is supported by several lines of evidence. The requirement of Scr and Ubx in T-row formation was suggested by prior reports that loss of Scr or Ubx function results in transformation of T1 or T3 legs, respectively, toward a T2 fate. This study shows that adult legs heterozygous for reduced function alleles of Scr (Scr^EdK6/Scr^EfW22) exhibit almost complete loss of T-rows in the adult. Moreover, ectopic expression of Scr or Ubx induces T-row formation on T2 legs, on which T-rows are never normally observed. The domains of elevated Scr and Ubx expression in T1 and T3 prepupal legs correspond to the respective positions of T-rows in adult T1 and T3 legs. Furthermore, comparison of Scr expression to that of an SOP marker, sca-Gal4, shows that T-row mCs are selected from groups of cells that express high-level Scr on T1 legs (Shroff, 2007).

Strong evidence is provided that Scr and Ubx repress Dl expression in the T-row primordia. First, a correlation is observed between up-regulated Scr and Ubx expression and domains of reduced Dl expression. Second, loss and gain of function studies indicate that Scr and Ubx negatively regulate Dl expression. In Scr^EdK6/Scr^EfW22, prepupal legs, Dl is expressed in two longitudinal stripes overlapping the region of high-level Scr expression, whereas in wild type legs Dl stripes flank but do not overlap this domain. In addition, Dl is ectopically expressed in either Scr or Ubx loss of function clones within the T-row primordia. Consistent with loss of function results, it was found that ectopic high-level expression of Scr or Ubx results in repression of Dl expression (Shroff, 2007).

Taken together, these observations suggest a function for Scr and Ubx in specification of a T-row fate. However, the finding that the formation of T-rows in response to ectopic Scr or Ubx is confined to ventro-lateral regions along the circumference implies that there may be other positional cues, in addition to elevated Scr or Ubx expression, that are required for T-row specification. Hence, it is plausible that these genes function combinatorially with other factors to induce T-row formation. Wg, for example, is a good candidate since it is expressed in ventro-lateral regions of the leg. In addition, ectopic Wg expression results in expansion of T-row bristles in T1 legs. It is also plausible that, in addition to or instead of T-row promoting factors in ventro-lateral leg regions, there are factors outside these domains that inhibit T-row formation (Shroff, 2007).

These studies have elucidated a general pathway for leg mC patterning in which an early event is establishment of position-specific expression of the prepattern genes hairy and Delta, presumably in response to the global regulators of limb patterning. The spatial regulation of hairy expression during leg development has been investigated ant it has been determined that hairy expression is controlled by modular enhancer elements that integrate patterning information provided by the signaling molecules known to pattern the leg along its circumference, Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). Periodic Dl expression is partially established by Hairy. However, it is likely that Dl expression in the mC proneural fields is also regulated, like hairy, by genes that pattern the leg along the circumference (circumferential patterning genes), such as hh, dpp and wg (Shroff, 2007).

Up-regulated expression of Scr and Ubx at specific positions along the circumference and P/D axis of T1 and T3 prepupal legs is key to generating the T-row pattern, raising the question of how Scr and Ubx expression in the T-row primordia is regulated. It is proposed that Scr and Ubx expression is controlled by the circumferential patterning genes and genes that pattern the leg along the P/D axis (P/D patterning genes). For example, Scr and/or Ubx expression might be regulated by Wg, which is known to specify ventral leg identity and patterns the ventral leg along the A/P axis in a concentration dependent manner. Along the P/D axis, a number of genes, such as Distal-less and dachshund, are expressed in defined and partially overlapping domains and might function to define the extent of up-regulated Scr and/or Ubx expression. This model would suggest that circumferential and P/D patterning information is integrated by Scr and Ubx, implying that these Hox genes link the global regulators of leg development to local acting genes, such as ac, that specify a neural fate (Shroff, 2007).

These studies indicate that Scr and Ubx function early in T-row development to repress Dl expression, which allows formation of the T-row proneural fields. It will be of interest to determine whether Dl is a direct target of these Hox genes, especially since few direct Hox-gene targets have been identified to date. However, it is probable that Scr and Ubx have other functions in T-row development. Establishment of ac expression in the T-row primordia is an early and essential step of T-row development, but, while ac specifies a neural fate, it does not specify sensory organ type. Hence, it is likely that Scr and Ubx function, in conjunction with other factors, to specify 'T-row-type' mCs. For example, since the T-row mCs are less pigmented than L-row mCs, a potential role of Scr and Ubx in T-row development is to regulate genes involved bristle pigmentation. A second potential function for these Hox genes is in controlling growth in the regions of the legs where T-rows are formed. In T1 legs, for example, the region between L-rows 7 and 8, in which the T-rows are found, is larger than the corresponding region on T2 legs, implying that there is additional growth in this domain. Inconsistent with this hypothesis, however, is the observation that posterior compartment clones lacking Ubx function in the T3 basitarsus did not have a significant effect on basitarsal width (Shroff, 2007).

Another potential role for Scr and Ubx in patterning T-row bristles is to implement a mechanism for selection and organization of T-row mCs into transverse rows. The mechanisms through which the L-row and T-row bristles are selected and organized within their respective proneural fields are likely to differ substantially. The regular spacing of L-row mCs suggests that the L-row SOPs send inhibitory signals in all directions to establish their proper spacing. This is also suggested by the observation that in hairy mutant legs, Ac is expressed in four broad domains, similar to the broad T-row proneural fields, and the supernumerary mCs that are formed on hairy mutant legs are well spaced along the leg circumference. This would suggest that the lateral inhibitory signals are sent along both the leg circumference and P/D axis. Unlike the L-row bristles, the T-rows mCs are positioned directly adjacent to one another in straight regularly spaced transverse rows. How the T-row precursors are selected from a broad field of ac-expressing cells and are arranged in tandem in straight rows is not understood. Previous studies have implicated N and EGFR signaling in formation of organized T-rows. Although the current studies indicate that N-signaling is down-regulated in the T-row primordia, it is conceivable that N functions at later stages of T-row development to pattern the T-row bristles. For example, N might function to set the register and spacing of the T-rows. Also of interest is how the T-rows are aligned in tandem within the rows. It has been suggested that homophilic adhesion between mC SOPs might be involved in organizing T-row bristles. Hence, it is plausible that Hox genes regulate expression of genes involved in adhesion, N signaling and/or EGFR signaling. Investigation of the mechanisms of T-row SOP selection and organization will provide an opportunity to uncover a potential connection between Hox gene function and morphogenesis (Shroff, 2007).

The proposed function for Scr and Ubx in T-row patterning bears some similarity to that described for Ubx in generation of diverse trichome patterns among the T2 legs of various Drosophila species. It has been shown that late pupal expression of Ubx in the T2 femur primordia correlates with lack of trichome formation in different Drosophila species, implying that Ubx inhibits formation of these structures. This role for Ubx, which has been termed a 'micromanaging role' is analogous to the function described here for Scr and Ubx in directing formation of T-rows in specific domains of T1 and T3 legs. Hence, micromanaging functions for Hox genes in generating complex and detailed morphologies may be a general phenomenon (Shroff, 2007).

Another common theme that has emerged from studies of the mechanisms through which Hox genes generate morphological diversity is that, in many cases, Hox genes function to suppress specific developmental pathways. For example, in legs, Antennapedia functions to repress expression of genes that promote antennal development, and Ubx functions to prevent development of specific macrochaete bristles on T3 legs. Furthermore, Ubx is known to act at several levels of the wing patterning hierarchy to suppress wing development in the haltere disc and as mentioned, Ubx functions late in leg development to suppress trichome formation. Less well understood, in contrast, is how and whether Hox genes act positively to direct the formation of morphological novelties among homologous structures, e.g., the T-row bristles on T1 and T3 legs. Further analysis of the mechanisms involved in T-row specification and morphogenesis is likely to provide insight into this question (Shroff, 2007).

Homeotic effects

Ectopically expressed Teashirt protein induces an almost complete transformation of the labial to prothoracic segmental identity, when expressed during the first 8 hours of development. Positive autoregulation of the endogenous teashirt gene and the presence of SCR protein in the labium explain this homeosis. Trunk patterns partially replace those patterns in the maxillary and a more anterior head segment. teashirt function is overridden by homeotic gene function acting in the posterior trunk. Strong heat-shock regimes provoke novel defects: ectopic sense organs differentiate in posterior abdominal segments and trunk pattern elements differentiate in the ninth abdominal segment. It has been suggested that Teashirt and HOM-C proteins regulate common sets of downstream target genes (de Zulueta, 1994).

Zygotic expression of modifier of variegation Modulo depends on the activity of genes which pattern the embryo along dorsoventral and anteroposterior axes and specify diversified morphogenesis. dorsal and the mesoderm-specific genes twist and snail direct modulo expression in the presumptive mesoderm. The homeotic genes Sex combs reduced and Ultrabithorax positively regulate modulo in the ectoderm of parasegment 2 and abdominal mesoderm (Graba, 1994).

Protein Interactions

Antennapedia and Sex combs reduced determine cell fates in the epidermis and internal tissues of the posterior head and thorax. Genes encoding chimeric Antp/Scr proteins have been introduced into flies and their effects on morphology and target gene regulation observed. The N-terminus of the homeodomain is critical for determining the specific effects of these homeotic proteins in vivo, but other parts of the proteins have some influence as well. The N-terminal part of the homeodomain has been observed to contact the minor groove of the DNA. The different effects of Antennapedia and Sex combs reduced proteins in vivo may depend on differences in DNA binding, protein-protein interactions, or both (Zeng, 1993).

Sex combs reduced (SCR) is a Drosophila Hox protein that determines the identity of the labial and prothoracic segments. In search of factors that might associate with SCR to control its activity and/or specificity, a yeast two-hybrid screen was performed. A Drosophila homolog of the regulatory subunit (B'/PR61) of serine-threonine protein phosphatase 2A (dPP2A,B') specifically interacts with the SCR homeodomain. The N-terminal arm within the SCR homeodomain has been shown to be a target of phosphorylation/dephosphorylation by cAMP-dependent protein kinase A and protein phosphatase 2A, respectively. In vivo analyses reveal that mutant forms of SCR mimicking constitutively dephosphorylated or phosphorylated states of the homeodomain are active or inactive, respectively. Inactivity of the phosphorylated mimic form is attributable to impaired DNA binding. Specific ablation of dPP2A,B' gene activity by double-stranded RNA-mediated genetic interference results in embryos without salivary glands, an SCR null phenotype. These data demonstrate an essential role for Drosophila PP2A,B' in positively modulating SCR function (Berry, 2000).

PP2A exists as a multisubunit enzyme complex in a variety of organisms and cell types. The enzyme complex is composed of a catalytic and a scaffold subunit, which together form a core dimer that then associates with one of a number of regulatory subunits to constitute a trimeric enzyme complex. Regulatory subunits of PP2A are encoded by at least three unrelated gene families: B (PR55), B' (PR61) and B" (PR72). Each family consists of several members, which in addition can give rise to a number of splice variants, thereby greatly increasing the variety of distinct trimeric enzyme complexes. Several lines of evidence suggest that the regulatory subunits of PP2A may serve as specific adaptors that confer substrate specificity to the core domain of PP2A. Specific interaction of dPP2A,B' with the SCR homeodomain, as documented here, therefore reflects its potential of reversibly recruiting SCR into the PP2A complex (Berry, 2000).

The two phosphorylatable residues (T and S) within the N-terminal arm of the SCR homeodomain appear to be conserved, since at least one such site has been found in all SCR homologs from other species, except for PS12-B of Atlantic salmon. The homeodomain of PS12-B in fact seems more closely related to ANTP than to SCR. In vivo results suggest that in developing embryos, SCR is functionally inactive when the N-terminal arm of its homeodomain is phosphorylated and is active upon dephosphorylation. These results may have important implications for the functional specificity of homeotic proteins in general: since ANTP has a glutamine instead of threonine at position 6 (which is well conserved in all the SCR homologs), it is proposed that the differential modification of this residue plays an important role in determining the functional specificity of these two homeotic proteins (Berry, 2000).

The data from the functional knockout of dPP2A,B' by dsRNA interference prove unequivocally that expression of dPP2A,B' is essential for the functional activity of SCR. Genetic studies in Drosophila have shown that Ras-1 activity positively modulates the function of Hox proteins such as proboscipedia (PB) and SCR -- a finding that suggests that covalent modifications triggered by Ras-1-mediated signals might influence the activity of PB and SCR. The catalytic subunit of dPP2A has been identified as a component operating downstream of Ras-1. The observation that the functional activity of SCR is dependent upon the presence of dPP2A,B' seems to provide a missing link, suggesting that Ras-1 might influence the activity of SCR via dPP2A (Berry, 2000).

A model is proposed to describe the regulation of SCR activity: in a cell, where SCR function is not required continuously, the protein is locked in an inactive state by phosphorylation of residues 6 and/or 7 within the N-terminal arm of the homeodomain. The fact that, in older embryos, SCR is present but is no longer able to induce the expression of its target gene forkhead, may be a case in point. In response to positive signals, SCR-specific protein phosphatase (dPP2A) becomes activated, possibly through a signaling cascade involving Ras-1. In the absence of positive signals, or when negative signals, e.g. DPP and SP1 prevail, specific dPP2A activity is inhibited and, as a result, SCR can no longer be maintained in its dephosphorylated state. PKA or PKA-like enzymes will phosphorylate residues 6/7 of the SCR homeodomain, thus abrogating the ability of SCR to bind to its target genes. A delicate balance between the activities of SCR-specific PP2A and specific protein kinases would thus allow a cell to fine-tune SCR activity (Berry, 2000).

Principles of microRNA-target recognition: Post-transcriptional regulation

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. This study systematically evaluates the minimal requirements for functional miRNA-target duplexes in vivo and classes of target sites with different functional properties are distinguished. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. Examples and genome-wide statistical support is presented to show that both classes of sites are used in biologically relevant genes. Evidence is provided that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families (Brennecke, 2005).

The 3′ UTR of the HOX gene Sex combs reduced (Scr) provides a good example of a 3′ compensatory site. Scr contains a single site for miR-10 with a 5mer seed and a continuous 11-base-pair complementarity to the miRNA 3′ end. The miR-10 transcript is encoded within the same HOX cluster downstream of Scr, a situation that resembles the relationship between miR-iab-5p and Ultrabithorax in flies and miR-196/HoxB8 in mice. The predicted pairing between miR-10 and Scr is perfectly conserved in all six drosophilid genomes, with the only sequence differences occurring in the unpaired loop region. The site is also conserved in the 3′ UTR of the Scr genes in the mosquito, Anopheles gambiae, the flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori. Conservation of such a high degree of 3′ complementarity over hundreds of millions of years of evolution suggests that this is likely to be a functional miR-10 target site. Extensive 5′ and 3′ sequence conservation is also seen for other 3′ compensatory sites, e.g., the two let-7 sites in lin-41 or the miR-2 sites in grim and sickle (Brennecke, 2005).

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