Interactive Fly, Drosophila



Promoter Structure

A promoter region of 287 bp, 2 kb upstream of the kni structural gene is sufficient to drive kni expression in both anterior and posterior stripes under the positive regulation of Bicoid and Caudal, and the negative regulation of Hunchback. Bicoid and Caudal binding elements overlap (Rivera-Pomar, 1995).

Caudal and Bicoid cooperate to form an activator system for giant and knirps The process of body prepatterning during Drosophila blastoderm formation relies on the localized activities of zygotic segmentation genes, controlled by asymmetrically distributed maternal determinants. The anterior determinant Bicoid forms an anterior-to-posterior concentration gradient. The posterior maternal system prevents the repressor Hunchback from acting in the posterior half; consequently the gap genes giant and knirps are activated. Caudal protein's posterior-to-anterior concentration gradient, in cooperation with Bicoid, forms a partly redundant activator system in the posterior region of the embryo (Rivera-Pomar, 1995). High levels of Giant act as a repressor of kni, thereby setting its posterior border (Eldon, 1991).

kni expression is repressed by tailless activity, whereas it is directly enhanced by Krüppel activity. Thus, Kr activity is present throughout the domain of kni expression and forms a long-range protein gradient, which in combination with kni activity is required for abdominal segmentation of the embryo. A construct containing 4.4 Kb of kni upstream sequence located at -0.9 Kb from the start of transcriptiongives the correct spatial pattern of expression in the anterior and posterior domains. Kr responsive elements in kni reside in this 4.4 kb fragment and more precisely in 800 bp fragment located at the 3' end of the 4.4 Kb. Two adjacent Kr protein binding regions are present approximately in the middle of this 0.8 Kb fragment. More sites become protected in footprint experiments when using higher concentration levels of the Kr protein (Pankratz, 1989)

radius incompletus (ri) is a well-known mutant that has a severely truncated L2 vein. ri maps very close to the neighboring and functionally equivalent kni and knrl genes. Four different embryonic lethal kni alleles fail to complement ri when the ri mutation is carried on a chromosome that is rearranged with respect to the kni mutant chromosome. The failure of multiple kni alleles to complement ri indicates that ri is likely to be an allele of the kni/knrl locus. However, these same kni alleles fully complement ri, when the ri and kni alleles are carried on non-rearranged chromosomes. In Drosophila, regulatory and coding region mutations in the same gene frequently complement, a phenomenon referred to as transvection. Unlike other forms of inter-allelic complementation, transvection requires that the two mutant chromosomes be co-linear. Transvection can be blocked by inverting one chromosome with respect to the other. The failure of ri and kni point mutations to complement when transvection is blocked by chromosomal rearrangement suggests that ri, is a cis-acting regulatory mutation in the kni/knrl locus. Since the L2 vein-loss phenotype is more variable and typically less complete in kni/TM3 ri trans-heterozygous flies than in ri/ri homozygotes, it is likely that both kni and knrl contribute to ri function. Consistent with kni and knrl providing overlapping functions in promoting L2 development, the L2 vein forms normally in wings containing kni- single mutant clones, which encompass the L2 vein on both the dorsal and ventral wing surfaces. Allelism between ri and the kni/knrl locus is further supported by the observation that low level ubiquitous expression of a kni cDNA transgene in UAS-kni EP flies can rescue the ri L2 truncation phenotype, although the position of the rescued L2 vein is displaced to the anterior, relative to the wild-type L2 vein (Lunde, 1998).

The adjacent knirps and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expressing high levels of the related spalt genes. A 1.4 kb cis-acting enhancer element from the kni locus has been identified that faithfully directs gene expression in the L2 primordium. Three independent ri alleles have alterations mapping within the L2-enhancer element; two of these observed lesions eliminate the ability of the enhancer element to direct gene expression in the L2 primordium. The L2 enhancer can be subdivided into distinct activation and repression domains. The activation domain mediates the combined action of the general wing activator Scalloped and a putative locally provided factor, the activity of which is abrogated by a single nucleotide alteration in the ri53j mutant. Misexpression of genes in L2 that are normally expressed in veins other than L2 results in abnormal L2 development. These experiments provide a mechanistic basis for understanding how kni and knrl link AP patterning to morphogenesis of the L2 vein by orchestrating the expression of a selective subset of vein-promoting genes in the L2 primordium (Lunde, 2003).

Wing vein development in Drosophila can be subdivided into two temporally distinct stages: initiation and differentiation. Vein initiation begins during the mid-third larval instar when the wing imaginal disc is a monolayer of cells. The wing blade proper derives from an oval region of the wing disc known as the wing pouch, while the remainder of the disc generates elements of the wing hinge and thoracic body wall. Vein differentiation, the second phase of vein development, occurs during metamorphosis as the wing disc buds out (or everts), folding along the future wing margin. Ultimately, this eversion leads to the apposition of the dorsal and ventral surfaces of the wing during pupal development, creating the bilayer of cells comprising the mature wing blade (Lunde, 2003).

Genes involved in initiating wing vein development (vein genes) are expressed during the third larval instar in narrow stripes, corresponding to vein primordia, or in broader 'provein' stripes, consisting of cells that are competent to become vein cells. Some vein genes are expressed in all vein primordia, while others are expressed in subsets of veins or in single veins. For example rhomboid (rho), which encodes an integral membrane protein, is expressed in all vein primordia and promotes vein formation throughout wing development by locally activating the Egfr signaling pathway. caupolican (caup) and the neighboring gene araucan (ara) encode related homeobox genes that promote expression of other vein genes such as rho in odd numbered veins. Proneural genes such as achaete (ac) and scute (sc) promote neural development in the L1 and L3 primordium. Delta (Dl) encodes a ligand for the Notch (N) receptor, which mediates lateral inhibitory interactions among cells in vein-competent domains during pupal development. Delta is also expressed earlier during larval stages of wing development in all longitudinal veins except L2, and is likely to play a role in limiting vein thickness at this stage as well, since loss of Notch function during larval development leads to greatly broadened expression of the vein marker rho. kni and knrl, encoding related zinc finger transcription factors in the steroid hormone superfamily, are expressed in a single stripe corresponding to the L2 primordium beginning in the mid-third larval instar, and are required to initiate L2 development (Lunde, 2003).

The L2 stripe of kni/knrl-expressing cells forms along the anterior border of a broad domain of cells expressing high levels of the related and functionally overlapping spalt-major (salm) zinc finger transcription factors. A variety of evidence indicates that central domain cells expressing the patterning genes salm and salr (together referred to as sal) induce their anterior neighbors, which express very low levels of sal, to become the L2 primordium. For example, in wings containing salm-mutant clones, ectopic branches of L2 are induced that track along and inside the salm- clone borders, mimicking the normal situation in which an L2 vein forms just outside the domain of high-level sal-expressing cells. This ability of sal-expressing cells to induce their anterior low-level sal-expressing neighbors to initiate L2 development requires the activity of the kni locus. The induction of kni/knrl expression in the L2 primordium therefore provides an excellent system for studying the transition from spatial patterning to tissue morphogenesis (Lunde, 2003).

Once activated along the anterior sal border, kni and knrl organize development of the L2 vein, in part by activating expression of the key vein-promoting gene rho and by suppressing expression of the intervein gene blistered (bs). The kni locus is highly selective in regulating downstream gene expression in the L2 primordium as revealed by the observation that several genes expressed in other veins, such as caup and ara, ac and sc, and Delta, are excluded from the L2 primordium. Thus, the kni locus links patterning to vein-specific morphogenesis by functioning downstream of sal and upstream of genes involved in vein versus intervein development (Lunde, 2003).

The role of the kni locus in L2 formation has been clarified by analysis of likely regulatory alleles of the kni locus, previously known as radius incompletus (ri). Flies with this mutation lack large sections of the L2 vein. kniri mutants are homozygous viable, in contrast to kni null mutants, which die as embryos with a gap gene phenotype. In support of kniri mutations being wing-specific regulatory alleles of the kni/knrl locus, expression of kni and knrl in the L2 primordium is absent in kniri[1] mutants, and the L2 vein-loss phenotype can be partially rescued by ubiquitous expression of kni in the wing (Lunde, 2003).

These findings suggest that kni and knrl organize the L2 vein developmental program by orchestrating expression of genes that execute distinct subsets of functions required for proper L2 development. Several important unanswered questions remain, including: what function(s) are disrupted in kniri[1] and other existing kniri mutants?; do these mutations eliminate the function of an L2-specific cis-regulatory element in the kni locus?; and finally, with respect to the definition of L2 versus other veins, is it important to exclude expression of genes expressed in veins other than the L2 primordium (Lunde, 2003)?

An enhancer element upstream of the kni coding region selectively directs gene expression in the L2 primordium in third instar larval wing discs. Three separate ri alleles have defects mapping within a minimal 1.4 kb L2 enhancer element. Two of these mutations eliminate activity of the L2 enhancer, kniri[1], which contains a 252 bp deletion, and kniri[53j], which harbors a single base-pair substitution. Truncation of the minimal L2 enhancer to a 0.69 kb fragment leads to ectopic reporter gene expression in the extreme anterior and posterior regions of the wing, indicating that repression contributes to restricting activation of the L2 enhancer. In addition, the general wing promoting transcription factor Scalloped (Sd) binds with high affinity to several sites in the L2 enhancer and sd is required for kni expression in the wing disc. The L2 enhancer element has been employed as a tool to drive expression of various UAS transgenes in the L2 primordium. The loss of the L2 vein in ri mutants can be rescued by L2-specific expression of either the kni or knrl genes, or the downstream target gene rho. In addition, misexpression of genes in the L2 primordium that are normally expressed in veins other than L2 results in abnormal L2 development. These results provide a framework for understanding how positional information is converted into morphogenesis of the L2 wing vein by 'vein organizing genes' such as kni and knrl (Lunde, 2003).

The results described in this study demonstrate definitively that ri mutations are regulatory alleles of the kni locus disrupting the function of a cis-regulatory enhancer element that drives gene expression in the L2 primordium of wing imaginal discs. A crucial line of evidence supporting this conclusion is that mutant versions of the L2 enhancer incorporating either the 252 bp deletion present in kniri[1] or the single base pair substitution present in kniri[53j] eliminate the ability of this element to direct gene expression in the L2 primordium. In addition, it is possible to completely rescue the vein-loss phenotype of kniri[1] by expressing either the UAS-kni or UAS-knrl transgenes with an L2-GAL4 driver. Consistent with activation of rho being one of the key effectors of kni/knrl function, it is also possible to rescue the L2 vein-loss phenotype of kniri[1] by expressing a UAS-rho transgene in L2, although rescue is less complete and penetrant than that observed with UAS-kni or UAS-knrl (Lunde, 2003).

The isolation of the L2 enhancer also addresses an unresolved question regarding the basis for the failure of Df(3L)kniri[XT2] and Df(3L)kniFC82 to complement. Since these two deletions have endpoints that break within the same 1.7 kb EcoRI fragment, one explanation for the failure of complementation could be that this 1.7 kb fragment contains the L2 enhancer. The alternative explanation for the ri phenotype of Df(3L)kniri[XT2]/Df(3L)kniFC82 is that transvection, which normally occurs between regulatory and coding region alleles of the kni locus, is interrupted by the large divergent deletions that have little if any overlap. Since the 4.8 EcoRI fragment containing the L2 enhancer maps nearly 10 kb upstream of the 1.7 kb EcoRI fragment, and a lacZ fusion construct containing the 1.7 kb EcoRI fragment does not drive any gene expression in the wing disc, the latter hypothesis that Df(3L)kniri[XT2] and Df(3L)kniFC82 are unable to engage in effective transvection is the most likely explanation for the failure of these two mutations to complement (Lunde, 2003).

The results of these and previous studies of L2 vein initiation led to a model in which localized vein inductive signaling acts against a background of regional repression and global wing-specific activation. Previous genetic experiments have suggested a model in which sal-expressing cells produce a short-range L2 inducing signal (X) to which sal-expressing cells themselves cannot respond. According to this 'for export only' signaling model, the signal X diffuses into adjacent anterior cells and activates expression of the kni/knrl genes in the L2 primordium, in much the same fashion as Hh, expressed from the refractory posterior compartment, activates expression of target genes such as dpp or ptc in the anterior compartment. Based on the analysis of the L2 enhancer element described in this study, the 252 bp region deleted in kniri[1] mutants may contain response element(s) to this putative factor X. It is worth noting, however, that lacZ expression is lost in all cells of the wing disc (posterior and circumferential cells as well as the L2 primordium) when these sequences are deleted from the 4.8 kb fragment. This observation suggests that sequences mediating more general activation (e.g. Sd binding sites) may also be contained within this 252 bp region. The sequence surrounding the single nucleotide alteration in kniri[53j] mutants (C596A), however, is a particularly intriguing candidate for mediating the L2-specific activation of kni since introducing this mutation in the context of the minimal 1.4 kb enhancer leads to selective loss of lacZ expression only in the stripe of cells adjacent to the sal domain (Lunde, 2003).

The hypothetical transcription factor X' that binds to the region surrounding the kniri[53j] point mutation and mediates the inductive signal X presumably collaborates with the more generally required wing selector Sd, since mutation of four of the Sd binding sites (the doublet and two single sites) in the L2 activation domain completely eliminates enhancer activity in the wing disc. Clonal analysis with a hypomorphic sd allele also indicates that sd is required for high-level expression of the full 4.8 kb L2 enhancer element in the wing disc. It is notable that the reduction in lacZ expression in these clones is not as dramatic as the complete loss of L2 activity observed when Sd binding sites in the activation domain are mutated. There are several possible explanations for this discrepancy. (1) The sd mutation used in these experiments is a hypomorphic allele and therefore has residual activity. Unfortunately, stronger sd alleles produce even smaller viable clones in the wing disc and thus were not used. (2) Since only small clones can be generated, they must typically have been produced with only two or three intervening cycles of cell division. Consequently, the sd- cells may still contain functional levels of wild type Sd (protein perdurance). (3) Another possibility is that other activators can partially substitute for Sd, at least in certain regions of the wing. Based on the absence of L2 activity when Sd binding sites are mutated and the reduction in L2 activity in sd- hypomorphic clones, it is concluded that Sd plays an important role as an activator of the L2 enhancer. These results support the view that Sd functions as a general transcriptional activator of genes expressed in the wing field (Lunde, 2003).

Repression also plays a key role in restricting L2 enhancer expression to a narrow stripe of wing disc cells. It has been shown previously that salm and salr, which are expressed strongly in the central region of the wing, repress expression of kni and knrl, although low levels of sal may also be required to activate kni expression. Evidence has been found for repression of L2-enhancer activity in peripheral wing disc cells abutting those expressing high levels of sal. Truncation of the minimal 1.4 kb (fragment EX) L2 enhancer element results in reporter gene expression expanding to fill the anterior and posterior regions of the wing pouch in a pattern complementary to that of sal. The region deleted from the EX fragment contains several consensus binding sites for Brinker, which may mediate this repression since the pattern of brinker (brk) expression in the wing pouch is very similar to that of lacZ expression driven by the 0.69 kb fragment EC. One way to integrate the action of the localized inductive signal X-->X' pathway with that of abutting central and peripheral repressive factors is to propose that the signal-dependent activator X' can overcome the repressive action of the peripheral inhibitor but not repression by Salm/Salr. One feature common to several prominent signaling pathways is that activation of the pathway converts a resting repressor into a transcriptional activator. In the kni L2 enhancer, activation of the hypothetical signal X pathway may relieve repression by a heterologous repressor since the putative activator (i.e. X') and repressor (e.g. Brk?) sequences in the L2 enhancer are separable. In addition to central and peripheral repression in the wing disc, there may also be a repressor in posterior compartment cells (e.g. En) to prevent activation of the L2 enhancer in cells posterior to the sal expression domain. Perhaps the 4.8 kb L2 enhancer element lacks some sites for this putative repressor since E-lacZ drives significantly higher levels of gene expression in the posterior stripe than those observed for endogenous kni or knrl expression (Lunde, 2003).

All longitudinal veins share several morphological characteristics such as being composed of densely packed cells on both the dorsal and ventral surfaces of the wing that secrete a thickened cuticle. The primordia of all longitudinal veins also express the rhomboid gene, which is required for activating the Egfr pathway in vein but not in intervein cells. In addition to these shared properties, each vein can be distinguished by expression of other vein genes. For example, vein L2-specific characteristics include: expression of kni and knrl, lack of Delta expression, lack of caup/ara expression, and lack of ac/sc expression. Given that all veins are ultimately quite similar morphologically, it is relevant to ask whether the differences in gene expression patterns observed in different veins are important (Lunde, 2003).

This study examines the necessity of excluding expression of non-L2 vein genes in the L2 primordium by forcing expression of genes such as Dl, ara, ac and sc in L2 and asking whether this manipulation had any impact on L2 development. These experiments strongly suggest that exclusion of non-L2 vein genes from the L2 primordium is indeed important since misexpression of each of these genes results in abnormal L2 development. The phenotypes resulting from misexpressing non-L2 vein genes in the L2 primordium can largely be reconciled with the normal functions of these genes. For example, forced expression of Dl leads to loss of L2, consistent with suppression of vein formation by Notch signaling. The ectopic bristles that form strictly along the L2 vein in wings misexpressing ac or sc are also consistent with the neural promoting function of proneural genes. It is less clear why misexpression of the ara gene causes thinning of the L2 primordium, since this gene normally activates expression of the vein-promoting gene rho in odd numbered veins and ac and sc in the L3 primordium. Perhaps expression of a gene normally involved in development of the odd numbered dorsal veins is somehow incompatible with development of the ventral L2 vein (Lunde, 2003).

The sharp stripe of gene expression driven by L2-GAL4 can also be used as a rapid assay to test the function or range of action of various genes in the wing. For example, in the case of the Egfr pathway, it is possible to distinguish components exerting cell-autonomous versus non cell-autonomous functions. In line with the fact that activation of the Egfr pathway in the wing leads to the formation of veins, L2-GAL4-driven expression of the secreted ligand sSpi generates ectopic veins in the neighborhood of L2 while expression of the equally potent constitutively active lambdaTop form of the EGF receptor does not result in a non-autonomous phenotype. The L2 expression system may also be used to compare the functions of related genes such as the nuclear receptors Kni, Knrl, and Eagle (Eg). Although these three transcription factors share nearly identical DNA binding domains and can all rescue the vein-loss phenotype of ri mutants, they differ in that misexpression of eg in the L2 primordium induces the formation of ectopic bristles along L2. This distinct activity of eg may relate to its normal embryonic function in directing cell fate choices in the CNS. Using the L2 expression system as an assay, it should be straightforward to map the domain in eagle that is responsible for its neural inducing capacity by constructing chimeric molecules composed of different domains of Eagle and Kni. Finally, the observation that L2-driven expression of Dl leads to a highly penetrant loss of L2 in males and a consistently weaker phenotype in females provides the basis for a modifier screen to identify mutations that either suppress the phenotype in males or enhance the phenotype in females. Some of the modifier loci identified in such a screen, that are not specifically involved in Notch signaling, may encode components of the hypothesized factor X-->X' pathway (Lunde, 2003).

Drosophila Brakeless interacts with Atrophin and is required for Tailless-mediated transcriptional repression in early embryos: Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA

Complex gene expression patterns in animal development are generated by the interplay of transcriptional activators and repressors at cis-regulatory DNA modules (CRMs). How repressors work is not well understood, but often involves interactions with co-repressors. Mutations were isolated in the brakeless gene in a screen for maternal factors affecting segmentation of the Drosophila embryo. Brakeless, also known as Scribbler, or Master of thickveins, is a nuclear protein of unknown function. In brakeless embryos, an expanded expression pattern was noted of the Krüppel (Kr) and knirps (kni) genes. Tailless-mediated repression of kni expression is impaired in brakeless mutants. Tailless and Brakeless bind each other in vitro and interact genetically. Brakeless is recruited to the Kr and kni CRMs, and represses transcription when tethered to DNA. This suggests that Brakeless is a novel co-repressor. Orphan nuclear receptors of the Tailless type also interact with Atrophin co-repressors. Both Drosophila and human Brakeless and Atrophin interact in vitro, and it is proposed that they act together as a co-repressor complex in many developmental contexts. The possibility is discussed that human Brakeless homologs may influence the toxicity of polyglutamine-expanded Atrophin-1, which causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA) (Haecker, 2007).

Repression plays a pivotal role in establishing correct gene expression patterns that is necessary for cell fate specification during embryo development. For example, in the early Drosophila embryo, repression by gap and pair-rule proteins is essential for specifying the positions of the 14 segments of the animal. The mechanisms by which transcriptional repressors delimit gene expression borders are not well understood. However, many repressors require co-repressors for function. In the Drosophila embryo, the CtBP and Groucho co-repressors are required for activity of many repressors. Atrophin has been identified as a co-repressor for Even-skipped and Tll. Still, co-regulators for several important transcription factors in the early embryo have not yet been identified. Therefore a screen was performed for novel maternal factors that are required for establishing correct gene expression patterns in the early embryo (Haecker, 2007).

From this screen, mutations were identified in the bks gene that cause severe phenotypes on gap gene expression and embryo segmentation. The Bks protein is evolutionarily conserved between insects and deuterostomes, but has not been characterized in any species except Drosophila, in which it has been shown to repress runt expression in photoreceptor cells and thickveins expression in wing imaginal discs. However, the molecular function of Bks has been unknown. This study shows that Bks interacts with the transcriptional repressor Tll, is recruited to target gene CRMs, and will repress transcription when targeted to DNA (Haecker, 2007).

Tll has been shown to utilize Atrophin as a co-repressor. Atrophin genetically interacts with Tll and physically interacts with its ligand binding domain. Atrophin binding is conserved in nuclear receptors within the same subfamily, such as Seven-Up in Drosophila as well as Tlx and COUP-TF in mammals. When expressed in mammalian cells, Drosophila Atrophin and mouse Atrophin-2 interact with the histone deacetylases HDAC1 and HDAC2. Histone deacetylation may therefore be part of the mechanism by which Atrophin functions as a co-repressor. Another recent report described genetic interactions among bks and atrophin mutants in the formation of interocellar bristles in adult flies. Furthermore, it was shown that atrophin mutants have virtually identical phenotypes as bks mutants, including de-repression of runt expression in the eye, thickveins expression in the wing, and Kr and kni expression in the embryo (Haecker, 2007).

Both proteins are recruited to the kni CRM, a Tll-regulated target gene, in the embryo. Importantly, Atrophin and Bks interact in vitro and that they can be co-immunoprecipitated from S2 cells. It is proposed that Bks and Atrophin function together as a co-repressor complex, and based on the similar bks and atrophin mutant phenotypes at several developmental stages, the complex may function throughout development. These results are compatible with the existence of a tripartite complex consisting of Tll, Bks, and Atrophin. Bks binding to Tll is enhanced by the Tll DNA binding domain, whereas the interaction of Tll with Atrophin is mediated through the C-terminal ligand binding domain. Tll may therefore simultaneously interact with Bks and Atrophin. Alternatively, Tll interacts separately with Bks and Atrophin on the kni CRM. In either case, both Bks and Atrophin are required for full Tll activity. However, at high enough Tll concentration, Bks activity is dispensable. Some bks embryos misexpressing Tll still repress kni expression, and overexpressing Tll from a heat-shock promoter can repress the posterior kni stripe in both wt and bks mutant embryos. For this reason, it is believed that Bks and Atrophin are cooperating as Tll co-repressors, so that Tll function is only partially impaired by the absence of either one. It was found that Tet-Bks-mediated repression in cells is insensitive to the deacetylase inhibitor trichostatin A (TSA). It is possible, therefore, that whereas Atrophin-mediated repression may involve histone deacetylation, Bks could repress transcription through a separate mechanism (Haecker, 2007).

These results have not revealed any differences between the molecular functions of the two Bks isoforms. Both Bks-A and Bks-B repress transcription when tethered to DNA, and the sequences that mediated binding to Tll and Atrophin are shared between the two isoforms. However, the bks339 allele that selectively affects the Bks-B isoform causes a weaker, but comparable phenotype to the stronger bks alleles that disrupt both isoforms. Therefore, the C-terminus of Bks-B provides a function that is indispensable for embryo development and regulation of kni expression. This part of Bks-B contains two regions (D3 and D4) that are highly conserved in insects and loosely conserved in deuterostome Bks sequences, but does not resemble any sequence with known function. The only sequence similarity to domains found in other proteins is a single zinc-finger motif in Bks-B. Preliminary results indicate that the zinc finger in isolation or together with the conserved D2 domain does not exhibit sequence-specific DNA binding activity. Indeed, multiple zinc fingers are generally required to achieve DNA binding specificity. Instead, Bks is likely brought to DNA through interactions with Tll and other transcription factors (Haecker, 2007).

Atrophins are required for embryo development in C. elegans, Drosophila, zebrafish, and mice. In vertebrates, two atrophin genes are present. Atrophin-1 is dispensable for embryonic development in mice, and lacks the N-terminal MTA-2 homologous domain that interacts with histone deacetylases . However, the homologous C-termini of Atrophin-1 and Atrophin-2 can interact, and it was found that this domain can also bind to the human Bks homolog ZNF608. Atrophin-1 interacts with another co-repressor-associated protein as well, ETO/MTG8, and can repress transcription when tethered to DNA. These data are consistent with the emerging view that deregulated transcription may be an important mechanism for the pathogenesis of polyglutamine diseases. Recent evidence indicates that interactions with the normal binding partners may cause toxicity of polyglutamine-expanded proteins such as Ataxin-1 . It will be interesting to investigate whether the interaction between human Bks homologs and Atrophin-1 is important for the neuronal toxicity of polyglutamine-expanded Atrophin-1 (Haecker, 2007).

Multiple enhancers ensure precision of gap gene-expression patterns in the Drosophila embryo

Segmentation of the Drosophila embryo begins with the establishment of spatially restricted gap gene-expression patterns in response to broad gradients of maternal transcription factors, such as Bicoid. Numerous studies have documented the fidelity of these expression patterns, even when embryos are subjected to genetic or environmental stress, but the underlying mechanisms for this transcriptional precision are uncertain. This study presents evidence that every gap gene contains multiple enhancers with overlapping activities to produce authentic patterns of gene expression. For example, a recently identified hunchback (hb) enhancer (located 5-kb upstream of the classic enhancer) ensures repression at the anterior pole. The combination of intronic and 5' knirps (kni) enhancers produces a faithful expression pattern, even though the intronic enhancer alone directs an abnormally broad expression pattern. Different models are presented for 'enhancer synergy,' whereby two enhancers with overlapping activities produce authentic patterns of gene expression (Perry, 2011).

Candidate gap enhancers were identified using ChIP-chip data. Specifically, clustered binding sites for maternal and gap proteins were identified within 100 kb of every gap gene. This survey identified each of the known enhancers, as well as putative shadow enhancers. For example, a potential distal shadow enhancer was identified for hb, located 4.5-kb upstream of the proximal transcription start site (designated 'P2' in earlier literature) and upstream of the later-acting distal promoter (designated 'P1') (Perry, 2011).

A 400-bp genomic DNA fragment from this newly identified region was attached to a lacZ reporter gene and expressed in transgenic embryos. The resulting hb/lacZ fusion gene exhibits localized expression in anterior regions of the embryo similar to that seen for the endogenous gene and 'classic' enhancer identified over 20 y ago. The classic proximal and distal shadow enhancers exhibit similar responses to increasing Bicoid copy number (Perry, 2011).

ChIP-chip data also identified potential pairs of enhancers for Kr and kni. There are two distinct clusters of transcription factor binding sites upstream of Kr. The previously identified Kr 'CD2' enhancer contains the proximal enhancer but also part of the distal binding cluster. Subsequent lacZ fusion assays identified each ChIP-chip peak and underlying binding sites as separable proximal and distal enhancers. Similarly, more refined limits were determined for the kni intronic enhancer, in addition to the previously identified 5' distal enhancer. Both the distal Kr enhancer and the intronic kni enhancer produce somewhat broader patterns of expression than the endogenous gene. Additional gap enhancers were also identified for giant, including an additional distal enhancer located ~35-kb downstream within a neighboring gene (Perry, 2011).

The survey of gap and maternal binding clusters was extended to include the so-called 'head' and 'terminal' gap genes, critical for the differentiation of head structures and the nonsegmented termini of early embryos. Additional enhancers were identified for empty-spiracles (ems), huckebein (hkb), and forkhead (fkh). More refined limits were also determined for the previously identified ocelliless/orthodenticle (oc/otd) intronic enhancer. For simplicity, the two enhancers regulating a given gap gene will be identified as proximal and distal, based on their relative locations to the transcription start site (Perry, 2011).

BAC recombineering, phiC31-targeted genome integration, and quantitative in situ hybridization assays were used to determine the contributions of the proximal and distal enhancers to the hb expression pattern. BACs containing ~20 kb of genomic DNA encompassing the hb gene and flanking sequences were integrated into the same position in the Drosophila genome. The hb transcription unit was replaced with the yellow gene, which permits quantitative detection of nascent transcripts using an intronic hybridization probe. The modified BAC retains the complete hb 5' and 3' UTRs. Additional BACs were created by inactivating the proximal or distal enhancers by substituting critical regulatory elements with 'random' DNA sequences (Perry, 2011).

BAC transgenes lacking either the distal or proximal enhancer continue to produce localized patterns of transcription in anterior regions of transgenic embryos in response to the Bicoid gradient. However, the patterns are not as faithful compared with the BAC transgene containing both enhancers. Embryos were double-labeled to detect both yellow and hb nascent transcripts. During nuclear cleavage cycle (cc) 13, a substantial fraction of nuclei (14%) expressing hb nascent transcripts lack yellow transcription upon removal of the shadow enhancer. An even higher fraction of nuclei (24%) lack yellow transcription when the proximal enhancer is removed. Control transgenic embryos containing both enhancers exhibit more uniform patterns of transcription, whereby only an average of ~3% of nuclei fail to match the endogenous pattern of transcription (Perry, 2011).

The pairwise Wilcoxon rank sum test (also called the Mann-Whitney u test) was used to determine the significance of the apparent variation in gene expression resulting from the removal of either the proximal or distal enhancer. Control embryos containing the hb BAC transgene with both enhancers exhibit some variation in the number of nuclei that lack yellow nascent transcripts. Despite this variation, the statistical analyses indicate that the loss of either the proximal or distal enhancer results in a significant change in yellow transcription patterns compared with the control BAC transgene (Perry, 2011).

The preceding analyses suggest that multiple enhancers produce more uniform patterns of de novo transcription than individual proximal or distal enhancers. Additional studies were done to determine whether multiple enhancers also help produce authentic spatial limits of transcription (Perry, 2011).

The expression of hb normally diminishes at the anterior pole of cc13 to 14 embryos. This loss in expression has been attributed to attenuation of Bcd activity by Torso RTK signaling. However, the proximal enhancer fails to recapitulate this loss. In contrast, the distal enhancer is inactive at the anterior pole, and the two enhancers together produce a pattern that is similar to endogenous expression, including reduced expression at the pole (Perry, 2011).

To examine the relative contributions of the proximal and distal enhancers in this repression, yellow nascent transcripts were measured in transgenic embryos expressing BAC reporter genes containing one or both hb enhancers. Particular efforts focused on the early phases of cc14, when repression of endogenous hb transcripts is clearly evident. For the transgene lacking the proximal, classic enhancer, but containing the newly identified distal enhancer, a median of 6% (std 6%) of nuclei exhibit expression of yellow nascent transcripts but lack expression of the endogenous gene. In contrast, a median of 24% (std 11%) of nuclei displays a similar discordance upon removal of the distal enhancer. In control embryos, 16% (std 11%) of nuclei express yellow but lack hb nascent transcripts. It should be noted that the BAC transgene lacking the proximal enhancer exhibits 'super-repression' because of reduced activation at the anterior pole (Perry, 2011).

Kr/lacZ and kni/lacZ fusion genes containing either one or two enhancers were inserted into the same position in the Drosophila genome. Transgenic embryos were double-labeled to detect the expression of the transgene (lacZ) as well as the endogenous gap gene (Perry, 2011).

The kni proximal (intronic) enhancer alone produces an abnormally broad pattern of expression, especially in posterior regions. In contrast, the kni distal (5') enhancer produces erratic lacZ activation within nearly normal spatial limits. An essentially normal pattern of lacZ transcription is observed when both enhancers are combined in a common transgene (intronic enhancer 5' and distal enhancer 3' of lacZ). It appears that lacZ transcription is slightly broader than the endogenous pattern, but considerably narrower than the pattern observed for the intronic enhancer alone, and not statistically different from the expression limits of the distal enhancer alone. There is no significant narrowing of the Kr/lacZ expression pattern when both the distal and proximal enhancers are combined within the same transgene. Perhaps additional Kr regulatory elements are required for the type of narrowing observed for the kni intronic enhancer. Alternately, all of these transgenes use the eve basal promoter and it is possible that promoter-specific interactions are important for establishing the normal limits of the Kr expression pattern (Perry, 2011).

As discussed earlier, long-range repressors bound to the distal hb enhancer might inhibit the activities of the proximal enhancer at the anterior pole of precellular embryos. The distal kni enhancer might function in a similar manner to sharpen the expression limits of the intronic enhancer. The spatial limits of gap gene-expression patterns have been shown to depend on cross-repressive interactions. The kni intronic enhancer might lack critical gap repression elements because it produces an abnormally broad expression pattern. Indeed, whole-genome ChIP assays identify more putative Tailless binding sites in the distal vs. intronic enhancer. These Tailless repression elements might function in a dominant fashion to restrict the limits of the intronic enhancer (Perry, 2011).

The modest anterior expansion of the expression pattern driven by the kni intronic enhancer is more difficult to explain because this boundary is probably formed by the Hb repressor, which is not known to function in a long-range and dominant manner. If the action of short-range repressors is also affected by stochastic processes (e.g., binding of the repressor to enhancer or looping of a bound enhancer to promoter), perhaps having two enhancers might improve the chances of maintaining proper repression (Perry, 2011).

This study has presented evidence that the robust and tightly defined patterns of gap gene expression do not arise from the unique action of individual enhancers. Rather, these patterns depend on multiple and separable enhancers with similar, but slightly distinct regulatory activities. This enhancer synergy produces more homogeneous patterns of transcriptional activity, as well as more faithful spatial limits of expression (Perry, 2011).

The enhancer synergy documented in this study is somewhat distinct from the proposed role of the shadow enhancer regulating snail expression in the presumptive mesoderm. The dual regulation of snail by the proximal and distal (shadow) enhancers was shown to ensure homogenous and reproducible expression in embryo after embryo in large populations of embryos, even when they are subject to increases in temperature. In contrast, dual regulation of hb expression by proximal and distal enhancers appears to ensure homogenous activation in response to limiting amounts of the Bicoid gradient. They are used as an obligatory patterning mechanism rather than buffering environmental changes. Despite these apparent differences, it is possible that dominant repression is also used as a mechanism of synergy for the regulation of snail expression. The distal enhancer contains repressor elements (e.g., Huckebein) that inhibit the expression of the proximal enhancer at the termini (Perry, 2011).

Different mechanisms can be envisioned to account for enhancer synergy. Perhaps the simplest is that there are fewer inactive nuclei within a given gap expression domain because of the diminished failure rate of successful enhancer-promoter interactions with two enhancers rather than one. If the rates at which enhancers fail to activate transcription are completely independent, then one would expect the combined action of two enhancers to yield a multiplicative reduction in how often a given cell fails to express the gene within a given window of time. This sort of synergy does not require any direct physical or cooperative interactions between the enhancers. Nonetheless, the effect can be significant (as seen for hb). For example, two enhancers, each with a 10% uncorrelated failure rate, may together be expected to have a 1% failure rate, a 10-fold reduction. For genes that produce strong bursts of mRNA expression, this change in frequency of transcription may have a dramatic effect on the variation of total mRNA levels (Perry, 2011).

A second but critical potential mechanism of enhancer synergy concerns long-range, dominant repression. Repressors (such as Tailless) bound to one enhancer are sufficient to restrict the spatial limits of the other enhancer. There is no need for long-range repressor elements to appear in both enhancers to achieve normal spatial limits of gene expression. It has been suggested that long-range repressors, such as Hairy, mediate the assembly of positioned nucleosomes at the core promoter. Such repressive nucleosomes should block productive enhancer-promoter interactions, even for enhancers lacking repressor sites (Perry, 2011).

Regardless of the detailed molecular mechanisms, the combined action of multiple enhancers helps explain why an individual enhancer sometimes fails to recapitulate an authentic expression pattern when taken from its native context. Enhancers that produce abnormal patterns of expression (e.g., kni intronic enhancer) can nonetheless contribute to homogeneous and robust patterns of gene expression in conjunction with the additional enhancers contained within the endogenous locus (Perry, 2011).

Transcriptional Regulation

Most of the thoracic and abdominal segments of Drosophila are specified early in embryogenesis by the overlapping activities of hunchback (hb), Krüppel, knirps, and giant. The orderly expression of these genes depends on two maternal determinants: bicoid, which activates anterior HB transcription, and Nanos, which blocks posterior translation of HB transcripts. The resulting gradient of HB protein dictates where Krüppel, knirps, and giant are expressed by providing a series of concentration thresholds that regulate each gene independently (Struhl, 1992).

The transactivating factor requirements for the expression of kni and knirps-related are identical for the two anterior expression domains. Requirements differ slightly for the posterior domain of expression in the blastoderm. Both the anterior/posterior morphogen bicoid and the dorsoventral morphogen dorsal are necessary but not sufficient for the activation of the kni and kni-r in the anterior cap domain, suggesting BIC and DL act together to bring about normal spatial limits kni (Rothe, 1994).

Zygotic expression of caudal forms an abdominal and a posterior domain. The cad domain functions by activating the expression of knirps and giant (Schulz, 1995).

When ectopically expressed, giant represses the expression of both Krüppel and knirps. Analysis of interactions among Krüppel, knirps and giant reveals a network of negative regulation. The apparent positive regulation of knirps expression by Krüppel is in fact mediated by a negative effect of Krüppel on giant and a negative effect of Giant on knirps expression (Capovilla, 1992).

Decapentaplegic controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. The requirement for Dpp is revealed by two manipulations: (1) the overexpression of Dpp using a heat-shock promoter and (2) use of mutations in the Dpp receptors thickveins and punt. The failure of tracheal cells to receive the DPP signal from adjacent dorsal and ventral cells results in the absence of dorsal and ventral migrations. Ectopic Dpp signaling can reprogram cells in the center of the placode to adopt a dorsoventral migration behavior. The effects observed in response to ectopic Dpp signaling are also observed upon the tracheal-specific expression of a constitutive active Dpp type I receptor (TKV[Q253D]). The alterations in migration behavior are similar for constitutively active receptor and for Dpp ectopic expression, indicating that the Dpp signal is received and transmitted in tracheal cells to control their migration behavior. Whereas, lack of Dpp signaling results in a failure of tracheal cells to migrate along the dorsoventral axis without significantly affecting anterior migrations, ubiquitous Dpp signaling suppresses anterior migrations without interfering with dorsoventral migration (Vincent, 1997).

Dpp signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the Branchless (Bnl) guidance molecule, the ligand of the Breathless (Btl) receptor. The gene knirps is activated in the developing tracheal system in all the branches (dorsal branch, ganglionic branch, and lateral trunk) that are thought to be under the control of Dpp. kni expression is lost in tkv mutants; (kni expression only persists in the visceral branches of tkv mutants). kni expression is turned on in all tracheal cells after constitutive Dpp signaling (Vincent, 1997).

Mutations in several Polycomb group (Pc-G) genes cause maternal-effect or zygotic segmentation defects, suggesting that Pc-G genes may regulate the segmentation genes of Drosophila. Individuals doubly heterozygous for mutations in polyhomeotic and six other Pc-G genes show gap, pair rule, and segment polarity segmentation defects. Posterior sex combs and polyhomeotic interact with Krüppel and enhance embryonic phenotypes of hunchback and knirps mutants (McKeon, 1994).

Suppressors of nanos have been isolated. In the absence of nanos high levels of HB protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high HB levels. The suppressors are alleles of Enhancer of zeste [E(z)]a member of the Polycomb group of genes. E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal HB protein gradient. A small region of the knirps promoter mediates the regulation by E(z) and HB. Because Pc-G genes are thought to control gene expression by regulating chromatin, it is proposed that imprinting at the chromatin level underlies the determination of anterior/posterior polarity in the early embryo (Pelegri, 1994).

Needs and targets for the multi sex combs gene product in Drosophila melanogaster

The requirements for the multi sex combs (mxc) gene during development have been examined to gain further insight into the mechanisms and developmental processes that depend on the important trans-regulators forming the Polycomb group (PcG) in Drosophila. Although mxc has not yet been cloned, it is known to be allelic with the tumor suppressor locus lethal (1) malignant blood neoplasm [l(1)mbn]. The mxc product is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc also has a strong maternal effect. Hypomorphic mxc mutations are found to enhance other PcG gene mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are cooperatively involved in repression of selector genes outside their normal expression domains. The mxc product is needed for imaginal head specification, through regulation of the ANT-C gene Deformed. This analysis reveals that mxc is involved in the maternal control of early zygotic gap gene expression known to involve some other PcG genes and suggests that the mechanism of this early PcG function could be different from the PcG-mediated regulation of homeotic selector genes later in development (Saget, 1998).

Induction of uncontrolled growth and deregulation of Hox genes are linked in mammals, where Hox products can induce leukemia. In Drosophila, modification of homeotic gene expression causes homeosis, sometimes associated with increased proliferation but not with uncontrolled tumorous growth, possibly because the identity of each segment is specified by a combination of HOM products. Loss or gain of one HOM gene will likely lead to a new combination that is found elsewhere in wild type, and cells expressing this combination could be expected to follow the corresponding developmental pathway and give rise to homeotic transformations. However, because each cellular identity apparently corresponds to a given proliferation rate, loss or ambiguity of identity due to deregulation of several selector genes in a single cell, such as mxc mutations apparently induce, could lead to loss of proliferation control. Identification of mxc partners and targets, as well as of the molecular nature of the mxc product, may help throw light on the genes and mechanisms involved in this process (Saget, 1998).

It has been proposed that certain PcG genes are required for the maintenance of the expression domains of knirps and giant, through a mechanism similar to the regulation of homeotic genes. The regionalization of the Drosophila embryo depends on the maternally supplied products of bicoid (bcd), hunchback (hb), and nanos (nos). Nos represses the translation of the maternal HB mRNA in the posterior embryonic region. This permits the expression of the zygotic gap genes knirps (kni) and giant (gt), which specify posterior identities. These genes would otherwise be repressed by Hb. Embryos from nos/nos mothers form no abdominal segments, but this phenotype can be rescued by a total lack of hb in the maternal germline. It can also be dominantly rescued by the mutation of maternally supplied regulator molecules that normally repress kni and gt in the zygote. Pelegri and Lehmann (1994) have shown that certain mutant products of the PcG genes E(z), Psc, and pleiohomeotic can partially rescue nos by such a maternal effect. To determine if mutation of mxc also affects this regulation, the cuticles of embryos were examined from mxc/+;hb nos/nos mothers that were heterozygous for different mxc mutations. This genetic background was used because a decrease in the amount of maternal hb product can partially rescue the nos phenotype in F1 embryos. Such embryos can differentiate a few abdominal denticle belts and form an adequate background to evaluate increased rescue of nos. Thus loss-of-function PcG mutations should have a strong effect on rescue, and the embryos from hb nos/nos mothers that have two PcG mutations in their genetic background should permit increased rescue of the nos phenotype (Saget, 1998).

Any of three E(z)son (suppressor of nanos) alleles or a hypomorphic pleiohomeotic allele partially rescue the phenotypes of hb nos/nos progeny by a maternal effect; deficiencies covering E(z) or the Psc/Su(z)2 complex also allow some maternal rescue of hb nos/nos progeny, yet the strongest effect is observed with the gain-of-function E(z)son alleles. The EMS-induced allele mxcG48 rescues the hb nos/nos progeny phenotype, whereas a deficiency of mxc does not. Some rescue with the Psc/Su(z)2 complex deletion Df(2)vgB is also observed and strong rescue (consistently >50%) is observed with an EMS-induced pleiohomeotic allele phob, described as amorphic. This suggests that phob and mxcG48 are probably not amorphic alleles, and that maternal rescue of hb nos/nos progeny by a PcG gene is most efficient with a non-null mutation (Saget, 1998).

Segmentation of embryos from transheterozygous mothers was also examined. Because neither a reduction of wild-type PcG product nor two PcG mutations in trans in the hb nos/nos mothers increases nos rescue, these data strongly suggest that, whatever the mechanism of gap gene regulation by these PcG mutations may be, it does not function like the PcG-mediated maintenance of homeotic gene expression in embryos and in imaginal discs. The strong rescue provided by several non-null EMS-induced mutations, which may produce mutant proteins, leads to a proposal that modified PcG proteins are poisoning a normal process. How this process depends on wild-type regulation by PcG products has yet to be established (Saget, 1998).

Thoracic patterning by the Drosophila gap gene hunchback

Localized gene expression patterns are critical for establishing body plans in all multicellular animals. In Drosophila, the gap gene hunchback is expressed in a dynamic pattern in anterior regions of the embryo. Hb protein is first detected as a shallow maternal gradient that prevents expression of posterior gap genes in anterior regions. HB mRNA is also expressed zygotically, first as a broad anterior domain controlled by the Bicoid morphogen, and then in a stripe at the position of parasegment 4 (PS4). The PS4-hb stripe changes the profile of the anterior Hb gradient by generating a localized peak of protein that persists until after the broad domain has started to decline. This peak is required specifically for the formation of the mesothoracic (T2) segment. At the molecular level, the PS4-hb stripe is critical for activation of the homeotic gene Antennapedia, but does not affect a gradient of Hb repressive activity formed by the combination of maternal and Bcd-dependent Hb. The repressive gradient is critical for establishing the positions of several target genes, including the gap genes Kruppel, knirps, and giant, and the homeotic gene Ultrabithorax. Different Hb concentrations are sufficient for repression of gt, kni, and Ubx, but a very high level of Hb, or a combinatorial mechanism, is required for repression of Kr. These results suggest that the individual phases of hb transcription, which overlap temporally and spatially, contribute specific patterning functions in early embryogenesis (Wu, 2001).

Primary zygotic expression of HB mRNA (P2-hb) covers much of the anterior half of wild-type embryos early in nuclear cleavage cycle 14. This pattern is soon transformed into the secondary pattern, which includes the PS4-hb stripe. This stripe appears just before midcycle 14, and persists until the onset of gastrulation. The distribution of Hb protein is similar to the mRNA profile, but the protein seems to degrade more slowly. Thus, in midcycle-14 embryos, the Hb pattern consists of a broad anterior domain, with a peak at the position of PS4. Later, when cellularization is complete, Hb in anterior regions degrades further, leaving a clear stripe at PS4 (Wu, 2001).

To specifically remove this stripe, a misexpression transgene (st2-kni) was used that directs barely detectable levels of the gap gene kni at the position of PS3. Ectopic kni expression completely abolishes the PS4-hb stripe, and the peak of Hb protein observed in wild-type embryos. By the end of cellularization, no protein is detectable at the PS4 position of st2-kni embryos. The relative levels of Hb were further quantified at midcycle 14 in wild-type and st2-kni embryos. The PS4 position was determined by simultaneous fluorescence in situ hybridization with an RNA probe directed against fushi tarazu, a pair-rule gene expressed in stripes that correspond to the even-numbered parasegments (ftz stripe 2 corresponds to PS4). In st2-kni embryos, nuclei at the center of ftz stripe 2 contain only about 50% of the Hb normally present at this position. Thus, in wild-type embryos, the PS4-hb stripe alters the profile of the anterior Hb gradient by creating a peak of protein concentration at midcycle 14, and also ensures the perdurance of high Hb levels at this position until the end of cellularization (Wu, 2001).

In contrast to previous hb rescue experiments, the st2-kni transgene removes the PS4-hb stripe without changing the normal maternal and P2-hb gradients. Embryos carrying this transgene were examined for morphological defects later in development. More than 75% of these embryos die by the end of embryogenesis and show deletions of the T2 denticle band, which is derived from cells in PS4. While approximately 50% show a complete T2 deletion in dorsal and ventral regions, the phenotype is more severe on the ventral side, with ~90% completely lacking the T2 ventral denticle band. The cause of the phenotypic difference between dorsal and ventral regions is not clear. These results indicate that high levels of Hb at the position of PS4 are critical for T2 development (Wu, 2001).

To investigate the role of PS4-hb in T2 development, the expression patterns of segmentation and homeotic genes was examined in st2-kni embryos, and those fully rescued by the st2DeltaK-hb-1 transgene (a transgenic line that directs high levels of hb in a wide stripe that overlaps the PS4 position). No changes in the expression patterns of the gap genes Kr, kni, or gt were detected in st2-kni embryos. This suggests that Hb-mediated repression of these genes is not dependent on the PS4-hb stripe. However, several genes normally expressed in PS4 were significantly altered. One such gene is ftz. In st2-kni embryos, activation of ftz stripe 2 is delayed, and reduced in intensity. To test whether this reduction affects ftz function, expression of the ftz target gene engrailed was examined. en stripe 4, which is normally activated by ftz stripe 2, is also significantly reduced in st2-kni embryos (Wu, 2001).

Previous experiments have implicated Hb as a repressor of the gap genes Kr, kni, and gt, but the expression domains of these genes are not affected by the removal of the PS4-hb stripe. Another gene controlled by Hb-mediated repression is the homeotic gene Ultrabithorax, which is strongly expressed as a stripe at the position of PS6 in late-blastoderm embryos. Since the PS4-hb stripe is also expressed at this stage, whether it is required for setting the anterior Ubx expression border was tested. Like the gap gene targets (Kr, kni, and gt), Ubx expression is undisturbed in embryos specifically lacking the PS4-hb stripe. Thus, the peak of protein provided by this stripe is not required for repression of any of these four genes (Wu, 2001).

In wild-type embryos, the anterior borders of Kr, kni, gt, and Ubx are located at different positions along the anterior-posterior axis, suggesting that they respond to different thresholds of Hb concentration. The position of the gt border, which lies most posteriorly, is established by the maternal Hb gradient. There is very little change in the position of this border in zygotic hb mutants, suggesting that the maternal gradient is sufficient for gt repression. The anterior kni border, which lies six to eight nuclear diameters from nuclei that produce zygotic HB mRNA, is also initially established by the maternal gradient. In mutants lacking zygotic hb, this border is correctly positioned early in cycle 14, but there is an anterior expansion at midcycle 14, possibly due to degradation of the maternal gradient. Whether this expansion is sensitive to ectopic Hb driven by the st2DeltaK-hb construct was tested. These experiments show a dose-dependent repression of kni back to its original position. Since only one copy of the transgene causes a strong repression, kni is sensitive to very low levels of Hb. The homeotic gene Ubx, which is normally expressed about four nuclear diameters from the posterior edge of the hb domain, also expands anteriorly in zygotic hb mutants. Addition of the st2DeltaK-hb transgene causes a dose-dependent repression of this gene as well. In this case, two copies of the transgene are significantly more effective than one, suggesting that Ubx repression requires a higher level of Hb than kni (Wu, 2001).

Function of the spalt/spalt-related gene complex in positioning the veins in the Drosophila wing

Spalt and Spalt-related encode conserved Zn-finger proteins that mediate the function of the TGF-beta molecule Decapentaplegic during the positioning of veins in the Drosophila wing. Spalt and Spalt-related regulate the vein-specific expression of the transcription factors of the knirps and iroquois gene complexes, delimiting their domains of expression in the wing pouch. The effects of spalt/spalt-related mutations on knirps and iroquois expression are cell-autonomous, suggesting that they could be direct. The regulation of iroquois involves transcriptional repression by Spalt and Spalt-related, whereas the regulation of knirps involves a combination of transcriptional activation and repression mediated by the same genes. It is suggested that the regulation of the iroquois and knirps gene complexes by Spalt and Spalt-related translates the Decapentaplegic morphogenetic gradient into precisely spaced pattern elements (de Celis, 2000).

Although the development of the four longitudinal veins of Drosophila (L2-L5) involves the same signaling systems, and vein cells show the identical type of differentiation, there are several characteristics that distinguish one vein from another. Thus, the veins L2 and proximal L4 differentiate predominantly in the ventral wing surface, whereas the veins L3, L5 and distal L4 do so in the dorsal surface. Furthermore, several genes are required for the formation of individual veins, suggesting that each vein is individually specified. Vein-specific genes include the transcription factors of the iroquois gene complex (iro-C), which are only expressed and required in L3 and L5, and the transcription factors of the knirps gene complex (kni-C), which are only expressed and required in L2. Vein-specific genes could be part of a combinatorial code of signals that activate a common vein-differentiation program in different parts of the wing. In addition, vein specific genes could also confer individual qualities to each longitudinal vein (de Celis, 2000 and references therein).

The heterogeneity in the levels of Sal/Salr could have a functional significance during vein patterning, and suggests that other factors, in addition to Dpp, participate in the regulation of sal/salr in the wing pouch. The expressions of Kni (L2) and Iro (L3 and L5) are also related to developing vein regions. Iro proteins are localized in L3 and L5, and are present in the vein and in the associated stripes of E(spl)m beta expression, both during imaginal and pupal development. In the larval disc, Kni is expressed in a domain broader than the vein L2 that corresponds to the region where E(spl)m beta is expressed at low levels. These observations indicate that Iro and Kni are expressed in vein competent regions, and that the loss of L2 and L3/L5 veins in kni and iro mutants, respectively, is due to failures in the specification of the corresponding vein competent region (de Celis, 2000).

The spatial relationships between the distribution of Kni, Iro and Sal were examined directly using appropriate antibodies. Kni is expressed within the anterior edge of the Sal/Salr expression domain, in the region where Sal/Salr are detected at lower levels. This differs from a previous report that placed the limit of Sal expression adjacent to but not overlapping with kni expression. Iro expression in the L5 vein competent region is, in contrast, immediately adjacent to the posterior limit of Sal expression. Thus, each individual vein expresses a unique combination of transcription factors (vein L2: Kni-C + Sal/Salr; veinL3: Iro + Sal/Salr; vein L4: Sal/Salr; L5: Iro) that are required for its formation and could confer individual characteristics to each longitudinal vein (de Celis, 2000).

The contrasting effects of sal/salr on the formation of specific veins (promoting L2 and suppressing L3, L4 and L5) indicate that these genes could both stimulate and antagonize the expression or activity of other vein-promoting genes. Thus, there is no evidence to indicate that sal and salr regulate vein differentiation directly; rather, they appear to influence vein development indirectly through regulating the expression of other genes that define individual veins. This regulation would require low levels of Sal/Salr to promote L2 and higher levels of Sal/Salr to inhibit L3 and L5 development. Two good candidates to be regulated by Sal/Salr are the kni-C and iro-C, because they are expressed in L2 and L3/L5, respectively. Furthermore, the function of kni-C and iro-C is required for the formation of these veins (de Celis, 2000).

To characterize the relationships of sal/salr with kni and iro-C, the effects of sal/salr mutant clones on kni and iro expression were examined. The expression of kni is eliminated in sal/salr clones that overlap the domain of Kni expression. These effects are cell-autonomous and can be observed in very small sal/salr clones. This suggests that, in contrast to the non-autonomous effect of Sal on kni, Sal/Salr regulate kni expression in a cell-autonomous fashion. All sal/salr clones localized between veins L2/L3 and L4/L5 are associated with ectopic expression of Iro proteins. Again, this effect is strictly cell-autonomous, suggesting that the repression of iro-C genes by Sal/Salr could be direct. Ectopic expression of Iro in sal/salr mutant clones is not observed in regions close to the dorso-ventral boundary, presumably because in this region iro-C expression is repressed by wingless. The effects of sal/salr on kni-C and iro-C expression have also been analyzed in experiments in which sal and salr are expressed ectopically using the GAL4 system. Widespread expression of sal or salr in the wing blade eliminates L2 and L5, and prevents expression, respectively, of Kni and Iro in the L2 and L5 territories of the corresponding imaginal discs. The expression of Iro in L3, which depends on Hh activity, is not affected by removal sal/salr functions and only slightly decreased by their ectopic expression. Taken together, these observations indicate that Sal/Salr negatively regulates iro-C expression in cells not exposed to Hh protein, and suggests that precise levels of Sal/Salr proteins are needed to activate kni expression in L2 (de Celis, 2000).

Grunge, related to human Atrophin-like proteins, has multiple functions in Drosophila development

To understand how loss of Grunge activity affects segmentation, the expression of hunchback (hb), Krüppel (Kr), knirps (kni) and fushi tarazu (ftz) was analyzed in embryos derived from Gug35 germline clones fertilized by Gug35 sperm. In wild-type embryos, the expression of these segmentation gene products localizes to discrete domains in the early embryo. In almost all of the expression domains, loss of Gug activity increases the number of cells expressing these segmentation genes, suggesting that Gug plays a role in their repression. Later the expression of ftz displays a more complex defective pattern with some stripes being broader, and others narrower, than wild type (Erkner, 2002).

Loss of Gug activity severely affects the process of segmentation and the expression of segmentation genes when missing from the female germ line. At the blastoderm stage, most of the expression domains of hb, Kr, kni and ftz genes are expanded compared with wild type. These observations indicate that maternal production of Gug is crucial for the repression of these genes to precise domains in the early embryo. Gap proteins, including Hb, Kr and Kni are known to be required to restrict each others domains of expression. It will be interesting to test if Gug acts with these proteins for these repression activities (Erkner, 2002).

Notch signaling controls cell fate specification along the dorsoventral axis of the Drosophila gut

The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).

The regionalization of the hindgut tube involves the formation of three major subregions: the small intestine, which localizes to the anterior end of the hindgut; the large intestine, which represents the middle part and the rectum, the posterior part. The formation of the small intestine and the rectum has been shown to depend on Hedgehog and Wingless activities, which coordinate morphogenesis and cell differentiation in the hindgut. The steroid receptor-encoding genes knirps and knirps-related, which are expressed in banded expression domains in the small intestine and the rectum, are target genes of the Hedgehog and Wingless signaling pathways required for restricting endoreduplication cycles to the middle part of the hindgut, the large intestine (Fusse, 2002).

While studying the role of the kni and knrl genes which act redundantly during hindgut development, it was observed that both genes are also coexpressed in the large intestine, from germ band extension stage onward in two rows of lateral cells (20 ± 1) on each side of the tube. This expression is maintained until late stage 16. In the lateral cell rows, kni and knrl are coexpressed with the rhomboid (rho) gene that encodes a transmembrane protein involved in epidermal growth factor receptor (Egfr) signaling. rho gene expression in the lateral cells appears slightly earlier than kni/knrl gene expression and is also maintained until late stage 16. At the transitions to the small intestine and the large intestine, rho is expressed in two circular expression domains. The transmembrane protein and apical polarity determinant Crumbs (Crb) becomes strongly upregulated in the lateral cell rows after germ band extension stage and displays an unusual cellular distribution. This contrasts with the dorsal and ventral cells of the hindgut, where Crb is located at the apical cell margins -- Crb is localized to the entire apical domain of the lateral cell rows, and expression is maintained until the end of embryogenesis. Similarly, Discs lost (now redefined as Drosophila Patj), another apical polarity organizer, is located to the entire apical cell surface in these cells (Fusse, 2002).

Using various cell shape and cell polarity markers, such as the septate junction markers Fas III, Neurexin IV, and Discs lost, it was determined that the cells of the lateral cell rows show a flat and long-shaped morphology and that these cells separate homogenous cell populations in the dorsal and the ventral halves of the large intestine. The cells of the lateral cell rows can thus be considered boundary cells separating dorsal from ventral cells in the large intestine. The dorsal cells, which are big and columnar, express the homeodomain protein Engrailed (En) from extended germ band stage onward until late embryogenesis. In contrast, the ventral cells, which are small and cuboidal, display expression of Delta from extended germ band stage onward until late embryogenesis. Double immunostainings reveal that En expression in the dorsal half of the large intestine is adjacent and nonoverlapping to the kni/knrl/rho expression domains in the boundary cells. Similarly, the Delta expression domain in the ventral half is adjacent to the boundary cells, although coexpression at a low level in the boundary cells cannot be excluded. In summary, dorsal cells express En; boundary cells kni/knrl, rho, crb, and ventral cells express Delta (Fusse, 2002).

To investigate the role of the genes expressed in the large intestine, lack- and gain-of-function studies were performed. In amorphic Notch and Delta mutant embryos, kni/knrl, rho, and high levels of Crb expression on the apical plate are absent in the large intestine, and the boundary cell fate is not established. In contrast, ventral cell morphologies are normal in Notch or Delta mutant embryos, and En expression and dorsal cell fates are unchanged. This indicates that Notch signaling is required to establish the boundary cells but not for dorsal or ventral cell fates. To further test this, gain-of-function experiments were performed using the UAS/Gal4 system. As driver lines, the G445.2 or the 14-3-fkhGal4 strains were used -- they mediate ubiquitous gene expression in the developing hindgut from the extended germ band stage onward until late stage 16. In order to ectopically activate the Notch signaling pathway, flies carrying the Notch intracellular domain fragment, Nicd, under the control of UAS sequences were used. Expressing Nicd ubiquitously in the hindgut results in an ectopic induction of kni and of rho. In addition, the cellular localization of the Crb protein is affected in these embryos. In dorsal and ventral cells of the large intestine of wild-type embryos, Crb is localized to the apical cell margins, whereas it is localized to the entire apical plates of the boundary cells. In the embryos, in which Nicd is ectopically expressed, Crb protein is found on the apical plates of all the hindgut cells; in addition, it is found in high concentrations in vesicles, especially on the baso/lateral sides of the cells. A similar but less intensive ectopic expression of Crb can also be induced if both Kni and Rho are coexpressed in all the hindgut cells, suggesting that crb may be a downstream effector gene of Kni/Knrl and Rho activities. This is consistent with the analysis of rho7M; Df(3L) riXT1 mutants [Df(3L) riXT1 is a deficieny encompassing the kni and knrl transcription units] in which the expression of crb in the boundary cells is strongly reduced. In summary, these results suggest that rho, kni/knrl, and Crb are target genes which are activated in response to Notch signaling in the boundary cells (Fusse, 2002).

To investigate the relationship between rho and kni/knrl in the boundary cells, the expression of the genes in the respective mutants was examined. rho expression is still present in kni mutants and Df(3L) riXT1 mutants. Similarly, kni and knrl expression are maintained in amorphic rho7M mutants or EGF receptor mutants, such as faint little ball (flb). In flbIK35 mutants, the hindgut tube is much shortened due to a reduction of the cell number. However, banded expression of both genes is found in the small intestine and the rectum along the AP axis of the hindgut; expression is also found in a few cells in the large intestine region. Ectopic expression of rho using the corresponding UAS-effector line combined with a driver line that mediates ubiquitous expression in the hindgut does not result in ectopic kni/knrl gene expression and vice versa. This points toward rho and kni/knrl being regulated independently of one another (Fusse, 2002).

To study whether En, which is expressed in the adjacent dorsal cells, contributes to the boundary cell fate, the expression of kni/knrl, rho, and crb was examined in en mutants and in en; invected double mutants (enE), since en and invected are known to act redundantly. Whereas the expression of the Notch target genes remains unchanged in en mutants, it is absent in the large intestine of en; invected double mutants. Morphological studies indicate that the dorsal and the boundary cell fates are not established in these mutants, and the large intestine seems to consist entirely of the ventral cell fates. To investigate the cause for this effect, the expression of Delta was studied in these mutants and it was found to be expressed ubiquitously in the large intestine. These data indicate that a boundary between Delta expressing and nonexpressing cells is required for Notch receptor activation. Ectopic expression of En in the large intestine using the 14-3 fkh driver and UAS-En effector lines results in a repression of kni/knrl and rho gene expression. This indicates that En bears the potential to act as a negative regulator of Notch target genes. Upon ectopic activation of Notch signaling in the entire hindgut by expressing Nicd, En is repressed on the dorsal side of the large intestine, thus allowing ectopic activation of Notch target genes (Fusse, 2002).

In order to investigate whether Notch signaling in the large intestine of wild-type embryos is activated beyond the boundary cells but actively repressed dorsally and ventrally, flies that carry the chimeric Notch receptor/transcription factor fusion construct N-Gal4/VP16 were used and the range of Notch signaling was determined. Upon heat shock, this fusion protein, which is membrane bound, becomes ubiquitously expressed in the embryo. In cells in which the Notch receptor is activated by ligand binding, the intracellular Gal4-VP16 transcription factor moiety is cleaved off and is able to subsequently activate reporter gene expression in cells that carry a UAS-lacZ construct. The ß-Gal expression pattern of such embryos reflects the range of Notch signaling. When anti-ß-Gal stainings of embryos that were heat shocked and carried the N-Gal4/VP16 and UAS-lacZ constructs was performed, ß-Gal expression was observed exclusively in the lateral boundary cells of the large intestine, demonstrating that Notch signaling is restricted to the boundary cells only. To further test this, flies were used carrying a lacZ-reporter construct in which multiple Su(H) binding sites from the Enhancer of Split m8 gene were combined with binding sites for the transcription factor Grainyhead (Grh). In cells, in which Notch signaling is active and Grh is expressed, Su(H) cooperates with Grh to yield high levels of reporter gene expression, whereas reporter gene expression is repressed in cells in which Notch is inactive. Determining the activity pattern of this construct in the hindgut using anti-ß-Gal antibody stainings demonstrates that activation of the reporter gene occurs exclusively in the boundary cells of the large intestine, consistent with the N-Gal4/VP16 data (Fusse, 2002).

In the Drosophila wing imaginal disc, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of cells that express Wingless and organize wing growth and patterning. The range of Notch signaling is determined by the spatial and temporal expression pattern of its ligands, Delta and the transmembrane protein Serrate (Ser), and by the activity of the glycosyltransferase Fringe (Fng). Fng controls ligand selectivity of Notch and plays a major role in the Notch-dependent positioning of sharp compartment boundaries. Fng has been shown to modify the glycosylation state of the receptor in the Golgi complex, thereby lowering its sensitivity to Ser and raising its sensitivity to Delta. To investigate whether Fng or Ser are also taking part in restricting Notch signaling to the boundary cells in the hindgut, expression studies and lack- and gain-of-function analyses were performed. In situ hybridization using a Ser antisense probe or ß-Gal expression studies of a Ser-lacZ enhancer trap line show that Ser is not expressed in the large intestine of the hindgut, and Ser mutants show a normal hindgut. Furthermore, ectopically expressing Ser in all the hindgut cells has no effect on Notch target gene expression. In contrast, Fng is expressed in boundary and dorsal cells as shown by double immunostainings of Fng and En. However, in amorphic fng80 mutants, misspecification of boundary cells occurs only at a low frequency, and ectopic expression of Fng using the 14-3 fkh driver and UAS-Fng flies does not induce an ectopic activation of the kni/knrl and rho genes in the hindgut. These results indicate that Ser seems not to be required, and fng may play only a minor role in restricting Notch signaling to the boundary cells (Fusse, 2002).

These results suggest that the activation of the Notch receptor in the boundary cells of the hindgut is triggered by the binding of Delta, which is expressed at high levels in adjacent ventral cells. If Delta levels are uniform and this boundary condition is lost, as in enE mutants, Notch signaling fails to occur. To further obtain insight into how the spatial control of Notch receptor activation is mediated, the localization of the receptor was determined using antibody stainings to Notch. In ventral and dorsal cells, Notch is expressed in the apical cell margins, as can be demonstrated using coimmunostainings with Neurexin IV. However, in the boundary cells, the Notch receptor is positioned to the entire apical plate where it is colocalized with Crb or Discs-lost. To test whether the apical localization of the receptor is necessary for its signaling activity, amorphic crb mutants were studied in which the sorting of proteins to the apical domain of the cells is affected. In these mutants, a strong reduction of the number of boundary cells was found, although hindgut morphogenesis is only slightly affected. In addition, the remaining boundary cells are mislocalized, and two rows of cells are often found instead of a single row as is found in wild-type. Anti-Notch/anti-Kni double immunostainings of crb mutants demonstrate the reduction of apical Notch receptor localization in crb mutants. Furthermore, in cells in which the Notch receptor is not localized along the apical plate of the cells, the activation of Notch target genes fails to occur. These results indicate that apical localization of the receptor may be important for boundary cell fate determination (Fusse, 2002).

These results further demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).

These results provide evidence that Notch signaling in the Drosophila hindgut controls the fate of a single row of boundary cells separating the dorsal and ventral halves of the gut tube. Activation of the Notch receptor in the boundary cells is mediated by its ligand Delta that is expressed in adjacent ventral cells. The induction of Notch target genes activate the expression of the apical polarity organizer Crb, which may be required, in turn, for apical Notch receptor localization. These findings identify a simple mechanism that controls the specification of a single row of DV boundary cells in an animal gut (Fusse, 2002).

In vivo imaging reveals different cellular functions for FGF and Dpp signaling in tracheal branching morphogenesis

In the developing tracheal system of Drosophila, six major branches arise by guided cell migration from a sac-like structure. The chemoattractant Branchless/FGF (Bnl) appears to guide cell migration and is essential for the formation of all tracheal branches, while Decapentaplegic signaling is strictly required for the formation of a subset of branches, the dorsal and ventral branches. Using in vivo confocal video microscopy, it has been found that the two signaling systems affect different cellular functions required for branching morphogenesis. Bnl/FGF signaling affects the formation of dynamic filopodia, possibly controlling cytoskeletal activity and motility as such, and Dpp controls cellular functions allowing branch morphogenesis and outgrowth (Ribeiro, 2002).

The formation of tracheal branches via directed cell migration requires input from other signaling systems in addition to Bnl/FGF. Activation of the Dpp signal transduction cascade is essential in dorsal and ventral tracheal cells prior to migration for the subsequent formation of dorsal and ventral (ganglionic and lateral trunk anterior and posterior) branches. In the absence of the Dpp receptors Thick veins (Tkv) or Punt (Put), dorsal branches completely fail to develop and ventral branches are strongly affected. Dpp induces the expression of the genes kni and knrl in the ventral and dorsal cells of the placode; in the absence of these two nuclear proteins, dorsal branches are absent and ventral branches are strongly abnormal (Ribeiro, 2002).

Knowing that Bnl/FGF acts as a chemoattractant for tracheal cells, and having shown above that Bnl/FGF signaling induces filopodial activity, one must wonder why cells need input from the Dpp signaling cascade for a directed movement to the Bnl/FGF source. Is the Dpp response a prerequisite for the subsequent induction of filopodia by Bnl/FGF? Or do dorsal branch cells respond to Bnl/FGF with the formation of filopodia even in the absence of Dpp signaling input, yet fail to migrate properly? In order to find out how these different signaling systems interact in vivo, the cytoskeletal activity of tracheal cells was examined in the absence of Dpp signaling, with particular emphasis on dorsal branches. However, both tkv and put mutants lack dorsal expression of bnl; therefore, they not only lack the Dpp signaling input but also the Bnl/FGF signaling input. In line with the absence of dorsal bnl expression, cellular extensions were not observed in dorsal tracheal cells in put mutants when analyzed in vivo using the GFP-actin fusion protein (Ribeiro, 2002).

To circumvent the problem of the absence of dorsal bnl expression in mutants defective in Dpp signaling, use was made of the inhibitory SMAD protein encoded by the Drosophila Daughters against dpp (Dad) gene. Specific inhibition of Dpp signaling in tracheal cells via trachea-specific ectopic expression of Dad led to the absence of dorsal branches, despite the presence of bnl expression on the dorsal side of the embryo. Consistent with the absence of dorsal branches upon ectopic expression of Dad, kni expression was not detectable in dorsal tracheal cells. Loss of dorsal branches was also readily visible in the later larval stages; no dorsal branches were observed in third instar larvae upon the expression of Dad in the tracheal system during embryogenesis. In embryos and in larvae expressing Dad, stump-like dorsal outgrowths were occasionally observed at positions where dorsal branches form in wild-type animals. It is argued that these stumps are misrouted dorsal trunk outgrowths; such outgrowths are never observed in tkv or put mutants, presumably due to the lack of bnl expression dorsal to the invaginating placode. It is concluded from these experiments that ectopic expression of Dad mimics the tkv and put mutant phenotypes with regard to the lack of dorsal branch formation, and that dorsal branches fail to form through guided cell migration in this particular Dpp loss-of-function situation despite the presence of dorsal bnl expression (Ribeiro, 2002).

To investigate the possible cell shape changes or cytoskeletal rearrangements in dorsal tracheal cells in the absence of Dpp signaling in vivo, confocal imaging was performed of living embryos expressing both a Dad and a GFP-tagged actin transgene in the developing trachea. Confirming the observations made in fixed embryos and in third instar larvae, the phenotype observed in late embryonic stages (stages 15 and 16) in vivo is the complete absence of dorsal branches. However, analysis of a time-lapse study of three-dimensional reconstructions, in which tracheal GFP-actin dynamics were recorded in an interval of 5 min for 135 min, revealed a strikingly different picture. Unlike put mutants, embryos in which Dpp signaling is inhibited specifically in tracheal cells by ectopic expression of Dad clearly show dorsal outgrowths and filopodial activities in positions where dorsal branches normally form. These outgrowths look bud-like and showed dynamic filopodial extensions, but never refine to single-cell diameter, tubular dorsal branches. Although tracheal cells migrated dorsally, they never migrated over a large distance, and in most cases all the cells forming these buds eventually reintegrated into the main dorsal trunk, leading to a general absence of dorsal branches (Ribeiro, 2002).

These results demonstrate that in the absence of Dpp signaling, tracheal cells close to the dorsal bnl-expressing ectodermal cells are able to form actin-containing filopodial extensions and initiate dorsal migration. However, the lack of Dpp signaling, which results in the lack of expression of the kni/knrl target genes, leads to failure to form a dorsal branch, and the short, bud-like dorsal outgrowths eventually reintegrate into the main dorsal trunk. Consistent with this interpretation, cells forming the initial dorsal outgrowth in Dad-expressing embryos in rare cases generated a dorsal trunk-sized lumen. These dorsally directed stumps of dorsal trunk were also visible in third instar larvae. Such dorsal trunk-like buds are also seen in mutants that lack Dpp-induced kni/knrl in the tracheal system, indicating that dorsal migration also takes place in these mutants. These buds are never observed in put mutants, presumably due to the lack of dorsal expression of the chemoattractant Bnl/FGF (Ribeiro, 2002).

Activation and repression activities of ash2 in Drosophila wing imaginal discs

Polycomb (PcG) and trithorax (trxG) group genes are chromatin regulators involved in the maintenance of developmental decisions. Although their function as transcriptional regulators of homeotic genes has been well documented, little is known about their effect on other target genes or their role in other developmental processes. The patterning of veins and interveins in the wing has been used as a model with which to understand the function of the trxG gene ash2 (absent, small or homeotic discs 2). ash2 is required to sustain the activation of the intervein-promoting genes net and blistered (bs) and to repress rhomboid (rho), a component of the EGF receptor (Egfr) pathway. Moreover, loss-of-function phenotypes of the Egfr pathway are suppressed by ash2 mutants, while gain-of-function phenotypes are enhanced. These results also show that ash2 acts as a repressor of the vein L2-organising gene knirps (kni), whose expression is upregulated throughout the whole wing imaginal disc in ash2 mutants and mitotic clones. Furthermore, ash2-mediated inhibition of kni is independent of spalt-major and spalt-related. Together, these experiments indicate that ash2 plays a role in two processes during wing development: (1) maintaining intervein cell fate, either by activation of intervein genes or inhibition of vein differentiation genes, and (2) keeping kni in an off state in tissues beyond the L2 vein. It is proposed that the Ash2 complex provides a molecular framework for a mechanism required to maintain cellular identities in the wing development (Angulo, 2004).

Loss of ash2 function causes differentiation of ectopic vein tissue, indicating that ash2 is required for intervein development, where it functions as an activator of the intervein-promoting genes net and bs, restricting rho expression to vein regions. In addition, the loss-of-function phenotypes of Egfr alleles are rescued in ash2 mutants, while the gain-of-function phenotypes are enhanced. Furthermore, rho mRNA exhibits an expanded expression pattern in ash2 mutant tissues. Thus, ash2 promotes the maintenance of intervein fate, either by activation of net and bs or by repression of the Egfr pathway. Since rho and bs/net expression is mutually exclusive, it cannot be determined whether the Ash2 complex interacts directly with one or all of them. However, since bs expression is inhibited by the loss-of-function of ash2 during larval and pupal stages, it can be proposed that ash2 acts as a long-term chromatin imprint of bs that is stable throughout development (Angulo, 2004).

The results in adult clones and from analysis of genetic interactions suggest that ash2 acts principally by maintaining B and D intervein regions, since the C intervein remains unaltered in ash2 mutants. This region is under the control of organising genes that respond to the Hh signal. One of these genes is kn, which prevents vein differentiation in the C intervein and is required for the expression of bs in this domain. bs expression is regulated by two enhancer elements: the boundary enhancer, which is dependent on hh and controls bs expression in the C intervein region through kn; and another enhancer dependent on Dpp activity, which controls bs expression in B and D intervein domains. Thus, the role of ash2 as a positive regulator of bs is mainly restricted to regions beyond the kn domain where the Dpp dependent bs enhancer is active (Angulo, 2004).

Dynamical analysis of regulatory interactions in the gap gene system of Drosophila

Genetic studies have revealed that segment determination in Drosophila melanogaster is based on hierarchical regulatory interactions among maternal coordinate and zygotic segmentation genes. The gap gene system constitutes the most upstream zygotic layer of this regulatory hierarchy, responsible for the initial interpretation of positional information encoded by maternal gradients. A detailed analysis of regulatory interactions involved in gap gene regulation is presented based on gap gene circuits, which are mathematical gene network models used to infer regulatory interactions from quantitative gene expression data. The models reproduce gap gene expression at high accuracy and temporal resolution. Regulatory interactions found in gap gene circuits provide consistent and sufficient mechanisms for gap gene expression, which largely agree with mechanisms previously inferred from qualitative studies of mutant gene expression patterns. These models predict activation of Kr by Cad and clarify several other regulatory interactions. This analysis suggests a central role for repressive feedback loops between complementary gap genes. Repressive interactions among overlapping gap genes show anteroposterior asymmetry with posterior dominance. Finally, these models suggest a correlation between timing of gap domain boundary formation and regulatory contributions from the terminal maternal system (Jaeger, 2004b).

Although activating contributions from Bcd and Cad show some degree of localization, positioning of gap gene boundaries during cycle 14A is largely under the control of repressive gap-gap cross-regulatory interactions. Thereby, activation is a prerequisite for repressive boundary control, which counteracts broad activation of gap genes in a spatially specific manner. In addition, gap genes show a tendency toward autoactivation, which increasingly potentiates activation by Bcd and Cad during cycle 14A. Autoactivation is involved in maintenance of gap gene expression within given domains and sharpening of gap domain boundaries during cycle 14A (Jaeger, 2004b).

Regulatory loops of mutual repression create positive regulatory feedback between complementary gap genes, providing a straightforward mechanism for their mutually exclusive expression patterns. Such a mechanism of 'alternating cushions' of gap domains has been proposed previously. The results suggest that this mechanism is complemented by repression among overlapping gap genes. Overlap in expression patterns of two repressors imposes a limit on the strength of repressive interactions between them. Accordingly, repression between neighboring gap genes is generally weaker than that between complementary ones. Moreover, repression among overlapping gap genes is asymmetric, centered on the Kr domain. Posterior to this domain, only posterior neighbors contribute functional repressive inputs to gap gene expression, while anterior neighbors do not. This asymmetry is responsible for anterior shifts of posterior gap gene domains during cycle 14A (Jaeger, 2004b).

Repression by Tll mediates regulatory input to gap gene expression by the terminal maternal system. Tll provides the main repressive input to early regulation of the posterior boundary of posterior gt, and activation by Tll is required for posterior hb expression. Note that these two features form only during cycle 13 and early cycle 14A, while other gap domain boundaries are already present at the transcript level during cycles 10-12 and largely depend on the anterior and posterior maternal systems for their initial establishment. The delayed formation of posterior patterning features and their distinct mode of regulation are reminiscent of segment determination in primitive dipterans and intermediate germ-band insects, supporting a conserved dynamical mechanism across different insect taxa (Jaeger, 2004b).

The set of regulatory interactions presented here provides a consistent and sufficient dynamical mechanism for gap gene expression. In summary, this set of interactions consists of the following five basic regulatory mechanisms: (1) broad activation by Bcd and/or Cad, (2) autoactivation, (3) strong repressive feedback between mutually exclusive gap genes, (4) asymmetric repression between overlapping gap genes, and (5) feed-forward repression of posterior domain boundaries by the terminal gap gene tll. In the following subsections, evidence is discussed concerning specific regulatory interactions involved in each of these basic mechanisms in some detail (Jaeger, 2004b).

Activation by Bcd and Cad: Activation of gap gene expression by Bcd and Cad is supported by the following. Bcd binds to the regulatory regions of hb, Kr, and kni. The kni regulatory region also contains binding sites for Cad. The anterior domains of gt and hb are absent in embryos from bcd mothers. The posterior domain of gt is missing in embryos mutant for both maternal and zygotic cad, while the posterior domain of kni is absent in embryos mutant for maternal bcd plus maternal and zygotic cad. These results suggest partial redundancy of activation of kni by Bcd, consistent with evidence from zygotic cad embryos from bcd mothers, where maternally provided Cad is sufficient to activate kni (Jaeger, 2004b).

Kr expression expands anteriorly in embryos from bcd mothers, which is due to the absence of the anterior gt and hb domains. Bcd has been shown to activate expression of Kr reporter constructs. The fact that Kr is still expressed in embryos from bcd mutant mothers has been attributed to activation by general transcription factors or low levels of Hb. In contrast, the models predict that this activation is provided by Cad. Although Kr expression is normal in embryos overexpressing cad, repressive control of Kr boundaries could account for the lack of expansion of the Kr domain in such embryos (Jaeger, 2004b).

The activating effect of Cad on hb found in gap gene circuits is likely to be spurious. The anterior hb domain is absent in embryos from bcd mutant mothers, which show uniformly high levels of Cad. Moreover, the complete absence of the posterior hb domain in tll mutants suggests activation of posterior hb by Tll rather than by Cad. It is believed that this spurious activation of hb by Cad is due to the absence of hkb in gap gene circuits. The posterior hb domain fails to retract from the posterior pole in hkb mutants, suggesting a repressive role of Hkb in regulation of the posterior hb border. Consistent with this, the posterior boundary of the posterior hb domain never fully forms in any of the circuits. Moreover, Tll is constrained to a very small or no interaction with hb due to the absence of the posterior repressor Hkb, since activation of hb by Tll would lead to increasing hb expression extending to the posterior pole (Jaeger, 2004b).

Autoactivation:: A role for autoactivation in the late phase of hb regulation is supported by the fact that the posterior border of anterior hb is shifted anteriorly in a concentration-dependent manner in embryos with decreasing doses of zygotic Hb. Weakened and narrowed expression of Kr in mutants encoding a functionally defective Kr protein suggests Kr autoactivation. Similarly, a delay in the expression of gt in mutants encoding a defective Gt protein indicates gt autoactivation. However, the results suggest that gt autoactivation is not essential. It is generally weaker than autoactivation of other gap genes, and circuits lacking gt autoactivation show no specific defects in gt expression. Finally, in the case of kni, there is no experimental evidence for autoactivation, while some authors have even suggested kni autorepression. No such autorepression has been detected in any gap gene circuit (Jaeger, 2004b).

Repression between complementary gap genes: Mutual repression of gt and Kr is supported by the following. gt expression expands into the region of the central Kr domain in Kr embryos. In contrast, Kr expression is not altered in gt mutants before germ-band extension. However, Gt binds to the Kr regulatory region, and the central domain of Kr is absent in embryos overexpressing gt. Moreover, Kr expression extends further anterior in hb gt double mutants than in hb mutants alone. The above is consistent with this analysis, which shows no significant derepression of Kr in the absence of Gt even though repression of Kr by Gt is quite strong (Jaeger, 2004b).

Hb binds to the kni regulatory region, and the posterior kni domain expands anteriorly in hb mutants. Embryos overexpressing hb show no kni expression at all, and embryos misexpressing hb show spatially specific repression of kni expression.There is no clear posterior expansion of kni in hb mutants. This could be due to the relatively weak and late repressive contribution of Hb on the posterior kni boundary or due to partial redundancy with repression by Gt and Tll. The posterior hb domain expands anteriorly in kni mutants, but anterior hb expression is not altered in these embryos. Nevertheless, a role of Kni in positioning the anterior hb domain is suggested by the fact that misexpression of kni leads to spatially specific repression of both anterior and posterior hb domains. Moreover, only slight posterior expansion of anterior hb is observed in Kr mutants, while hb is completely derepressed between its anterior and posterior domains in Kr kni double mutants (Jaeger, 2004b).

Repression between overlapping gap genes: gt, kni, and Kr show repression by their immediate posterior neighbors hb, gt, and kni, respectively. Retraction of posterior Gt from the posterior pole during midcycle 14A fails to occur in hb mutants, and no gt expression is observed in embryos overexpressing hb. The posterior kni boundary is shifted posteriorly in gt mutant embryos, and kni expression is reduced in embryos overexpressing gt. Note that these effects are very subtle and were not reported in similar studies by different authors. A weak but functional interaction of Gt with kni is consistent with these results. This interaction was found to be essential even in a circuit where it was deemed below significance level. Finally, Kni has been shown to bind to the Kr regulatory region, and the central Kr domain expands posteriorly in kni mutants (Jaeger, 2004b).

In contrast, no effect of Kr on hb was detected. However, hb expression expands posteriorly in Kr mutants. This effect is likely to involve repression of hb by Kni. Kni levels are reduced in Kr embryos. hb is completely derepressed between its anterior and posterior domains in Kr kni double mutants, whereas anterior hb does not expand at all in kni mutants alone. Taken together these results suggests that there is direct repression of hb by Kr in the embryo, but it is at least partially redundant with repression of hb by Kni (Jaeger, 2004b).

Unlike repression by posterior neighbors, no or only weak repression was found of posterior kni, gt, and hb by their anterior neighbors Kr, kni, and gt, respectively. Most gap gene circuits show weak activation of hb by Gt. Graphical analysis failed to reveal any functional role for such activation. Moreover, no functional interaction was found between gt and Kni. Although relatively weak repression of kni by Kr was found in 6 out of 10 circuits, no specific patterning defects could be detected in the other 4. Consistent with the above, expression of posterior hb is normal in gt mutants, and both the anterior boundaries of posterior gt and kni are positioned correctly in kni and Kr mutant embryos, respectively (Jaeger, 2004b).

Note that activation of kni by Kr, which has been proposed to explain decreased expression levels of kni in Kr mutants, was never found. The results strongly support the view that this interaction is indirect through Gt, which is further corroborated by the fact that kni expression is completely restored in Kr gt double mutants compared to that in Kr mutants alone (Jaeger, 2004b).

A significant repressive effect of Hb on Kr was found. Consistent with this, Hb has been shown to bind to the Kr regulatory region, and the central Kr domain expands anteriorly in hb mutants. However, partial redundancy of this interaction is suggested by correct positioning and shape of the anterior Kr domain in a circuit that does not show repression of Kr by Hb (Jaeger, 2004b).

It has been proposed that Hb plays a dual role as both activator and repressor of Kr. In the framework of the gene circuit model, concentration-dependent switching of regulative action could be implemented by allowing genetic interconnection parameters to switch sign at certain regulator concentration thresholds. The current model explicitly does not include such a possibility. Nevertheless, circuits have been obtained that reproduce Kr expression faithfully, suggesting that a dual role of Hb is not required for proper Kr expression. Moreover, activation of Kr by Hb was ever observed in any of the circuits. Therefore, the results support a mechanism in which the activation of Kr by Hb is indirect through derepression of kni (Jaeger, 2004b).

Repression by Tll: Only a few earlier theoretical approaches have considered terminal gap genes. Gap gene circuits accurately reproduce tll expression. However, in gene circuits, tll is subject to regulation by other gap genes, which is inconsistent with experimental evidence. In contrast, the correct expression pattern of tll in gap gene circuits allows its effect on other gap genes to be studied in great detail. Strong repressive effects of Tll on Kr, kni, and gt have been found. Tll binding sites have been found in the regulatory regions of Kr and kni. In tll mutants, Kr expression is normal, whereas expression of kni expands posteriorly, and the posterior gt domain fails to retract from the posterior pole. No expression of Kr, kni, or gt can be detected in embryos overexpressing tll under a heat-shock promoter (Jaeger, 2004b).

Reverse engineering the gap gene network of Drosophila

A fundamental problem in functional genomics is to determine the structure and dynamics of genetic networks based on expression data. A new strategy is described for solving this problem and it is applied to recently published data on early Drosophila development. The method is orders of magnitude faster than current fitting methods and allows fitting of different types of rules for expressing regulatory relationships. Specifically, this approach is sused to fit models using a smooth nonlinear formalism for modeling gene regulation (gene circuits) as well as models using logical rules based on activation and repression thresholds for transcription factors. The technique also allows inference of regulatory relationships de novo or testing network structures suggested by the literature. A series of models is fitted to test several outstanding questions about gap gene regulation, including regulation of and by hunchback and the role of autoactivation. Based on the modeling results and validation against the experimental literature, a revised network structure is proposed for the gap gene system. Interestingly, some relationships in standard textbook models of gap gene regulation appear to be unnecessary for, or even inconsistent with, the details of gap gene expression during wild-type development (Perkins, 2006).

The regulatory structure of the Combined model is itself sufficient to reproduce all six gap gene domains using either the gene circuit or logical formalisms for production rate functions. Support is cited for the Combined model, and then consider the results of the individual models in light of several outstanding questions about gap gene regulation are discussed (Perkins, 2006).

The maternal proteins Bcd and Cad are largely responsible for activating the trunk gap genes, with Bcd being more important for the anterior domains and Cad more important for the posterior domains. Bcd is a primary activator of the anterior hb domain, the anterior gt domain, and the Kr domain. Cad activates posterior gt. The kni domain is present in bcd mutants and in cad mutants, but not in bcd;cad double mutants. This suggests redundant activation by the two maternal factors. Such redundant activation of kni is present in the Unc-GC model. For the other models, the optimization selected one or the other as activators, but not both. Tll is crucial for activating the posterior hb domain, while it represses Kr, kni, and gt, preventing their expression in the extreme posterior. All the regulatory relationships between the gap genes in the Combined model are repressive. The complementary gap gene pairs, hb-kni and Kr-gt are known to be strongly mutually repressive, as was found in nearly all the models. [Repression of hb by Kni is not part of the Rivera-Pomar and Jäckle (RPJ) regulatory relationships (Rivera-Pomar, 1996), but the unconstrained gene circuit (Unc-GC) model and Unc-Logic model (that employs the regulatory structure discovered by the unconstrained gene circuit fit, except that Gt activation of hb and Kni activation of gt were removed) included the link.] The models also suggest that mutual repression between hb and Kr helps to set the boundary between those two domains. A chain of repressive relationships, hb-gt-kni-Kr, causes the shifts in the Kr, kni, and posterior gt domains. Autoactivation by hb is well-established, and there is also some evidence for autoactivation by Kr and gt (Perkins, 2006).

Does Hb have a dual regulatory effect on Kr? There is a long-running debate about whether or not low levels of Hb activate Kr. In hb mutants, the Kr domain expands anteriorly, suggesting that Hb represses Kr. However, Kr expression in these mutants is lower than in wild-type and expands posteriorly in embryos overexpressing Hb. Further, in embryos lacking Bcd and Hb, the Kr domain is absent, but can be restored in a dosage-dependent manner by reintroducing Hb. These observations suggest that Hb activates Kr. It has been suggested, therefore, that low levels of Hb activate Kr while high levels repress it. An alternative explanation, however, is that the apparently activating effects of Hb are indirect, via Hb's repression of kni and Kni's repression of Kr. Optimization of the Unc-GC model, which could have resulted in activation or repression of Kr by Hb, but not both, resulted in repression. The RPJ models allow for a dual effect, but activation by Hb was eliminated during optimization of the RPJ-Logic model. The RPJ-GC model retained functional activation and repression of Kr by Hb. However, Kr expression in this model is defective. Kr is not properly repressed in the anterior. Further, Kr is ectopically expressed in a small domain in the posterior of the embryo. Thus, the current models provide no support for activation of Kr by Hb. The only support found, which is crucial in all models except Unc-Logic and also consistent with the mutant and overexpression studies, is for repression of Kr by Hb (Perkins, 2006).

What represses hb between the anterior and posterior domains? Another point of disagreement in the literature is what prevents the expression of hb between its two domains. In the model of Rivera-Pomar and Jäckle (1996), repression by Kr is the explanation. The RPJ models confirm that this mechanism is sufficient. Specifically, in these models Kr repression prevents hb expression just to the posterior of the anterior hb domain. Between the Kr and posterior hb domains, there is no explicit repression of hb. Rather, Hb is not produced simply because of a lack of activating factors. In contrast, the models of Jaeger (2004a and b) detected no effect of Kr and attributed repression solely to Kni. The Unc-GC and Unc-Logic models found repression by Kni, but in addition to repression by Kr, not instead of it. Kr is more responsible for repression near the anterior hb domain and Kni is more responsible for repression near the posterior hb domain. This is consistent with observations of expression in mutant embryos. Embryos mutant for Kr show slight expansion of the anterior hb domain, while kni embryos show expansion of the posterior hb domain. In Kr;kni double mutants, hb is completely derepressed between its two usual domains. This suggests, as seen in the Unc-GC and Unc-Logic models, that Kr and Kni are both repressors of hb, that their activity is redundant in the center of the trunk, and that Kr and Kni are the dominant repressors for setting the boundaries of the anterior and posterior domains, respectively. This interpretation was also favored by Jaeger (2004a and b), on the basis of the mutant data, even though Jaeger's models did not find repression by Kr (Perkins, 2006).

The posterior hb domain. In all of the current models, the posterior hb domain is activated by Tll and sustained by Tll and hb autoactivation. Rivera-Pomar (1996) did not consider the posterior hb domain, and did not include activation by Tll in his model. That link was added to the RPJ network structure because otherwise it was not possible to capture the posterior hb domain. The model of Jaeger (2004a and b) captured the domain without Tll activation by substituting activation from cad. However, there is no confirming evidence for such an interaction. The absence of posterior hb in tll mutants and the inability of the models to explain posterior hb by other means, leads to the straightforward hypothesis that Tll activates posterior hb. Posterior hb is unique in that the domain begins to form later than the other five domains modeled. In the RPJ models, this happens simply because high levels of Tll are needed to activate hb -- levels that are reached only at about t = 30 min. The Unc-GC and Unc-Logic models also employ repression by Cad to slightly delay Hb production in the posterior. However, there is no confirming evidence for such repression, and it is omitted from the Combined model (Perkins, 2006).

Shifting of the Kr, kni, and posterior gt domains. Domain shifting was first observed by Jaeger (2004a and b) and attributed to a chain of repressive regulatory relationships, hb-gt-kni-Kr. The current models largely support the importance of this regulatory chain, particularly the final two links. Repression of Kr by Kni was significant in all of the current models. Repression of kni by Gt was present in all models except RPJ-Logic, where it would be of little impact anyway, since RPJ-Logic has a defective posterior gt domain. Consistent with these findings, Kni binds to the regulatory region of Kr, and the Kr domain expands towards the posterior in kni mutants. Similarly, the kni domain expands posteriorly in gt mutants, while embryos overexpressing gt show reduced kni expression (Perkins, 2006).

Repression of gt by Hb is not as well supported by the current models. The Unc-GC model included the link, though the regulatory weight was the smallest of all those in the model. The link was eliminated from Unc-Logic and, of course, not present in the RPJ network structure. Instead, the models utilized decreasing activation by Cad (Unc-GC, Unc-Logic) and repression by Tll (Unc-GC, RPJ-GC) to shift the posterior gt domain. Even with these links, however, shifting of the domain is not well-captured. RPJ-GC appears to capture the posterior gt shift best (Figure 3E). However, it relies on its small ectopic Kr domain to repress gt, a completely incorrect mechanism. Interestingly, a gene circuit fit using the network structure of Sanchez and Thieffry (2001), captured the shift of posterior gt better than any of the other current models, and it did so using repression of gt by Hb, providing additional modeling support for the relationship. There also is strong mutant evidence in favor of the relationship. In hb mutants, the posterior gt domain does not retract from the posterior pole. Further, Gt is absent in embryos that have ubiquitous Hb, such as maternal oskar or nanos mutants or embryos expressing Hb ubiquitously under a heat-shock promoter. Thus, sufficient evidence was found to include a repressive link from hb to gt in the Combined model (Perkins, 2006).

Activating or repressing links that oppose the direction of the repressive chain were eliminated by optimization of the Unc-Logic, RPJ-GC, and RPJ-Logic models. In agreement with this result, the boundaries of the kni and posterior gt domains are correctly positioned in Kr and kni mutants, respectively. Thus, the simplest picture supported by the current models and consistent with the mutant studies is that there is no regulation from Kr, kni, or posterior gt to any of their immediate posterior neighbors, and that the repressive chain highlighted by Jaeger (2004a and b) is indeed responsible for domain shifting (Perkins, 2006).

Do gap genes autoregulate? All four of the current models include autoactivation by hb. This is supported by the observation that late anterior hb expression is absent in embryos lacking maternal and early zygotic Hb 47. The models suggest hb autoactivation also plays a crucial role in sustaining the posterior domain, once it has been initiated by Tll, a role not previously emphasized. Autoactivation for the other genes was found by the Unc-GC model, but is not part of the RPJ network structure. It included autoactivation only for Kr and gt in the Combined model, on the basis of a weakened and narrowed Kr domain in embryos producing defective Kr protein and a delay in gt expression in embryos producing defective gt protein. Interestingly, the gene circuit models of Jaeger (2004a and b) also found autoactivation for all four gap genes, but they considered autoactivation by gt to be the weakest and least certain. In contrast, the Unc-Logic model retained gt autoactivation while eliminating autoactivation for Kr and kni. The RPJ-Logic model was unable to reproduce the posterior gt domain. However, it was found that by adding gt autoactivation to the model, it was able to create and sustain posterior gt correctly, bringing the error of the model down to 15.34. This suggests that, after hb, gt is the most likely candidate for autoactivation. However, even this is not strictly necessary. The RPJ-GC model is able to reproduce and sustain the posterior gt domain without autoactivation by relying on cooperative activation from Bcd and Cad (Perkins, 2006).

Comparison of regulatory architectures. The regulatory relationships proposed by Rivera-Pomar and Jäckle (1996) are not fully consistent with the data and require amending. Repression of gt by Kni, which contradicts the mechanism of domain shifts described by Jaeger (2004a and b), was eliminated by the optimization in both of the current models based on the RPJ regulators. Activation of kni by Kr was never observed. No support was found for a dual regulatory effect of Hb on Kr. Activation of Kr at low levels of Hb was eliminated in the RPJ-Logic model. It was retained in the RPJ-GC model, but resulted in serious patterning defects. Inclusion of Tll as an activator of hb was sufficient to produce the posterior hb domain. Based on the current fits and the primary experimental literature, there are likely other regulatory links missing from the model of Rivera-Pomar and Jäckle, though they are not strictly required to reproduce the wild-type gap gene patterns. Foremost is repression of hb by Kni, which appears important for eliminating hb expression anterior of the posterior domain. Fits based on the Sanchez and Thieffry (2001) regulatory relationships also support these conclusions (Perkins, 2006).

In contrast, the regulatory relationships in the Combined model and both the Unc-GC and Unc-Logic models are able to capture the wild-type gap patterns without gross defects. The relationships in the Unc-GC model are very similar to those obtained by Jaeger (2004a and b). For example, the regulation of Kr and kni is qualitatively equivalent in both models, and there is a single minor difference in the regulation of gt. The optimizations correctly identified activation of hb by Tll, which was missed by Jaeger (2004a and b), though the current models did less well at capturing shifting of the posterior gt domain. These regulatory relationships are also similar to those found by Gursky (2004), though that study was based on gap gene expression data with much lower accuracy and temporal resolution than the data used in this study. These similarities show that differences in the mathematical formulations of these models-as ordinary versus partial differential equations, how diffusion and nuclei doubling are modeled, and choice of boundary conditions and other simulation parameters-are not important for the reproduction of the gap gene patterns nor for the inference of regulatory relationships from the data (Perkins, 2006).

Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes

A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. A new negative regulator of JAK/STAT signaling is described and a potential mechanism identified by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, Ken & Barbie (Ken), which is an ortholog of the mammalian proto-oncogene BCL6, has been identified as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, endogenous Ken is shown specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo (Arbouzova, 2006).

Analysis of phenotypes associated with mutations in Drosophila JAK/STAT pathway components have identified a wide variety of requirements for the pathway during embryonic development and in adults. What is less clear is how the repeated stimulation of a single pathway is able to generate this pleiotropy of developmental functions. In order to identify modulators of JAK/STAT signaling that may be involved in this process, a genetic screen was undertaken for modifiers of the dominant phenotype caused by the ectopic expression of the pathway ligand Unpaired (Upd) in the developing eye imaginal disc. Such misexpression by GMR-updΔ3′ results in overgrowth of the adult eye, a phenotype sensitive to the strength of pathway signaling activity. With this assay, one genomic region, defined by Df(2R)Chig320, was found to enhance the GMR-updΔ3′-induced eye overgrowth phenotype. Of the genes deleted by Df(2R)Chig320, only mutations in ken showed consistent and reproducible enhancement of the phenotype. In addition, other dominant phenotypes induced by transgene expression from the GMR promoter are not modulated by ken mutations, indicating that Ken is unlikely to interact with the misexpression construct used (Arbouzova, 2006).

The enhancement of the GMR-updΔ3′ phenotype after removal of one copy of ken implies that Ken normally functions antagonistically to JAK/STAT signaling. Therefore phenotypes associated with mutations in other pathway components were tested to establish the reliability of this initial observation. Consistent with this, genetic interaction assays between ken mutations and the hypomorphic loss-of-function allele stat92EHJ show a reduction in the frequency of wing vein defects normally associated with this stat92E allele. Moreover, the degree of suppression is consistent with the strength of ken alleles tested. Similarly, the frequency of “strong” posterior spiracle phenotypes caused by the dome367 allele of the pathway receptor is also reduced when crossed to ken alleles or the Df(2R)Chig320 deficiency, with a concomitant increase in “weak” phenotypes (Arbouzova, 2006).

Thus, multiple independent ken alleles all modify diverse phenotypes caused by both gain- and loss-of-function mutations in multiple JAK/STAT pathway components. Each of these components acts at different levels of the signaling cascade and show interactions indicating that Ken consistently acts as an antagonist of the pathway (Arbouzova, 2006).

The ken locus contains three exons encoding a 601 aa protein. Ken possesses an N-terminal BTB/POZ domain between aa 17 and 131 and three C-terminal C2H2 zinc finger motifs from aa 502 to 590. Strikingly, a number of Zn finger-containing proteins that also contain BTB/POZ domains have also been shown to function as transcriptional repressors—often via the recruitment of corepressors such as SMRT, mSIN3A, N-CoR, and HDAC-1 (Arbouzova, 2006).

Searches for proteins similar to Ken identified homologs in Drosophila pseudoobscura and the mosquito Anopheles gambiae. In vertebrates, human B-Cell Lymphoma 6 (BCL6) was the closest full-length homolog. Drosophila Ken and human BCL6 share the same domain structure and show 20.3% overall identity. Proteins listed as potential vertebrate homologs of Ken in Flybase are more distantly related (Arbouzova, 2006).

Expression of ken was also examined during development, where it is detected in a dynamic pattern from newly laid eggs, throughout embryogenesis, and in imaginal discs. As such, endogenous Ken is present in all tissues and stages in which genetic interactions were observed (Arbouzova, 2006).

Given the presence of potentially DNA binding Zn finger domains and the nuclear localization of GFPKen, the DNA binding properties of Ken was determined by using an in vitro selection technique termed SELEX (systematic evolution of ligands by exponential enrichment). With a GST-tagged Ken Zn finger domain and a randomized oligonucleotide library, ten successive rounds of selection were undertaken. Sequencing of the resulting oligonucleotide pool and alignment of 43 independent clones showed that all recovered plasmids were unique and each contained one, or occasionally two, copies of the motif GNGAAAK (K = G/T) (Arbouzova, 2006).

To confirm the SELEX results, GFPKen was expressed in tissue culture cells and these were used for electromobility shift assays (EMSA). A radioactively labeled probe containing the wild-type (wt) consensus binding site GAGAAAG gives a specific band, which can be supershifted by an anti-GFP antibody and therefore represents a GFPKen/DNA complex. In order to identify positions essential for binding, a competition assay was used in which unlabeled oligonucleotides containing single substitutions in each position from 1 to 7 were added to binding reactions. 10-fold excess of unlabeled wild-type consensus oligonucleotide greatly diminished the intensity of the GFPKen band, while 50- and 100-fold excess totally blocked the original signal. By contrast, competition with unlabeled m3 oligonucleotides containing a G to A substitution at position 3 failed to significantly reduce the intensity of the band even at 100-fold excess. With this approach, the positions 1 and 7 are found dispensable for DNA binding, whereas the central GAAA core is absolutely required. Similar results were obtained with the converse experiment with labeled mutant probes, although in this case the wt probe produces a stronger signal than the m1 and m7 mutant oligonucleotides. Taken together, these experiments not only define the core sequence for Ken binding, but also demonstrate the specificity of Ken as a site-specific DNA binding molecule. Interestingly, the core consensus bound by Ken is very similar to that identified for human BCL6, with the Zn fingers of the latter binding to a DNA sequence containing a core GAAAG motif (Arbouzova, 2006).

One initial observation made is that the core GAAA essential for Ken binding overlaps the sequence recognized by STAT92E. Consistent with this overlap, a 100-fold excess of unlabeled oligonucleotide containing the STAT92E consensus is sufficient to fully compete for Ken in EMSA assays. Given this finding, it is hypothesized that the negative regulation of JAK/STAT signaling by Ken observed in genetic interaction assays may occur via a mechanism of competitive DNA binding site occupation. Due to the incomplete overlap between the STAT92E and Ken core sequences, this hypothesis also implies the existence of STAT92E DNA binding sites to which both STAT92E and Ken could bind (STAT+/Ken+) as well as sites with which Ken cannot associate (STAT+/Ken) (Arbouzova, 2006).

To test this hypothesis, a cell culture-based assay was set up by using a luciferase-expressing reporter containing four STAT92E binding sites originally identified in the promoter of the Draf locus. In addition to this STAT+/Ken+ wild-type reporter, STAT+/Ken and STAT/Ken variants identical but for the binding sequences were generated. When transfected into the hemocyte-like Kc167 Drosophila cell line, both STAT+/Ken+ and STAT+/Ken reporters showed strong stimulation upon coexpression with the pathway ligand Upd, an assay previously shown to require an intact JAK/STAT cascade. When cotransfected with KenGFP, the activity of the STAT+/Ken+ reporter was reduced, an effect reproduced in three independent experiments with both KenGFP and Ken. While the reduction in reporter activity for the STAT+/Ken+ assay shown is statistically significant, the STAT+/Ken reporter was unaffected by the coexpression of Ken. Reporters containing binding sites mutated to prevent binding of both STAT92E and Ken (STAT/Ken) showed no activation after pathway stimulation and did not respond to Ken (Arbouzova, 2006).

These results indicate that Ken functions as a transcriptional repressor in this cell-culture system and shows that this effect is specific to the DNA sequence determined by SELEX and EMSA. This result is also consistent with a recent whole-genome RNAi-based screen, which used a reporter containing STAT+/Ken+ binding sites and includes Ken among the list of JAK/STAT regulators identified. In addition, recent reports have also demonstrated BCL6 binding to STAT6 sites in vitro and have shown that BCL6 can act as a repressor of STAT6-dependent target gene expression in cell culture. Although this repression is mediated by the binding to corepressors to the BTB/POZ domain of BCL6, no link between BCL6 and STAT activity has been demonstrated in vivo (Arbouzova, 2006).

Finally, it should also be noted that both the STAT+/Ken+ and STAT+/Ken reporters contain additional GAAA sequences that are not part of the characterized STAT92E binding sequences. However, despite the presence of these potential Ken binding sites within 15 bp of the STAT92E site, Ken expression did not affect the STAT+/Ken reporter, suggesting that Ken may require STAT92E to influence gene expression. Although no direct association between Ken and STAT92E has been demonstrated, this possibility cannot be excluded, and further analysis remains to be undertaken (Arbouzova, 2006).

Having established that Ken functions at the level of DNA binding in cell culture, it was asked whether Ken also acts as a transcriptional repressor of JAK/STAT pathway target genes in vivo. For this, the effect of ectopically expressed Ken on the expression of putative JAK/STAT pathway target genes was examined and, given the high levels of maternally loaded STAT92E present at blastoderm stage, focus was placed on targets expressed later in embryogenesis. These include the hindgut-specific expression of vvl, the expression of trachealess (trh) and knirps (kni) in the tracheal placodes, and the dynamic expression of socs36E throughout the embryo (Arbouzova, 2006).

First, the effect of Ken was addressed on trh, whose expression precedes the formation of the tracheal pits in the embryonic segments T2 to A8. Levels of trh are greatly reduced in embryos uniformly misexpressing Ken driven by the daughterless-GAL4 (da-GAL4) line. Many tracheal placodes express little or no trh, and tracheal pits fail to form even in the presence of residual trh. Similar effects are seen in updOS1A mutant embryos lacking all pathway activity. Likewise, downregulation of Kni expression is also observed in embryos misexpressing ken. These results show that both endogenous trh and kni are downregulated by ectopically expressed Ken (Arbouzova, 2006).

Whether Ken can modulate the expression of socs36E, a Drosophila homolog of mouse SOCS-5, was tested. socs36E expression closely mirrors that of upd, showing JAK/STAT pathway-dependent upregulation in segmentally repeated stripes, tracheal pits, and the hindgut. By contrast to trh and kni, ectopically expressed Ken does not affect any aspect of socs36E transcription. However, controls expressing a dominant-negative form of the pathway receptor DomeΔCyt, using the same Gal4 driver line, show a strong downregulation of socs36E, an effect reproduced by the complete removal of all JAK/STAT pathway activity by the updOS1A allele. Taken together, these results illustrate that ectopic expression of Ken during Drosophila development is sufficient to downregulate the expression of only a subset of putative JAK/STAT pathway target genes (Arbouzova, 2006).

As part of this analysis, modulation of vvl by Ken was tested. In wild-type embryos, vvl is expressed in the developing trachea and lateral ectoderm (in a JAK/STAT-independent manner) and in the hindgut of stage 12–14 embryos, where it requires JAK/STAT signaling. In updOS1A mutants, no vvl expression in the hindgut can be detected, indicating that this locus is a target of pathway activation. When Ken is uniformly misexpressed throughout the embryo, vvl expression is no longer detectable in the hindgut. Thus vvl, like trh and kni, can be a target of Ken-mediated repression (Arbouzova, 2006).

Having established that ectopic Ken is sufficient to downregulate vvl in the hindgut, whether endogenous Ken performs a similar role was determined. One overlap between ken expression and regions known to require JAK/STAT signaling are the developing posterior spiracles, structures in which both the pathway ligand upd and ken are simultaneously expressed. However, vvl is never detected in the posterior spiracle primordia in wild-type embryos, despite JAK/STAT pathway activity induced by upd expression in these tissues. Intriguingly, in a heteroallelic combination of the strongest kenk11035 allele and Df(2R)Chig320, vvl transcript was detected not only in its normal expression domain within the hindgut but also in the posterior spiracles. This ectopic expression is initially detected from late stage 13 and rapidly strengthens during stage 14–15. When kenk11035/Df(2R)Chig320 embryos simultaneously mutant for the amorphic updOS1A allele were analyzed, upregulation of vvl in the presumptive posterior spiracles was never observed at the stage by which ectopic vvl expression was first detected in the ken mutant embryos. At later stages, JAK/STAT pathway activity is required for posterior spiracle morphogenesis, posterior spiracles do not form, and upregulated vvl is not present (Arbouzova, 2006).

These results demonstrate that Ken is not only sufficient to downregulate the JAK/STAT pathway-dependent expression of vvl in the hindgut, but its endogenous expression is also necessary for vvl repression in the posterior spiracles. In ken mutants, ectopic vvl expression in the posterior spiracles results from a derepression of endogenous STAT92E activity (Arbouzova, 2006).

The overlap between the consensus sequences bound by STAT92E and Ken, together with the analysis of reporters containing STAT+/Ken+ and STAT+/Ken binding sites, indicate that Ken is likely to selectively regulate only a subset of JAK/STAT target genes. In this model, some target genes are regulated by binding sites compatible with both STAT92E and Ken, while others contain sequences to which only STAT92E can associate. While the DNA binding site is critical in cell-culture systems, similar proof is more difficult to establish in vivo. In particular, only a limited number of JAK/STAT pathway target genes have been rigorously demonstrated to require STAT92E binding in vivo (Arbouzova, 2006).

Although studied in some detail, the regulatory domains controlling vvl expression in the developing hindgut have not been identified. Therefore, although these results predict that such a domain would contain STAT+/Ken+ binding sequences, further analysis is required to confirm this hypothesis. By contrast, the regulatory domain of socs36E required to drive gene expression in the blastoderm, tracheal pits, and hindgut comprises a 350 bp region containing three STAT+/Ken+ and two STAT+/Ken binding sites. Although not conclusive, the presence of STAT92E-exclusive sites in this region may explain the inability of Ken to downregulate socs36E in vivo (Arbouzova, 2006).

The findings also draw a parallel between Drosophila Ken and BCL6. The data presented demonstrate that both proteins show similar abilities to bind DNA and to mediate transcriptional repression with some evidence also linking BCL6 to JAK/STAT signaling as described here. Taken together, these similarities suggest that Ken and BCL6 represent functional orthologs of one another. Given this evolutionary conservation, it is tempting to speculate that the selective regulation of JAK/STAT pathway target genes is also conserved and may represent a general mechanism by which the pathway is modulated to elicit diverse developmental roles in vivo. Although many STAT targets undoubtedly remain to be identified, it will be intriguing to see which may also be coregulated by Ken/BCL6-dependent mechanisms (Arbouzova, 2006).

Histone deacetylase-associating Atrophin proteins are nuclear receptor corepressors: Repression of knirps by Tll involves Atrophin

Drosophila Tailless (Tll) is an orphan nuclear receptor involved in embryonic segmentation and neurogenesis. Although Tll exerts potent transcriptional repressive effects, the underlying molecular mechanisms have not been determined. Using the established regulation of knirps by tll as a paradigm, it is reported that repression of knirps by Tll involves Atrophin, which is related to vertebrate Atrophin-1 and Atrophin-2. Atrophin interacts with Tll physically and genetically, and both proteins localize to the same knirps promoter region. Because Atrophin proteins interact with additional nuclear receptors and Atrophin-2 selectively binds histone deacetylase 1/2 (HDAC1/2) through its ELM2 (EGL-27 and MTA1 homology 2)/SANT (SWI3/ADA2/N-CoR/TFIII-B) domains, this study establishes that Atrophin proteins represent a novel class of nuclear receptor corepressors (Wang, 2006).

Since SMRT is a transcriptional corepressor for many NRs and SMRTER is the Drosophila cognate of SMRT, the first step in identifying Tll/Tlx-interacting corepressors was to test whether Tll and Tlx interact with SMRT and SMRTER. Using yeast two-hybrid assays, it was found that, whereas EcR, TR, and RAR interact with both SMRTER and SMRT, both Tll and Tlx fail to interact with SMRTER or SMRT (Wang, 2006).

To find potential corepressors of Tll/Tlx, a yeast two-hybrid screen was used, in which a Tll-expressing bait construct was deployed against a Drosophila embryonic library. A positive clone was identified, whose insert codes for the (1301-1966) region of Atro. This clone was selected for further investigation for several reasons: (1) In yeast, this clone also interacts strongly with chick and human Tlx, but not with RAR or TR; (2) Atro encodes a SANT domain, a RERE stretch, and an ELM2 domain; (3) Atro is a transcriptional corepressor of the Drosophila segmentation gene even-skipped; (4) two Atro-related proteins, Atr1 and Atr2, exist in vertebrates; and (5) Atr2 interacts with HDAC1 in mouse embryos. These properties of Atro proteins highlight the possibility that they are corepressors for Tll and Tlx (Wang, 2006).

To determine which region in Tll is required for Atro association, a series of truncated Tll expression constructs was prepared and their interactions with Atro were tested in yeast. The (192-452) region of Tll was found to be sufficient to mediate its interaction with Atro. Since this region of Tll harbors its ligand-binding domain (LBD), it suggested that an intact LBD is required for Tll to bind Atro. Indeed, no association between Tll variants lacking an intact LBD [e.g., Tll(33-161) or Tll(132-352)] and Atro could be detected (Wang, 2006).

A LBD-dependent interaction between Tll and Atro was further confirmed in human cells by using an immunostaining approach. CFP-tagged Atro (CFP-Atro) localizes to subnuclear regions when expressed in cells. This nuclear focal pattern of Atro resembles the nuclear pattern known for Atr2. Expressing Atro with Tll or Tlx in the same cells alters the nuclear distribution of Tll and Tlx: Both Tll and Tlx shift from their evenly distributed nuclear patterns to punctate nuclear patterns virtually identical to that displayed by CFP-Atro. Deleting the LBD from Tll and Tlx abrogates their localization to Atro-positive nuclear foci, confirming that Atro-Tll/Tlx interactions are mediated through the LBD of Tll and Tlx (Wang, 2006).

The regions in Atro responsible for Tll or Tlx interaction were mapped, using serial deletion Atro constructs. Two regions in Atro were found to mediate its interaction with Tll: Atro(965-1511) interacts weakly with Tll, whereas Atro(1711-1966) interacts strongly with both Tll and Tlx. The latter finding is of great interest, since the 1711-1966 region of Atro contains sequences conserved in the C-terminal regions of vertebrate Atr1 and Atr2. This correlation prompted an investigation of whether Atr1 and Atr2 interact with Tll or Tlx (Wang, 2006). Accordingly, two constructs expressing the C-terminal regions of Atr1 and Atr2 were generated and tested individually against Tll- or Tlx-expressing plasmids. As expected, both Atr1(846-1191) and Atr2(1224-1566), like Atro(1711-1966), interact strongly with Tll and Tlx in yeast, confirming that all Atro proteins are commonly targeted by Tll/Tlx (Wang, 2006).

Because the mapped Tll/Tlx-interacting regions in Atro, Atr1, and Atr2 share a stretch of highly conserved residues, whether mutations created within this region, which is called in this study the Atrobox, affect Tll/Tlx interaction was examined. Indeed, substitution of two leucine residues with alanine abolishes the interaction between Atro(1711-1966) and Tll or Tlx in yeast. Atro-Tll/Tlx interactions are, therefore, in part mediated through the Atro-box (Wang, 2006).

Tll/Tlx belong to the NR2 subfamily of the NR superfamily. The similarity shared by members of the NR2 subfamily suggests that additional NR2 proteins may interact with Atro proteins as well. This possibility was tested first with GST pull-down assays, in which several 35S-methionine-labeled NR2 and NR1 proteins were tested for their interactions with GST or GST-Atro fusion proteins. Atro proteins specifically bind Tll, Tlx, human chicken ovalbumin upstream promoter-transcription factor (COUP-TF), and Seven-Up1 (SVP1) (the Drosophila COUP-TF homolog), but not TRß and Ultraspiracle (USP). A similar interacting profile was observed between Atro proteins and COUP-TF or SVP1 in yeast. Therefore, Atro proteins do not interact with all NRs; rather, they preferentially bind a subset of NR2, including Tll, Tlx, SVP1, and COUP-TF (Wang, 2006).

Having demonstrated that Atro physically interacts with various NRs, the biological relevance of these interactions was examined. In this study, focus was placed on the in vivo relationship between Atro and Tll in flies by exploiting the known role of Tll in the segmentation process during Drosophila early embryogenesis. At this stage, Atro is expressed as a nuclear protein throughout the embryos. Consistent with previous observations that tll represses kni expression at the posterior end of the embryo, in situ hybridization for tll1 embryos shows a posterior expansion of kni stripe, especially in the ventral region of the embryos. Removal of zygotic Atro alone, as in the P-element excision line Atro35, does not cause such expansion, due to the presence of maternally deposited Atro. When maternal alone or both maternal and zygotic Atro are depleted using the dominant female sterile-FLP method, however, kni expression expands posteriorly in embryos. Because mutations of Atro and tll alter kni expression similarly, these in vivo observations suggest that Atro and tll are involved in overlapping transcriptional pathways (Wang, 2006).

To address the genetic interaction between tll and Atro further, advantage was taken of the hypomorphic nature of the tll1 allele, and it was asked whether the tll1-mediated phenotype is aggravated by additional Atro mutation. Specifically, whether the observed posterior expansion of kni stripe in tll1 embryos becomes more prominent when zygotic Atro is removed was investigated. Accordingly, a tll1, Atro35 double-mutant fly line was generated, in which both tll1 and Atro35 alleles were recombined to the same chromosome, and kni expression was tested in the resulting homozygous mutant embryos. Indeed, a further posterior expansion of kni stripe was observed in tll1, Atro35 double-mutant embryos, mimicking that found in tlle embryos. Therefore it is concluded that Atro is required for Tll to repress kni (Wang, 2006).

Since Atro is a binding factor of another terminal gap gene product, Huckebein (Hkb), the expression of kni was examined in hkb2 mutant embryos. No significant posterior expansion of kni was observed, therefore indicating that the repression of kni in the posterior-terminal region primarily results from the combined effect of Tll and Atro (Wang, 2006).

The genetic interaction between tll and Atro was further assessed by monitoring the expression of the pair-rule gene fushi tarazu (ftz) in the posterior region of the mutant embryos described above. In wild-type and in Atro35 zygotic mutant embryos, ftz is expressed as seven stripes in the central region. In tll1 embryos, however, the posterior stripes of ftz (mostly the fifth, sixth, and seventh stripes) shift toward the posterior end. In the most severely affected tll1 embryos, the seventh stripe of ftz is lost. This altered ftz pattern is known to be the consequence of cell fate changes, partly owing to the posterior expansion of kni, when tll is mutated. In tll1, Atro35 double-mutant embryos and in tlle embryos, additional loss of the sixth stripe of ftz was observed. Because the cell fate change is more pronounced in tll1, Atro35 double mutants than in tll1 or Atro35 embryos, it is concluded that Atro participates with Tll in determining posterior-terminal cell fates in early Drosophila embryos (Wang, 2006).

To verify the involvement of Atro in the regulation of kni by Tll at the chromatin level, chromatin immunoprecipitation (ChIP) assays were carried out for 0- to 4-h-old Drosophila embryos using Atro antibody, Tll antibody, and control IgG, respectively. The immunoprecipitated (IP) chromatin was subjected to PCR using primers corresponding to two separate regions, P1 and P2, in the kni gene, and a region in a randomly selected control (CG11562) promoter. In the kni promoter, P1 resides 2.5 kb upstream of the transcription initiation site and has a defined Tll-binding site. P2 corresponds to the 3' untranslated region of the kni gene, where no Tll-binding site is found (Wang, 2006).

In vivo ChIP assays revealed that both Atro and Tll antibodies, but not the control IgG, specifically precipitated chromatin that harbors the P1 site, but not chromatin containing P2 or the CG11562 promoter. These results establish that Atro, by forming protein complexes with Tll, is present naturally on the kni promoter (Wang, 2006).

Many transcriptional corepressors, including SMRT and N-CoR, are associated with HDAC activity. Because the results indicate that Atro proteins are corepressors of Tll/Tlx, the following was further investigated: (1) whether Atro proteins also show HDAC activity; (2) whether Atro proteins bind selected HDACs; and, if so, (3) which regions/domains in Atro proteins mediate their HDAC binding. To address these interconnected questions, fluorometric HDAC assays and Western blot analysis were performed on protein complexes immunoprecipitated by Flag-tagged Atro, Atr1, Atr2, or truncated Atr2 variants expressed in HEK293 cells. In parallel experiments, Flag and Flag-SMRT were used as a negative and a positive control, respectively. The expression of tested Flag fusion proteins was first examined using Western blot analysis (Wang, 2006).

As expected, Flag-SMRT is associated with potent HDAC activity that is sensitive to trichostatin A (TSA), an HDAC inhibitor. Robust levels of TSA-sensitive HDAC activity were also observed for both Atro and Atr2, confirming that both proteins' properties involve HDACs. surprisingly, Atr1 displays no prominent HDAC activity. Since Atr1 lacks the conserved ELM2 and SANT domains found in the N-terminal regions of Atr2 and Atro, it is suspected that the missing N-terminal region in Atr1 might be important for the HDAC activity of Atro proteins (Wang, 2006).

To determine whether the HDAC activity of Atro or Atr2 depends on its N-terminal region, the BAH (Bromo adjacent homology), the ELM2, and the SANT domains in this region of Atr2 were deleted sequentially. Note that the BAH domain is absent in Atro. Whereas Atr2DeltaBAH still exerts a robust level of HDAC activity, a dramatic reduction of HDAC activity was observed with Atr2DeltaBAH-ELM2. A further deletion of the SANT domain, Atr2DeltaBAH-ELM2-SANT, causes a complete loss of HDAC activity, indicating that both the ELM2 and SANT domains are central to Atr2's HDAC activity (Wang, 2006).

Next, which HDACs Atro proteins interact with was investigated, and whether Atr2's association with HDACs involves its ELM2/SANT domains. Protein complexes immunoprecipitated by Flag-tagged Atro proteins and Atr2 variants were examined by Western blot for a panel of potential associating proteins, including HDAC1, HDAC2, HDAC3, and Sin3A. Sin3A was not detected in any of the IP complexes. In contrast, a significant level of HDAC3 was precipitated along with SMRT. Although SMRT also interacts with HDAC1 or HDAC2, these interactions are considerably weaker. Conversely, abundant HDAC1 and HDAC2 (but only minimal HDAC3) are present in the protein complexes associated with Atr2. Similarly, Atro, but not Atr1, also precipitates HDAC1/2 specifically, indicating that Atro-family (except Atr1) and SMRT-family proteins display distinct preferences for different HDACs (Wang, 2006).

Consistent with HDAC assay results, removing the ELM2 domain or both the ELM2 and SANT domains from Atr2DeltaBAH impairs or disrupts its ability to associate with HDAC1/2. Given the fact that similar results were also obtained when the distribution of endogenous HDAC1 was examined in cells expressing different CFP-Atr2 variants, it is therefore concluded that the ability of Atr2 to exert HDAC activity and to recruit HDAC1/2 depends on its ELM2 and SANT domains (Wang, 2006).

In many respects, the transcriptional properties that discover in this study for Atro proteins parallel those found for SMRT, N-CoR, and SMRTER. (1) These two classes of corepressors share a SANT domain and RERE stretch; (2) they are conserved in vertebrates and in flies; (3) they bind NRs, albeit selectively, and (4) they associate with HDACs, also selectively. Additionally, Atr1, like SMRT and N-CoR, also interacts with ETO/MTG8, which is known to be a transcriptional repressor involved in acute myeloid leukemia. Considering that SMRT and N-CoR interact with a large number of NRs, with a wide variety of transcriptional factors, and also with type II HDACs, it is predicted that Atro proteins may have similar qualities as well. Therefore, more Atro-interacting factors still await discovery (Wang, 2006).

In the context of human diseases, it is known that polyglutamine expansion in human Atr1 causes DRPLA. It has been shown that Atr1 lacks HDAC activity, yet it binds Atr2 through their RERE stretches, and it associates with both Tlx and COUP-TF, two known NRs with key roles in CNS development and functioning. It is therefore proposed that mutant Atr1 may cause its pathological effects by interfering with the normal transcriptional properties of Atr2 and its associated nuclear receptors (Wang, 2006).

Precise registration of gene expression boundaries by a repressive morphogen in Drosophila

Morphogen gradients are thought to create concentration thresholds that differentially position the expression boundaries of multiple target genes. Despite intensive study, it is still unclear how the concentration profiles within gradients are spatially related to the critical patterning thresholds they generate. This study used a combination of quantitative measurements and ectopic-misexpression experiments to examine the transcriptional-repression activities of the Hunchback (Hb) protein gradient in Drosophila embryos. The results define five expression boundaries that are set primarily by differences in Hb concentration and two boundaries that are set by combinatorial mechanisms involving Hb and at least one other repressor. Hb functions as a repressive morphogen, but only within a specific range of concentrations (~40% to ~4.4% of maximum Hb concentration), within which there are at least four distinct concentration thresholds. The lower limit of the range reflects a position where the slope of the gradient becomes too shallow for resolution by specific target genes. Concentrations above the upper limit do not contribute directly to differential-repression mechanisms, but they provide a robust source that permits proper functioning of the gradient in heterozygous embryos that contain only one functional hb gene (Yu, 2008).

This study measured the relative Hb concentrations associated with the positions of seven expression boundaries and tested whether different Hb concentrations can account for the differential positioning of these boundaries along the AP axis of the Drosophila embryo. These experiments lead to the following conclusions. 1. The Hb gradient functions as a bona fide repressive morphogen for five target-gene expression boundaries, eve 3, nub, pdm2, eve 4, and kni, all of which appear to be positioned primarily, if not exclusively, by specific thresholds of Hb concentration. These boundaries move anteriorly in concert with the dynamic changes of the Hb gradient in wild-type embryos, they shift anteriorly in zygotic hb mutants, and their sensitivities to repression by ectopically expressed Hb are consistent with their relative positions in wild-type embryos. Two other boundaries, the anterior boundary of Kr and the anterior boundary of the posterior gt domain, are established by combinatorial mechanisms involving Hb and Gt, and Hb and Kr, respectively. 2. There is a specific concentration range (~40% to ~4.4% [Hb]max) that mediates the major morphogenetic activities of the Hb repression gradient. Within this range, four thresholds were detected, one at ~40% [Hb]max that sets the anterior boundary of eve 3, one at ~12% [Hb]max that sets the anterior boundaries of both nub and pdm2, one at ~8% [Hb]max that sets the anterior boundary of eve 4, and one at ~4.4% [Hb]max that sets the anterior boundary of kni. These results suggest that these five target genes are exquisitely sensitive to small changes in Hb concentration. Hb also acts as a direct repressor to position the anterior boundary of the Hox gene Ultrabithorax (Ubx), which is first activated in late cycle 14 just before the initiation of gastrulation. The anterior Ubx boundary is positioned between the eve 3 and eve 4 boundaries and thus may represent a fifth threshold within the morphogenetic range described in this study. Ventral misexpression of Hb causes a strong repression of Ubx. However, it was not possible to directly compare the sensitivity of Ubx with the other target genes because the patterns of these genes have begun to decay when Ubx is first activated (Yu, 2008).

Previous studies have identified discrete regulatory elements that recapitulate the five expression patterns within the morphogenetic range described in this study. All of these elements contain multiple Hb binding sites, and one attractive model is that differences in sensitivity are determined by the quantity and/or quality of their Hb binding sites. The more sensitive eve 4+6 enhancer seems to contain a stronger cluster of Hb binding sites than the less sensitive eve 3+7 enhancer, which is consistent with this hypothesis. However, in preliminary experiments, it was found that this simple model cannot be applied to the five target genes shown in this study to be differentially sensitive. For example, the kni expression pattern is more sensitive to Hb-mediated repression than either eve 3 or eve 4, but its enhancer sequence does not appear to have a stronger cluster of Hb binding sites than either the eve 3+7 or the eve 4+6 enhancer. Similarly, two enhancer elements have been found to be associated with the pdm locus, which contains both nub and pdm2. When tested in reporter genes, both enhancers drive patterns of gene expression similar to the endogenous nub and pdm2 patterns, but they do not appear to contain similar clusters of Hb sites (Yu, 2008).

If differential sensitivity cannot be linked to differences in the number or affinity of Hb binding sites for this set of regulatory elements, other architectural features may control the level of Hb required for repression. These features may include changes in spacing between adjacent Hb sites, or specific site orientations that affect cooperative binding. Also, specific arrangements between repressor and activator sites may influence the apparent sensitivities. Consistent with this, it has been shown that specific arrangements between Dl and Twi sites are critical for Dorsal-dependent target-gene expression in the prospective neuroectodermal region along the DV axis. A careful analysis of the enhancer elements that respond to Hb-mediated repression will be required to fully understand the molecular rules that govern differential sensitivity (Yu, 2008).

At the low end of the effective morphogenetic range, there is a ~2-fold difference between the Hb concentration at the eve 4 boundary (~8% [Hb]max) and the amount at the kni boundary (~4.4% [Hb]max). Moving farther posteriorly, from the kni boundary to the gt boundary, does not correlate with a significant drop in the relative Hb concentration (~4.4% [Hb]max to ~3.7% [Hb]max). It is proposed that the slope of the gradient in this region is too shallow for differential target-gene positioning. However, by participating in a combinatorial mechanism with Kr, the very low concentrations of Hb in this region can set the gt boundary in a more posterior position than the kni boundary. Hb and Kr both bind to the regulatory element that drives posterior expression of gt, suggesting that these interactions may be direct (Yu, 2008).

Within the morphogenetic range, the anterior-most boundary is that of eve 3, which corresponds to ~40% [Hb]max. Outside this range on the anterior side is the Kr boundary, which was previously shown to expand anteriorly in zygotic hb mutants. In these experiments, Kr appeared to be quite resistant to repression by ectopic Hb, which seemingly contradicts a previous study that showed that high levels of Hb were sufficient for repression. However, in the previous study, ectopic Hb was provided maternally, significantly before the sna-hb transgene used in this study would be activated. Together, the two studies support the idea that the Kr boundary is initially set independently by Hb, and that maintenance of the boundary requires both Hb and Gt activities. The results suggest that maintenance is mediated primarily by Gt, but that Gt is an effective repressor only in the presence of Hb. The potentiating effect of Hb on Gt-mediated repression may involve direct binding of Hb and Gt to the Kr promoter, which contains binding sites for both proteins (Yu, 2008).

One of the most important findings from this study is that the effective range of Hb's morphogenetic activity is between 40% [Hb]max and 4.4% [Hb]max. This range may seem surprising in light of the fact that Hb is expressed at much higher levels throughout the anterior half of the embryo. Previous studies suggest that anteriorly expressed Hb is required for activation of most Bcd-dependent target genes, which are expressed in a variety of anterior patterns, and that the zygotic stripe of Hb expressed at the position of PS4 is required for the activation of the Hox gene Antennapedia. It is proposed that the high level of Hb protein in anterior regions also provides a reservoir, or buffer, that ensures that the repressive gradient, with all of its thresholds, remains intact in individual embryos that vary in their absolute levels of Hb expression. Such a buffering mechanism could explain how heterozygous embryos, which contain roughly half the concentration of Hb, can nonetheless develop normally (Yu, 2008).

It is proposed further that most other morphogens will function via concentration ranges similar to the one measured in this study. The two best-studied morphogens in Drosophila are Bcd and Dorsal (Dl), both of which are viable and fertile in the heterozygous state. In embryos laid by bcd/+ females, there are dramatic shifts in the positioning of target genes in the early embryo, but the order of gene positioning remains unchanged, the embryos survive to adulthood, and the adults are fertile. Survival would not be possible if activation of a critical target gene required a threshold greater than 50% of the maximum concentration of Bcd (Yu, 2008).

Establishment of cell fate during early Drosophila embryogenesis requires transcriptional Mediator subunit dMED31

During early Drosophila embryogenesis, formation of the anterior-posterior (A/P) axis depends on spatial gradients of maternal morphogens. It is well recognized that positional information is transmitted from these morphogens to the gap genes. However, how this information is being transmitted is largely unknown. The transcriptional Mediator complex is involved in the fine tuning of the signaling between chromatin status, transcription factors and the RNA polymerase II transcription machinery. This study found that a mutation in the conserved subunit of the Mediator complex, dMED31, hampers embryogenesis prior to gastrulation and leads to aberrant expression of the gap genes knirps and Krüppel and the pair-rule genes fushi tarazu and even-skipped along the A/P axis. Expression of the maternal morphogens dorsal and hunchback was not affected in dMED31 mutants. mRNA expression of dMED31 exactly peaks between the highest expression levels of the maternal genes and the gap genes. Together, these results point to a role for dMED31 in guiding maternal morphogen directed zygotic gap gene expression and provide the first in vivo evidence for a role of the Mediator complex in the establishment of cell fate during the cellular blastoderm stage of Drosophila (Bosveld, 2008).

Proper fine tuning of the eukaryotic transcriptome depends on numerous cis and trans acting factors that modulate the chromatin environment of genes and influence the RNA polymerase II (RNAPII) transcription machinery. The Mediator complex is a core processor in the signaling between RNAPII and transcription factors. This complex is an evolutionary conserved protein assembly of 25-30 subunits (Bosveld, 2008).

Support for a specific role during development of Mediator subunits is provided by several studies in Drosophila. These studies describe mostly functions of Mediator subunits during late developmental stages, but a role of the subunits during early embryonic development is largely unknown. The Mediator consists of more then 25 subunits, pointing to a multifaceted role of this complex during metazoa development. Understanding this complexity starts with the identification of the function of each subunit (Bosveld, 2008).

Drosphila MED31 was identified by bioinformatics analysis (Boube, 2000) and its presence in the Mediator complex was confirmed in purified complexes from embryos and cells (Park, 2001; Gu, 2002). In a pull down assay, the Mediator (containing dMED31) complex binds to the transcription factors Bicoid (Bcd), Krüppel (Kr), Fushi tarazu (Ftz), Dorsal (Dl) and HSF, but not Twist (Twi), Hunchback (Hb) and Even-skipped (Eve) (Park, 2001). Moreover, the Mediator complex is required for in vitro transcription from developmentally important promoters regulated by these transcription factors. Despite these in silico and in vitro results, to date the functional role of MED31 in eumetazoa remains elusive. This study reports the identification of the highly conserved Drosophila Mediator subunit dMED31 as a novel maternal-effect gene necessary for proper segment specification during early embryogenesis. dMED31 mutant females have fecundity defects and embryos deposited by homozygous mothers display severe defects along the anterior-posterior (A/P) axis when gastrulation is initiated. Whereas expression of maternal morphogens is not affected, alterations in gap and pair-rule gene expression during the proceeding blastoderm stage correlate with these defects observed in dMED31 mutant embryos. Remarkably, a small percentage of the progeny of homozygous mutant females escape from embryonic death and develop into adults. These escapers have defects in their abdominal segmentation pattern, a phenotype enhanced by mutations in dMED13. These findings provide the first in vivo evidence for a specific role of dMED31 in establishing cell fate in the cellular blastoderm and point to a role for the Mediator in guiding maternal morphogen directed zygotic gap gene expression (Bosveld, 2008).

These findings identify a component of the conserved eukaryotic transcriptional Mediator complex, dMED31, that is required for normal initiation of zygotic gene expression during the blastoderm stage of Drosophila embryogenesis. Female flies that carry a mutation in the dMED31 gene suffer from fecundity defects and the embryos deposited by these females display abnormal embryogenesis due to aberrant cell migration events upon gastrulation. Impaired embryogenesis coincided with changes in kni, Kr, ftz and eve expression along the A/P axis. Furthermore, adult flies derived from embryos that escaped from embryonic death displayed severe defects in their abdominal segmentation. Because mRNA production was hampered in dMED311/1, these abdominal defects were likely the result of abnormal maternal and zygotic dMED31 mRNA production. A mutation in the Mediator subunit dMED13 also caused segmentation defects and this mutant enhanced the dMED31 mutant maternal effect phenotype. Therefore, these data indicate that the Mediator complex directs zygotic gene expression upon egg deposition to establish cell fate in the embryonic blastoderm (Bosveld, 2008).

In order to accomplish cell fate determination, cells gain a transcriptional poised state during early embryogenesis that is maintained throughout development and requires many cis and trans acting factors that modulate the chromatin environment of the genes involved. In Drosophila, cell identity along the A/P is established in the blastoderm stage when the pair-rule genes are expressed. A/P polarity is controlled by the maternal morphogenes cad, nos, bcd and hb whose activity results in the spatio-temporal expression of the gap genes gt, kni, tll and Kr. These gap genes are the first genes expressed along the A/P axis and encode transcription factors that in turn govern patterned expression of the pair-rule genes. Pair-rule gene expression occurs in distinct stripes and is accompanied by cellularization. Thus when cellularization takes place, large clusters of cells gain an imprint that defines the primordial segments. Cell identity is fine tuned when expression of the segment polarity and Hox genes is activated. Although this cascade of maternal, gap, pair-rule and segment polarity genes is well studied, much remains unknown how the maternal morphogens regulate RNAPII activity at their cognate promoters in order to establish regional domains of gap gene expression (Bosveld, 2008).

Because segmentation defects in escaper flies derived from dMED311/1 mothers were restricted to the abdomen, it is possible that the bithorax complex (BX-C) is abnormally expressed. This complex contains the homeotic genes Ultrabithorax, abdominal A and Abdominal B, which control the identity of the posterior two-thirds of the fly. Mutations in hb, Kr, tll and kni affect expression of BX-C and result in homeotic transformation. No complete homeotic transformations of entire parasegments were observed, suggesting an indirect effect of dMED311 on Hox activation. Since segment identity is established during early embryogenesis, this implies that only groups of cells and not whole primordial segments gained abnormal imprinting. Regional errors in cellular imprinting are supported by the variety of the abdominal defects observed in adult flies. Moreover, defects in embryogenesis were accompanied by cell loss at the embryonic poles and aberrant migratory behavior of cells upon gastrulation, processes which occur prior to the activation of the segment polarity and the Hox genes. Finally, early developmental defects coincided with abnormal expression of the gap genes kni and Kr and subsequently the pair-rule genes ftz and eve. These genes are expressed prior to Hox gene expression and are required for activation/repression of the Hox cluster. Although it is possible that the abdominal region is preferentially sensitive for a mutation in dMED31, it is more likely that random defects during formation of the abdomen are tolerated, whereas defects in other regions of the embryo are incompatible with adult viability and these adults never eclose (Bosveld, 2008).

Several intriguing questions remain: why is the embryonic phenotype so variable (>90% of the mutant embryos die, while a small percentage of embryos is able to reach the adult stage), why are mainly embryos affected by a mutation in dMED31 and what is the primary embryonic defect caused by a mutation in dMED31? Answers to these questions can be derived from studies of the Mediator in yeast in combination with the current data. The budding yeast MED31 protein is part of the Mediator transcription initiation complex. Although a mutation in yeast MED31 affects gene expression, mutants display no sensitivity to transcriptional inhibition by 6-azauracil and MED31 is not essential for growth. However, yeast MED31 mutants have a synthetic growth defective phenotype when combined with mutations in genes encoding for the two largest subunits of RNAPII (RPB1, RPB2) and the transcription initiation factors TFIIB and TFIIS. As in yeast, depletion of dMED31 in Drosophila SL2 cells by RNAi does not interfere with the Mediator composition and no growth alterations have been reported. Thus, Drosophila MED31, like yeast MED31, might not be essential for RNAPII activity per se, but could be an auxiliary factor involved in the signaling between specific transcription factors and the RNAPII machinery. Together, these findings and the current data suggest that dMED31 is not required for transcription in general, but is merely required for the fine tuning of transcription of specific genes (Bosveld, 2008).

Based on studies in yeast, it has been proposed that the Mediator functions as a platform that allows rapid regulation of transcription at (re)initiation. Fast regulation and (re)initiation of transcription might be key during the interphase periods of the final syncytial cell cycles when zygotic transcription is initiated, while such large scale, strict and 'fast' control over transcription would not be essential during subsequent stages of development and thus may explain why dMED31 function is essential during early embryogenesis. The observation that a small percentage of embryos derived from dMED311/1 mothers is able to develop into an adult, while the majority of the embryos displayed severe defects during embryogenesis, might also be attributed to such auxiliary function(s) of the dMED31 protein. Minor differences in dMED31 protein levels, due to the hypomorphic dMED311 allele, may result in subtle changes in the expression of the gap and pair-rule genes and allow embryos to progress throughout embryogenesis, but with the formation of segmentation defects. On the other hand, in the majority of embryos, more severe changes in gap and pair-rule patterning occur, which results in embryonic death (Bosveld, 2008).

In summary, this study demonstrated that dMED31 is essential to establish regional domains of expression of cell fate determinants kni, Kr, ftz and eve. mRNA expression of dMED31 peaks exactly between maternal morphogen and gap gene expression and it has been demonstrated that the Mediator complex is able to bind to several maternal transcription factors. Together this indicates that the Mediator complex constitutes an interface between the maternal morphogens and the RNAPII machinery to guide zygotic gene expression of cell fate determinants that specify primordial segment identity. These findings provide the first in vivo evidence for a role of the Mediator complex in establishing cell fate during early embryogenesis and since MED31 resembles one of the most conserved subunits within the Mediator complex this protein could serve a crucial role in the control of RNAPII activity during early developmental processes in all higher eukaryotes (Bosveld, 2008).

knirps: Biological Overview | Targets of Activity and Protein Interactions | Developmental Biology | Effects of Mutation | References

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