Genetic mechanism regulating diversity in the placement of eyes on the head of animals.

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Founding the Wnt gene family: How wingless was found to be a positional signal and oncogene homolog.

The Wnt family of developmental regulators were named after the Drosophila segmentation gene wingless and the murine proto-oncogene int-1. Homology between these two genes connected oncogenesis to cell-cell signals in development. I review how wingless was initially characterized, and cloned, as part of the quest to identify developmental cell-to-cell signals, based on predictions of the Positional Information Model, and on the properties of homeotic and segmentation gene mutants. The requirements and cell-nonautonomy of wingless in patterning multiple embryonic and adult structures solidified its status as a candidate signaling molecule. The physical location of wingless mutations and transcription unit defined the gene and its developmental transcription pattern. When the Drosophila homolog of int-1 was then isolated, and predicted to encode a secreted proto-oncogene homolog, it's identity to the wingless gene confirmed that a developmental cell-cell signal had been identified and connected cancer to development.

Regulation of morphogen pathways by a Drosophila chondroitin sulfate proteoglycan Windpipe.

Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.

Ecdysone exerts biphasic control of regenerative signaling, coordinating the completion of regeneration with developmental progression.

In Drosophila melanogaster, loss of regenerative capacity in wing imaginal discs coincides with an increase in systemic levels of the steroid hormone ecdysone, a key coordinator of their developmental progression. Regenerating discs release the relaxin hormone Dilp8 (Drosophila insulin-like peptide 8) to limit ecdysone synthesis and extend the regenerative period. Here, we describe how regenerating tissues produce a biphasic response to ecdysone levels: lower concentrations of ecdysone promote local and systemic regenerative signaling, whereas higher concentrations suppress regeneration through the expression of broad splice isoforms. Ecdysone also promotes the expression of wingless during both regeneration and normal development through a distinct regulatory pathway. This dual role for ecdysone explains how regeneration can still be completed successfully in dilp8- mutant larvae: higher ecdysone levels increase the regenerative activity of tissues, allowing regeneration to reach completion in a shorter time. From these observations, we propose that ecdysone hormone signaling functions to coordinate regeneration with developmental progression.

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.

Circulating Hedgehog: a fresh view of a classic morphogen.

Members of the Hedgehog family of morphogens mediate the intercellular communication necessary for the organisation and development of many animal tissues. They are modified by various lipid adducts, rendering them insoluble in hydrophilic environments and leading to the contentious question of how these molecules travel in the aqueous extracellular space. Seminal work carried out by Suzanne Eaton and her colleagues has shed light on how these morphogens can spread over long distances through their association with lipoprotein particles. In this Spotlight article, we discuss Suzanne's pioneering work and her contribution to our understanding of the transport and activity of morphogens, in particular Hedgehog. We also describe two other essential aspects of her work: the discovery and characterisation of endogenously present Hedgehog variants, as well as her proposition that, in addition to its role as a morphogen, Hedgehog acts as an endocrine hormone.

Wingless counteracts epithelial folding by increasing mechanical tension at basal cell edges in Drosophila.

The modulation of mechanical tension is important for sculpturing tissues during animal development, yet how mechanical tension is controlled remains poorly understood. In Drosophila wing discs, the local reduction of mechanical tension at basal cell edges results in basal relaxation and the formation of an epithelial fold. Here, we show that Wingless, which is expressed next to this fold, promotes basal cell edge tension to suppress the formation of this fold. Ectopic expression of Wingless blocks fold formation, whereas the depletion of Wingless increases fold depth. Moreover, local depletion of Wingless in a region where Wingless signal transduction is normally high results in ectopic fold formation. The depletion of Wingless also results in decreased basal cell edge tension and basal cell area relaxation. Conversely, the activation of Wingless signal transduction leads to increased basal cell edge tension and basal cell area constriction. Our results identify the Wingless signal transduction pathway as a crucial modulator of mechanical tension that is important for proper wing disc morphogenesis.

The molecular mechanisms of remodeling in asthma, COPD and IPF with a special emphasis on the complex role of Wnt5A.

INTRODUCTION: Chronic inflammatory lung diseases are a common cause of suffering and death. Chronic obstructive pulmonary disease (COPD) is the reason for 6% of all deaths worldwide. A total of 262 million people are affected by asthma and 461,000 people died in 2019. Idiopathic pulmonary fibrosis (IPF) is diagnosed in 3 million people worldwide, with an onset over the age of 50 with a mean survival of only 24-30 months. These three diseases have in common that remodeling of the lung tissue takes place, which is responsible for an irreversible decline of lung function. Pathological lung remodeling is mediated by a complex interaction of different, often misguided, repair processes regulated by a variety of mediators. One group of these, as has recently become known, are the Wnt ligands. In addition to their well-characterized role in embryogenesis, this group of glycoproteins is also involved in immunological and structural repair processes. Depending on the combination of the Wnt ligand with its receptors and co-receptors, canonical and noncanonical signaling cascades can be induced. Wnt5A is a mediator that is described mainly in noncanonical Wnt signaling and has been shown to play an important role in different inflammatory diseases and malignancies. OBJECTIVES: In this review, we summarize the literature available regarding the role of Wnt5A as an immune modulator and its role in the development of asthma, COPD and IPF. We will focus specifically on what is known about Wnt5A concerning its role in the remodeling processes involved in the chronification of the diseases. CONCLUSION: Wnt5A has been shown to be involved in all three inflammatory lung diseases. Since the ligand affects both structural and immunological processes, it is an interesting target for the treatment of lung diseases whose pathology involves a restructuring of the lung tissue triggered in part by an inflammatory immune response.

Cell sorting and morphogenesis in early Drosophila embryos.

The regionalisation of growing tissues into compartments that do not mix is thought to be a common motif of animal development. Compartments and compartmental boundaries were discovered by lineage studies in the model organism Drosophila. Since then, many compartment boundaries have been identified in developing tissues, from insects to vertebrates. These are important for animal development, because boundaries localize signalling centres that control tissue morphogenesis. Compartment boundaries are boundaries of lineage restriction, where specific mechanisms keep boundaries straight and cells segregated. Here, we review the mechanisms of cell sorting at boundaries found in early Drosophila embryos. The parasegmental boundaries, separating anterior from posterior compartments in the embryo, keep cells segregated by increasing actomyosin contractility at boundary cell-cell interfaces. Differential actomyosin contractility in turn promotes fold formation and orients cell division. Earlier in development, actomyosin differentials are also important for cell sorting during axis extension. Specific cell surface asymmetries and signalling pathways are required to initiate and maintain these actomyosin differentials.

A conserved role for arrow in posterior axis patterning across Arthropoda.

Segmentation is a key characteristic of Arthropoda that is linked to the evolutionary success of this lineage. It has previously been shown in both vertebrates and short germ insects that posterior segmentation requires canonical Wnt (cWnt) signaling, which maintains the expression of Caudal and the posterior growth zone; disruption of cWnt signaling incurs posterior truncations in these lineages due to the loss of the tail bud. However, comparable datasets for Wnt signaling are limited outside of holometabolous insects, due to incomparable phenotypic spectra and inefficacy of gene misexpression methods in certain model species. We applied RNA interference (RNAi) against the Wnt co-receptor arrow (arr), a key member of the cWnt signaling pathway in holometabolous insects and vertebrates, to examine posterior axis elongation of the cobweb spider Parasteatoda tepidariorum (short germ embryogenesis; one Wnt8 homolog), the cricket Gryllus bimaculatus (intermediate germ; one Wnt8 homolog), and the milkweed bug Oncopeltus fasciatus (short germ; two Wnt8 homologs). Knockdown of arr in insects resulted in posterior truncations affecting the gnathos through the abdomen in O. fasciatus, whereas posterior truncations only affected the T3 segment through the abdomen in G. bimaculatus. Spider embryos with disrupted arr expression exhibited defects along the entire axis, including segmentation defects throughout the germband. RNA-Seq-based differential gene expression analysis of severe Ptep-arr loss-of-function phenotypes at two developmental stages was used to confirm that knockdown of Ptep-arr results in systemic disruption of the Wnt pathway. Intriguingly, we found that knockdown of arr did not abrogate Wnt8 expression in any of the three species, with cad expression additionally retained in severe loss-of-function phenotypes in the cricket and the spider. Together with data from a holometabolous insect, our results suggest that cWnt signaling is not required for maintenance of Wnt8 expression across Arthropoda. These outcomes underscore the diagnostic power of differential gene expression analyses in characterizing catastrophic phenotypes in emerging model species.

Axin proteolysis by Iduna is required for the regulation of stem cell proliferation and intestinal homeostasis in Drosophila.

Self-renewal of intestinal stem cells is controlled by Wingless/Wnt-ß catenin signaling in both Drosophila and mammals. As Axin is a rate-limiting factor in Wingless signaling, its regulation is essential. Iduna is an evolutionarily conserved ubiquitin E3 ligase that has been identified as a crucial regulator for degradation of ADP-ribosylated Axin and, thus, of Wnt/ß-catenin signaling. However, its physiological significance remains to be demonstrated. Here, we generated loss-of-function mutants of Iduna to investigate its physiological role in Drosophila Genetic depletion of Iduna causes the accumulation of both Tankyrase and Axin. Increase of Axin protein in enterocytes non-autonomously enhanced stem cell divisions in the Drosophila midgut. Enterocytes secreted Unpaired proteins and thereby stimulated the activity of the JAK-STAT pathway in intestinal stem cells. A decrease in Axin gene expression suppressed the over-proliferation of stem cells and restored their numbers to normal levels in Iduna mutants. These findings suggest that Iduna-mediated regulation of Axin proteolysis is essential for tissue homeostasis in the Drosophila midgut.

Molecular phylogeny and biogeography of the genus Symbrenthia (Lepidoptera, Nymphalidae) correlates with the past geography of the Oriental region.

Jesters, butterflies in the genus Symbrenthia Hübner, 1819, comprise 14 species mainly distributed in the Oriental region. Although this genus has attracted the attention of many researchers in the past, its taxonomy and biogeographic history remain unclear. In this study, we investigate phylogenetic and biogeographic relationships inferred from one mitochondrial (COI) and two nuclear genes (ArgKin, wingless), using both likelihood and Bayesian approaches. With the exception of S. hippalus, which we find to be either sister to Mynes Boisduval, 1832 or sister to Symbrenthia + Mynes + Araschnia, all species of Symbrenthia form a single monophyletic group. We describe a new genus Mynbrenthia Fric & Rindos gen. nov. to accommodate the taxon hippalus. The genus Symbrenthia splits into four sub-groups, "Brensymthia" (with S. niphanda and S. sinoides), "hypselis" (with S. hypselis, S. brabira, S. leoparda and S. doni), "intricata" (with S. intricata and S. hypatia) and "hippoclus" group (including S. platena and a complex of S. hippoclus and S. lilaea). The genus probably originated in Sundaland or continental Asia during the Eocene. The history of the genus Symbrenthia was more influenced by dispersal events and then by subsequent vicariances. Whereas the "hypselis" group colonised the Indo-Australian Archipelago from the Asian continent, the "hippoclus" group dispersed to continental Asia from the Indo-Australian Archipelago.

V-ATPase subunit a is required for survival and midgut development of Locusta migratoria.

The vacuolar-type H+ -ATPase (V-ATPase) is an ATP-dependent proton pump, which regulates various cellular processes. To date, most functional studies on V-ATPases of insects have focused on subunits of the V1 complex, and there is little information on the VO genes. In this study, two cDNA sequences of LmV-ATPase a were identified in Locusta migratoria. RT-qPCR analysis revealed that LmV-ATPase a1 and LmV-ATPase a2 are differentially expressed in various tissues and developmental stages. Injection of dsRNA for the common region of LmV-ATPase a1 and LmV-ATPase a2 into third-instar nymphs resulted in a significant suppression of LmV-ATPase a. The injected nymphs ceased feeding, lost body weight and finally died at a mortality of 98.6%. Furthermore, aberrations of midgut epithelial cells, the accumulation of electron-lucent vesicles in the cytoplasm, and a partially damaged brush border were observed in dsLmV-ATPase a-injected nymphs using transmission electron microscopy. Especially, the mRNA level of wingles, and notch genes were dramatically down-regulated in the dsLmV-ATPase a-injected nymphs. Taken together, our results suggest that LmV-ATPase a is required for survival and midgut development of L. migratoria. Hence, this gene could be a good target for RNAi-based control against locusts.

Inhibited HDAC3 promotes microRNA-376c-3p to suppress malignant phenotypes of gastric cancer cells by reducing WNT2b.

OBJECTIVE: Our study aims to identify the impact of histone deacetylase 3 (HDAC3) and microRNA-376c-3p (miR-376c-3p) on gastric cancer (GC) by targeting wingless-type MMTV integration site family member 2b (WNT2b). METHODS: Levels of miR-376c-3p, HDAC3 and WNT2b were assessed. GC cells were treated with altered HDAC3 or miR-376c-3p to evaluate their biological functions, and rescue experiment was performed to assess the effect of WNT2b on GC cells. The tumor growth in vivo was observed. RESULTS: HDAC3 and WNT2b were up-regulated while miR-376c-3p was reduced in GC tissues and cell lines. The inhibited HDAC3 or elevated miR-376c-3p could restrain malignant behaviors of GC cells in vitro, and also suppress the xenograft growth. WNT2b silencing reduced the effect of miR-376c-3p inhibition while WNT2b overexpression mitigated that of miR-376c-3p promotion on GC cell growth. CONCLUSION: Inhibiting HDAC3 promotes miR-376c-3p to suppress malignant phenotypes of GC cells via reducing WNT2b, thereby restricting GC development.

FGF signaling promotes myoblast proliferation through activation of wingless signaling.

Indirect flight muscles (IFMs) are the largest muscles in Drosophila and are made up of hundreds of myonuclei. The generation of these giant muscles requires a large pool of wing disc associated adult muscle precursors (AMPs), however the factors that control proliferation to form this myoblast pool are incompletely known. Here, we examine the role of fibroblast growth factor (FGF) signaling in the proliferation of wing disc associated myoblasts. We find that the components of FGF signaling are expressed in myoblasts and surrounding epithelial cells of the wing disc. Next, we show that attenuation of FGF signaling results in a diminished myoblast pool. This reduction in the pool size is due to decreased myoblast proliferation. By contrast, activating the FGF signaling pathway increases the myoblast pool size and restores the proliferative capacity of FGF knockdown flies. Finally, our results demonstrate that the FGF receptor Heartless acts through up-regulating ß-catenin/Armadillo signaling to promote myoblast proliferation. Our studies identify a novel role for FGF signaling during IFM formation and uncover the mechanism through which FGF coordinates with Wingless signaling to promote myoblast proliferation.

Wnt gene regulation and function during maxillary palp development in Drosophila melanogaster.

Wnt genes encode secreted ligands that play many important roles in the development of metazoans. There are thirteen known Wnt gene subfamilies and seven of these are represented in Drosophila melanogaster. While wingless (wg) is the best understood and most widely studied Wnt gene in Drosophila, the functions of many of the other Drosophila Wnt genes are less well understood. For example, relatively little is known about Wnt6, which is an ancient paralog of wg and they form a conserved Wnt cluster together with Wnt9 (Dwnt4) and Wnt10. Wg and Wnt6 encode similar proteins and exhibit overlapping expression in several tissues during development. Both wg and Wnt6 were previously shown to regulate the development of maxillary palps, important olfactory organs in flies, but it remained unclear how these two ligands may combine to carry out specific functions and how this is regulated. Here, we have further analysed Wnt6 function in the context of maxillary palp development. Surprisingly, we found that Wnt6 does not appear to be necessary for development of maxillary palps. While a deletion of the 5' region of Wnt6 results in very small maxillary palps, we show that this effect is more likely to be a consequence of removing cis-regulatory elements that may regulate wg expression in this tissue rather than through the loss of Wnt6 function. Although, we cannot completely exclude the possibility that Wnt6 may subtly regulate maxillary palp development in combination with wg, our analysis of Wnt6 loss of function mutants suggests this ligand plays a more general role in regulating growth during development. Taken together our results provide new insights into maxillary palp formation and Wnt6 functions in Drosophila, and further evidence for a complex cis-regulatory landscape in the Wnt9-wg-Wnt6-Wnt10 cluster, which may help explain its evolutionary conservation.

Wnt8B, transcriptionally regulated by ZNF191, promotes cell proliferation of hepatocellular carcinoma via Wnt signaling.

Dysregulation of wingless-type (Wnt) signaling is implicated in hepatocellular carcinoma (HCC). Wnt family member 8B (Wnt8B), one of the canonical Wnt ligands, is implicated in oncogenesis. However, the role of Wnt8B in human HCCs and its transcriptional regulation mechanism are presently unknown . Here, we report that Wnt8B expression was frequently increased in HCCs and was significantly associated with poorer patient prognosis. Wnt8B knockdown suppresses HCC cell growth both in vitro and in vivo via inhibiting the canonical Wnt signaling. Zinc finger transcription factor 191 (ZNF191) can positively regulate Wnt8B mRNA and protein expression, and promoter luciferase assay indicated that ZNF191 can increase the transcription activity of the 2-Kbps WNT8B promoter. Chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assay showed that ZNF191 protein directly binds to the WNT8B promoter, and the binding sites are at nt-1491(ATTAATT) and nt-1178(ATTCATT). Moreover, Wnt8B contributes to the effect of ZNF191 on cell proliferation, and Wnt8B expression correlates positively with ZNF191 in human HCCs. Our findings suggested that Wnt8B, directly transcriptionally regulated by ZNF191, plays a pivotal role in HCC proliferation via the canonical Wnt pathway and may serve as a new prognostic biomarker and a potential therapeutic target for HCC patients.

Naked cuticle inhibits wingless signaling in Drosophila wing development.

Wnt signaling is one of the major signaling pathways that regulate cell differentiation, tissue patterning and stem cell homeostasis and its dysfunction causes many human diseases, such as cancer. It is of tremendous interests to understand how Wnt signaling is regulated in a precise manner both temporally and spatially. Naked cuticle (Nkd) acts as a negative-feedback inhibitor for Wingless (Wg, a fly Wnt) signaling in Drosophila embryonic development. However, the role of Nkd remains controversial in later fly development, particularly on the canonical Wg pathway. In the present study, we show that nkd is essential for wing pattern formation, such that both gain and loss of nkd result in the disruption of Wg target expression in larvae stage and abnormal adult wing morphologies. Furthermore, we demonstrate that a thirty amino acid fragment in Nkd, identified previously in Wharton lab, is critical for the canonical Wg signaling, but is dispensable for Wg/planar cell polarity pathway. Putting aside the pleiotropic nature of nkd function, i.e. its role in the Decapentaplegic signaling, we conclude that Nkd universally inhibits the canonical Wg pathway across a life span of Drosophila development.

Deltex cooperates with TRAF6 to promote apoptosis and cell migration through Eiger-independent JNK activation in Drosophila.

JNK signaling is a highly conserved signaling pathway that regulates a broad spectrum of cellular processes including cell proliferation, migration, and apoptosis. In Drosophila, JNK signaling is activated by binding of the tumor necrosis factor (TNF) Eiger to its receptor Wengen, and a conserved signaling cascade operates that culminates into activation of dual phosphatase Puckered thereby triggering apoptosis. The tumor necrosis factor receptor (TNFR) associated factor 6 (TRAF6) is an adaptor protein, which transduces the signal from TNFRs and Toll-like receptor/interleukin-1 receptor superfamily to induce a wide spectrum of cellular responses. TRAF6 also acts as the adaptor protein that mediates Eiger/JNK signaling in Drosophila. In a genetic interaction study, deltex (Dx) was identified as a novel interactor of TRAF6. Dx is well known to regulate Notch signaling in a context-dependent manner. Our data suggest that combinatorial action of Dx and TRAF6 enhances the Dx-induced wing nicking phenotype by inducing caspase-mediated cell death. Co-expression of Dx and TRAF6 also results in enhanced invasive behavior and perturbs the normal morphology of cells. The cooperative action of Dx and TRAF6 is attributed to JNK activation, which also leads to ectopic wingless (Wg) and decapentaplegic (Dpp) expression. Our results also reveal that the endocytic pathway component Rab7 may play a pivotal role in the regulation of Dx-TRAF6-mediated activation of JNK signaling. Here, we present the fact that Dx and TRAF6 together activate JNK signaling in an Eiger-independent mechanism.

Two pair-rule responsive enhancers regulate wingless transcription in the Drosophila blastoderm embryo.

BACKGROUND: While many developmentally relevant enhancers act in a modular fashion, there is growing evidence for nonadditive interactions between distinct cis-regulatory enhancers. We investigated if nonautonomous enhancer interactions underlie transcription regulation of the Drosophila segment polarity gene, wingless. RESULTS: We identified two wg enhancers active at the blastoderm stage: wg 3613u, located from -3.6 to -1.3 kb upstream of the wg transcription start site (TSS) and 3046d, located in intron two of the wg gene, from 3.0 to 4.6 kb downstream of the TSS. Genetic experiments confirm that Even Skipped (Eve), Fushi-tarazu (Ftz), Runt, Odd-paired (Opa), Odd-skipped (Odd), and Paired (Prd) contribute to spatially regulated wg expression. Interestingly, there are enhancer specific differences in response to the gain or loss of function of pair-rule gene activity. Although each element recapitulates aspects of wg expression, a composite reporter containing both enhancers more faithfully recapitulates wg regulation than would be predicted from the sum of their individual responses. CONCLUSION: These results suggest that the regulation of wg by pair-rule genes involves nonadditive interactions between distinct cis-regulatory enhancers.