The product of the Drosophila melanogaster segment polarity gene armadillo is highly conserved in sequence and expression in the housefly Musca domestica

Summary Segmental pattern in Drosophila melanogaster is set up via a set of cell-cell interactions mediated by the products of the segment polarity genes. Among these is the armadillo gene, whose product seems to be required for the reception of an intercellular signal encoded by the wingless gene. As part of our effort to relate the structure of the armadillo protein to its function within the cell, we have examined the evolutionary conservation of the armadillo gene during insect evolution. We have cloned the armadillo gene from the housefly, Musca domestica, which diverged from Drosophila 100 million years ago. The Musca protein is 97.5% identical to that in Drosophila, while the noncoding sequences have diverged extensively. This remarkable degree of conservation at the protein level is mirrored in the expression pattern of the armadillo protein. Antibodies against the Drosophila protein cross-react with a Musca protein of the appropriate size. We have also used these antibodies to show that the Musca armadillo protein has a pattern of expression in larval and adult tissues similar to that of Drosophila armadillo. We discuss the implications of conservation of structure and expression for the cellular role of the armadillo protein and its mammalian homologs.Offprint requests to: M. Peifer     

The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin

The Drosophila segment polarity gene armadillo is required for pattern formation within embryonic segments and imaginal discs. We have found that armadillo is highly conserved during evolution; it is 63% identical to human plakoglobin, a protein found in adhesive junctions joining epithelial and other cells. We have examined arm protein localization in a number of larval tissues and found that arm protein accumulation within cells shares many features with the accumulation of plakoglobin. We have compared the phenotype and molecular lesions responsible for the different arm mutations. Surprisingly, severely truncated proteins retain some function; the degree of function is strictly correlated with the length of the truncated protein, suggesting that the internally repetitive arm protein is modular in function. We present a possible model for the cellular role of arm.     

Drosophila segment polarity gene product porcupine stimulates the posttranslational N-glycosylation of wingless in the endoplasmic reticulum

Wnt is a family of cysteine-rich secreted glycoproteins, which controls the fate and behavior of the cells in multicellular organisms. In the absence of Drosophila segment polarity gene porcupine (porc), which encodes an endoplasmic reticulum (ER) multispanning transmembrane protein, the N-glycosylation of Wingless (Wg), one of Drosophila Wnt family, is impaired. In contrast, the ectopic expression of porc stimulates the N-glycosylation of both endogenously and exogenously expressed Wg. The N-glycosylation of Wg in the ER occurs posttranslationally, while in the presence of dithiothreitol, it efficiently occurs cotranslationally. Thus, the cotranslational disulfide bond formation of Wg competes with the N-glycosylation by an oligosaccharyl transferase complex. Porc binds the N-terminal 24-amino acid domain (residues 83-106) of Wg, which is highly conserved in the Wnt family and stimulates the N-glycosylation at surrounding sites. Porc is also necessary for the processing of Drosophila Wnt-3/5 in both embryos and cultured cells. Thus, Porc binds the N-terminal specific domain of the Wnt family and stimulates its posttranslational N-glycosylation by anchoring them at the ER membrane possibly through acylation.     

Spatial regulation of segment polarity gene expression in the anterior terminal region of the Drosophila blastoderm embryo

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of the segment polarity genes, wg and hh, were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression. Additionally, btd is required for the first segmental stripe (mandibular segment) of both hh and wg at blastoderm stages. The subsequent segmentation of the cephalic segments (preantennal, antennal and intercalary) appears to be dependent on the overlap of the wg and hh cephalic domains as defined by these gap genes at the blastoderm stage. None of these five known gap genes are required for the activation of the labral segment domains of hh and wg, which are presumably either activated directly by maternal pathways or by an unidentified gap gene.     

Planar polarity is positively regulated by casein kinase Iepsilon in Drosophila

Members of the casein kinase I (CKI) family have been implicated in regulating canonical Wnt/Wingless (Wg) signaling by phosphorylating multiple pathway components. Overexpression of CKI in vertebrate embryos activates Wg signaling, and one target is thought to be the cytoplasmic effector Dishevelled (Dsh), which is an in vitro target of CKI phosphorylation. Phosphorylation of Dsh by CKI has also been suggested to switch its activity from noncanonical to canonical Wingless signaling. However, in vivo loss-of-function experiments have failed to identify a clear role for CKI in positive regulation of Wg signaling. By examining hypomorphic mutations of the Drosophila CKIepsilon homolog discs overgrown (dco)/double-time, we now show that it is an essential component of the noncanonical/planar cell polarity pathway. Genetic interactions indicate that dco acts positively in planar polarity signaling, demonstrating that it does not act as a switch between canonical and noncanonical pathways. Mutations in dco result in a reduced level of Dishevelled phosphorylation in vivo. Furthermore, in these mutants, Dishevelled fails to adopt its characteristic asymmetric subcellular localisation at the distal end of pupal wing cells, and the site of hair outgrowth is disrupted. Finally, we also find that dco function in polarity is partially redundant with CKIalpha.     

Unique establishment of procephalic head segments is supported by the identification of cis-regulatory elements driving segment-specific segment polarity gene expression in Drosophila

Anterior head segmentation is governed by different regulatory mechanisms than those that control trunk segmentation in Drosophila. For segment polarity genes, both initial mode of activation as well as cross-regulatory interactions among them differ from the typical genetic circuitry in the trunk and are unique for each of the procephalic segments. In order to better understand the segment-specific gene network responsible for the procephalic expression of the earliest active segment polarity genes wingless and hedgehog, we started to identify and analyze cis-regulatory DNA elements of these genes. For hedgehog, we could identify a cis-regulatory element, ic-CRE, that mediates expression specifically in the posterior part of the intercalary segment and requires promoter-specific interaction for its function. The intercalary stripe is the last part of the metameric hedgehog expression pattern that appears during embryonic development, which probably reflects the late and distinct establishment of this segment. The identification of a cis-regulatory element that is specific for one head segment supports the mutant-based observation that the expression of segment polarity genes is governed by a unique gene network in each of the procephalic segments. This provides further indication that the anterior-most head segments represent primary segments, which are set up independently, in contrast to the secondary segments of the trunk, which resemble true repetitive units.     

The vertebrate adhesive junction proteins beta-catenin and plakoglobin and the Drosophila segment polarity gene armadillo form a multigene family with similar properties

Three proteins identified by quite different criteria in three different systems, the Drosophila segment polarity gene armadillo, the human desmosomal protein plakoglobin, and the Xenopus E-cadherin-associated protein beta-catenin, share amino acid sequence similarity. These findings raise questions about the relationship among the three molecules and their roles in different cell-cell adhesive junctions. We have found that antibodies against the Drosophila segment polarity gene armadillo cross react with a conserved vertebrate protein. This protein is membrane associated, probably via its interaction with a cadherin-like molecule. This cross-reacting protein is the cadherin-associated protein beta-catenin. Using anti-armadillo and antiplakoglobin antibodies, it was shown that beta-catenin and plakoglobin are distinct molecules, which can coexist in the same cell type. Plakoglobin interacts with the desmosomal glycoprotein desmoglein I, and weakly with E-cadherin. Although beta-catenin interacts tightly with E-cadherin, it does not seem to be associated with either desmoglein I or with isolated desmosomes. Anti-armadillo antibodies have been further used to determine the intracellular localization of beta-catenin, and to examine its tissue distribution. The implications of these results for the structure and function of different cell-cell adhesive junctions are discussed.     

Cell patterning in the Drosophila segment: spatial regulation of the segment polarity gene patched

Intrasegmental patterning in the Drosophila embryo requires the activity of the segment polarity genes. The acquisition of positional information by cells during embryogenesis is reflected in the dynamic patterns of expression of several of these genes. In the case of patched, early ubiquitous expression is followed by its repression in the anterior portion of each parasegment; subsequently each broad band of expression splits into two narrow stripes. In this study we analyse the contribution of other segment polarity gene functions to the evolution of this pattern; we find that the first step in patched regulation is under the control of engrailed whereas the second requires the activity of both cubitus interruptusD and patched itself. Furthermore, the products of engrailed, wingless and hedgehog are essential for maintaining the normal pattern of expression of patched.     

Isolation of a human gene with protein sequence similarity to human and murine int-1 and the Drosophila segment polarity mutant wingless.

An expressed gene sequence which was identified by the isolation of a methylation free CpG island from human chromosome 7 has been cloned from a human lung cDNA library. The deduced protein sequence contains 360 amino acids and has several features of a secreted protein; it is cysteine rich with a signal peptide sequence and two potential asn-linked glycosylation sites. The protein sequence shows marked similarity with human and murine int-1 and their Drosophila homolog wingless (Dint-1). This human int-1 related protein, int-1 and Dint-1 have diverse patterns of expression, but the inferred structural similarities suggest that some of the functional characteristics of these proteins may be shared.     

The wingless gene is required for embryonic brain development in Drosophila

 We have studied the role of the wingless gene in embryonic brain development of Drosophila. wingless is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller domains in the anlagen of the deutocerebrum and tritocerebrum. Elimination of the wingless gene in null mutants has dramatic effects on the developing protocerebrum; although initially generated, approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death at late embryonic stages. Using temperature sensitive mutants, a rescue of the mutant phenotype can be achieved by stage-specific expression of functional wingless protein during embryonic stages 9–10. This time period correlates with that of neuroblast specification but preceeds the generation and subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants results in dramatically oversized CNS. We conclude that wingless is required for the development of the anterior protocerebral brain region in Drosophila. We propose that an important role of wingless in this part of the developing brain is the determination of neural cell fate. Received: 7 October 1997 / Accepted: 30 December 1997     

Bar homeobox genes are latitudinal prepattern genes in the developing Drosophila notum whose expression is regulated by the concerted functions of decapentaplegic and wingless

In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. Here, we show that two homeobox genes at the Bar locus (BarH1 and BarH2) may belong to a different class of prepattern genes expressed latitudinally, and suggest that the developing notum consists of checker-square-like subdomains, each governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activating achaete-scute. Presutural macrochaetae formation also requires Bar homeobox gene activity. Bar homeobox gene expression is restricted dorsally and posteriorly by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar homeobox genes is determined by wingless whose expression is under the control of Decapentaplegic signaling.     

Drosophila segment borders result from unilateral repression of hedgehog activity by wingless signaling

Body structures of Drosophila develop through transient developmental units, termed parasegments, with boundaries lying between the adjacent expression domains of wingless and engrailed. Parasegments are transformed into the morphologically distinct segments that remain fixed. Segment borders are established adjacent and posterior to each engrailed domain. They are marked by single rows of stripe expressing cells that develop into epidermal muscle attachment sites. We show that the positioning of these cells is achieved through repression of Hedgehog signal transduction by Wingless signaling at the parasegment boundary. The nuclear mediators of the two signaling pathways, Cubitus interruptus and Pangolin, function as activator and symmetry-breaking repressor of stripe expression, respectively.