Drosophila Segment Polarity Gene Research Articles

A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos

The segment polarity gene hedgehog (hh) encodes a novel signaling protein that mediates local cell-cell interactions in the developing Drosophila embryo. Here we describe the existence of an hh-related gene family in the zebrafish, Brachydanio rerio. One of these genes, sonic hedgehog (shh), is expressed in the notochord, floor plate, and posterior fin mesoderm, tissues associated with polarizing activities in various vertebrate embryos. The pattern of shh expression in zebra-fish mutants affecting axial structures, together with the consequences of its ectopic expression in normal embryos, is consistent with a role for shh in floor plate induction. By expressing shh in transgenic Drosophila embryos, we also demonstrate a strong functional conservation between the fish and fly hh genes.

Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity

We have identified three members of a mouse gene family related to the Drosophila segment polarity gene, hedgehog(hh). Like hh, they encode putative secreted proteins and are thus implicated in cell-cell interactions. One of these, Sonic hh (Shh), is expressed in the notochord, the floor plate, and the zone of polarizing activity, signaling centers that are thought to mediate central nervous system (CNS) and limb polarity. Ectopic expression of Shh in the mouse CNS leads to the activation of floor plate-expressed genes. These results suggest that Shh may play a role in the normal inductive interactions that pattern the ventral CNS.

A model system for cell adhesion and signal transduction in Drosophila

Cells must cooperate and communicate to form a multicellular animal. Information about the molecules required for these processes have come from a variety of sources; the convergence between the studies of particular molecules by vertebrate cell biologists and the genes identified by scientists investigating development in Drosophila has been especially fruitful. We are interested in the connection between cadherin proteins that regulate cell-cell adhesion and the wingless/wnt-1 cell-cell signaling molecules controlling pattern formation during development. The Drosophila segment polarity gene armadillo, homolog of the vertebrate adherens junction protein beta-catenin, is required for both cell adhesion and wg signaling. We review what is known about wingless signaling in Drosophila, and discuss the role of cell-cell junctions in both cell adhesion and cell communication. We then describe the results of our preliminary structure-function analysis of Armadillo protein in both cell adhesion and wingless signaling. Finally, we discuss evidence supporting a direct role for Armadillo and adherens junction in transduction of wingless signal.

Responses to Wnt signals in vertebrate embryos may involve changes in cell adhesion and cell movement.

Wnt genes encode secreted glycoproteins, and, because of their homology with the Drosophila segment polarity gene wingless, are likely to play important roles as modulators of local intercellular signalling during embryonic development. Although little is known of the mechanisms by which Wnts signal in an autocrine or paracrine manner, it is increasingly clear that cells can respond rapidly to Wnt signals in the absence of transcription, and that these responses may include changes in cell adhesion and cell movement. We review recent evidence from studies on Xenopus laevis and other systems, which demonstrate that (1) a subset of Wnts modulate gap junctional permeability, which may be a reflection of changes in cadherin-mediated cell adhesion, (2) embryos express beta-catenin and plakoglobin, which are homologs of the armadillo gene products, known to be involved in the wingless signalling pathway, and known to be found at cell junctions, and (3) overexpression of specific Wnts in Xenopus embryos leads to clear changes in cell behavior and movement.

Molecular organisation and expression pattern of the segment polarity gene fused of Drosophila melanogaster

The Drosophila segment-polarity gene fused (fu) is required for pattern formation within embryonic segments and imaginal discs. We previously reported that the 5′ part of the fused gene is homologous to the catalytic domain of serine/threonine kinases. We present here the sequence of the complete transcription unit, which predicts a 805 amino acid long protein. The kinase domain actually corresponds to 268 amino acids in the N-terminal part, and no known function can be attributed to the rest of the putative FUSED protein. Transcripts from the fused gene have been characterized: a unique 3.2 kb fused transcript is produced in nurse cells, in low abundance, from stage 8 of oogenesis, and persistently through the rest of oogenesis. In embryos, this transcript is evenly distributed in all embryonic cells until the extended germ band stage, after which its amount strongly decreases. Ubiquitous expression is detected later in imaginal wing and leg discs. Possible roles of the FUSED protein in signal transduction pathways required for intercellular communication at different stages of development are discussed.

Induction of a secondary body axis in Xenopus by antibodies to beta-catenin.

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.

A role for the Drosophila segment polarity gene armadillo in cell adhesion and cytoskeletal integrity during oogenesis

The epithelial sheet is a structural unit common to many tissues. Its organization appears to depend on the function of the multi-protein complexes that form adherens junctions. Elegant cell biological experiments have provided support for hypotheses explaining the function of adherens junctions and of their components. These systems, however, lack the ability to test function within an entire organism during development. The realization that the product of the Drosophila segment polarity gene armadillo is related to the vertebrate adhesive junction components plakoglobin and beta-catenin led to the suggestion that armadillo might provide a genetic handle to study adhesive junction structure and function. An examination of the potential function of Armadillo in cell-cell adhesive junctions was initiated using the Drosophila ovary as the model system. We examined the distribution of Armadillo in the Drosophila ovary and demonstrated that this localization often parallels the location of cell-cell adhesive junctions. The consequences of removing armadillo function from the germ-line cells of the ovary were also examined. Germ-line armadillo mutations appear to disrupt processes requiring cell adhesion and integrity of the actin cytoskeleton, consistent with a role for Armadillo in cell-cell adhesive junctions. We have also used armadillo mutations to examine the effects on ovarian development of altering the stereotyped cell arrangements of the ovary. The implications of these results for the role of adhesive junctions during development are discussed.

Separable regulatory elements mediate the establishment and maintenance of cell states by the Drosophila segment-polarity gene gooseberry.

During Drosophila embryogenesis, position along the anteroposterior axis is specified within each segment by the products of the segment-polarity genes which include wingless (wg) and gooseberry (gsb). The striped expression of these genes in each segment is initially established by the pair-rule gene products during late blastoderm. This pattern is subsequently maintained after germ band extension by interaction among the segment-polarity genes themselves. Here we show that the maintenance of gsb, a PHox gene encoding a paired-domain and a homeodomain, is controlled by the wg signal, the homolog of the murine Wnt-1 protein. A control element responsible for wg-dependent maintenance of gsb expression, gsb-late element, is separable from an element required for the initial activation of gsb by pair-rule transcription factors, gsb-early element. The significance of such a regulatory strategy is discussed with respect to the establishment and maintenance of cell states within each segment by segment-polarity genes.

In pursuit of the functions of the Wnt family of developmental regulators: insights from Xenopus laevis.

Wnts are a recently described family of secreted glycoproteins related to the Drosophila segment polarity gene, wingless, and to the proto-oncogene, int-1. Wnts are thought to function as developmental modulators, with signalling distances of only a few cell diameters. In Xenopus, at least six Wnts, including Xwnts-1, -3A, and -4, are expressed initially in the developing central nervous system, with some regions expressing multiple Xwnts. Xwnt-8 is expressed by mid-blastula stage, in ventral and lateral mesoderm. Xwnt-5A mRNAs are stored in the egg, and later are expressed throughout the embryo in both ectoderm and mesoderm, but with a pronounced enrichment in the head and tail. Recent studies in Xenopus have pursued the diverse roles of Xwnts in early development, the mechanisms by which Xwnts signal information between cells, and the cell physiological responses to Xwnt signals.

Structure and expression of Hedgehog, a Drosophila segment-polarity gene required for cell-cell communication

The complete nucleotide sequence of the coding region of hedgehog (hh), a segment-polarity gene in Drosophila melanogaster, was determined. The gene was found to include three exons which would encode a 421- (or 471-) amino acid (aa) polypeptide with a long hydrophobic stretch. The hh mRNA was about 2.3 kb long and expressed throughout development. The hh expression in an embryo occurred in stripes, while that in imaginai discs occurred in the posterior compartment. As a whole, the spatial expression pattern of hh mRNA was very similar to that of Drosophila ( en), a homeobox gene required for the formation of the anterior-posterior compartment boundary. Unlike en, no hhexpression was observed in the central nervous system.

The product of Drosophila segment polarity gene armadillo is part of a multi-protein complex resembling the vertebrate adherens junction

The product of Drosophila segment polarity gene armadillo is part of a multi-protein complex resembling the vertebrate adherens junction

The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation.

cDNAs were isolated that represent transcripts of the Drosophila segment polarity gene, hedgehog (hh). Sequence analysis reveals a motif characteristic of a transmembrane domain, suggesting that the hh protein is membrane-associated. hh expression in epidermal cells is confined to the posterior compartments and coincides precisely with that of engrailed (en). Despite the similar patterns of expression in the cellular blastoderm, hh expression is independent of en, but hh expression becomes sensitive to and dependent on en during the extended germ band stage. The ectopic expression of hh that is normally induced in patched (ptc) mutant embryos does not appear in ptc en double mutants. We discuss these findings in terms of the relationship between en and hh, and the role of the hh function.

Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila.

The stable maintenance of expression patterns of homeotic genes depends on the function of a number of negative trans-regulators, termed the Polycomb (Pc) group of genes. We have examined the pattern of expression of the Drosophila segment polarity gene, engrailed (en), in embryos mutant for several different members of the Pc group. Here we report that embryos mutant for two or more Pc group genes show strong ectopic en expression, while only weak derepression of en occurs in embryos mutant for a single Pc group gene. This derepression is independent of two known activators of en expression: en itself and wingless. Additionally, in contrast to the strong ectopic expression of homeotic genes observed in extra sex combs- (esc-) mutant embryos, the en expression pattern is nearly normal in esc- embryos. This suggests that the esc gene product functions in a pathway independent of the other genes in the group. The data indicate that the same group of genes is required for stable restriction of en expression to a striped pattern and for the restriction of expression of homeotic genes along the anterior-posterior axis, and support a global role for the Pc group genes in stable repression of activity of developmental selector genes.

The armadillo homologs β-catenin and plakoglobin are differentially expressed during early development of Xenopus laevis

Plakoglobin and β-catenin are cytoplasmic proteins associated with the intracellular plaques of cell adhesive junctions. While plakoglobin is present in both adherens junctions and desmosomes, β-catenin is associated with the cadherins that accumulate only in adherens junctions. Both β-catenin and plakoglobin are homologs of armadillo, a Drosophila segment polarity gene that is considered to be in the wingless signaling pathway. We have characterized the expression and distribution of both plakoglobin and β-catenin in Xenopus embryos. As shown by RNA blot analysis, β-catenin and plakoglobin transcripts are present in fertilized eggs and in embryos through to tadpole stage. Whole-mount in situ hybridization indicates that both genes are expressed in the dorsal ectoderm and mesoderm of tailbud- and tadpolestage embryos and that β-catenin is expressed in the midbrain. Both plakoglobin and β-catenin polypeptides are present during early Xenopus development; however, differences exist in the timing of maximal expression. Plakoglobin is present in the fertilized egg, increases in abundance by neurula stage, then declines at the tailbud and tadpole stages. β-Catenin, recognized by an anti- arm antibody, is also present in the fertilized egg and in blastula-stage embryos. However, β-catenin continues to be detected at the neurula, tailbud, and tadpole stages when levels of plakoglobin decline. The presence of multiple homologs of armadillo in Xenopus embryos and the differences in their patterns of expression suggest distinct roles for these proteins in processes affected by cell adhesion.

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.

Cloning of segment polarity gene homologues from the unsegmented brachiopod Terebratulina retusa (Linnaeus)

We have used the polymerase chain reaction (PCR) to amplify, clone and sequence homologues of the Drosophila segment polarity genes engrailed ( en), cubitus interruptus Dominant ( ci D ) and wingless ( wg) from the genome of the brachiopod, Terebratulina retusa (Linnaeus). The deduced translation products of brachiopod en and ci D share high levels of sequence identity with their Drosophila homologues. The brachiopod wg-related clone is divergent from Drosophila wg, although clearly a member of the wg/Wnt gene family. These results indicate that structural diversity of Drosophila segment polarity genes has been evolutionarily conserved in a divergent, ancient and unsegmented animal phylum.

Genomic structure and restricted neural expression of the zebrafish wnt-1 (int-1) gene.

The Wnt-1 (int-1) gene was originally identified as an oncogene, but its normal function is in embryogenesis. The gene is the vertebrate homologue of the Drosophila segment polarity gene wingless, and encodes a secretory protein. In mouse embryos, Wnt-1 expression is necessary for proper development of the midbrain and anterior hindbrain. Here we describe the molecular cloning and primary structure of the zebrafish Wnt-1 gene (denoted wnt-1). Comparison with its mouse homologue reveals that both the genomic organization of wnt-1 and the amino acid sequence of the corresponding gene product have been extensively conserved during vertebrate evolution. Moreover, there is probably at least one Wnt-1-related sequence in the zebrafish genome. In zebrafish embryos, wnt-1 is expressed during differentiation of the neural tube. In situ hybridization analysis reveals that the transcripts are confined to the dorsal surfaces of the midbrain, hindbrain and spinal cord, and to lateral cells at the midbrain-hindbrain junction. Thus, the pattern of wnt-1 expression in the developing central nervous system of zebrafish is virtually identical to that seen in mouse embryos. Unexpectedly, despite the striking similarities of Wnt-1 structure and expression in fish and higher vertebrates, we could not identify sequences of obvious homology outside the coding regions, neither in the promoter nor in the introns.

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.

Spatial expression of the Drosophila segment polarity gene armadillo is posttranscriptionally regulated by wingless

armadillo ( arm) is one of a group of Drosophila segment polarity genes that are required for normal patterning within the embryonic segment. Although arm RNA is uniformly distributed in embryos, arm protein accumulates at higher levels in regions that contain wingless, another segment polarity gene which encodes a secreted protein that regulates patterning via cell-cell communication. These local increases in arm protein require wingless activity, and mutations that alter wingless distribution produce corresponding changes in the arm protein pattern. These results suggest that wingless regulates accumulation of arm protein by a posttranscriptional mechanism. Two other segment polarity genes, porcupine and dishevelled, are required for this effect. We also show that arm protein is closely associated with the plasma membrane in virtually all cell types and often colocalizes with F-actin.

GLI3 Encodes a 190-Kilodalton Protein with Multiple Regions of GLI Similarity

The GLI oncogene, discovered by virtue of its amplification in human tumors, encodes a sequence-specific DNA-binding protein containing five zinc fingers. We have now characterized one member of a family of GLI-related zinc finger genes. A previously identified fragment of GLI3 genomic DNA was used to localize GLI3 to chromosome 7p13 and to isolate cDNA clones. Sequence analysis of these clones and identification of the GLI3 protein by using polyclonal antisera demonstrated that GLI3 encodes a protein of 1,596 amino acids and an apparent molecular mass of 190 kilodaltons. Amino acid sequence comparison with GLI demonstrated seven regions of similarity (53 to 88% identity), with the zinc fingers representing the most similar region. Furthermore, when produced in vitro, the GLI3 protein bound specifically to genomic DNA fragments containing GLI-binding sites. Amino acid sequence comparison with the product of another member of the GLI family, the Drosophila segment polarity gene cubitus interruptus Dominant, revealed additional similarity that was not shared with GLI. These studies suggest that the GLI-related genes encode a family of DNA-binding proteins with related target sequence specificities. In addition, sequence similarity aside from the zinc finger region suggests that other aspects of function are shared among the members of this gene family.