Exploring myriapod segmentation: the expression patterns of even-skipped,engrailed, and wingless in a centipede

Segment formation is critical to arthropod development, yet there is still relatively little known about this process in most arthropods. Here, we present the expression patterns of the genes even-skipped (eve), engrailed, and wingless in a centipede, Lithobius atkinsoni. Despite some differences when compared with the patterns in insects and crustaceans, the expression of these genes in the centipede suggests that their basic roles are conserved across the mandibulate arthropods. For example, unlike the seven pair-rule stripes of eve expression in the Drosophila embryonic germband, the centipede eve gene is expressed strongly in the posterior of the embryo, and in only a few stripes between newly formed segments. Nonetheless, this pattern likely reflects a conserved role for eve in the process of segment formation, within the different context of a short-germband mode of embryonic development. In the centipede, the genes wingless and engrailed are expressed in stripes along the middle and posterior of each segment, respectively, similar to their expression in Drosophila. The adjacent expression of the engrailed and wingless stripes suggests that the regulatory relationship between the two genes may be conserved in the centipede, and thus this pathway may be a fundamental mechanism of segmental development in most arthropods.     

Functional conservation of the wingless-engrailed interaction as shown by a widely applicable baculovirus misexpression system

BACKGROUND: The expression patterns of the segment polarity genes wingless and engrailed are conserved during segmentation in a variety of arthropods, suggesting that the regulatory interactions between these two genes are also evolutionarily conserved. Hypotheses derived from such comparisons of gene expression patterns are difficult to test experimentally as genetic manipulation is currently possible for only a few model organisms. RESULTS: We have developed a system, using recombinant baculoviruses, that can be applied to a wide variety of organisms to study the effects of ectopic expression of genes. As a first step, we studied the range and type of infection of several reporter viruses in the embryos of two arthropod and one vertebrate species. Using this system to express wingless, we were able to induce expression of engrailed in the anterior half of each parasegment in embryos of the fruit fly Drosophila melanogaster. Virus-mediated wingless expression also caused ectopic naked ventral cuticle formation in wild-type Drosophila larvae. In the flour beetle, Tribolium castaneum, ectopic wingless also induced engrailed expression. As in Drosophila, this expression was only detectable in the anterior half of the parasegment. CONCLUSIONS: The functional interaction between wingless and engrailed, and the establishment of cells competent to express engrailed, appears to be conserved between Drosophila and Tribolium. The data on the establishment of an engrailed-competent domain also support the idea that prepatterning by pair-rule genes is conserved between these two insects. The recombinant baculovirus technology reported here may help answer other long-standing comparative evolutionary questions.     

Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.

The segment polarity genes engrailed and wingless are expressed in neighboring stripes of cells on opposite sides of the Drosophila parasegment boundary. Each gene is mutually required for maintenance of the other's expression; continued expression of both also requires several other segment polarity genes. We show here that one such gene, hedgehog, encodes a protein targeted to the secretory pathway and is expressed coincidently with engrailed in embryos and in imaginal discs; maintenance of the hedgehog expression pattern is itself dependent upon other segment polarity genes including engrailed and wingless. Expression of hedgehog thus functions in, and is sensitive to, positional signaling. These properties are consistent with the non-cell autonomous requirement for hedgehog in cuticular patterning and in maintenance of wingless expression.     

Evolutionary flexibility of pair-rule patterning revealed by functional analysis of secondary pair-rule genes, paired and sloppy-paired in the short-germ insect, Tribolium castaneum

In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning.     

Differential requirements of the fused kinase for hedgehog signalling in the Drosophila embryo.

The two signalling proteins, Wingless and Hedgehog, play fundamental roles in patterning cells within each metamere of the Drosophila embryo. Within the ventral ectoderm, Hedgehog signals both to the anterior and posterior directions: anterior flanking cells express the wingless and patched Hedgehog target genes whereas posterior flanking cells express only patched. Furthermore, Hedgehog acts as a morphogen to pattern the dorsal cuticle, on the posterior side of cells where it is produced. Thus responsive embryonic cells appear to react according to their position relative to the Hedgehog source. The molecular basis of these differences is still largely unknown. In this paper we show that one component of the Hedgehog pathway, the Fused kinase accumulates preferentially in cells that could respond to Hedgehog but that Fused concentration is not a limiting step in the Hedgehog signalling. We present direct evidence that Fused is required autonomously in anterior cells neighbouring Hedgehog in order to maintain patched and wingless expression while Wingless is in turn maintaining engrailed and hedgehog expression. By expressing different components of the Hedgehog pathway only in anterior, wingless-expressing cells we could show that the Hedgehog signalling components Smoothened and Cubitus interruptus are required in cells posterior to Hedgehog domain to maintain patched expression whereas Fused is not necessary in these cells. This result suggests that Hedgehog responsive ventral cells in embryos can be divided into two distinct types depending on their requirement for Fused activity. In addition, we show that the morphogen Hedgehog can pattern the dorsal cuticle independently of Fused. In order to account for these differences in Fused requirements, we propose the existence of position-specific modulators of the Hedgehog response.     

Role of segment polarity genes in the definition and maintenance of cell states in the Drosophila embryo

Segment polarity genes are expressed and required in restricted domains within each metameric unit of the Drosophila embryo. We have used the expression of two segment polarity genes engrailed (en) and wingless (wg) to monitor the effects of segment polarity mutants on the basic metameric pattern. Absence of patched (ptc) or naked (nkd) functions triggers a novel sequence of en and wg patterns. In addition, although wg and en are not expressed on the same cells absence of either one has effects on the expression of the other. These observations, together with an analysis of mutant phenotypes during development, lead us to suggest that positional information is encoded in cell states defined and maintained by the activity of segment polarity gene products.     

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.     

The consequences of ubiquitous expression of the wingless gene in the Drosophila embryo.

The segment polarity gene wingless has an essential function in cell-to-cell communication during various stages of Drosophila development. The wingless gene encodes a secreted protein that affects gene expression in surrounding cells but does not spread far from the cells where it is made. In larvae, wingless is necessary to generate naked cuticle in a restricted part of each segment. To test whether the local accumulation of wingless is essential for its function, we made transgenic flies that express wingless under the control of a hsp70 promoter (HS-wg flies). Uniform wingless expression results in a complete naked cuticle, uniform armadillo accumulation and broadening of the engrailed domain. The expression patterns of patched, cubitus interruptus Dominant and Ultrabithorax follow the change in engrailed. The phenotype of heatshocked HS-wg embryos resembles the segment polarity mutant naked, suggesting that embryos that overexpress wingless or lack the naked gene enter similar developmental pathways. The ubiquitous effects of ectopic wingless expression may indicate that most cells in the embryo can receive and interpret the wingless signal. For the development of the wild-type pattern, it is required that wingless is expressed in a subset of these cells.     

Autocatalytic ftz activation and metameric instability induced by ectopic ftz expression

Inappropriate expression of the Drosophila pair-rule gene, fushi tarazu (ftz), causes cuticular pattern deletions apparently complementary to those in ftz larvae. We show that the two patterns actually originate similarly, in both cases affecting the even-numbered parasegmental boundaries. The reciprocal cuticular patterns derive from differing patterns of selector gene expression (homoeotic transformations). The primary effect of ectopic ftz activity is to broaden ftz domains by autocatalytic activation of endogenous ftz expression in an additional anterior cell. This activates engrailed (en) and represses wingless (wg) expression, consistent with their proposed combinatorial control by ftz (and other pair-rule genes) to define parasegmental primordia. We propose that the anterior margin of each ftz stripe is normally defined by the posterior even-skipped (eve) boundary.     

Parasegmental organization of the spider embryo implies that the parasegment is an evolutionary conserved entity in arthropod embryogenesis

Spiders belong to the chelicerates, which is a basal arthropod group. To shed more light on the evolution of the segmentation process, orthologs of the Drosophila segment polarity genes engrailed, wingless/Wnt and cubitus interruptus have been recovered from the spider Cupiennius salei. The spider has two engrailed genes. The expression of Cs-engrailed-1 is reminiscent of engrailed expression in insects and crustaceans, suggesting that this gene is regulated in a similar way. This is different for the second spider engrailed gene, Cs-engrailed-2, which is expressed at the posterior cap of the embryo from which stripes split off, suggesting a different mode of regulation. Nevertheless, the Cs-engrailed-2 stripes eventually define the same border as the Cs-engrailed-1 stripes. The spider wingless/Wnt genes are expressed in different patterns from their orthologs in insects and crustaceans. The Cs-wingless gene is expressed in iterated stripes just anterior to the engrailed stripes, but is not expressed in the most ventral region of the germ band. However, Cs-Wnt5-1 appears to act in this ventral region. Cs-wingless and Cs-Wnt5-1 together seem to perform the role of insect wingless. Although there are differences, the wingless/Wnt-expressing cells and en-expressing cells seem to define an important boundary that is conserved among arthropods. This boundary may match the parasegmental compartment boundary and is even visible morphologically in the spider embryo. An additional piece of evidence for a parasegmental organization comes from the expression domains of the Hox genes that are confined to the boundaries, as molecularly defined by the engrailed and wingless/Wnt genes. Parasegments, therefore, are presumably important functional units and conserved entities in arthropod development and form an ancestral character of arthropods. The lack of by engrailed and wingless/Wnt-defined boundaries in other segmented phyla does not support a common origin of segmentation.     

The glypican Dally-like is required for Hedgehog signalling in the embryonic epidermis of Drosophila

The Drosophila genes dally and dally-like encode glypicans, which are heparan sulphate proteoglycans anchored to the cell membrane by a glycosylphosphatidylinositol link. Genetic studies have implicated Dally and Dally-like in Wingless signalling in embryos and imaginal discs. Here, we test the signalling properties of these molecules in the embryonic epidermis. We demonstrate that RNA interference silencing of dally-like, but not dally, gives a segment polarity phenotype identical to that of null mutations in wingless or hedgehog. Using heterologous expression in embryos, we uncoupled the Hedgehog and Wingless signalling pathways and found that Dally-like and Dally, separately or together, are not necessary for Wingless signalling. Dally-like, however, is strictly necessary for Hedgehog signal transduction. Epistatic experiments show that Dally-like is required for the reception of the Hedgehog signal, upstream or at the level of the Patched receptor.     

Interactions between segment polarity genes and the generation of the segmental pattern in Drosophila.

Although mutations in the segment polarity genes wingless, engrailed, hedgehog, gooseberry and cubitus-interruptusD all affect the region of naked cuticle within each segment of the Drosophila larva, subtle phenotypic differences suggest that these genes play different roles in segmental patterning. In this paper, the regulative interactions between these genes are analysed. They have revealed that the products of most of these genes accomplish more than one function during embryogenesis. Whereas early on a positive feed-back loop involving wg, en and hh maintains the expression of wg and en in the extremes of each parasegment, later on wg and en become independent from each other. en appears to regulate the expression of hh and ptc, while wg depends on gsb and ciD.     

hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen

Like the Drosophila embryo, the abdomen of the adult consists of alternating anterior (A) and posterior (P) compartments. However the wing is made by only part of one A and part of one P compartment. The abdomen therefore offers an opportunity to compare two compartment borders (A/P is within the segment and P/A intervenes between two segments), and ask if they act differently in pattern formation. In the embryo, abdomen and wing P compartment cells express the selector gene engrailed and secrete Hedgehog protein whilst A compartment cells need the patched and smoothened genes in order to respond to Hedgehog. We made clones of cells with altered activities of the engrailed, patched and smoothened genes. Our results confirm (1) that the state of engrailed, whether 'off' or 'on', determines whether a cell is of A or P type and (2) that Hedgehog signalling, coming from the adjacent P compartments across both A/P and P/A boundaries, organises the pattern of all the A cells. We have uncovered four new aspects of compartments and engrailed in the abdomen. First, we show that engrailed acts in the A compartment: Hedgehog leaves the P cells and crosses the A/P boundary where it induces engrailed in a narrow band of A cells. engrailed causes these cells to form a special type of cuticle. No similar effect occurs when Hedgehog crosses the P/A border. Second, we look at the polarity changes induced by the clones, and build a working hypothesis that polarity is organised, in both compartments, by molecule(s) emanating from the A/P but not the P/A boundaries. Third, we show that both the A and P compartments are each divided into anterior and posterior subdomains. This additional stratification makes the A/P and the P/A boundaries fundamentally distinct from each other. Finally, we find that when engrailed is removed from P cells (of, say, segment A5) they transform not into A cells of the same segment, but into A cells of the same parasegment (segment A6).     

Generating patterns from fields of cells: Examples from Drosophila segmentation

In Drosophila, a cascade of maternal, gap, pair-rule and segment polarity genes subdivides the antero/posterior axis of the embryo into repeating segmental stripes. This review summarizes what happens next, i.e. how an intrasegmental pattern is generated and controls the differentiation of specific cell types in the epidermis. Within each segment, cells secreting the signalling molecules Wingless (the homologue of vertebrate Wnt-1) and Hedgehog are found in narrow stripes on both sides of the parasegmental boundary. The Wingless and Hedgehog organizing activities help to establish two more stripes per segment that localize ligands for the Epidermal Growth Factor and the Notch signalling pathways, respectively. These four signals then act at short range and in concert to control epidermal differentiation at the single cell level across the segment. This example from Drosophila provides a paradigm for how organizers generate precise patterns, and ultimately different cell types, in a naïve field of cells.     

Arthropod-like expression patterns of engrailed and wingless in the annelid Platynereis dumerilii suggest a role in segment formation

The origin of animal segmentation, the periodic repetition of anatomical structures along the anteroposterior axis, is a long-standing issue that has been recently revived by comparative developmental genetics. In particular, a similar extensive morphological segmentation (or metamerism) is commonly recognized in annelids and arthropods. Mostly based on this supposedly homologous segmentation, these phyla have been united for a long time into the clade Articulata. However, recent phylogenetic analysis dismissed the Articulata and thus challenged the segmentation homology hypothesis. Here, we report the expression patterns of genes orthologous to the arthropod segmentation genes engrailed and wingless in the annelid Platynereis dumerilii. In Platynereis, engrailed and wingless are expressed in continuous ectodermal stripes on either side of the segmental boundary before, during, and after its formation; this expression pattern suggests that these genes are involved in segment formation. The striking similarities of engrailed and wingless expressions in Platynereis and arthropods may be due to evolutionary convergence or common heritage. In agreement with similarities in segment ontogeny and morphological organization in arthropods and annelids, we interpret our results as molecular evidence of a segmented ancestor of protostomes.