Comparison of the Structure and Expression of Odd-Skipped and Two Related Genes That Encode a New Family of Zinc Finger Proteins in Drosophila

The odd-skipped (odd) gene, which was identified on the basis of a pair-rule segmentation phenotype in mutant embryos, is initially expressed in the Drosophila embryo in seven pair-rule stripes, but later exhibits a segment polarity-like pattern for which no phenotypic correlate is apparent. We have molecularly characterized two embryonically expressed odd-cognate genes, sob and bowel (bowl), that encode proteins with highly conserved C(2)H(2) zinc fingers. While the Sob and Bowl proteins each contain five tandem fingers, the Odd protein lacks a fifth (C-terminal) finger and is also less conserved among the four common fingers. Reminiscent of many segmentation gene paralogues, the closely linked odd and sob genes are expressed during embryogenesis in similar striped patterns; in contrast, the less-tightly linked bowl gene is expressed in a distinctly different pattern at the termini of the early embryo. Although our results indicate that odd and sob are more likely than bowl to share overlapping developmental roles, some functional divergence between the Odd and Sob proteins is suggested by the absence of homology outside the zinc fingers, and also by amino acid substitutions in the Odd zinc fingers at positions that appear to be constrained in Sob and Bowl.     

The segmentation cascade in the centipede Strigamia maritima: involvement of the Notch pathway and pair-rule gene homologues

The centipede Strigamia maritima forms all of its segments during embryogenesis. Trunk segments form sequentially from an apparently undifferentiated disk of cells at the posterior of the germ band. We have previously described periodic patterns of gene expression in this posterior disc that precede overt differentiation of segments, and suggested that a segmentation oscillator may be operating in the posterior disc. We now show that genes of the Notch signalling pathway, including the ligand Delta, and homologues of the Drosophila pair-rule genes even-skipped and hairy, show periodic expression in the posterior disc, consistent with their involvement in, or regulation by, such an oscillator. These genes are expressed in a pattern of apparently expanding concentric rings around the proctodeum, which become stripes at the base of the germ band where segments are emerging. In this transition zone, these primary stripes define a double segment periodicity: segmental stripes of engrailed expression, which mark the posterior of each segment, arise at two different phases of the primary pattern. Delta and even-skipped are also activated in secondary stripes that intercalate between primary stripes in this region, further defining the single segment repeat. These data, together with observations that Notch mediated signalling is required for segment pattern formation in other arthropods, suggest that the ancestral arthropod segmentation cascade may have involved a segmentation oscillator that utilised Notch signalling.     

Expression of pair-rule gene homologues in a chelicerate: early patterning of the two-spotted spider mite Tetranychus urticae

Embryo segmentation has been studied extensively in the fruit fly, DROSOPHILA: These studies have demonstrated that a mechanism acting with dual segment periodicity is required for correct patterning of the body plan in this insect, but the evolutionary origin of the mechanism, the pair-rule system, is unclear. We have examined the expression of the homologues of two Drosophila pair-rule genes, runt and paired (Pax Group III), in segmenting embryos of the two-spotted spider mite (Tetranychus urticae Koch). Spider mites are chelicerates, a group of arthropods that diverged from the lineage leading to Drosophila at least 520 million years ago. In T. urticae, the Pax Group III gene Tu-pax3/7 was expressed during patterning of the prosoma, but not the opisthosoma, in a series of stripes which appear first in even numbered segments, and then in odd numbered segments. The mite runt homologue (Tu-run) in contrast was expressed early in a circular domains that resolved into a segmental pattern. The expression patterns of both of these genes also indicated they are regulated very differently from their Drosophila homologues. The expression pattern of Tu-pax3/7 lends support to the possibility that a pair-rule patterning mechanism is active in the segmentation pathways of chelicerates.     

The nuclear receptor E75A has a novel pair-rule-like function in patterning the milkweed bug, Oncopeltus fasciatus

Genetic studies of the fruit fly Drosophila have revealed a hierarchy of segmentation genes (maternal, gap, pair-rule and HOX) that subdivide the syncytial blastoderm into sequentially finer-scale coordinates. Within this hierarchy, the pair-rule genes translate gradients of information into periodic stripes of expression. How pair-rule genes function during the progressive mode of segmentation seen in short and intermediate-germ insects is an ongoing question. Here we report that the nuclear receptor Of'E75A is expressed with double segment periodicity in the head and thorax. In the abdomen, Of'E75A is expressed in a unique pattern during posterior elongation, and briefly resembles a sequence that is typical of pair-rule genes. Depletion of Of'E75A mRNA caused loss of a subset of odd-numbered parasegments, as well as parasegment 6. Because these parasegments straddle segment boundaries, we observe fusions between adjacent segments. Finally, expression of Of'E75A in the blastoderm requires even-skipped, which is a gap gene in Oncopeltus. These data show that the function of Of'E75A during embryogenesis shares many properties with canonical pair-rule genes in other insects. They further suggest that parasegment specification may occur through irregular and episodic pair-rule-like activity.     

Pair-rule genes cooperate to activate en stripe 15 and refine its margins during germ band elongation in the D. melanogaster embryo

Patterns of gene expression have been well documented during embryogenesis for the Drosophila melanogaster trunk segments. The same is not the case for the terminal segments. Here, gene expression patterns are followed during embryogenesis in the caudal segments (A8-A10 and the anal plate), with special attention paid to the novel regulation of engrailed (en). Chosen for this study are the pair-rule genes even-skipped (eve), fushi tarazu (ftz), runt (run), hairy (h), paired (prd) and odd-skipped (odd), and the segment polarity gene (en). The results demonstrate a progressive and coupled translocation of gene expression distally for all genes studied, suggesting that the most posterior segments are determined later than trunk segments.     

Pax group III genes and the evolution of insect pair-rule patterning

Pair-rule genes were identified and named for their role in segmentation in embryos of the long germ insect Drosophila. Among short germ insects these genes exhibit variable expression patterns during segmentation and thus are likely to play divergent roles in this process. Understanding the details of this variation should shed light on the evolution of the genetic hierarchy responsible for segmentation in Drosophila and other insects. We have investigated the expression of homologs of the Drosophila Pax group III genes paired, gooseberry and gooseberry-neuro in short germ flour beetles and grasshoppers. During Drosophila embryogenesis, paired acts as one of several pair-rule genes that define the boundaries of future parasegments and segments, via the regulation of segment polarity genes such as gooseberry, which in turn regulates gooseberry-neuro, a gene expressed later in the developing nervous system. Using a crossreactive antibody, we show that the embryonic expression of Pax group III genes in both the flour beetle Tribolium and the grasshopper Schistocerca is remarkably similar to the pattern in Drosophila. We also show that two Pax group III genes, pairberry1 and pairberry2, are responsible for the observed protein pattern in grasshopper embryos. Both pairberry1 and pairberry2 are expressed in coincident stripes of a one-segment periodicity, in a manner reminiscent of Drosophila gooseberry and gooseberry-neuro. pairberry1, however, is also expressed in stripes of a two-segment periodicity before maturing into its segmental pattern. This early expression of pairberry1 is reminiscent of Drosophila paired and represents the first evidence for pair-rule patterning in short germ grasshoppers or any hemimetabolous insect.     

Double-segment defining role of even-skipped homologs along the evolution of insect pattern formation

Recent studies on insect patterning suggest that the genetic hierarchy may be roughly conserved in phylogenetically divergent species, but pair-rule genes may not function identically in all insects. In order to understand potential evolutionary changes in the role of the pair-rule genes, a Bombyx even-skipped homolog was cloned and its expression pattern during early embryogenesis studied. Eight stripes of Bombyx even-skipped were progressively expressed in an antero–posterior order. Later, these stripes disappeared anteriorly. Under this detection system, Bombyx even-skipped stripes clearly do not resolve into the corre sponding secondary stripes, an obvious difference from Drosophila and Tribolium . These results suggest that Bombyx even-skipped may serve a double-segment defining role and may determine the odd-numbered engrailed stripes.     

even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera)

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.     

Drosophila segmentation: supercomputer simulation of prepattern hierarchy

Spontaneous prepattern formation in a two level hierarchy of reaction-diffusion systems is simulated in three space co-ordinates and time, mimicking gap gene and primary pair-rule gene expression. The model rests on the idea of Turing systems of the second kind, in which one prepattern generates position dependent rate constants for a subsequent reaction-diffusion system. Maternal genes are assumed responsible for setting up gradients from the anterior and posterior ends, one of which is needed to stabilize a double period prepattern suggested to underly the read out of the gap genes. The resulting double period pattern in turn stabilizes the next prepattern in the hierarchy, which has a short wavelength with many characteristics of the stripes seen in actual primary pair-rule gene expression. Without such hierarchical stabilization, reaction-diffusion mechanisms yield highly patchy short wave length patterns, and thus unreliable stripes. The model yields seven stable stripes located in the middle of the embryo, with the potential for additional expression near the poles, as observed experimentally. The model does not rely on specific chemical reaction kinetics, rather the effect is general to many such kinetic schemes. This makes it robust to parameter changes, and it has good potential for adapting to size and shape changes as well. The study thus suggests that the crucial organizing principle in early Drosophila embryogenesis is based on global field mechanisms, not on particular local interactions.