Expression of pair rule gene orthologs in the blastoderm of a myriapod: evidence for pair rule-like mechanisms?

ABSTRACT: BACKGROUND: A hallmark of Drosophila segmentation is the stepwise subdivision of the body into smaller and smaller units, and finally into the segments. This is achieved by the function of the well-understood segmentation gene cascade. The first molecular sign of a segmented body appears with the action of the pair rule genes, which are expressed as transversal stripes in alternating segments. Drosophila development, however, is derived, and in most other arthropods only the anterior body is patterned (almost) simultaneously from a pre-existing field of cells; posterior segments are added sequentially from a posterior segment addition zone. A long-standing question is to what extent segmentation mechanisms known from Drosophila may be conserved in short-germ arthropods. Despite the derived developmental modes, it appears more likely that conserved mechanisms can be found in anterior patterning. RESULTS: Expression analysis of pair rule gene orthologs in the blastoderm of the pill millipede Glomeris marginata (Myriapoda: Diplopoda) suggests that these genes are generally involved in segmenting the anterior embryo. We find that the Glomeris pairberry-1 (pby-1) gene is expressed in a pair rule pattern that is also found in insects and a chelicerate, the mite Tetraynchus urticae. Other Glomeris pair rule gene orthologs are expressed in double segment wide domains in the blastoderm, which at subsequent stages split into two stripes in adjacent segments. CONCLUSIONS: The expression patterns of the millipede pair rule gene orthologs resemble pair rule patterning in Drosophila and other insects, and thus represent evidence for the presence of an ancestral pair rule-like mechanism in myriapods. We discuss the possibilities that blastoderm patterning may be conserved in long-germ and short-germ arthropods, and that a posterior double segmental mechanism may be present in short-germ arthropods.     

Maternal-effect genes that alter the fate map of the Drosophila blastoderm embryo

The pattern of segmentation in the Drosophila embryo is controlled by at least 25 zygotically active genes and at least 20 maternally active genes. We have examined the pattern of expression of the protein product of the zygotically active segmentation gene fushi tarazu (ftz) at the cellular blastoderm stage in progeny of mutant females homozygous for each of six maternal-effect segmentation genes to observe the early effects of the maternal-effect genes on zygotic gene expression. The genes included exuperantia (a member of the anterior class of maternal-effect segmentation genes); staufen and vasa (members of the posterior class); and torso, trunk, and fs(1)N (members of the terminal class). Mutations in the genes caused a disruption of the normal pattern of ftz stripes in regions of the embryo where gene activity is known to be required. The ftz stripes provide a marker for segmental determination at the cellular blastoderm stage, making it possible to correlate aberrant patterns of ftz protein with defects in cuticle morphology at the end of embryogenesis. ftz protein expression in progeny of females mutant for combinations of the above genes was also examined. The changes in the ftz pattern in progeny of females doubly mutant for genes of the anterior and terminal classes or of the posterior and terminal classes can largely be understood as the result of the additive effects of the single mutations. In contrast, clearly nonadditive effects on the ftz pattern were seen when a mutation in a gene of the anterior class (exuperantia) was combined with mutations in posterior class genes.     

Determinants of Drosophila fushi tarazu mRNA instability.

The fushi tarazu gene is essential for the establishment of the Drosophila embryonic body plan. When first expressed in early embryogenesis, fushi tarazu mRNA is uniformly distributed over most of the embryo. Subsequently, fushi tarazu mRNA expression rapidly evolves into a pattern of seven stripes that encircle the embryo. The instability of fushi tarazu mRNA is probably crucial for attaining this localized pattern of expression. mRNA stability in transgenic embryos was measured by a new method that does not use drugs or external interference. Experiments using hybrid genes that fuse fushi tarazu sequences to those of the stable ribosomal protein A1 mRNA provide evidence for at least two destabilizing elements in the fushi tarazu mRNA, one located within the 5' one-third of the mRNA and the other near the 3' end (termed FIE3 for ftz instability element 3'). The FIE3 lies within a 201-nucleotide sequence just upstream of the polyadenylation signal and can act autonomously to destabilize a heterologous mRNA. Further deletion constructs identified an essential 68-nucleotide element within the FIE3. Lack of homology between this element and other previously identified destabilization sequences suggests that FIE3 contains a novel RNA destabilization element.     

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.     

Pattern formation in the Drosophila embryo: allocation of cells to parasegments by even-skipped and fushi tarazu

The first sign of metamerization in the Drosophila embryo is the striped expression of pair-rule genes such as fushi tarazu (ftz) and even-skipped (eve). Here we describe, at cellular resolution, the development of ftz and eve protein stripes in staged Drosophila embryos. They appear gradually, during the syncytial blastoderm stage and soon become asymmetric, the anterior margins of the stripes being sharply demarcated while the posterior borders are undefined. By the beginning of germ band elongation, the eve and ftz stripes have narrowed and become very intense at their anterior margins. The development of these stripes in hairy-, runt-, eve-, ftz- and engrailed- embryos is illustrated. In eve- embryos, the ftz stripes remain symmetric and lack sharp borders. Our results support the hypothesis (Lawrence et al. Nature 328, 440-442, 1987) that individual cells are allocated to parasegments with respect to the anterior margins of the eve and ftz stripes.     

Zygotically active genes that affect the spatial expression of the fushi tarazu segmentation gene during early Drosophila embryogenesis

The establishment of the segmental body pattern of Drosophila requires the coordinated functions of three classes of zygotically active genes early in development. We have examined the effects of mutations in these genes on the spatial expression of the fushi tarazu (ftz) pair-rule segmentation gene. Mutations in four gap loci and in three pair-rule loci dramatically affect the initial pattern of transverse stripes of ftz-containing nuclei. Five other pair-rule genes and several other loci that affect the larval cuticular pattern do not detectably affect ftz expression. No simple regulatory relationships can be deduced. Rather, expression of the ftz gene depends upon the interactions among the different segmentation genes active at each position along the anterior-posterior axis of the early embryo.     

Control of the initiation of homeotic gene expression by the gap genes giant and tailless in Drosophila

The process of segmentation in Drosophila is controlled by both maternal and zygotic genes. Members of the gap class of segmentation genes play a key role in this process by interpreting maternal information and controlling the expression of pair-rule and homeotic genes. We have analyzed the pattern of expression of a variety of homeotic, pair-rule, and gap genes in tailless and giant gap mutants. tailless acts in two domains, one anterodorsal and one posterior. In its anterior domain tailless exerts a repressive effect on the expression of fushi tarazu, hunchback, and Deformed. In its posterior domain of action, tailless is responsible for the establishment of Abdominal-B expression and demarcating the posterior boundary of the initial domain of expression of Ultrabithorax. giant is an early zygotic regulator of the gap gene hunchback: in giant- embryos, alterations in the anterior domain of hunchback expression are visible by the beginning of cycle 14. giant also regulates the establishment of the expression patterns of Antennapedia and Abdominal-B. In particular, giant is the factor that controls the anterior limit of early Antennapedia expression.     

Regulation and function of the Drosophila segmentation gene fushi tarazu

The Drosophila segmentation gene fushi tarazu (ftz) is expressed in a pattern of seven stripes at the blastoderm stage. Two cis-acting control elements are required for this expression: the zebra element, which confers the striped pattern by mediating the effects of a subset of segmentation genes; and the upstream element, an enhancer element requiring ftz+ activity for its action. Fusion of the upstream element to a basal promoter results in activation of the heterologous promoter in a ftz-dependent striped pattern, supporting the idea that ftz regulates itself by acting through its enhancer. The upstream element can also confer expression patterns similar to that of the homeotic gene Antennapedia, suggesting that a similar element may play a role in the activation of Antennapedia.     

Drosophila fushi tarazu. a gene on the border of homeotic function

BACKGROUND: Hox genes specify cell fate and regional identity during animal development. These genes are present in evolutionarily conserved clusters thought to have arisen by gene duplication and divergence. Most members of the Drosophila Hox complex (HOM-C) have homeotic functions. However, a small number of HOM-C genes, such as the segmentation gene fushi tarazu (ftz), have nonhomeotic functions. If these genes arose from a homeotic ancestor, their functional properties must have changed significantly during the evolution of modern Drosophila. RESULTS: Here, we have asked how Drosophila ftz evolved from an ancestral homeotic gene to obtain a novel function in segmentation. We expressed Ftz proteins at various developmental stages to assess their potential to regulate segmentation and to generate homeotic transformations. Drosophila Ftz protein has lost the inherent ability to mediate homeosis and functions exclusively in segmentation pathways. In contrast, Ftz from the primitive insect Tribolium (Tc-Ftz) has retained homeotic potential, generating homeotic transformations in larvae and adults and retaining the ability to repress homothorax, a hallmark of homeotic genes. Similarly, Schistocerca Ftz (Sg-Ftz) caused homeotic transformations of antenna toward leg. Primitive Ftz orthologs have moderate segmentation potential, reflected by weak interactions with the segmentation-specific cofactor Ftz-F1. Thus, Ftz orthologs represent evolutionary intermediates that have weak segmentation potential but retain the ability to act as homeotic genes. CONCLUSIONS: ftz evolved from an ancestral homeotic gene as a result of changes in both regulation of expression and specific alterations in the protein-coding region. Studies of ftz orthologs from primitive insects have provided a "snap-shot" view of the progressive evolution of a Hox protein as it took on segmentation function and lost homeotic potential. We propose that the specialization of Drosophila Ftz for segmentation resulted from loss and gain of specific domains that mediate interactions with distinct cofactors.     

Segmentally repeated pattern of expression of a cell surface glycoprotein in Drosophila embryos

We report here that a previously described cell surface antigen (Brower, Smith & Wilcox, 1980) is expressed in a segmentally repeating pattern of stripes in the epidermis and nervous system of segmented Drosophila embryos. We also report that the antigenic activity is found on two closely related cell surface glycoproteins. The pattern of expression of this antigen is reminiscent of the expression of some segmentation genes and is affected by mutation of at least two of these genes, fushi tarazu and paired. Thus these glycoproteins are candidates for cell surface molecules involved in carrying out the patterning processes controlled by segmentation genes.     

Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus

In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, a long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, we have investigated the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb). Here, we have focused on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, we found the following: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of our results with the results for other derived species revealed that the canonical hb function may have evolved from the non-canonical hb functions during evolution.     

Expression patterns of the rogue Hox genes Hox3/zen and fushi tarazu in the apterygote insect Thermobia domestica

Many embryonic patterning genes are remarkably conserved between vertebrates and invertebrates, and the Hox genes are paradigmatic examples of this conservation. Yet even Hox genes can change dramatically in evolution. Two genes in particular--Hox3 and fushi tarazu--lost their ancestral roles as homeotic genes and play very different developmental roles in the fruit fly Drosophila melanogaster. The Drosophila Hox3 homologs zerknullt and bicoid act in extraembryonic tissues and in establishment of the anteroposterior axis, respectively, whereas fushi tarazu acts in segmentation and neurogenesis. It would be valuable to know what mechanisms allowed Hox3 and ftz to abandon their ancestral roles as homeotic genes and take on new roles. To explore the evolutionary transition of these genes, we analyzed their expression in a primitive insect, the firebrat Thermobia domestica. The expression patterns seem to represent a stage of evolution intermediate between the ancestral state seen in basal arthropods and the derived expression patterns in Drosophila. These expression data help us to narrow the period in which the gene transitions took place. Hox3 appears to have evolved directly into zen within the insects, whereas ftz seems to have adopted the expression patterns of a segmentation and neurogenesis gene earlier in the mandibulate arthropods.     

Evolution: hox genes and the cellared wine principle

Two Drosophila Hox genes involved in segmentation, fushi tarazu and bicoid, appear to have acquired these roles by functional divergence from classical homeotic genes. Recent results indicate how genes with critical functions in development can evolve completely different functions among species.     

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.     

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.