Pair-rule and gap gene mutants in the flour beetle Tribolium castaneum

 Early pattern formation in the Drosophila embryo occurs in a syncytial blastoderm where communication between nuclei is unimpeded by cell walls. During the development of other insects, similar gene expression patterns are generated in a cellular environment. In Tribolium, for instance, pair-rule stripes are transiently expressed near the posterior end of the growing germ band. To elucidate how pattern formation in such a situation deviates from that of Drosophila, functional data about the genes involved are essential. In a genetic screen for Tribolium mutants affecting the larval cuticle pattern, we isolated 4 mutants (from a total of 30) which disrupt segmentation in the thorax and abdomen. Two of these mutants display clear pair-rule phenotypes. This demonstrates that not only the expression, but also the function of pair-rule genes in this short-germ insect is in principle similar to Drosophila. The other two mutants appear to identify gap genes. They provide the first evidence for the involvement of gap genes in abdominal segmentation of short-germ embryos. However, significant differences between the phenotypes of these mutants and those of known Drosophila gap mutants exist which indicates that evolutionary changes occurred in either the regulation or action of these genes. Received: 8 May 1998 / Accepted: 17 June 1998     

Zebrafish segmentation and pair-rule patterning

Segmentation in the vertebrate embryo is evident within the paraxial mesoderm in the form of somites, which are repeated structures that give rise to the vertebrae and muscle of the trunk and tail. In the zebrafish, our genetic screen identified two groups of mutants that affect somite formation and pattern. Mutations of one class, the fss-type mutants, disrupt the formation of the anterior-posterior somite boundaries during somitogenesis. However, segmentation within the paraxial mesoderm is not completely eliminated in these mutants. Irregular somite boundaries form later during embryogenesis and, strikingly, the vertebrae are not fused. Here, we show that formation of the irregular somite boundaries in these mutants is dependent upon the activity of a second group of genes, the you-type genes, which include sonic you, the zebrafish homologue of the Drosophila segment polarity gene, sonic hedgehog. Further to characterize the defects caused by the fss-type mutations, we examined their effects on the expression of her1, a zebrafish homologue of the Drosophila pair-rule gene hairy. In wild-type embryos, her1 is expressed in a dynamic, repeating pattern, remarkably similar to that of its Drosophila and Tribolium counterparts, suggesting that a pair-rule mechanism also functions in the segmentation of the vertebrate paraxial mesoderm. We have found that the fss-type mutants have abnormal pair-rule patterning. Although a her1 mutant could not be identified, analysis of a double mutant that abolishes most her1 expression suggests that a her1 mutant may not display a pair-rule phenotype analogous to the hairy phenotype observed in Drosophila. Cumulatively, our data indicate that zebrafish homologues of both the Drosophila segment polarity genes and pair-rule genes are involved in segmenting the paraxial mesoderm. However, both the relationship between these two groups of genes within the genetic heirarchy governing segmentation and the precise roles that they play during segmentation likely differ significantly between the two organisms. Dev. Genet. 23:65–76, 1998. © 1998 Wiley-Liss, Inc.     

The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

Giant, Krüppel, and caudal act as gap genes with extensive roles in patterning the honeybee embryo

In Drosophila, gap genes translate positional information from gradients of maternal coordinate activity and act to position the periodic patterns of pair-rule gene stripes across broad domains of the embryo. In holometabolous insects, maternal coordinate genes are fast-evolving, the domains that gap genes specify often differ from their orthologues in Drosophila while the expression of pair-rule genes is more conserved. This implies that gap genes may buffer the fast-evolving maternal coordinate genes to give a more conserved pair-rule output. To test this idea, we have examined the function and expression of three honeybee orthologues of gap genes, Krüppel, caudal, and giant. In honeybees, where many Drosophila maternal coordinate genes are missing, these three gap genes have more extensive domains of expression and activity than in other insects. Unusually, honeybee caudal mRNA is initially localized to the anterior of the oocyte and embryo, yet it has no discernible function in that domain. We have also examined the influence of these three genes on the expression of honeybee even-skipped and a honeybee orthologue of engrailed and show that the way that these genes influence segmental patterning differs from Drosophila. We conclude that while the fundamental function of these gap genes is conserved in the honeybee, shifts in their expression and function have occurred, perhaps due to the apparently different maternal patterning systems in this insect.     

JAK-STAT signalling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning.     

Segmental expression of Pax3/7 and Engrailed homologs in tardigrade development

How morphological diversity arises through evolution of gene sequence is a major question in biology. In Drosophila, the genetic basis for body patterning and morphological segmentation has been studied intensively. It is clear that some of the genes in the Drosophila segmentation program are functioning similarly in certain other taxa, although many questions remain about when these gene functions arose and which taxa use these genes similarly to establish diverse body plans. Tardigrades are an outgroup to arthropods in the Ecdysozoa and, as such, can provide insight into how gene functions have evolved among the arthropods and their close relatives. We developed immunostaining methods for tardigrade embryos, and we used cross-reactive antibodies to investigate the expression of homologs of the pair-rule gene paired (Pax3/7) and the segment polarity gene engrailed in the tardigrade Hypsibius dujardini. We find that in H. dujardini embryos, Pax3/7 protein localizes not in a pair-rule pattern but in a segmentally iterated pattern, after the segments are established, in regions of the embryo where neurons later arise. Engrailed protein localizes in the posterior ectoderm of each segment before ectodermal segmentation is apparent. Together with previous results from others, our data support the conclusions that the pair-rule function of Pax3/7 is specific to the arthropods, that some of the ancient functions of Pax3/7 and Engrailed in ancestral bilaterians may have been in neurogenesis, and that Engrailed may have a function in establishing morphological boundaries between segments that is conserved at least among the Panarthropoda. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

<Emphasis Type="Italic">odd-skipped</Emphasis> homologs function during gut development in<Emphasis Type="Italic"> C. elegans</Emphasis>

Genes in the odd-skipped (odd) family encode a discrete subset of C2H2 zinc finger proteins that are widely distributed among metazoan phyla. Although the initial member (odd) was identified as a Drosophila pair-rule gene, various homologs are expressed within each of the three germ layers in complex patterns that suggest roles in many pathways beyond segmentation. To further investigate the evolutionary history and extant functions of genes in this family, we have initiated a characterization of two homologs, odd-1 and odd-2, identified in the genome of the nematode, Caenorhabditis elegans. Sequence comparisons with homologs from insects (Drosophila and Anopheles) and mammals suggest that two paralogs were present within an ancestral metazoan; additional insect paralogs and both extant mammalian genes likely resulted from gene duplications that occurred after the split between the arthropods and chordates. Analyses of gene function using RNAi indicate that odd-1 and odd-2 play essential and distinct roles during gut development. Specific expression of both genes in the developing intestine and other cells in the vicinity of the gut was shown using GFP-reporters. These results indicate primary functions for both genes that are most like those of the Drosophila paralogs bowel and drumstick, and support a model in which gut specification represents the ancestral role for genes in this family.Edited by C. Desplan     

Elimination of EVE protein by CALI in the short germ band insect Tribolium suggests a conserved pair-rule function for even skipped

The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments. Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. We conclude that the primary function of eve as a pair rule gene is conserved between the two species.     

Expression of <Emphasis Type="Italic">Pax group III</Emphasis> genes in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Pax group III genes are involved in a number of processes during insect segmentation. In Drosophila melanogaster, three genes, paired, gooseberry and gooseberry-neuro, regulate segmental patterning of the epidermis and nervous system. Paired acts as a pair-rule gene and gooseberry as a segment polarity gene. Studies of Pax group III genes in other insects have indicated that their expression is a good marker for understanding the underlying molecular mechanisms of segmentation. We have cloned three Pax group III genes from the honeybee (Apis mellifera) and examined their relationships to other insect Pax group III genes and their expression patterns during honeybee segmentation. The expression pattern of the honeybee homologue of paired is similar to that of paired in Drosophila, but its expression is modulated by anterior–posterior temporal patterning similar to the expression of Pax group III proteins in Tribolium. The expression of the other two Pax group III genes in the honeybee indicates that they also act in segmentation and nervous system development, as do these genes in other insects.     

Regulation of Krüppel expression in the anlage of the Malpighian tubules in the Drosophila embryo

The expression of most Drosophila segmentation genes is not limited to the early blastoderm stage, when the segmental anlagen are determined. Rather, these genes are often expressed in a variety of organs and tissues at later stages of development. In contrast to the early expression, little is known about the regulatory interactions that govern the later expression patterns. Among other tissues, the central gap gene Krüppel is expressed and required in the anlage of the Malpighian tubules at the posterior terminus of the embryo. We have studied the interaction of Krüppel with other terminal genes. The gap genes tailles and huckebein, which repress Krüppel in the central segmentation domain, activate Krüppel expression in the posterior Malpighian tubule domain. The opposite effect on the posterior Krüppel expression is achieved by the interposition of another factor, the homeotic gene fork head, which is not involved in the control of the central domain. In addition, Krüppel activates different genes in the Malpighian tubules than in the central domain. Thus, both the regulation and the function of Krüppel in the Malpighian tubules differ strikingly from its role in segmentation.     

Genetic regulation of engrailed and wingless in Tribolium segmentation and the evolution of pair-rule segmentation

In Drosophila, primary pair-rule genes establish the parasegmental boundaries and indirectly control the periodic expression of the segment polarity genes engrailed (en) and wingless (wg) via regulation of secondary pair-rule genes. Although orthologs of some Drosophila pair-rule genes are not required for proper segmentation in Tribolium, segmental expression of Tc-en and Tc-wg is conserved. To understand how these segment polarity genes are regulated, we examined the results of expressing one or two pair-rule genes in the absence of the other known pair-rule genes. Expression of one or both of the secondary pair-rule genes, Tc-sloppy-paired (Tc-slp) and Tc-paired (Tc-prd), activated Tc-wg in the absence of the primary pair-rule genes, Tc-even-skipped (Tc-eve), Tc-runt (Tc-run) and Tc-odd-skipped (Tc-odd). Tc-eve alone failed to activate Tc-wg or Tc-en, but in combination with Tc-run or Tc-prd activated Tc-en. These results, interpreted within the pair-rule gene expression patterns, suggest separate models for the genetic regulation of the juxtaposed expression of Tc-wg and Tc-en at odd- and even-numbered parasegmental boundaries, respectively. Conserved interactions between eve and prd at the anterior boundary of odd-numbered parasegments may reflect an ancestral segmentation mechanism that functioned in every segment prior to the evolution of pair-rule segmentation.