Non-periodic cues generate seven ftz stripes in the Drosophila embryo

We have examined the expression pattern of the segmentation gene fushi tarazu (ftz) by in situ hybridization to whole mount embryos using digoxygenin labeled probes. This method has revealed previously undetected stages in the development of the ftz RNA pattern. The ftz stripes arise individually in a distinct, non-linear order along the anterior-posterior axis of the embryo. In addition, the stripes develop differentially along the dorsal-ventral axis; most stripes emerge on the ventral side and then gradually spread dorsally until they surround the entire circumference of the embryo. The order of appearance of ftz stripes is not inversely correlated with the order of appearance of hairy (h) stripes as would be expected if ftz stripes were generated by h repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, eve or runt, with normal expression patterns decaying only after cellularization. Thus, the so called primary pair-rule genes are involved in the refinement rather than establishment of the ftz stripes. The contribution of cis-acting regulatory elements to the ftz pattern was examined. The zebra and upstream elements interact to generate seven correctly positioned stripes at the end of cellularization. However, stripe establishement is not correctly mimicked by any ftz/lac fusion gene: stripes arise in an order drastically different from the endogenous ftz gene suggesting the existence of ftz regulatory elements outside the 10-kb region examined to date. These observations suggest that the ftz pattern is directed by at least two independent regulatory systems: first, stripe establishment is directed by regionally distributed factors that act differentially in individual stripes along both anterior-posterior and dorsal-ventral axes of the egg and, second, stripe refinement and maintenance are mediated by pair-rule gene products that interact with previously identified ftz regulatory elements. This multi-level regulation provides a back-up system that ensures the development of seven stripes in the blastoderm.     

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.     

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.     

Pax3/7 genes reveal conservation and divergence in the arthropod segmentation hierarchy

Several features of Pax3/7 gene expression are shared among distantly related insects, including pair-rule, segment polarity, and neural patterns. Recent data from arachnids imply that roles in segmentation and neurogenesis are likely to be played by Pax3/7 genes in all arthropods. To further investigate Pax3/7 genes in non-insect arthropods, we isolated two monoclonal antibodies that recognize the products of Pax3/7 genes in a wide range of taxa, allowing us to quickly survey Pax3/7 expression in all four major arthropod groups. Epitope analysis reveals that these antibodies react to a small subset of Paired-class homeodomains, which includes the products of all known Pax3/7 genes. Using these antibodies, we find that Pax3/7 genes in crustaceans are expressed in an early broad and, in one case, dynamic domain followed by segmental stripes, while myriapods and chelicerates exhibit segmental stripes that form early in the posterior-most part of the germ band. This suggests that Pax3/7 genes acquired their role in segmentation deep within, or perhaps prior to, the arthropod lineage. However, we do not detect evidence of pair-rule patterning in either myriapods or chelicerates, suggesting that the early pair-rule expression pattern of Pax3/7 genes in insects may have been acquired within the crustacean-hexapod lineage.     

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.     

The origin and evolution of segmentation

Arthropods, annelids and chordates all possess segments. It remains unclear, however, whether the segments of these animals evolved independently or instead were derived from a common ancestor. Considering this question involves examining not only the similarities and differences in the process of segmentation between these phyla, but also how this process varies within phyla, where the homology of segments is generally accepted. This article reviews what is known about the segmentation process and considers various proposals to explain its evolution.