letters to nature

4. Urick, R. J. Principles of Underwater Sound (McGraw Hill, New York, 1983). as , and , that are not closely related in 5. Henson, O. W. Jr The activity and function of the middle ear muscles in echolocating bats. J. Physiol. current phylogenies10. The complexity of generating a segmented (Lond.) 180, 871–887 (1965). 6. Suga, N. & Jen, P. H. S. Peripheral control of acoustic signals in the auditory system of echolocating might argue for a common origin of segmentation and a bats. J. Exp. Biol. 62, 277–331 (1975). common genetic programme1,2. In the past two decades, however, 7. Au, W. W. L. The Sonar of Dolphins (Springer, New York, 1993). genetic analyses in the fruitfly Drosophila3,4 and in various vertebrates 8. Au, W. W. L., Ford, J. K. B. & Allman, K. A. Echolocation signals of foraging killer whales (Orcinus 7–9,11–14 orca). J. Acoust. Soc. Am. 111, 2343–2344 (2002). such as mouse, chick and zebrafish suggest that fundamentally 9. Rasmussen, M. H., Miller, L. A. & Au, W. W. L. Source levels of clicks from free-ranging white beaked different mechanisms and gene networks are involved in dolphins (Lagenorhynchus albirostris Gray 1846) recorded in Icelandic waters. J. Acoust. Soc. Am. 111, and segmentation. segments are generated by a 1122–1125 (2002). successive spatial refinement along the anterior–posterior axis under 10. Ketten, D. R. in Hearing by Whales and Dolphins (eds Au, W. W. L., Popper, A. N. & Fay, R. R.) 43–108 3,4 (Springer, New York, 2000). the control of a hierarchical cascade of transcription factors .By 11. Heithaus, M. R. & Dill, L. M. in Encyclopedia of Marine Mammals (eds Perrin, W. F., Wursig, B. & contrast, the formation of vertebrate involves a molecular Thewissen, J. G. M.) 411–422 (Academic, San Diego, 2002). oscillator, known as the segmentation clock, that drives the periodic 12. Evans, W. W. & Powell, B. A. in Sonar Systems: Biology and Bionics (ed. Busnel, R. G.) 363–382 expression of genes in the presomitic mesoderm7,8,13,14. Each wave of (Laboratoire de Physiologie Acoustique, Jouy-en-Josas, 1967). 13. Morozov, B. P., Akapiam, A. E., Burdin, V., Zaitseva, K. A. & Solovkh, Y. A. Tracking frequency of the oscillatory results in the formation of one . location signals of dolphins as a function of distance to the target. Biofizika 17, 139–145 (1972). Notch and Delta genes are crucial components of this vertebrate 14. Cranford, T. in Animal Sonar: Processes and Performance (eds Nachtigall, P. E. & Moore, P. W. B.) segmentation clock8,15. 67–77 (Plenum, New York, 1988). One Notch gene and two Delta genes (Delta-1 and Delta-2)have 15. Cranford, T. in Hearing by Whales and Dolphins (eds Au, W. W. L., Popper, A. N. & Fay, R. R.) 109–155 16 (Springer, New York, 2000). been identified in the C. salei . Here we have analysed their 16. Ridgway, S. H. et al. Hearing and whistling in the deep sea: Depth influences whistle spectra but does role in segmentation. In the spider, segments are sequentially not attenuate hearing by white whales (Delphinapterus leucas)(Odontoceti cetaceia). J. Exp. Biol. 204, generated from a posterior growth zone17.TheNotch gene is 3829–3841 (2001). 17. Penner, R. H. in Animal Sonar: Processes and Performance (eds Nachtigall, P. E. & Moore, P. W. B.) expressed in this growth zone and resolves into segmental 707–713 (Plenum, New York, 1988). expression just before the segments form (Fig. 1a, e). There is a stronger accumulation of Notch transcripts at the posterior border Acknowledgements We thank the following individuals for their assistance in collecting data in of the newly formed stripes. Notch is expressed in the newly formed the field: D. Herzing (with S. frontalis), L. Miller and M. Rasmussen (with L. albirostris), and segments in the same register as the gene, which defines J. Ford and K. Allman (with O. orca). This work was supported, in part, by the Office of Naval Research. the parasegment boundary (ref. 6 and data not shown). The segmental expression of Notch fades in more anterior segments. Competing interests statement The authors declare that they have no competing financial The spider Delta-1 gene is expressed in a dynamic pattern in the interests. growth zone (Fig. 1c, d). Initially Delta-1 is expressed in a posterior

Correspondence and requests for materials should be addressed to W.A. ([email protected]).

...... Involvement of Notch and Delta genes in spider segmentation

Angelika Stollewerk*, Michael Schoppmeier* & Wim G. M. Damen

Institute for Genetics, Evolutionary Genetics, University of Cologne, Weyertal 121, D-50931 Ko¨ln, Germany * These authors contributed equally to this work ...... It is currently debated whether segmentation in different animal phylahasacommonoriginandsharesacommongenetic mechanism1,2. The apparent use of different genetic networks in arthropods and vertebrates has become a strong argument against a common origin of segmentation. Our knowledge of arthropod segmentation is based mainly on the Droso- phila, in which a hierarchical cascade of transcription factors controls segmentation3,4. The function of some of these genes seems to be conserved among arthropods, including spiders5,6, but not vertebrates1,6–8. The Notch pathway has a key role in vertebrate segmentation () but is not involved in Drosophila body segmentation1,7,9. Here we show that Notch and Delta genes are involved in segmentation of another arthropod, the spider Cupiennius salei. Expression patterns of Notch and Delta, coupled with RNA interference experiments, identify Figure 1 Expression of Notch, Delta-1 and Delta-2 genes during segmentation in the many similarities between spider segmentation and vertebrate spider C. salei. a–d, In situ hybridization shows the expression of Notch (a), Delta-2 (b) somitogenesis. Our data indicate that formation of the segments and Delta-1 (c, d) in the posterior growth zone of . The stripe corresponding to the in arthropods and vertebrates may have shared a genetic pro- just forming second opisthosomal segment is labelled O2. Arrowheads in c and d point to gramme in a common ancestor and that parts of this programme the posterior end of the proper. e, f, Posterior end of slightly older embryos have been lost in particular descendant lineages. stained for Notch (e)orDelta-1 (f). The most recently formed segment, the fifth Segmented body plans are found in different animal groups, such opisthosomal, is labelled O5. In all embryos anterior is to the left.

NATURE | VOL 423 | 19 JUNE 2003 | www.nature.com/nature © 2003 Nature Publishing Group 863 letters to nature domain (Fig. 1c), which resolves into a stripe while the posterior Table 1 Effect of Notch, Delta-1 and Delta-2 RNAi in spider embryos end becomes clear of Delta-1 transcripts (Fig. 1d). Delta-1 is then Total (n) Segmentation No effect Unspecific effects expressed again at the posterior end and a new stripe of Delta-1 ...... No injection 89 0 (0%) 76 (85%) 13 (15%) expression forms. In this way, the stripes of Delta-1 expression form GFP dsRNA 50 0 (0%) 44 (88%) 6 (12%) sequentially from the posterior end of the growth zone. This aspect Notch dsRNA 160 101 (63%) 38 (24%) 21 (13%) of Delta-1 expression resembles the expression pattern of the spider Delta-1 dsRNA 148 89 (60%) 37 (25%) 22 (15%) Delta-2 dsRNA 156 89 (57%) 48 (31%) 19 (12%) orthologues of the Drosophila pair rule genes hairy, even-skipped ...... 5 Shown are the number (and percentages) of embryos that show segmentation defects after the and runt . It remains to be determined whether the dynamic injection of dsRNA18. Control embryos were not injected (‘no injection’) or were injected with dsRNA expression of these genes is due to oscillation, as it is in vertebrates. targeted to the jellyfish GFP gene encoding green fluorescent protein. The more anterior Delta-1 stripes show a strong and sharp posterior border (Fig. 1f). Again, engrailed is expressed in the same register as the Delta-1 stripes (data not shown). The spider Delta-2 gene is dynamically expressed in stripes in the growth zone of the embryo expressed in the posterior growth zone and forms stripes towards (ref. 5 and Fig. 3a) in a pattern comparable to that of Delta-1 (Fig. 1c, anterior (Fig. 1b). But this expression is rather weak and the borders d). In a single-colour double in situ hybridization assay for both hairy of the stripes are fuzzy. and Delta-1, there is no more staining than in a single staining assay To analyse the functional role of Notch and Delta genes in spider for hairy, implying that the staining for Delta-1 expression must be segmentation, we used an approach based on RNA-mediated covered by the staining for hairy in this in situ assay (data not shown). interference (RNAi)18. Embryos injected with dsRNA targeted at Delta-1 is thus expressed in a subset of hairy-expressing cells during Notch, Delta-1 or Delta-2 genes showed severe defects in segment the dynamic phase in the growth zone. After the spider Notch or Delta patterning and formation of the segment borders (Fig. 2 and genes are silenced by RNAi, the hairy-expressing cells are no longer Table 1). The for the three genes seem to be identical. organized in stripes but are scattered throughout the growth zone Patterning defects include malformation of segments: the size, (Fig. 3b, c). Thus, Notch signalling is required to organize the shape and width of the segments are affected and vary. In addition, expression of hairy in stripes in the growth zone of the spider. the segment borders are irregular and not well defined, as can be The expression of vertebrate hairy/ of split (E(spl))-type seen by the disturbed stripes of engrailed gene expression, which no bHLH genes, such as zebrafish her1 and her7, mouse Hes1, Hes5 and longer form straight sharp borders (Fig. 2e–l) as compared with Hes7, and chicken c-hairy, in stripes in the presomitic mesoderm their straight sharp borders in control embryos (ref. 6 and Fig. 2a– also depends on Notch, and the expression of these bHLH genes in d). An additional defect found in the RNAi embryos is the apparent stripes is disturbed in embryos deficient for Notch signalling8,11–14,19– larger and malformed posterior growth zone; this seems to be the 21. By contrast, the Notch pathway does not regulate hairy in result of patterning or morphogenetic defects rather than additional Drosophila body segmentation, although it does in eye develop- cell divisions, as there are no detectable differences in cell prolifer- ment22 and it acts on other bHLH genes, such as the E(spl) genes, ation between RNAi and control embryos (see Supplementary during neurogenesis23. The disturbance of the hairy expression Information). pattern in Notch and Delta RNAi embryos suggests that these The spider basic helix–loop–helix (bHLH) gene hairy (Cs-h)is genes are incorporated into one genetic network for body segmen-

Figure 2 Segmentation defects in Notch and Delta RNAi embryos. Embryos were injected phenylindole dihydrochloride (DAPI)-stained DNA (b, d, f, h, j, l), respectively. The left with dsRNA targeted to GFP (control) (a–d), Notch (e–h) and Delta-1 (i–l). Embryos are images show the prosomal domain of the germ band, the right images show the posterior stained for the segmental marker engrailed6. For each embryo the bright field image is end of the opisthosoma. Arrowhead marks the fourth walking leg. In all embryos anterior is shown (a, c, e, g, i, k) together with the epi-fluorescence image of 4 0 ,6-diamidino-2- to the left.

864 © 2003 Nature Publishing Group NATURE | VOL 423 | 19 JUNE 2003 | www.nature.com/nature letters to nature

In situ hybridization and RNAi Whole-mount in situ hybridization with modifications for spider embryos was done as described6. RNAi was done as described18. Received 18 February; accepted 14 April 2003; doi:10.1038/nature01682.

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Genes Evol. 210, 329–336 (2000). the lineage leading to Drosophila and might be replaced by other 30. Wiellette, E. L. & McGinnis, W. Hox genes differentially regulate Serrate to generate segment-specific genes. Consequently, the observed similarities between spider and structures. Development 126, 1985–1995 (1999). vertebrates suggest that the formation of the segments in arthropods and vertebrates may have shared a genetic programme involving Supplementary Information accompanies the paper on www.nature.com/nature. Notch signalling in a common ancestor and that parts of this Acknowledgements We thank D, Tautz for support and discussion; S. Roth for comments on the programme have been lost in particular lineages, such as the one manuscript; T. Klein for discussion; A. Pozhitkov for help with the statistic analysis; and R. Janßen leading to the higher insects. A for care of the spider culture. This work was partially supported by the Deutsche Forschungsgemeinschaft. Methods Competing interests statement The authors declare that they have no competing financial Spiders interests. Colonies of the Central American wandering spider C. salei Keys (, Aranida, Ctenidae) are maintained in Cologne. We obtained embryos as described6. Correspondence and requests for materials should be addressed to W.D. ([email protected]).

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