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signalling. Separation behaviour induced by Fz7 in FGF-stimulated animal cap cells was inhibited by pertussis toxin, and this block was released by coexpression of PKCa. This indicates that, as in the lower lip, the PKC branch of the Fz signalling cascade is involved, and that the animal cap system is a valid model for endogenous involuted mesoderm. Induction of separation behaviour is speci®c to Xenopus Fz7, as similar ectopic expression of Fz3 or Fz8 did not promote it.

The response to Fz7 expression is ligand-dependent. A Fz7 deletion construct lacking the extracellular amino terminus (hNFz7) induced a low level of separation behaviour, which could not be further increased by FGF. Coexpression of Fz7 with a secreted, putative ligand-binding domain (NXfz7-fun) or a con- struct lacking the intracellular carboxy-terminus (hCFz7) pre- vented the separation response in FGF-stimulated animal cap cells, demonstrating that these constructs could act as dominant- negative proteins.

Together, our results with dorsal lips and animal caps show that the PCP and the Fz/PKC branches are clearly separable pathways. In particular, the Fz/PKC pathway does not involve Dsh function (Fig. 4c). During gastrulation this pathway controls tissue separa- tion in the anterior mesoderm through the Xenopus Fz7 receptor, which is critical for normal development. This type of boundary formation is an important process in embryogenesis and its regula- tion is of general signi®cance. Further experiments will clarify whether Fz/PKC signalling is also involved in other tissue separation events in Xenopus as well as in other vertebrate and invertebrate species. w

Methods DNA constructs

Two hundred and one base pairs of the 5W UTR and the complete coding sequence of Xenopus Fz7 were ampli®ed by polymerase chain reaction (PCR) and cloned into BamHI/ClaI sites of the expression plasmid pCS2+EMT to generate Myc-tagged UTR Fz7. For the N-terminal deletion of Fz7 (hNFz7), a fragment from the coding region (residues 215±549) containing the seven transmembrane domains and the carboxyl tail was ampli®ed by PCR and cloned into the EcoRI/XbaI sites of the pCS2+ vector. A fragment coding for the signal peptide (residues 1±52) was joined with this clone (residues 215± 549) at the BamHI/EcoRI sites. Sense RNA for hNFz7 was transcribed from template that was linearized with Asp 718, using the SP6 promoter.

Sequence of Fz7Mo and transcription/translation of Fz7

Fz7Mo oligonucleotide (5W-CCAACAAGTGATCTCTGGACAG CAG-3W) and unspeci®c control morpholino oligonucleotide (Gene-Tools). The target sequence for Fz7Mo is not present in the 5W UTR and the coding region of the Fz8 complementary DNA. For in vitro transcription/translation of Fz7 we used the TNT Coupled Reticulocyte Lysate System (Promega) according to the manufacturer's instructions.

Whole-mount analysis and immuno ̄uorescence

Expression of brachyury and chordin was analysed in embryos at the gastrula stage as described7. Immunostaining and colorimetric detection using horseradish peroxidase has been described17. Protein extracts were separated on SDS±polyacrylamide gel electrophoresis (PAGE) gels, and EGFP-Myc and Fz7-Myc protein was detected with the 9E10 antibody as described17.

Embryos were ®xed in Dent's solution (methanol/DMSO; 4:1). Cryosections (10 mm) and immunodetection of the phosphorylated form of PKCa (anti-phospho-PKCa

Ser 657; Upstate Biotechnology) and Myc-epitope (9E10) was performed as described18.

Separation behaviour and dorsal lip elongation

In vitro separation assay was performed as described8. Convergent extension in dorsal lips was assayed as described2.

Received 26 June; accepted 5 September 2001.

1. Miller, J. R., Hocking, A. M., Brown, J. D. & Moon, R. T. Mechanism and function of signal transduction by the Wnt/b-catenin and Wnt/Ca2+ pathways. Oncogene 18, 7860±7872 (1999).

2. Wallingford, J. B. et al. Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405, 81± 85 (2000).

3. Sumanas, S., Strege, P., Heasman, J. & Ekker, S. C. The putative wnt receptor Xenopus frizzled-7 functions upstream of b-catenin in vertebrate dorsoventral mesoderm patterning. Development 127, 1981±1990 (2000).

4. Tada, M. & Smith, J. C. Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development 127, 2227± 2238 (2000).

5. Heisenberg, C. P. et al. Silberblick/Wnt11 mediates convergent extension movements during zebra®sh gastrulation. Nature 405, 76±81 (2000).

6. Djiane, A., Riou, J., Umbhauer, M., Boucaut, J. & Shi, D. Role of frizzled 7 in the regulation of convergent extension movements during gastrulation in Xenopus laevis. Development 127, 3091±3100 (2000).

7. Medina, A., Reintsch, W. & Steinbeisser, H. Xenopus frizzled 7 can act in canonical and non-canonical

Wnt signaling pathways: implications on early patterning and morphogenesis. Mech. Dev. 92, 227±

237 (2000).

8. Wacker, S., Grimm, K., Joos, T. & Winklbauer, R. Development and control of tissue separation at

gastrulation in Xenopus. Dev. Biol. 224, 428±439 (2000).

9. Winklbauer, R. & SchuÈrfeld, M. Vegetal rotation, a new gastrulation movement involved in the

internalization of the mesoderm and endoderm in Xenopus. Development 126, 3703±3713 (1999). 10. He, X., Saint-Jeannet, J. P., Woodgett, J. R., Varmus, H. E. & Dawid, I. B. Glycogen synthase kinase-3

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14. Slusarski, D. C., Corces, V. G. & Moon, R. T. Interaction of Wnt and a Frizzled homologue triggers

G-protein-linked phosphatidylinositol signalling. Nature 390, 410±413 (1997).

15. Sheldahl, L. C., Park, M., Malbon, C. C. & Moon, R. T. Protein kinase C is differentially stimulated by

Wnt and Frizzled homologs in a G-protein-dependent manner. Curr. Biol. 9, 695±698 (1999). 16. Otte, A. P. & Moon, R. T. Protein Kinase C isozymes have distinct roles in neural induction and

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17. MuÈ nchberg, S. R. & Steinbeisser, H. The Xenopus Ets transcription factor XER81 is a target of the FGF

signaling pathway. Mech. Dev. 80, 53±65 (1999).

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and plays a role in early morphogenesis. Development 125, 2687±2700 (1998).

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regulate its role in early embryogenesis. EMBO J. 19, 1010±1022 (2000).

21. Shi, D. L., Goisset, C. & Boucaut, J. C. Expression of Xfz3, a Xenopus frizzled family member, is

restricted to the early nervous system. Mech. Dev. 70, 35±47 (1998). Acknowledgements

We thank A. BraÈndli, I. Dawid, M. Hammerschmidt, P. Klein, M. KuÈhl, R. Moon,

W. Reintsch, U. RothbaÈcher, R. Rupp, D. L. Shi and S. Sokol for providing plasmids;

U. MuÈller for technical assistance; and S. Cramton for critically reading the manuscript. This work was supported by a pre-doctoral fellowship from the Deutscher Akademischer Austanschdienst (DAAD) to A.M. and by a Deutsche Forschungsgemeinschaft (DFG) grant to R.W.

Correspondence and requests for materials should be addressed to H.S. (e-mail:


Gene expression in

Pseudomonas aeruginosa bio®lms

Marvin Whiteley*, M. Gita Bangera2, Roger E. Bumgarner3,

Matthew R. Parsek§, Gail M. Teitzel§, Stephen Lory2 & E. P. Greenberg*

* Department of Microbiology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA

2 Department of Microbiology and Molecular Genetics, Harvard University, Boston, Massachusetts 02115, USA

3 Department of Microbiology, University of Washington, Seattle, Washington 98195, USA

§ Department of Civil Engineering, Northwestern University, Chicago, Illinois 60208, USA

.............................................................................................................................................. Bacteria often adopt a sessile bio®lm lifestyle that is resistant to antimicrobial treatment1±5. Opportunistic pathogenic bacteria like Pseudomonas aeruginosa can develop persistent infections1±3. To gain insights into the differences between free-living P. aeru- ginosa cells and those in bio®lms, and into the mechanisms underlying the resistance of bio®lms to antibiotics, we used DNA microarrays. Here we show that, despite the striking differ- ences in lifestyles, only about 1% of genes showed differential

860 © 2001 Macmillan Magazines Ltd NATURE | VOL 413 | 25 OCTOBER 2001 |

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