Brefeldin A or monensin inhibits the 3D organizer in gastropod, polyplacophoran, and scaphopod molluscs
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In molluscs, the 3D vegetal blastomere acts as a developmental signaling center, or organizer, and is required to establish bilateral symmetry in the embryo. 3D is similar to organizing centers in other metazoans, but detailed comparisons are difficult, in part because its organizing function is poorly understood. To elucidate 3D function in a standardized fashion, we used monensin and brefeldin A (BFA) to rapidly and reversibly interfere with protein processing and secretion, thereby inhibiting the signaling interactions that underlie its specification and patterning. In the gastropods, Patella vulgata and Lymnaea stagnalis, the polyplacophoran, Mopalia muscosa, and the scaphopod, Antalis entalis, treatments initiated before the organizer-dependent onset of bilateral cleavage resulted in radialization of subsequent development. In radialized P. vulgata, L. stagnalis, and M. muscosa, organizer specification was blocked, and embryos failed to make the transition to bilateral cleavage. In all four species, the subsequent body plan was radially symmetric and was similarly organized about a novel aboral–oral axis. Our results demonstrate that brefeldin A (BFA) and monensin can be used to inhibit 3D’s organizing function in a comparative fashion and that, at least in M. muscosa, the organizer-dependent developmental architecture of the embryo predicts subsequent patterns of morphogenetic movements in gastrulation and, ultimately, the layout of the adult body plan.
KeywordsDevelopmental signaling center Monensin Brefeldin A Spiral cleavage 3D organizer
We wish to thank A. Klerkx, L. Nederbragt, and A. van Loon for assistance in collecting and culturing P. vulgata, G. Zwaan for performing the experiments and initial analysis of L. stagnalis, L. Nederbragt, R. Rubicz, S. Santagata, and A. Wanninger for assistance in collecting, spawning, or fixing M. muscosa, and O. Lespinet for assistance in collecting and spawning A. entalis. We thank J. Priano and M. Rice for guidance and assistance with scanning electron micrographs, V. Foe, G. von Dassow, and others at the FHL NIH CCD for advice and the generous use of their confocal. We also thank D-H. Kuo, A. van Hook, K. O’Day, and D. Weisblat for their helpful comments and advice on earlier drafts of the manuscript. E. Gonzales wishes to thank M. Rice, B. Swalla, and D. Rokhsar for their guidance and support in the analysis and writing of this work. Finally, we greatly appreciate the kindness and support of staff and scientists at the University of Washington Friday Harbor Laboratories, the University of Paris VI Station Biologique de Roscoff, the Smithsonian Marine Station in Fort Pierce, the University of Göteberg Kristineberg Marine Station, the University of California at Berkeley Center for Integrative Genomics, and the University of Utrecht’s former Department of Experimental Embryology. E. Gonzales was supported by NSF and Smithsonian predoctoral fellowships, two European Union Large Scale Facility grants, a Seaver Foundation grant to B. Swalla, and the NIH Genomics Training Grant to D. Rokhsar and the Center for Comparative Genomics.
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