INTRODUCTION Neurulation is the process by which progenitors of the central nervous system are shaped, separated from and brought beneath the epidermis. The cellular basis of these movements have been investigated in amphibians (Jacobson, 1981; Jacobson and Gordon, 1976; Keller et al., 1992b), chicken (Schoenwolf and Smith, 1990b; Smith and Schoenwolf, 1997) and mice (Bush et al., 1990; Sausedo and Schoenwolf, 1994; Smith et al., 1994) but remains poorly understood. In the chick, which has been used extensively as a paradigm for vertebrate neurulation, a broad neural plate folds and the margins of the plate are raised and brought into apposition at the neural folds as cells take on stereotypical shapes. Analysis of serial sections and electron microscopy have identified cell movements and shape changes accompanying neural tube formation in chick (Schoenwolf and Alvarez, 1989; Schoenwolf and Smith, 1990a) where these cell behaviors (apical contraction and interkinetic nuclear migration) result in the formation of a ‘medial hinge’ overlying the notochord and a ‘dorsal lateral hinge’ near the prospective sulcus limitans, that together bring the neural folds into opposition. The epidermis then fuses, the neural ectoderm fuses and the neural crest is released from neural epithelium of the newly formed neural tube. In this manner, it is thought that the flat neural plate rolls into a tube with the lumenal face of the neural tube forming from the apical face of the neural plate. However, additional processes appear to be involved in chick neurulation. By following the rapid events at the start of neurulation, van Straaten and coworkers (1996) found that the lateral face of the two neural folds ‘zip’ into apposition, starting near the floorplate and proceeding dorsally. They found that the lumen of the neural tube nearly disappears after apposition and then re-opens to form the lumen after the neural 4547 Development 126, 4547-4556 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 DEV6419 We have characterized the cell movements and prospective cell identities as neural folds fuse during neural tube formation in Xenopus laevis. A newly developed whole- mount, two-color fluorescent RNA in situ hybridization method, visualized with confocal microscopy, shows that the dorsal neural tube gene xpax3 and the neural-crest- specific gene xslug are expressed far lateral to the medial site of neural fold fusion and that expression moves medially after fusion. To determine whether cell movements or dynamic changes in gene expression are responsible, we used low-light videomicroscopy followed by fluorescent in situ and confocal microscopy. These methods revealed that populations of prospective neural crest and dorsal neural tube cells near the lateral margin of the neural plate at the start of neurulation move to the dorsal midline using distinctive forms of motility. Before fold fusion, superficial neural cells apically contract, roll the neural plate into a trough and appear to pull the superficial epidermal cell sheet medially. After neural fold fusion, lateral deep neural cells move medially by radially intercalating between other neural cells using two types of motility. The neural crest cells migrate as individual cells toward the dorsal midline using medially directed monopolar protrusions. These movements combine the two lateral populations of neural crest into a single medial population that form the roof of the neural tube. The remaining cells of the dorsal neural tube extend protrusions both medially and laterally bringing about radial intercalation of deep and superficial cells to form a single-cell-layered, pseudostratified neural tube. While ours is the first description of medially directed cell migration during neural fold fusion and re-establishment of the neural tube, these complex cell behaviors may be involved during cavitation of the zebrafish neural keel and secondary neurulation in the posterior axis of chicken and mouse. Time-lapse sequences online: http://www.people.virginia.edu/~lad4x/tubeclosure.html and http://www.biologists.com/Development/movies/dev6419.html Key words: Tyramide, Fluorescent in situ hybridization, Whole- mount confocal microscopy, xk81, Epidermal cytokeratin, xslug, Neural crest, xpax3, n-tubulin, neuron, xash3, Mediolateral intercalation, Radial intercalation, Directed protrusive activity, Convergent extension SUMMARY Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension L. A. Davidson* and R. E. Keller Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903, USA *Author for correspondence (e-mail: lance_davidson@virginia.edu) Accepted 21 July; published on WWW 27 September 1999