INTRODUCTION Within the developing vertebrate head, the organisation of skeletal structures and peripheral nerves depends on the orchestrated migration of pluripotent cranial neural crest cells

Within the developing vertebrate head, the organisation of skeletal structures and peripheral nerves depends on the orchestrated migration of pluripotent cranial neural crest cells (NCCs) through the cranial mesenchyme (Bronner-Fraser, 1995; Le Douarin, 1982). Cranial NCCs are generated throughout the dorsal hindbrain (Sechrist et al., 1993) but their emigration into the adjacent mesenchyme is patterned from the outset into three distinct streams (Lumsden et al., 1991) that mirror the transient segmentation of the neural tube into lineage-restricted units, called rhombomeres (r) (Lumsden and Krumlauf, 1996; Trainor and Krumlauf, 2000a). Thus, only at the level of r1+r2, r4 and r6 do NCCs migrate into the cranial mesenchyme. Fewer NCCs leave r3 and r5, owing to increased cell death (Graham et al., 1996), and they do not exit laterally, directly into the mesenchyme, but instead migrate rostrally and caudally along the dorsal surface of the neural tube to join NCCs in the neighbouring even-numbered rhombomeres (Kulesa and Fraser, 1998; Sechrist et al., 1993). After leaving the neural tube, NCCs still avoid entering r3 and r5 mesenchyme, suggesting that the cranial mesenchyme is segmented molecularly, as no anatomical segmentation has been observed (Freund et al., 1996). The idea that NCC ‘exclusion zones’ exist within the mesenchyme adjacent to r3 and r5 is supported by experiments demonstrating that quail cranial NCCs grafted into chick cranial mesenchyme migrate only into host mesenchyme adjacent to even-numbered rhombomeres (Farlie et al., 1999). The generation of NCC ‘exclusion zones’ may depend on cues from r3 and r5 neuroepithelium and/or on interactions among the NCCs themselves. In ovo grafting experiments to alter relative positions of rhombomeres and mesenchyme (Kuratani and Eichele, 1993; Sechrist et al., 1994) provide evidence for cues from neuroepithelium, while dorsal hindbrain ablation studies (Kulesa et al., 2000) suggest a role for NCC cell-cell interactions. Although the origins, migration pathways and destinations of cranial NCCs are well documented (Koentges and Lumsden, 1996; Lumsden et al., 1991), relatively few molecules have been found that influence their pathfinding. These include specific ephrins and their Eph receptors (Adams et al., 2001; Helbling et al., 1998; Holder and Klein, 1999; Robinson et al., 1997; Smith et al., 1997), Collapsin 1 (Eickholt et al., 1999), FGF2 (Kubota and Ito, 2000) and an uncharacterised chemoattractant released from the otic vesicle (Sechrist et al., 1994). Where studied in vivo, these factors appear to be involved in maintaining the segregation of NCC streams at the level of the branchial arches. However, the cues that enforce NCC segregation further dorsally, adjacent to the neuroepithelium, remain unknown. In this study, we show that cues from r3 neuroepithelium and the overlying surface ectoderm are required to exclude subpopulations of NCCs from r3-adjacent mesenchyme. 1095 Development 129, 1095-1105 (2002) Printed in Great Britain © The Company of Biologists Limited 2002 DEV2728