JCB_201612169 1..16

Neural crest cells are multipotent and highly invasive, yet they follow stereotypical migratory pathways. In the vertebrate head, neural crest cells move collectively in discrete streams to reach precise targets. In the hindbrain region, neural crest cells exit the neural tube and are sculpted into three distinct streams adjacent to rhombomeres 2 (r2), r4, and r6 (Trainor and Krumlauf, 2000) such that neural crest cell exclusion zones form adjacent to r3 and r5 (Farlie et al., 1999). Aberrant cranial neural crest cell migration may result from respecification of rhombomere segment identity (Trainor and Krumlauf, 2000) or disruption of signals within the hindbrain (Kulesa and Gammill, 2010). Thus, the proper anterior-to-posterior formation of neural crest– derived tissues of the face and neck (Le Douarin and Kalcheim, 1999) crucially relies on neural crest cell migration. A small number of signaling molecules have been identified as expressed in the hindbrain region and shown to control initial neural crest cell trajectories into the paraxial mesoderm. These include members of the Eph/ephrin family (Smith et al., 1997; Mellott and Burke, 2008), ErbB4/neuregulin (Dixon and Lumsden, 1999; Golding et al., 2000, 2002, 2004), versican (Landolt et al., 1995; Perris et al., 1996; Kerr and Newgreen, 1997; Perissinotto et al., 2000; Dutt et al., 2006a,b; Szabó et al., 2016), and neuropilin2/semaphorin3F (Eickholt et al., 1999; Osborne et al., 2005; Yu and Moens, 2005; Gammill et al., 2007). Several of these members are secreted factors that are thought to diffuse from the dorsal hindbrain into the local paraxial mesoderm (for example, ErbB4/neuregulin and neuropilin2/semaphorin3F); however, it is largely unknown what signals exist within the paraxial mesoderm that inhibit uncontrolled neural crest cell invasion. Although some neural crest cell inhibitory signals have been identified in several different model organisms, there has not been a systematic approach to isolate tissue and compare gene expression within cranial neural crest cell–free zones that would identify and test the function of novel inhibitory factors and unify existing hypotheses. Paralleling the invasive ability of the embryonic neural crest, and ancestrally related to this process, melanoma is among the most aggressive human metastatic cancers (Kulesa et al., 2013). However, efforts to constrain melanoma cell invasion have yielded minimal results, making this disease often fatal (Flaherty et al., 2012; Holderfield et al., 2014; Millet et al., 2017). Interestingly, human metastatic melanoma cells transplanted into the chick embryonic neural crest microenvironment Neural crest cells are both highly migratory and significant to vertebrate organogenesis. However, the signals that regulate neural crest cell migration remain unclear. In this study, we test the function of differential screening-selected gene aberrant in neuroblastoma (DAN), a bone morphogenetic protein (BMP) antagonist we detected by analysis of the chick cranial mesoderm. Our analysis shows that, before neural crest cell exit from the hindbrain, DAN is expressed in the mesoderm, and then it becomes absent along cell migratory pathways. Cranial neural crest and metastatic melanoma cells avoid DAN protein stripes in vitro. Addition of DAN reduces the speed of migrating cells in vivo and in vitro, respectively. In vivo loss of function of DAN results in enhanced neural crest cell migration by increasing speed and directionality. Computer model simulations support the hypothesis that DAN restrains cell migration by regulating cell speed. Collectively, our results identify DAN as a novel factor that inhibits uncontrolled neural crest and metastatic melanoma invasion and promotes collective migration in a manner consistent with the inhibition of BMP signaling. DAN (NBL1) promotes collective neural crest migration by restraining uncontrolled invasion