To better understand how and when BMP, Wnt and FGF signals specify the NPB in zebrafish, we knocked down Wnt and FGF signaling, and overexpressed BMP ligand at various developmental time points and monitored the expression of the NPB

INTRODUCTION A major theme in developmental biology is the need for cells to integrate multiple signals to make decisions about their fate. To achieve this, regulatory DNA must be structured so developmental control genes are expressed only when a cell is in the correct signaling environment. Furthermore, this regulation must be robust so development can proceed normally even when signaling levels fluctuate with environmental variability. Neural crest development is a classic example of how multiple signals are integrated to make cell fate decisions. Neural crest precursors are initially specified at the border between the neural plate and the epidermal ectoderm, a region called the neural plate border (NPB). This specification involves signals from the Wnt, FGF and BMP pathways. Wnt and FGF ligands are secreted by the neural plate and underlying mesoderm (Bang et al., 1999; Monsoro-Burq et al., 2003; García-Castro et al., 2002), while the epidermal ectoderm produces BMP ligands and the neural plate secretes BMP antagonists, resulting in a BMP signaling gradient along the dorsoventral axis (Marchant et al., 1998; Smith and Harland, 1992; Sasai et al., 1995; Endo et al., 2002). The NPB is specified in the region of the ectoderm that receives an intermediate dose of BMP signaling, as well as Wnts and/or FGFs (LaBonne and Bronner-Fraser, 1998; Streit and Stern, 1999; Villanueva et al., 2002). The combination of these signals leads to the expression of NPB specifier genes, including members the Pax3/7 and Zic families (Sato et al., 2005; Monsoro-Burq et al., 2003; Bang et al., 1999). Experiments in Xenopus indicate that the combination of Pax3/7 and Zic genes is sufficient to induce neural crest (Sato et al., 2005). How Wnt, FGF and BMP signaling specify the NPB is an area of active research. Much of what we know about this process comes from experiments in Xenopus. It is generally agreed that an intermediate level of BMP signaling is crucial for establishing the NPB and recent studies have shed light on the time dependence of the BMP and Wnt requirement (Steventon et al., 2009; Steventon and Mayor, 2012; Patthey et al., 2009). The relative importance of Wnts and FGFs in this process has been controversial. Studies in the past decade have fuelled a debate about whether FGF and Wnt signaling act in parallel to induce NPB genes (Monsoro-Burq et al., 2005) or whether FGF acts indirectly by activating the expression of Wnt ligands (Hong et al., 2008). The interactions of these pathways in NPB specification have been less well studied in zebrafish. The ease of transgenic zebrafish generation (Kawakami, 2004), coupled with excellent methods for temporally controlled gene overexpression make zebrafish an attractive model for studying NPB gene regulation. To better understand how and when BMP, Wnt and FGF signals specify the NPB in zebrafish, we knocked down Wnt and FGF signaling, and overexpressed BMP ligand at various developmental time points and monitored the expression of the NPB specifiers pax3a and zic3. We find that full pax3a NPB expression requires all three signals during mid-to-late gastrulation, whereas zic3 NPB expression only requires Wnt signaling and attenuation of BMP Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO 80309-0334, USA.