The neural ectoderm (NE) forms on the dorsal side of the Xenopus embryo in response to factors secreted from the blastula Chordin- and Noggin-expressing (BCNE) signaling center and the Organizer (De Robertis and Kuroda,2004; Levine and Brivanlou,2007). By gastrula stages, the NE expresses a large number of transcription factors whose expression domains widely overlap and that coordinately promote a neural fate, expand the neural plate, and regulate the onset of neural differentiation (Fig. 1; Sasai,1998; Moody and Je,2002). These include: (1) foxD5, a forkhead/winged helix gene (Sölter et al.,1999; Fetka et al.,2000; Sullivan et al.,2001); (2) geminin (gem), which interacts with the SWI/SNF complex (Kroll et al.,1998; Seo and Kroll,2006); (3) the high-mobility group (HMG)-box genes sox2, sox3, soxD, and sox11 (Uwanogho et al.,1995; Mizuseki et al.,1998a,b; Kishi et al.,2000; Hyodo-Miura et al.,2002; Wegner and Stolt,2005; Dee et al.,2008); (4) the zinc-finger genes zic1, zic2, and zic3 (Brewster et al.,1998; Kuo et al.,1998; Mizuseki et al.,1998a; Nakata et al.,1997,1998); and (5) the Iroquois genes Xiro1, Xiro2, and Xiro3 (Bellefroid et al.,1998; G&oaucte;mez-Skarmeta et al.,1998,2001). However, the interactions between these several genes during the establishment of the neural plate are largely unknown. Recently, we proposed a regulatory network, based on loss- and gain-of-function assays, that places foxD5 in an upstream position. It appears to directly regulate gem, sox11, and zic2, which together regulate the expression patterns of the other NE transcription factors (Fig. 1; Yan et al.,2009). This model, however, does not account for signaling pathways that might mediate the interactions.
Notch signaling, which is highly conserved from Drosophila to humans, plays essential roles in many processes during development including neurogenesis (Artavanis-Tsakonas et al.,1999; Lai,2004; Bray,2006; Chitnis,2007). When the Notch transmembrane receptor on the cell surface is bound by DSL (Delta/Serrate/Lag-2) ligands on the neighboring cells, the Notch intracellular domain (NICD) is cleaved, released into the cytoplasm, and translocated into the nucleus. There, it combines with its transcriptional cofactor CSL (CBF1/ Suppressor of Hairless/ Lag-1) to activate downstream target genes, mainly members of the HES (Hairy/ Enhancer of Split) family of basic helix-loop-helix (bHLH) transcriptional regulators (Mumm and Kopan,2000; Weinmaster,2000). During early vertebrate development, Notch is expressed broadly throughout the neural plate, and its signaling pathway plays an essential role in maintaining the neural progenitor state and regulating the diversification of cell fate (Yoon and Gaiano,2005; Louvi and Artavanis-Tsakonas,2006; Chitnis,2007). The classical view is that increasing Notch signaling within a cell up-regulates HES genes that subsequently repress neural differentiation bHLH genes (such as ngn and neuroD) and Notch DSL ligand genes.
The preponderance of work on Notch signaling during neural development focuses on the onset of neurogenesis, when progenitor cells become post-mitotic and acquire a particular neural cell fate. For example, at Xenopus and zebrafish neurula stages, increasing Notch signaling by expressing a constitutively activating form of Notch1 prevents expression of the neural differentiation genes and formation of primary neurons, whereas decreasing Notch signaling by interfering with Delta promotes neurogenesis (Chitnis et al.,1995; Itoh et al.,2003). However, Notch signaling also affects the size of the NE during Xenopus gastrulation (Coffman et al.,1993). Because Notch is expressed throughout the neural plate at a similar time as the NE transcription factors (Coffman et al.,1990,1993), and several of those factors both expand the neural plate and inhibit neural differentiation (Fig. 1), we investigated whether Notch signaling has a role in regulating the expression of these genes. We show that: (1) foxD5 acts upstream of notch1; (2) increasing Notch signaling phenocopies the effects of foxD5 on gem, sox11, sox2, sox3, and zic2, but it does not alter the expression of NE genes that are inhibited by foxD5 (soxD, zic1, zic3, Xiro1-3); and (3) Notch signaling is required for the foxD5-mediated expansion of sox2, sox3, and sox11 expression domains but not for the up-regulation of gem and zic2. These studies demonstrate that: (1) Notch signaling acts downstream of foxD5 to promote the expression of a subset of NE transcription factors; and (2) the Notch signaling pathway and the foxD5 transcriptional pathway together maintain the neural plate in an undifferentiated state.