type six1 with activating or repressing co-factors (eya1 and groucho, respectively), we demonstrate that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains.Key words: Pre-placodal ectoderm, Neural crest, foxD3, zic2, sox2, sox3, keratin, dlx5, dlx6 Research article 5872 Woda et al., 2003). Zic genes are initially expressed throughout the neural plate in response to anti-BMP factors, and as they become restricted to its lateral border they initiate neural crest fates (Nakata et al., 1997;Nakata et al., 1998; Brewster et al., 1998;Kuo et al., 1998;Mizuseki et al., 1998). The roles that border genes play to specify the fates of the different ectodermal subdomains remain to be elucidated. Although placodes have long been recognized as important embryonic structures, their transient nature and the lack of specific molecular markers have made it difficult to study the mechanisms by which they form. Recently, however, markers of the PPE during the initial induction of the placodes have been identified in Xenopus. six1 is homologous to Drosophila sine oculis; it is characterized by a homeobox DNA-binding domain and a protein-protein interaction domain called the Six domain. It is initially expressed in a band surrounding the anterior neural plate and later in all neurogenic placodes (Pandur and Moody, 2000). eya1 is homologous to Drosophila eyes absent (eya); it functions as a co-factor for Six genes of the Six1/2 and Six4/5 subfamilies (Pignoni et al., 1997;Ohto et al., 1999;Ikeda et al., 2002) and is expressed in a pattern very similar to that of six1 (David et al., 2001). We have used these markers to demonstrate that gradients of both neural inducer and anteroposterior signals are required for proper PPE formation. Moreover, we show that six1 expression is required for the establishment of the PPE, and it promotes the PPE at the expense of the neural crest and epidermis by both activating and repressing target gene expression. Finally, we demonstrate that several genes expressed in the embryonic ectoderm mutually influence each other to define its distinct subdomains.
Materials and methods
Expression constructsThe full open-reading frames of Xenopus six1 and Drosophila groucho (Dgroucho; LD33829, Berkeley Drosophila Genome Project) were cloned into expression vectors (pDH105, pCS2+). To generate a chimeric transactivating six1 construct, the Six domain plus the homeodomain (SDHD; amino acids 9-183) was amplified by PCR and ligated upstream of the VP16 activation domain in pCS2VP16 (from M. Whitman). To generate a chimeric repressive six1 construct, the SDHD region was ligated downstream of the Engrailed repressor (EnR) domain in pCS2EnR (from D. Kessler).
RNA microinjectionTranscripts of six1 (400-600 pg), six1VP16 (100 ...