The Function of Sia and Twn Conserved Domains 124

Sia/Twn are essential for the formation of the Spemann organizer (Bae et al., 2011), yet no vertebrate orthologs outside of amphibians have been identified. One possibility is that Sia/Twn carry out amphibian specific aspects of development, and may

be unnecessary in organizer formation in other species. Alternatively, other proteins may function as Sia/Twn, but the sequence of the protein could have diverged significantly. If this is the case, elucidation of functional domains of Sia/Twn could help identify proteins that function in a similar role in other species. Because of the high level of conservation of the Sia/Twn binding site within the Gsc proximal element in other vertebrates (Bae et al., 2011), we would predict that a paired type homeodomain protein, like Sia/Twn, would regulate some aspect of Gsc expression in other vertebrates. In the sequence of the homeodomain, Sia and Twn are most highly similar to the Mix family of paired-type homeodomain proteins (Laurent et al., 1997; Lemaire et al., 1995), which are downstream effectors of the Nodal signaling pathway (Hart et al., 2005). Mix family members are found in higher vertebrates, including mouse and human, and MIxl1 has been shown to be involved in early embryogenesis in mouse (Hart et al., 2002). However, Sia/Twn lie downstream of Wnt signaling (Bae et al., 2011; Brannon et al., 1997; Brannon and Kimelman, 1996; Carnac et al., 1996; Crease et al., 1998; Fan et al., 1998; Ishibashi et al., 2008; Kessler, 1997), while Mixl1 is downstream of Nodal

signaling (Hart et al., 2005). We find a similar situation in zebrafish, where the

homeodomain transcriptional repressor, bozozok (Fekany et al., 1999; Koos and Ho, 1999; Yamanaka et al., 1998), is essential for organizer formation and expression of organizer genes such as gsc (Shimizu et al., 2000; Solnica-Krezel and Driever, 2001), yet no vertebrate orthologs have been identified. It remains to be determined whether Sia/Twn and bozozok represent unique requirements in the development of the frog and the fish, or whether emerging genomic tools will help identify putative orthologs in other model systems.

appear to do so now. A comparison of the Sia A domain sequence in a closely related amphibian, Xenopus tropicalis, reveals conservation of a serine at position 12,

suggesting that the A domain of Xenopus tropicalis Sia is also inactive. Since Sia and Twn are highly similar in sequence and in function, the pressure to preserve conserved activation domains in these proteins might not be as great. Conversely, Sia and Twn could have both overlapping and distinct functions during organizer formation that may not be discernible with our experimental approaches. Similarly, the Sia A domain may function in an as yet unidentified manner to modulate protein stability, dimerization or other important proteins functions. A similar question persists for the Sia and Twn C domain. The high conservation in this domain suggests it might contribute to protein function; what function, however, remains to be determined. Removal of this domain does not affect transcriptional activity, but elimination of this domain from a Gal4-Sia construct leads to significant enhancement in transcriptional activity (Fig. 4.2A, compare Gal4-SiaN to Gal4-Sia1-75), suggesting that the C domain may function in negatively regulating transcriptional activity. Mutation of one conserved lysine residue within Sia or Twn led to a decrease in transcriptional activity, but mutation of two other lysine

residues, or the mutation of combination of all three conserved lysine residues did not have a significant effect on transcription. These results suggest that these residues may play multiple roles in modulating Sia/Twn function and it will be interesting to see how this conserved domain functions in the restriction of organizer gene expression.

In Xenopus, Gsc is regulated by inputs from both the Wnt and Nodal pathways, through a Nodal responsive Distal Element (DE) and a Wnt responsive Proximal Element (PE). The mouse PE retains Wnt responsiveness in Xenopus (Watabe et al., 1995), suggesting that Wnt signals are involved in Gsc expression in the mouse. A

search of the mouse genome for homeodomain proteins containing domains similar to the A, B or C domains did not reveal potential candidates. It is likely that combining data from expression profiles, bioinformatics and ChIP sequencing data may reveal promising candidates that could act during organizer formation in other species. The elucidation of the promoter regions of more organizer genes may also reveal transcription factors that fulfill the role of Sia/Twn in other vertebrate species.

Taken together, we have identified important domains within the transcriptional activators Sia and Twn. While further work is needed to elucidate the mechanism of Sia/Twn transcriptional activity, the identification of these domains is important in understanding the function of other proteins containing domains similar to Sia/Twn. A single amino acid within the Sia A domain, which confers transcriptional activity to an otherwise inactive domain, is an important finding and future work will focus on how this residue might contribute to protein structure and co-factor recruitment. Furthermore, future work should also focus on the post-translational modifications of Sia/Twn and their role in modulating protein activity. These modifications could restrict organizer gene expression to the dorsal side of the embryo, merely by limiting the spatial expression of the Sia/Twn modifier. Determining the mechanism of action of Sia/Twn is important in elucidating how transcriptional inputs can result in the restriction of gene expression to the Spemann organizer.