Meeting Abstract
P3.63 Thursday, Jan. 6 Origins of the Pax/Six Gene Regulatory Network DING, Siyu; SASSOON, Daniel*; RUED, Anna; WINTERS, Ian; HILL, April; RIVERA, Ajna; Univ of Richmond; Univ of the Pacific; Univ of Richmond; Univ of Richmond; Univ of Richmond; Univ of the Pacific dsassoon@pacific.edu
Pax and Six genes are two members of the PSED network, which regulates, among other processes, sensory organ development. Sponges are basally branching metazoans without a nervous system but with sensory perception. Thus, sponges are an excellent model to study the origins of the nervous system. Comparisons of the PSED network between sponges and other well-studied metazoans will help reveal how gene regulatory networks originate and evolve over time. To this end, we investigated the origins of the Pax/Six regulatory network in Ephydatia muelleri, a globally distributed demosponge. While demosponges, including Amphimedmon queenslandica and E. muelleri, appear to lack Eya and Dac representatives (the other two members of the PSED network), they have single Pax and Six orthologues (Hill et al, 2010). Gene expression analysis reveals overlapping patterns for Em-Pax and Em-Six, suggesting that they may be involved in a regulatory relationship similar to other metazoans. RNAi knockdowns also support this hypothesis. To further analyze the gene regulatory network, we performed gel-shift (EMSA) experiments and found that the paired domain of Em-Pax binds to canonical Pax2/5/8 binding sequences and, more specifically, to putative binding sequences upstream of sponge Six. To further characterize the sponge Pax/Six GRN, we performed Chromatin Immunoprecipitation (ChIP) with a commercial Pax 2/5/8 antibody and E. muelleri genomic DNA, and analyzed the results using a PCR based method followed by genome walking to identify putative natural targets of Pax in sponges. In conjunction with this, we also analyzed the newly available A. queenslandica genome for genes with flanking DNA sequences enriched with Pax 2/5/8 consensus binding sites. Future experiments include testing these potential targets via RNAi.
