Supplemental Figure S1
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Fig. S1. Microarray analysis for the brachyury mutant. Microarray analysis was used to investigate genes up- and down-regulated at the larval stage in bra−/− mutants in comparison to wild type. The microarrays covered approximately 75-80% of the C. intestinalis genes in the genome. (A) Scatter plot of up- and down-regulated genes in the bra−/− larva. Using a threshold of a 1.5-fold difference, 825 genes were categorized as being downregulated and 757 genes were categorized as being upregulated in the bra−/− larva. Downregulated genes included a number of known brachyury downstream targets such Ci-noto4 and Ci-noto8. Six genes, which are listed as being expressed in endoderm on the Ghost EST Database (Kyoto), were highly upregulated, although these have not been confirmed by RT-PCR. We identified three previously uncharacterized genes that were downregulated in the bra−/− larva. The first of these is a cub-domain protein with similarity to the tolloid-like/BMP1/Polce metalloproteases (KyotoGrail2005.1723.1.1). The two other genes (KyotoGrail2005.218.3.1 and KyotoGrail2005.44.15.1) encode novel proteins that contain latrophilin/CL-1-like GPS, and serine/threonine kinase domains, respectively. (B) RT-PCR confirmation of the microarray result for five notochord-specific genes downregulated in bra−/− larvae. Left panel shows the results from wild-type larvae and right panel shows the result from bra−/− larvae. Primers used for PCR are available on request. (C-N) Temporal and spatial expression patterns of three newly identified brachyury downregulated genes. (C,G,K) Wild-type early tailbud stage. (D,H,L) Wild-type mid-tailbud stage. (E,I,M) Wild-type larvae. (F,J,N) bra−/− larvae. (C-F) KyotoGrail2005.1723.1.1. This transcript was expressed exclusively in the notochord of wild-type larvae (E), but not at earlier stages (C,D). A faint signal for this transcript could be detected in notochord cells of bra−/− larvae (E). (G-J) KyotoGrail2005.218.3.1. This transcript was found to be expressed exclusively in the notochord of larvae (I), starting at the mid-tailbud stage (H), but not at the early tailbud stage (G). No in situ hybridization signal could be found in bra−/− larvae (J). (K-N) KyotoGrail2005.44.15.1. This transcript was found to be expressed exclusively in the notochord of larvae (M), starting at the mid-tailbud stage (L), but not at the early tailbud stage (K). No in situ hybridization signal could be found in bra−/− larvae (N). Scale bar: 100 µm.
