DEV01638 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. CEH-16/ENGRAILED domains (Engrailed Homology domains, EH), ClustalW alignments, protein structure and phylogenetic analysis. (A-D) Alignments of the EH domains (Burglin, 1995; Ekker et al., 1992; Hui et al., 1992; Logan et al., 1992). The degree of conservation is highest in EH2 and EH4 (homeodomain). The degree of protein identity reflects the subdivision in two groups, Deuterostomia and Protostomia; see also phylogenetic analysis of the EH4 (homeodomain) in H. (E) EH5, which is adjacent to EH4 in all species but strongly diverged in C. elegans. No other sequence stretches elsewhere in CEH-16 were found that could account for an EH5 domain. (F) Organization of the domains within the proteins, three examples (mouse, fruitfly, C. elegans). The sequence of the domains is conserved, although EH5 has probably lost its function (marked with ‘?’). (G) Conservation of an intron between the splice sites of EH2 and EH3 in all three species. Interestingly, this intron is unique in humans. (H) Phylogenetic analysis of Engrailed class EH4/homeodomains based on ClustalW alignment software. Abbreviations, accession numbers of engrailed genes: xenopus (frog), Xenopus laevis en-2 (X62973); artemia (shrimp), Artemia franciscana (X70937); bombyx (silkworm), Bombyx mori (M64335); periplaneta (cockroach), Periplaneta Americana Pa-en1 (AJ43883); Drosophila (fruitfly), Drosophila melanogaster (M10017); danio (zebrafish), Danio rerio en-2 (X68151); gallus (chicken), Gallus gallus en-2 (L12697); human, Homo sapiens en-2 (E48423); musculus (mouse), Mus musculus en-1 (A48423); amphioxus (lancelet), Brachiostoma floridae (U82487); oryzias (medaka - fish), Oryzias latipes (AF112141); chaetopterus (annelid), Chaetopterus sp. (AAK67707); elegans, Caenorhabditis elegans (CEH-16, this work, AY647457).

• Supplemental Figure 2 -

  Fig. S2. Expression pattern of ceh-16::gfp during morphogenesis, Z projection of confocal images. ceh-16::gfp is expressed in seam cells (red arrows), neurons (blue arrows). The green arrows point to the expression of ajm-1::gfp. Scale bar: 10 mm.

• Supplemental Figure 3 -

  Fig. S3. Ectopic expression of eff-1::gfp as a result of downregulation of either ceh-16 or elt-5: elt-5 and ceh-16 both repress eff-1::gfp expression. The arrows show how in both experiments eff-1::gfp can be ectopically detected in seam cells, when RNAi of the respective transcription factors is performed. Scale bar: 10 mm.

• Movie 1 -

  Movie S1. Epidermal development during bean to 1.5-fold stage in a wild-type animal transgenic for ajm-1::gfp (integrated). The dorsal epidermis fuses to form a syncytium (hyp7).

• Movie 2 -

  Movie S2. Epidermal development during bean to 1.5-fold stage in an eff-1(hy21) animal transgenic for a ajm-1::gfp and for a transcriptional eff-1::gfp construct. All epidermal cell fusions fail (Mohler et al., 2002).

• Movie 3 -

  Movie S3. Epidermal development during bean to 1.5-fold stage in an eff-1(hy21), ceh-16(RNAi) animal transgenic for an ajm-1::gfp and for a transcriptional eff-1::gfp construct. ceh-16 (–) seam cells, marked by the expression of eff-1::gfp, migrate dorsally and ventrally, disrupting their linear organization.