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Fig. S1. A phylogenetic tree of LIM homeodomain proteins. A phylogenetic tree was constructed from the comparison of protein sequences by the Maximum Likelihood (ML) method using Treefinder with the protein matrix JTTG. Protein sequences of two LIM domains including a short linker in between and a homeodomain were used for this analysis, with a partition between the LIM domains and the homeodomain. Before constructing a tree, these sequences were aligned using the ClustalW alignment tool with the Gonnet series protein weight matrix with manual correction based on conserved motifs. The tree was rooted by the homeodomain of Xl_Otx2. Expected likelihood weights were indicated in each node. A scale bar of branch length indicates substitutions per site. Genes cloned in this paper are boxed. A partial coding sequence of Nv_lhx3 was constructed from three exons, in which four bases were artificially inserted to adjust a frame-shift in the first LIM-domain-coding exon and to restore the LIM motif. The translated amino acid sequence of Nv_Lhx3 from the first LIM domain to the homeodomain is shown at the bottom. Bold, the LIM motif; underline, predicted residues histidine (H) and valine (V) corresponding to the inserted four bases. See Table S1 for species abbreviations.
Fig. S2. Distance trees of Lhx1 and Lhx3 proteins. A distance tree of Lhx1 (A) or Lhx3 (B) proteins was constructed using the distance data. Each branch was constrained by the phylogeny of organisms (Dunn et al., 2008), and branch lengths indicate distances between proteins. Distance data were obtained from phylogenetic trees constructed with partitions between each two regions in the same way as in Fig. S1. Full-length protein sequences were divided into nine partitions for Lhx1 or eight partitions for Lhx3 as follows: for Lhx1, N-terminal region, LIM domains, LH linker, homeodomain, CCR1, a region between CCR1 and CCR2, CCR2, a region between CCR2 and CCR5, and CCR5; and for Lhx3, N-terminal region, LIM domains, LH linker, homeodomain, R-rich region, a region between the R-rich region and the Lhx3-specific domain, and Lhx3-specific domain. Before constructing a tree, these sequences were aligned in the same way as in Fig. S1. The Ci_Lhx1 protein sequence lacking the N-terminal sequence upstream of the first LIM domain was used. See Table S1 for species abbreviations.
Fig. S3. RT-PCR analysis of Hp_lhx1 and Hp_lhx3 expression in sea urchin embryos. Hp_lhx3 was amplified by using a primer set of Sp_lhx3, which has one base mismatch for Hp_lhx3 in the reverse primer. Genes and PCR cycles are indicated on the left and right sides, respectively. MitCO1, loading control. Stages of embryos are indicated above the panels. Neither Hp_lhx1 nor Hp_lhx3 was detected in the prism and pluteus larvae. UF, unfertilized eggs; 16cell, 16-cell stage; HB, hatched blastula; G, gastrula; PR, prism larva; PL, pluteus larva. In Hp_lhx3, the zygotic upper band (arrowhead) turned out to be a splicing variant in the LH linker as examined by PCR cloning and sequencing. The Hp_lhx3 coding sequence, corresponding to the shorter variant from which the lower band originated, was used for in functional analyses because its predicted protein sequence has the LH linker of 14 amino acids, which is similar to those of other Lhx3 proteins, whereas the longer variant has the LH linker of 26 amino acids.
Fig. S4. Analysis of structure and function of Nv_Ldb protein. (A) Protein structure of Nv_Ldb. DD, dimerization domain; LCCD, Ldb1-Chip conservative domain (interacting with Ssbp); NLS, nuclear localization sequence; LID, LIM-interaction domain; percentages shown in each domain indicate amino acid identities against Xl_Ldb1; exon number, alternative exons. These domains of Ldb were based on a previous study (Enkhmandakh et al., 2006). (B) Alternative splicing of the Nv_Ldb gene. The exon-intron organization was constructed from EST, cDNA and genomic sequences. A single ldb gene exists in the genome data of Nematostella vectensis (http://genome.jgi-psf.org/Nemve1/Nemve1.home.html). Alternative start site (1b) and stop codon (asterisk in exon 9b) are consistent with those of vertebrate ldb genes (Tran et al., 2006). Other alternative exons 5b and 8b have not been reported for vertebrate. Numbered boxes, exons; black boxes, the coding sequence corresponding to vertebrate Ldb1a; gray boxes, alternative coding sequences; white boxes, UTRs.
Fig. S5. Comparison of organizer activities between vertebrate LIM homeodomain subfamilies. (A) An embryo injected with Lhx1* mRNA, which possessed a secondary axis with a large amount of somites. (B) A normal-looking embryo injected with Xenopus β-globin mRNA (negative control). Somites were stained with the monoclonal antibody 12/101 (brown), with nlacZ (blue) as a lineage tracer for injected cells. (C) Comparison of secondary axis-inducing (SAI) activity. Dr, zebrafish; Ma, Syrian hamster; Xl, frog (see Table S1 for full species abbreviations). (D) Comparison of organizer gene-inducing (OGI) activity. Dr_Lhx2* showed almost no SAI and OGI activities, and Xl_Lhx3*, Dr_Isl3* and Ma_Lmx1a* showed some SAI and OGI activities, but weaker than those of Xl_Lhx1*. It should be noted that lower SAI activity of Xl_Lhx3* appears to be consistent with weak OGI activity for chordin and cerberus. Exceptionally, Dr_Isl3* has weak SAI activity but high OGI activity for all five genes examined. Dr_Isl3* may be less functional in the ventral marginal zone than in the animal cap.
Fig. S6. Sequence alignments. (A) Sequence alignment of Xl_Lhx1 and Nv_Lhx1. Dots, identical amino acids; dashes, spaces for alignment; boxes, LIM domains and the homeodomain; bold lines, CCR1-CCR5 of Xl_Lhx1; yellow boxes, LIM motif coordinating to zinc ion; asterisks, cysteine mutated to glycine in LIM domain mutants; magenta boxes, tyrosine and phenylalanine in CCR2; boxed Ys, five tyrosines indispensable for the activity of CCR2 (Hiratani et al., 2001). (B) Sequence alignment of CCR1, CCR2+ and CCR5. Thin lines, flanking regions of CCR2; green boxes, amino acids highly conserved in the C-termini of Lhx1 proteins.
Fig. S7. Luciferase reporter assays for activation of the gsc or cer promoter by Xl_Lhx1 and Xl_Lhx3. (A) The gsc promoter (Mochizuki et al., 2000). Xl_Ldb1 was coexpressed as indicated. (B) The cer promoter (Yamamoto et al., 2003). Siamois and Mix.1 were coexpressed as indicated. Dosages of injected mRNAs were indicated in pg/embryo. Relative luciferase activity represents a ratio against the mean value of Xl_Lhx1 and Xl_Ldb1 (100 pg each) (A) or Siamois (3 pg) and Mix.1 (6 pg) (B). Bars represent the mean ± s.e.m. *, P<0.01 (t-test); n, total number of samples; exp, number of independent experiments.
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