Identification of zebrafish foxi2, foxi3a, and foxi3b genes
We used the zebrafish Foxi1 protein sequence, excluding the forkhead domain, as a query in a BLAST search of the Danio rerio translated trace repository on the Ensembl Trace Server Web site (http://trace.ensembl.org/). Nucleotide sequences of hits containing identity for at least five amino acids were retrieved and assembled into contigs using LASERGENE Navigator sequence alignment software. Although most sequences failed to fall into alignments, four separate contigs containing multiple sequences were identified. One of these contigs defined the foxi1 sequence. Consensus sequences were derived for the other three contigs and were used as translated queries in BLAST searches of protein databases on the NCBI Web site (http://www.ncbi.nlm.nih.gov/BLAST/). All three sequences showed the closest similarity to other FoxI class proteins, as was the case for the original analysis performed with foxi1 (Solomon et al., 2003). Potential complete coding sequences were derived for these contigs through searches of the partially assembled Danio rerio Ensembl database and comparisons with other FoxI coding sequences that showed high similarity by BLAST search, and we have named these sequences Foxi2, Foxi3a, and Foxi3b.
Expression of Zebrafish foxi2
foxi2 is expressed faintly in the notochord at the three-somite stage (3s), and this expression extends to a circular region corresponding to the position of the tailbud (Fig. 1A,B). By 18s, strong expression is detected in the pharyngeal arch region (Fig. 1C,D) and the anterior portion of the optic primordium (Fig. 1C,E), and expression is maintained in both of these domains at 2 days postfertilization (2d; Fig. 1F–H). Expression in the eye at 2d is detected ventrally to the lens and extends along the choroid fissure (Fig. 1F,G), and it is also detected more faintly in the ventral cells of the retina surrounding the choroid fissure (Fig. 1G). However, foxi2 is not expressed in the optic nerve or stalk. Four distinct stripes of expression are detected in the pharyngeal arch domain at this stage, potentially corresponding to either the cartilaginous precursor cells or the adjacent pharyngeal arch endoderm (Fig. 1F–H). Higher magnification reveals that at least some cartilaginous cells are included within this expression domain (Fig. 1H). Also at 2d, foxi2 is expressed bilaterally as two ventral, circular domains immediately anterior to the first somite, and in the region surrounding the mouth, including strong bilateral expression in cartilage adjacent to the ventro-lateral boundary of this structure (Fig. 1G). The identity of the tissue expressing the two ovoid domains remains unclear at this time. At 4d, foxi2 is expressed ventrally to the lens and bilaterally in a single stripe through a noncartilaginous region of the gill arches (Fig. 1I,J).
Figure 1. Expression of zebrafish foxi2. A,B: A three-somite (3s) embryo, dorsal view with anterior to the top of panel (A) and dorsoposterior view (B). Arrowheads denote expression in notochord (A) and tailbud region (B). C–E: An 18s embryo, lateral view, anterior to the left(C), dorsal view with anterior to the top (D), and rostral view (E). F–H: A 2 days postfertilization (2d) embryo, lateral (F) and ventral (G) views with anterior to the left of the panels, dorsolateral view of pharyngeal arches, anterior to the left, medial toward the bottom (H). I,J: Lateral views of 4d embryo, anterior to the left. pa, pharyngeal arches; op, optic primordium; cf, choroids fissure of the eye; vr, ventral retina; m, mouth.
Download figure to PowerPoint
Expression of Zebrafish foxi3a and foxi3b
foxi3a and foxi3b display nearly identical expression patterns, in accordance with our proposal that these FoxI homologs represent a recently duplicated gene pair. At 3s, both genes are expressed in a punctate pattern over a large portion of the yolk sac (Fig. 2A,B), and this pattern is detectable as early as 90% epiboly for foxi3a, but not foxi3b (data not shown). A similar pattern is observed at 18s, although in some cases the expression extends from the yolk sac to include the lateral portions of the trunk and tail (Fig. 2C,D). This punctate pattern is very similar to the mucous cell expression reported for several Na/K ATPase subunit genes (Blasiole et al., 2002; Canfield et al., 2002) and the parvalbumin genes pvalb3a and pvalb3b (Hsiao et al., 2002). Based on this comparison, it is likely that foxi3a and foxi3b are expressed within the mucous cell population of the epidermis, and this is the first report of expression within this cell type during late gastrulation/early somitogenesis in zebrafish. At 2d, expression is detected in the regions surrounding the pharyngeal arches and the posterior border of the eye and extends posteriorly along the trunk/yolk contact site, ending near the anterior-most portion of the hind-yolk (Fig. 2E,F). At 4d, extensive expression is detected in the gill epithelium for both genes (Fig. 2G,H). foxi3a expression also extends along the yolk sac/trunk border, similar to the pattern observed at 2d, whereas foxi3b expression is weaker in this domain.
Figure 2. Expression of zebrafish foxi3a and foxi3b. Expression of foxi3a (A,C,E,G) and foxi3b(B,D,F,H) at three somites (3s; A,B), 18s (C,D), 2 days postfertilization (2d; E,F), and 4d (G,H). A–F: Lateral views with anterior to the left. G,H: Ventral views with anterior to the top. pa, pharyngeal arches; ge, gill epithelium.
Download figure to PowerPoint
Phylogenetic Analysis of FoxI Genes
A data set was assembled including 26 available amino acid sequences comprising the zebrafish FoxI genes, the previously reported mouse (Hulander et al., 1998), human (Pierrou et al., 1994), rat (Clevidence et al., 1993), Xenopus homologs (Lef et al., 1994, 1996), and additional sequences derived from our database searches. Alignment of the winged-helix domains of these proteins with representatives of other Fox classes confirmed their identity as members of the FoxI class (Fig. 3).
Figure 3. Amino acid alignment of winged helix/forkhead domains. The forkhead domains of the 26 FoxI class sequences analyzed here are shown aligned with representative sequences (from human genes, given in parentheses) of Fox classes A–N. Sequences were arbitrarily selected from the list available at http://www.biology.pomona.edu/fox.html. All of the sequences are aligned to the top sequence (mouse Foxi1); residues identical to the top sequence are indicated by dots, whereas dashes indicate gaps introduced for alignment. GenBank accession numbers for the zebrafish FoxI class genes identified in this analysis are as follows: foxi2, AY331582; foxi3a, AY331583; foxi3b, AY331584.
Download figure to PowerPoint
To address the phylogenetic relationships within the FoxI genes, a multiple sequence alignment was initially constructed by using ClustalW (Thompson et al., 1994), and then carefully edited by eye using MacClade (Maddison and Maddison, 1989). The resulting sequence alignment consisted of 215 residues that could be aligned with confidence; this alignment was subjected to phylogenetic analyses using both distance and parsimony methods as available in the PAUP*, Tree-Puzzle, and PHYLIP packages (Felsenstein, 1995; Swofford, 1999; Schmidt et al., 2002). The tree shown in Figure 4 represents a summary of these analyses.
Figure 4. Phylogenetic analysis of FoxI homologs. The tree shown is based on a maximum likelihood distance analysis of a 215-residue amino acid alignment of 26 FoxI homologs. Evolutionary distances were determined with Tree-Puzzle version 5.0 (Schmidt et al., 2002), and the tree was constructed by Neighbor-Joining using PHYLIP version 3.6a3 (Felsenstein, 1995). Numbers above the branches represent percentage bootstrap support from 1,000 pseudoreplicates: left number, from a distance analysis (using Protdist in PHYLIP), and right number, a maximum parsimony analysis (using PAUP* version 4.0b10; Swofford, 1999). Dashes represent bootstrap support less than 50%.
Download figure to PowerPoint
Although multiple FoxI homologs were identified in all of the vertebrates, only one gene was identified from each of the two available urochordate genomes of Ciona intestinalis and Ciona savignyi. This finding suggests that gene duplications of vertebrate FoxI genes occurred after their divergence from urochordates. This parsimonious scenario indicates that the singular Ciona foxi represents the ancestral gene copy.
Inspection of the resulting trees (e.g., Fig. 4) indicates three major subgroups of FoxI genes in vertebrates. One subgroup, indicated as A in Figure 5, includes Danio foxi1 and its apparent Fugu ortholog (which we have named foxi1) together with putative orthologs from Xenopus laevis and X. tropicalis (which we have named X. laevis and tropicalis FoxI2, due to the previously described Xenopus FoxI1 genes). Our analysis suggests that this assemblage is an orthologous group that arose at the base of vertebrate evolution. If so, the absence of this ortholog in completed mammalian genomes would indicate a gene loss in the lineage leading to mammals. However, it is also possible that the sequence of mammalian FoxI1 proteins are substantially divergent, making it impossible for us to recognize as orthologs. The only expression data reported within this group is for zebrafish foxi1 (Solomon et al., 2003), and, as we have previously noted, this gene shares some similarity in expression with Xenopus laevis FoxI1c of subgroup B.
Figure 5. Evolutionary relationships and expression patterns of FoxI genes. Left, simplified schematic of the phylogenetic tree shown in Figure 4. Branch lengths are arbitrary. Three subgroups of vertebrate FoxI genes are indicated as A, B, and C, and the dashed line joining the mammalian members of subgroup B with the amphibian and fish members represents a hypothesized relationship (see text). A hypothesized loss of a mammalian ortholog of subgroup A is also indicated. Two instances are indicated where there is evidence for two very similar “pseudoalleles” from Xenopus laevis (FoxI3a/3b and 2a/2b) but for which only one was used in the phylogenetic analysis. Right, summary of available expression data for previously reported human (Pierrou et al., 1994), mouse (Hulander et al., 1998, 2003), rat (Clevidence et al., 1993), Xenopus laevis (Lef et al., 1994, 1996; Pohl et al., 2002), and zebrafish (Solomon et al., 2003) FoxI genes, together with data from this analysis, is shown for selected structures. + and −, present or absent, respectively, as determined by whole-mount in situ hybridization; (+) present as determined by Northern blot analysis; asterisks indicate unpublished observation.
Download figure to PowerPoint
Subgroup B is composed of two well-supported groups: one contains only mammalian genes (FoxI2 and FoxI3) and another is composed of amphibian (FoxI1) and fish (FoxI2) genes. Our phylogenetic analysis does not explicitly support this group, but it does not strongly reject it either. Based on the observed phylogeny and the representation of vertebrate lineages, we propose that the mammalian, amphibian, and fish members actually comprise this single orthologous subgroup. Clear similarities in expression exist between fish and amphibian members of this subgroup (Fig. 5), consistent with their evolutionary relationship. However, no expression data for the mammalian members have been reported, which may provide additional support for this grouping.
Subgroup C has the strongest support of all the groups, and contains two zebrafish genes, foxi3a and foxi3b. These two genes are highly similar (56% amino acid identity) and appear to represent a recently duplicated gene pair. Also in this group are the previously described mouse, human, and rat orthologs (FoxI1) and additional Xenopus laevis, X. tropicalis, and Fugu members (FoxI3 genes). Of interest, this highly supported subgroup shows no conservation of expression pattern between mammals and fish (Fig. 5), although the expression patterns of several members of this subgroup, particularly the amphibians, remain uncharacterized.