Evolution of the Alx homeobox gene family: parallel retention and independent loss of the vertebrate Alx3 gene

Abstract

The Alx gene family is implicated in craniofacial development and comprises two to four homeobox genes in each vertebrate genome analyzed. Using phylogenetics and comparative genomics, we show that the common ancestor of jawed vertebrates had three Alx genes descendent from the two-round genome duplications (Alx1, Alx3, Alx4), compared with a single amphioxus gene. Later in evolution one of the paralogues, Alx3, was lost independently from at least three different vertebrate lineages, whereas Alx1 and Alx4 were consistently retained. Comparison of spatial gene expression patterns reveals that the three mouse genes have equivalent craniofacial expression to the two chick and frog genes, suggesting that redundancy compensated for gene loss. We suggest that multiple independent loss of one Alx gene was predisposed by extensive and persistent overlap in gene expression between Alx paralogues. Even so, it is unclear whether it was coincidence or evolutionary bias that resulted in the same Alx gene being lost on each occasion, rather than different members of the gene family.

Fig. 1

Molecular phylogenetic trees showing the relationships between deuterostome Alx proteins based on neighbor-joining (NJ, A) and maximum-likelihood (ML, B) analyses. Echinoderm Alx proteins were used as outgroups. Branch lengths are proportional to evolutionary distance corrected for multiple substitutions; the scale bar denotes 0.1 underlying amino acid substitutions per site. Figures on branches indicate robustness of each node (>70%), estimated from 1000 bootstrap replicates for NJ (A) and 100 replicates for ML (B). AmphiAlx (GenBank: JF460798) is clearly shown to be an ortholog of vertebrate Alx genes by both the NJ (100%) and ML (98%) methods. Furthermore, all extant bony vertebrates share at least two Alx gene duplication events: one that gave rise to the Alx4 and Alx1/3 paralogy groups (100% for both trees), and a second that gave rise to the Alx1 and Alx3 paralogy groups (89.8%, 95%). Further duplication of Alx4 genes is observed in zebrafish and other teleost fishes (see text).

Fig. 2

Schematic diagram showing the location of Alx3 and neighboring genes in syntenic regions of Homo sapiens, Mus musculus, Gallus gallus, Taeniopygia guttata, Anolis carolinensis, Xenopus tropicalis, and Danio rerio. Arrows indicate genes and their direction of transcription. The blue/black arrows represent loci for which syntenic conservation is confidently identified (zebra finch Kcna genes are denoted by blue arrows because their orthologs in other vertebrates are located immediately downstream of Prok1). Alx3 genes are highlighted with red arrows/gray shadows. The green/gray arrows denote loci for which orthologs are not located in the syntenic region; these are probably inserted secondarily. The gray arrows denote predicted pseudogenes. The dotted crosses indicate inversions within this region in zebrafish genome. The double oblique lines in the zebrafish and chicken genomic regions indicate presence of other genes between the loci shown.

Fig. 3

Alx expression in the developing head of mouse, chick, and frog embryos. Expression of mouse, chick, and Xenopus Alx genes during craniofacial development was compared using whole mount in situ hybridization. Lateral (A, C, E, G, I, K, L, M, N) and frontal (B, D, F, H, J) views of the developing head are shown. At E10.5-11 in mouse, Alx1 (A, B), Alx3 (C, D), and Alx4 (E, F) are expressed in the mesenchyme of the facial prominences, particularly around the nasal region (n), and at the distal tip of the mandible (md). At this stage, Alx3 is additionally expressed in the mesenchyme (C, white arrow) encircling the eye. (). At stages 22–24 of chick development, Alx1 (G, H) and Alx4 (I, J) are also expressed in the facial prominences, again with strong expression around the nasal region (n), and at the distal tip of the mandible (md). In the chick, however, both Alx1 (G) and Alx4 (I) are expressed in the periocular mesenchyme (white arrows). Periocular expression of Alx1 is observed in frog embryos at the stage of 24 (K, white arrow). At this stage, Alx4 is not expressed in frog embryos (M) but later at stage 35, both Alx1 (L) and Alx4 (N) are expressed in frontal mesenchyme above the cement gland (cg).

Fig. 4

“Gene family tree” of vertebrate Alx homeobox genes in chordate evolution. After the two whole genome duplications (WGD, two green arrows at bottom of diagram) in early vertebrate evolution, four Alx homeobox genes were generated. The “Alx2” gene (yellow line) may have been swiftly lost, whereas the Alx3 genes were lost independently in at least three lineages (shown as the stars). Further modification in the teleost fish lineage followed the third WGD. The diagram depicts the phylogenetic location of each Alx3 gene loss, but note that the timing of loss within each lineage has not been determined.