Supplementary material from "Loss of intermediate regions of perpendicular body axes contributed to miniaturization of tardigrades"

Tardigrades have a miniaturized body plan. Miniaturization in tardigrades is associated with the loss of several organ systems and an intermediate region of their anteroposterior (AP) axis. However, how miniaturization has affected tardigrade legs is unclear. In arthropods and in onychophorans, the leg gap genes are expressed in regionalized proximodistal (PD) patterns in the legs. Functional studies indicate that these genes regulate growth in their respective expression domains and establish PD identities, partly through mutually antagonistic regulatory interactions. Here, we investigated the expression patterns of tardigrade orthologs of the leg gap genes. Rather than being restricted to a proximal leg region, as in arthropods and onychophorans, we detected coexpression of orthologs of homothorax and extradenticle broadly across the legs of the first three trunk segments in the tardigrade Hypsibius exemplaris. We could not identify a dachshund ortholog in tardigrade genomes, a gene that is expressed in an intermediate region of developing legs in arthropods and onychophorans, suggesting that this gene was lost in the tardigrade lineage. We detected Distal-less expression broadly across all developing leg buds in H. exemplaris embryos, unlike in arthropods and onychophorans, in which it exhibits a distally restricted expression domain. The broad expression patterns of the remaining leg gap genes in H. exemplaris legs may reflect the loss of dachshund and the accompanying loss of an intermediate region of the legs in the tardigrade lineage. We propose that the loss of intermediate regions of both the AP and PD body axes contributed to miniaturization of Tardigrada.