Loss of intermediate regions of perpendicular body axes contributed to miniaturization of tardigrades

Abstract

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 orthologues 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 orthologue 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.

Keywords: Panarthropoda; Tardigrada; body plan; leg gap genes; miniaturization; progenesis.

Figure 1.

Phylogeny of TALE-class homeodomain proteins HTH and EXD. Bootstrap support values of interest are shown above branches (500 replicates). Posterior probabilities of interest are shown below branches. Taxa names and accession numbers are provided in electronic supplementary material, table S1. (Online version in colour.)

Figure 2.

In situ hybridization results for He-exd3 and He-hth. Anterior is to the top, and embryos are facing right. (a,d) DIC micrographs. (a′–c, d′–e′) Three-dimensional reconstructions, except for c, which is a maximum projection. Embryos are counterstained with DAPI. (a–c) He-exd3 expression. a and a′ show the same embryo. (d–e′) He-hth expression. d and d′ show the same embryo. e and e′ show the same embryo. L1–L4, leg 1–leg 4; ga1–ga4, ganglion 1–ganglion 4; ph, pharynx. (Online version in colour.)

Figure 3.

In situ hybridization results for He-Dll and He-al. Anterior is to the top, and embryos are facing right in all panels besides a, which is a dorsoventral mount. (a,b) Maximum projections. b is a composite of two different maximum projections so that gene expression in all legs of the right side can be seen. (a′, c–e) three-dimensional reconstructions. (a–e) Embryos are counterstained with DAPI. (a–c) He-Dll expression. a′ is the same embryo as shown in a, but has been rotated in Volume Viewer in order to visualize the legs of the right side of the body. (d–e) He-al expression. L1–L4, leg 1–leg 4. (Online version in colour.)

Figure 4.

Model for the evolution of PD axes. (a) Evolutionary model. For simplicity, only lineages relevant to the co-option hypothesis are shown. AP represents the evolution of the anteroposterior nervous system patterning role of the leg gap genes. PD represents co-option of the leg gap genes for the evolution of PD limb axes. (b) The presence of a leg gap gene orthologue in the genome of a species in a is represented by a coloured box. (c) Expression patterns of orthologues of leg gap genes in developing limbs in the species in a are diagrammed. The colour gradient in the H. exemplaris models indicates uncertainty in how far proximally or distally a gene is expressed. Citations for these diagrams and the topology of the phylogenetic tree are provided in the text. (Online version in the colour.)