Expression of distal limb patterning genes in Hypsibius exemplaris indicate regionalization and suggest distal identity of tardigrade legs.

Abstract

Background: Panarthropods, a major group of invertebrate animals comprised of arthropods, onychophorans, and tardigrades, are the only limb-bearing members of Ecdysozoa. The complexity and versatility of panarthropod paired limbs has prompted great interest in their development to better understand the formation of these structures and the genes involved in this process. However, studies of limb patterning and development are overwhelmingly focused on arthropods, followed by select work on onychophorans but almost entirely lacking for tardigrades. This model organism bias is inherently limited and precludes a comparative analysis of how panarthropod legs originated, have evolved, and the likely limb patterning genes present in the earliest panarthropod ancestors. In this study, we investigated tardigrade homologs of seven arthropod distal limb patterning genes (apterous, aristaless, BarH1, clawless, Lim1, rotund, and spineless) to better characterize tardigrade limb development in a comparative context.

Results: We detected homologs of all seven genes in the eutardigrade Hypsibius exemplaris and heterotardigrade Echiniscoides cf. sigismundi suggesting their conservation in both tardigrade lineages. Hybridization chain reaction experiments in H. exemplaris reveal a regionalized expression pattern for the genes aristaless, BarH1, clawless, rotund and spineless.

Conclusion: The observed regionalized expression of the distal limb patterning genes in H. exemplaris might reflect the external morphological features of tardigrade legs, such as the distal claws, sensory organs in the proximal region, and specific muscle attachment sites. The comparison between the expression of these limb patterning genes in H. exemplaris relative to other panarthropods suggests their conserved role in the last common panarthropod ancestor, such as establishing the distal limb end and the distribution of sensory structures. Our results support the hypothesis that tardigrade legs are homologous to the distal region of other panarthropod limbs, as suggested by previous work on the expression of leg gap genes in H. exemplaris.