In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada

Abstract

Water is essential for life, but anhydrobiotic tardigrades can survive almost complete dehydration. Anhydrobiosis has been a biological enigma for more than a century with respect to how organisms sustain life without water, but the few choices of genetic toolkits available in tardigrade research have been a challenging circumstance. Here, we report the development of an in vivo expression system for tardigrades. This transient transgenic technique is based on a plasmid vector (TardiVec) with promoters that originated from an anhydrobiotic tardigrade Ramazzottius varieornatus. It enables the introduction of GFP-fused proteins and genetically encoded indicators such as the Ca²⁺ indicator GCaMP into tardigrade cells; consequently, the dynamics of proteins and cells in tardigrades may be observed by fluorescence live imaging. This system is applicable for several tardigrades in the class Eutardigrada: the promoters of anhydrobiosis-related genes showed tissue-specific expression in this work. Surprisingly, promoters functioned similarly between multiple species, even for species with different modes of expression of anhydrobiosis-related genes, such as Hypsibius exemplaris, in which these genes are highly induced upon facing desiccation, and Thulinius ruffoi, which lacks anhydrobiotic capability. These results suggest that the highly dynamic expression changes in desiccation-induced species are regulated in trans. Tissue-specific expression of tardigrade-unique unstructured proteins also suggests differing anhydrobiosis machinery depending on the cell types. We believe that tardigrade transgenic technology opens up various experimental possibilities in tardigrade research, especially to explore anhydrobiosis mechanisms.

Keywords: anhydrobiosis; in vivo expression; live imaging; tardigrades.

Fig. 1.

Tardigrade vector system enables in vivo expression in tardigrades. (A) Tardigrade vector named TardiVec. TardiVec consists of a promoter and terminator that originate from the genome of tardigrades. (B) Microinjection into tardigrades. To perform microinjection, a thin cover glass is affixed on the slide glass, and tardigrades are soaked in halocarbon oil with a small amount of water for fixation. (C) Tardigrade, R. varieornatus, expressing mEGFP-NLS under the actin promoter of R. varieornatus, pRvACT. White arrowheads indicate the mEGFP signal in muscle cells, and a red arrowhead indicates the signal in a neuron. (D) R. varieornatus expressing GCaMP6s and mCherry with pRvACT. White arrowheads indicate the GCaMP signal in muscle cells, and a red arrowhead indicates the signal in a neuron. (E) Another anhydrobiotic tardigrade, H. exemplaris, expressing mEGFP-NLS under the actin promoter that originates from the genome of H. exemplaris. (F) R. varieornatus and H. exemplaris expressing mEGFP-NLS by interchange introduction of TardiVec. The merged images were obtained by adding transmission light to observe tardigrade body under the condition of observing GFP signal. (Scale bars, 100 μm.)

Fig. 2.

The tissue-specific expression of the tardigrade-specific genes CAHS and SAHS in R. varieornatus and H. exemplaris. (A) R. varieornatus expressing mEGFP-NLS under pRvCAHS3. (B) R. varieornatus expressing mEGFP-NLS under pRvSAHS1. (C) Comparison of transcripts of CAHS (red circle) and SAHS (purple circle) gene families between storage cells and the whole body of R. varieornatus (D) H. exemplaris expressing mEGFP-NLS under pRvCAHS3. (E) H. exemplaris expressing mEGFP-NLS under pRvSAHS1. (F) Comparison of transcripts of CAHS (red circle) and SAHS (purple circle) gene families between storage cells and the whole body of H. exemplaris without preconditioning. (G) T. ruffoi expressing mEGFP-NLS under pRvCAHS3. (E) T. ruffoi expressing mEGFP-NLS under pRvSAHS1. (I) M. inceptum expressing mEGFP-NLS under pRvCAHS3. D, E, G, H, and I are the results in heterologous expression experiment. The merged images were obtained by adding transmission light to observe tardigrade body under the condition of observing GFP signal. (Scale bars, 100 μm.)

Fig. 3.

Subcellular localization of tardigrade-specific proteins fused with mEGFP in tardigrade cells. (A) Overexpression of CAHS3-mEGFP and mEGFP-only in R. varieornatus. (B) Overexpression of SAHS1-mEGFP and mCherry-NLS by cotransfection in R. varieornatus. (C) Observation of a punctured tardigrade expressing SAHS1-mEGFP and mCherry-NLS. White arrowheads indicate cells in which mEGFP and mCherry signals were merged. (Scale bars, 50 μm.)