RNA interference can be used to disrupt gene function in tardigrades

Abstract

How morphological diversity arises is a key question in evolutionary developmental biology. As a long-term approach to address this question, we are developing the water bear Hypsibius dujardini (Phylum Tardigrada) as a model system. We expect that using a close relative of two well-studied models, Drosophila (Phylum Arthropoda) and Caenorhabditis elegans (Phylum Nematoda), will facilitate identifying genetic pathways relevant to understanding the evolution of development. Tardigrades are also valuable research subjects for investigating how organisms and biological materials can survive extreme conditions. Methods to disrupt gene activity are essential to each of these efforts, but no such method yet exists for the Phylum Tardigrada. We developed a protocol to disrupt tardigrade gene functions by double-stranded RNA-mediated RNA interference (RNAi). We showed that targeting tardigrade homologs of essential developmental genes by RNAi produced embryonic lethality, whereas targeting green fluorescent protein did not. Disruption of gene functions appears to be relatively specific by two criteria: targeting distinct genes resulted in distinct phenotypes that were consistent with predicted gene functions and by RT-PCR, RNAi reduced the level of a target mRNA and not a control mRNA. These studies represent the first evidence that gene functions can be disrupted by RNAi in the phylum Tardigrada. Our results form a platform for dissecting tardigrade gene functions for understanding the evolution of developmental mechanisms and survival in extreme environments.

Fig. 1

Phylogenetic position and anatomy of H. dujardini. a Evolutionary position of tardigrades, with relationships to selected taxa shown. Within Ecdysozoa, arthropods, onychophorans and tardigrades are proposed to form a Panarthropoda sub-clade. Some studies place tardigrades as a sister clade to the nematodes and nematomorphs. Drawn from (Dunn et al. 2008; Telford et al. 2008; Hejnol et al. 2009; Rota-Stabelli et al. 2010; Campbell et al. 2011). b Scanning electron micrograph of an H. dujardini adult, ventral view, ~150 μm long. Scale bar = 10 μM c Schematic drawing of injection slide showing coverslip corner (triangle) affixed to glass slide. Up to five tardigrades (black ovals) were positioned lengthwise against the straight edge of the coverslip, and an injection needle was positioned perpendicularly to each animal. d Lateral view of adult with needle (black arrow) inserted into midsection. The coverslip edge (white arrows) is positioned vertically to the left of the animal. The head of this animal is at the top of the image. Scale bar = 10 μM

Fig. 2

RNAi of selected targets affects embryo viability and adult fecundity. a Percent embryonic lethality observed for progeny of females injected with dsRNA compared to progeny from uninjected control animals. For each condition, data is shown for progeny from all broods (dark pink bars), and for progeny from the first brood only (light pink bars). b Average number of broods for injected females. Error bars indicate 95% confidence interval. c Embryonic lethality in Hd-mag-1/mago nashi(RNAi) progeny decreased with each successive brood. Numbers over each bar indicate the total number of embryos observed in each brood.

Fig. 3

RNAi results in target-specific depletion of gene function. a–c Representative images of wild-type embryos. a Stage 13 embryo (~24 hours after egg-laying), showing elongation along the anterior-posterior axis, with ectodermal segmentation (red arrows). b Same embryo as in (a), at late Stage 15 (~48 hrs after egg-laying), showing three developing limb buds (asterisks). Intestinal birefringent granules are visible in a higher focal plane. c Stage 19 embryo (~4 days after egg laying), prior to hatching. The pharynx and intestine are outlined (white dotted lines). Yellow arrows in this panel and in panel (e) mark birefringent granules, a marker of intestine differentiation. Note that this embryo is different from the one shown in a, b. d Hd-act-1(RNAi) embryo, ~24 hours after egg-laying. e Hd-mag-1(RNAi) embryo, ~48 hrs after egg-laying. The embryo has not elongated along the anterior-posterior axis, but birefringent granules are visible. f Hd-dlc-1(RNAi) embryo, ~4 days after egg-laying. The pharynx and part of the intestine are outlined (white dotted lines). The intestine appears to lack birefringent granules. A=anterior; P=posterior. Scale bar = 10 μm. Embryos were staged as described in (Gabriel et al. 2007).

Fig. 4

RNAi leads to decrease in Hd-mag-1 expression level. a Schematic of the Hd-mag-1 coding sequence, showing the Hd-mag-1(FL) product amplified by RT-PCR (black bar). The positions of the start (ATG) and stop (TGA) codons are marked with an asterisk and filled circle, respectively. Below the box, the sequences used as templates for dsRNA synthesis are indicated: Hd-mag-1(399bp) (grey bar), Hd-mag-1(5′L) (blue bar), Hd-mag-1(3′L) (purple bar), Hd-mag-1(5′S) (cyan bar), and Hd-mag-1(3′S) (magenta bar). b Relative expression of Hd-tbb-1/β-tubulin and Hd-mag-1/mago nashi amplified by RT-PCR in three wild-type and three Hd-mag-1(RNAi) embryos. c Histogram comparing ratios of amplified Hd-mag-1 and Hd-tbb-1 levels in the wild-type and Hd-mag-1(RNAi) embryos shown in (b). Averages were calculated from pixel intensities measured for each band. Error bars indicate standard deviation.