Microinjection of dsRNA in Tardigrades.

Classical genetic analysis in the tardigrade Hypsibius exemplaris is a challenge because these animals are parthenogens. The publication of the H. exemplaris genome has facilitated the study of targeted genes by RNA interference (RNAi), a robust mechanism to disrupt gene function. This protocol describes microinjection of double-stranded RNA (dsRNA) in tardigrades using techniques adapted from protocols originally developed in Caenorhabditis elegans. A DNA template (either genomic or cDNA) is used to prepare dsRNA, to which T7 polymerase binding sites are added at the 5' end of each strand. The dsRNA is injected into adult tardigrades, preferably targeting the gonad or intestine. Injected adults are allowed to recover in spring water and then transferred to culture dishes or individual wells of a 96-well plate.

Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.

Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes.

RNA interference can be used to disrupt gene function in tardigrades.

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.

MEX-5 asymmetry in one-cell C. elegans embryos requires PAR-4- and PAR-1-dependent phosphorylation.

Anteroposterior polarity in early C. elegans embryos is required for the specification of somatic and germline lineages, and is initiated by a sperm-induced reorganization of the cortical cytoskeleton and PAR polarity proteins. Through mechanisms that are not understood, the kinases PAR-1 and PAR-4, and other PAR proteins cause the cytoplasmic zinc finger protein MEX-5 to accumulate asymmetrically in the anterior half of the one-cell embryo. We show that MEX-5 asymmetry requires neither vectorial transport to the anterior, nor protein degradation in the posterior. MEX-5 has a restricted mobility before fertilization and in the anterior of one-cell embryos. However, MEX-5 mobility in the posterior increases as asymmetry develops, presumably allowing accumulation in the anterior. The MEX-5 zinc fingers and a small, C-terminal domain are essential for asymmetry; the zinc fingers restrict MEX-5 mobility, and the C-terminal domain is required for the increase in posterior mobility. We show that a crucial residue in the C-terminus, Ser 458, is phosphorylated in vivo. PAR-1 and PAR-4 kinase activities are required for the phosphorylation of S458, providing a link between PAR polarity proteins and the cytoplasmic asymmetry of MEX-5.

Notch signaling and morphogenesis of single-cell tubes in the C. elegans digestive tract.

During organogenesis of the C. elegans digestive system, epithelial cells within a cyst-like primordium develop diverse shapes through largely unknown mechanisms. We here analyze two adjacent, dorsal epithelial cells, called pm8 and vpi1, that remodel their shapes and apical junctions to become donut-shaped, or toroidal, single-cell tubes. pm8 and vpi1 delaminate from the dorsal cyst epithelium and migrate ventrally, across the midline of the cyst, on a transient tract of laminin. pm8 appears to encircle the midline by wrapping around finger-like projections from neighboring cells. Finally, pm8 and vpi1 self-fuse to become toroids by expressing AFF-1 and EFF-1, two fusogens that are each sufficient to promote crossfusion between other cell types. Notch signaling in pm8 induces AFF-1 expression, while simultaneously repressing EFF-1 expression; vpi1 expresses EFF-1 independent of Notch. Thus, the adjacent pm8 and vpi1 cells express different fusogens, allowing them to self-fuse into separate, single-cell tubes while avoiding crossfusion.

EEL-1, a Hect E3 ubiquitin ligase, controls asymmetry and persistence of the SKN-1 transcription factor in the early C. elegans embryo.

During early divisions of the C. elegans embryo, many maternally supplied determinants accumulate asymmetrically, and this asymmetry is crucial for proper cell fate specification. SKN-1, a transcription factor whose message is maternally supplied to the embryo, specifies the mesendodermal cell fate. In the 2-cell embryo, SKN-1 is expressed at a higher level in the posterior cell. This asymmetry becomes more pronounced at the 4-cell stage, when SKN-1 is high in the posterior cell's daughters and low in the daughters of the anterior blastomere. To date, the direct mechanisms that control SKN-1 distribution remain unknown. In this report, we identify eel-1, which encodes a putative Hect E3 ubiquitin ligase that shares several domains of similarity to the mammalian E3 ligase Mule. EEL-1 binds SKN-1 and appears to target SKN-1 for degradation. EEL-1 has two functions in regulating SKN-1 during early embryogenesis. First, eel-1 promotes the spatial asymmetry of SKN-1 accumulation at the 2- and 4-cell stages. Second, eel-1 acts in all cells to downregulate SKN-1 from the 12- to the 28-cell stage. Although loss of eel-1 alone causes a reduction in SKN-1 asymmetry at the 2-cell stage, the function of eel-1 in both the spatial and temporal regulation of SKN-1 is redundant with the activities of other genes. These data strongly suggest that multiple, functionally redundant pathways cooperate to ensure precise control of SKN-1 asymmetry and persistence in the early embryo.

Reduced dosage of pos-1 suppresses Mex mutants and reveals complex interactions among CCCH zinc-finger proteins during Caenorhabditis elegans embryogenesis.

Cell fate specification in the early C. elegans embryo requires the activity of a family of proteins with CCCH zinc-finger motifs. Two members of the family, MEX-5 and MEX-6, are enriched in the anterior of the early embryo where they inhibit the accumulation of posterior proteins. Embryos from mex-5 single-mutant mothers are inviable due to the misexpression of SKN-1, a transcription factor that can specify mesoderm and endoderm. The aberrant expression of SKN-1 causes a loss of hypodermal and neuronal tissue and an excess of pharyngeal muscle, a Mex phenotype (muscle excess). POS-1, a third protein with CCCH motifs, is concentrated in the posterior of the embryo where it restricts the expression of at least one protein to the anterior. We discovered that reducing the dosage of pos-1(+) can suppress the Mex phenotype of mex-5(-) embryos and that POS-1 binds the 3'-UTR of mex-6. We propose that the suppression of the Mex phenotype by reducing pos-1(+) is due to decreased repression of mex-6 translation. Our detailed analyses of these protein functions reveal complex interactions among the CCCH finger proteins and suggest that their complementary expression patterns might be refined by antagonistic interactions among them.

C. elegans MOM-5/frizzled functions in MOM-2/Wnt-independent cell polarity and is localized asymmetrically prior to cell division.

C. elegans embryonic cells have a common anterior/posterior (a/p) polarity that is apparent in the localization of the transcription factor POP-1. The level of nuclear POP-1 remains high in the anterior daughters of dividing cells but is lowered in the posterior daughters. To generate POP-1 asymmetry, most early embryonic cells require contact with signaling cells that express the ligand MOM-2/Wnt; the point of cell contact specifies the daughter with low nuclear POP-1. In contrast, slightly older embryonic cells that have no apparent prior exposure to Wnt signaling can generate POP-1 asymmetry, provided these cells express MOM-5/Frizzled. We show here that MOM-5::GFP is enriched at the posterior pole of cells prior to division and that a similar asymmetry is observed in cultured cells with no apparent prior exposure to Wnt signaling. While depleting these latter cells of MOM-5/Frizzled causes both daughter cells to have high levels of POP-1, we show that both daughter cells have low levels of POP-1 in embryos with atypically high levels of MOM-5::GFP. These results suggest that MOM-5/Frizzled asymmetry leads to POP-1 asymmetry. In later embryogenesis, we find that MOM-5::GFP localizes to the leading edges of epidermal cells during ventral enclosure. These localization patterns suggest a parallel between MOM-5/Frizzled and the roles of Drosophila Frizzled in planar polarity and dorsal enclosure.