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 show 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

Linking PAR polarity proteins to cell fate regulation: analysis of MEX-5 localization in Caenorhabditis elegans embryos

Thesis (Ph. D.)--University of Washington, 2007.Specification of somatic and germline lineages in the nematode Caenorhabditis elegans requires the establishment of anterior-posterior polarity in early embryos. Polarization begins by a sperm-induced cue in 1-cell embryos that cortically localizes PAR polarity proteins, including the Ser/Thr kinases PAR-1 and PAR-4/LKB1. Capping at the anterior pole of non-muscle myosin and several PAR proteins leads to asymmetric localization of proteins such as MEX-5 and MEX-6. MEX-5,-6 are closely-related CCCH zinc finger proteins required for germline specification that are anteriorly localized in 1-cell embryos. While no direct targets of the PAR proteins have been described in C. elegans, MEX-5,-6 are proposed to function as key intermediaries in the transduction of polarity cues from PAR proteins to downstream cell fate regulators. To understand how MEX-5 asymmetry is established, I constructed a series of fusion proteins containing Green Fluorescent Protein (GFP) fused to all or part of the MEX-5 protein; these fusion proteins allowed me to monitor asymmetry in living embryos. Deletion analysis of GFP:MEX-5 identified a single residue, Ser458, that is necessary for anterior localization of GFP:MEX-5 in 1-cell embryos. MEX-5 is phosphorylated at Ser458 in vivo, and this phosphorylation occurs at the onset of MEX-5 expression in the gonad. In a screen of 41 Ser/Thr kinases, I found that only PAR-1 and PAR-4 are necessary for MEX-5 phosphorylation. PAR-1 kinase activity is required for the initial phosphorylation of MEX-5, as a kinase-dead allele of par-1 abolished all staining with anti-MEX-5(pSer458) in oocytes and embryos. PAR-4 kinase activity is required to maintain MEX-5 phosphorylation in mature oocytes; anti-MEX-5(pSer458) staining decreased progressively in par-4 mutant oocytes, and was present only at low levels in 1-cell embryos. While phosphorylation of MEX-5 is necessary for its asymmetry in 1-cell embryos, it is not sufficient; in par-1 alleles with mutations outside the kinase domain, MEX-5 was phosphorylated but remained symmetric. In summary, my research has described multiple roles for the PAR-1 and PAR-4 kinases in MEX-5 phosphorylation and localization. These results provide the first evidence of a role for the PAR kinases in setting up embryonic asymmetries prior to fertilization

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

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 show 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