Chapter 14 Whole-Mount in Situ Hybridization for the Detection of RNA in Caenorhabditis elegans Embryos

doi:10.1016/S0091-679X(08)61394-1

Methods in Cell Biology

Volume 48, 1995, Pages 323-337

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This chapter discusses the whole-mount in situ hybridization for the detection of RNA in Caenorhabditis elegans embryos. In situ hybridization to RNA is an effective tool for the analysis of gene expression during development. This technique is particularly important for Caenorhabditis elegans, as isolation of RNA from specific tissues or developmental stages is generally not possible in this organism. The availability of the complete cell lineage and the reproducibility of cell positions from one animal to the next allow RNA expression patterns to be analyzed at the level of individual cells. A number of in situ hybridization protocols have been developed for the detection of RNA in squashed, dissected, or sectioned tissues of C. elegans. More recently, protocols using whole-mount preparations of C. elegans have also been described. By preserving the three-dimensional structure of the specimen, whole-mount preparations facilitate the identification of specific cells and the analysis of complex expression patterns. This chapter describes a protocol for detection of RNA in whole-mount C. elegans embryos. This procedure is based on protocols for Drosophila, which makes use of highly sensitive digoxigenin-labeled probes.

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In situ hybridisation of juveniles and adults was performed according to the procedure of de Boer et al. (1998), with some modifications. Briefly, more than 10,000 mixed-stage B. xylophilus were fixed in 4% paraformaldehyde at 4 °C for 18 h, followed by an additional incubation at 25 °C for 5 h. Furthermore, in situ hybridisation of eggs was conducted as described by Seydoux and Fire (1995) with some changes; the fixation and hybridisation of eggs were carried out in 1.5 mL tubes instead of the polylysine-coated slides. Hybridisation signals within the nematodes were detected with alkaline phosphatase-conjugated anti-DIG antibody and substrate, and observed using an Olympus BX51 microscope equipped with a DP72 Digital Camera (Olympus, Japan).
The glycoside hydrolase family 18 (GH18) of chitinases is a gene family widely expressed in archaes, prokaryotes and eukaryotes, and hydrolyzes the β-1,4-linkages in chitin. The pinewood nematode Bursaphelenchus xylophilus is one of the organisms that produces GH18 chitinases. Notably, B. xylophilus has a higher number of GH18 chitinases compared with the obligate plant-parasitic nematodes Meloidogyne incognita and Meloidogyne hapla. In this study, seven GH18 chitinases were identified and cloned from B. xylophilus based on genomic analyses. The deduced amino acid sequences of all these genes contained an N-terminal signal peptide and a GH18 catalytic domain. Phylogenetic analysis showed that the origin of B. xylophilus GH18 chitinases was independent of those from fungi and bacteria. Real-time quantitative reverse transcription PCR analysis indicated that GH18 chitinase genes had discrete expression patterns, representing almost all the life stages of B. xylophilus. In situ hybridisation showed that the mRNA of GH18 chitinase genes of B. xylophilus were detected mainly in the spermatheca, esophageal gland cells, seminal vesicle and eggs. RNA interference (RNAi) results revealed different roles of GH18 chitinase genes in B. xylophilus. Bx-chi-1, Bx-chi-2 and Bx-chi-7 were associated with reproduction, fungal cell-wall degradation and egg hatching, respectively. Bx-chi-5 and Bx-chi-6 may be involved in sperm metabolism. In conclusion, this study demonstrates that GH18 chitinases have multiple functions in the life cycle of B. xylophilus.
Stabilization of RNT-1 protein, runt-related transcription factor (RUNX) protein homolog of Caenorhabditis elegans, by oxidative stress through mitogen-activated protein kinase pathway
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Worms were fed by each RNAi E. coli strain on NGM plates containing 1 mm isopropyl 1-thio-β-d-galactopyranoside. Detection of rnt-1 transcripts in embryos, larvae, and adult worms was performed as described in Ref. 25. Digoxigenin-dideoxy UTP-labeled oligonucleotide was synthesized, followed by the protocol (Bionex, Korea).
RUNX proteins are evolutionarily conserved transcription factors known to be involved in various developmental processes. Here we report a new role for a RUNX protein: a role in stress response. We show that RNT-1, the Caenorhabditis elegans RUNX homolog, is constantly produced and degraded by the ubiquitination-proteasome pathway in the intestine of the nematode. RNT-1 was rapidly stabilized by oxidative stress, and the rnt-1-mutant animals were more sensitive to oxidative stress, indicating that rapid RNT-1 stabilization is a defense response against the oxidative stress. The MAP kinase pathway is required for RNT-1 stabilization, and RNT-1 was phosphorylated by SEK-1/PMK-1 in vitro. ChIP-sequencing analysis revealed a feedback loop mechanism of the MAP kinase pathway by the VHP-1 phosphatase in the RNT-1-mediated oxidative stress response. We propose that rnt-1 is regulated at the protein level for its role in the immediate response to environmental challenges in the intestine.
Immunofluorescence Microscopy
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Immunolocalization also complements other molecular studies of gene expression. Whereas Northern blots and RNA in situ hybridization studies (Seydoux and Fire, 1995) provide important information on the transcriptional regulation of particular genes, protein immunofluorescence reveals where the majority of the endogenous protein is actually localized and presumably functions. In some cases, striking differences between the two patterns arise from posttranscriptional regulatory mechanisms (Merritt et al., 2008, Evans et al., 1994).
Immunofluorescence microscopy is a powerful technique that is widely used by researchers to assess both the localization and endogenous expression levels of their favorite proteins. The application of this approach to C. elegans, however, requires special methods to overcome the diffusion barrier of a dense, collagen-based outer cuticle. This chapter outlines several alternative fixation and permeabilization strategies for overcoming this problem and for producing robust immunohistochemical staining of both whole animals and freeze-fractured samples. In addition, we provide an accounting of widely used antibody reagents available to the research community. We also describe several approaches aimed at reducing non-specific background often associated with immunohistochemical studies. Finally, we discuss a variety of approaches to raise antisera directed against C. elegans antigens.
In situ Hybridization of Embryos with Antisense RNA Probes
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Citation Excerpt :
Newer transgene protocols may overcome some of these limitations (Giordano-Santini et al., 2010; Praitis et al., 2001; Schlager et al., 2009; Semple et al., 2010); however, it remains to be seen whether transgenes in other nematode species will in general be as reliable as those seen in C. elegans. Historically, in situ detection of mRNA has relied on detection of colorimetric or fluorescent signals from localized antisense DNA probes in whole-mount embryos (Seydoux and Fire, 1995; Tabara et al., 1996). For low-abundance transcripts, signal amplification can be used (Bobrow and Moen, 2001).
Detection of transcripts in situ is a rapid means by which gene expression can be characterized in many systems. In the nematode, Caenorhabditis elegans, the ease with which transgenics can be made and the general reliability of reporter fusion expression patterns, have made this technique comparatively less popular than in other systems. There are, however, still applications in which in situ hybridization is desired, such as for maternally expressed genes, or in related species without established transgene methods. The most frequently used method of in situ hybridization uses DNA probes and formaldehyde fixation. A newer approach that permits single-transcript detection has been reported and will not be described here (Raj and Tyagi, 2010). Rather, we describe an alternative protocol that uses RNA probes with a different fixative. This approach has been applied to C. elegans and related nematodes, providing reliable, sensitive detection of endogenous transcripts.
Structure and evolution of the C. elegans embryonic endomesoderm network
2009, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
The specification of the Caenorhabditis elegans endomesoderm has been the subject of study for more than 15 years. Specification of the 4-cell stage endomesoderm precursor, EMS, occurs as a result of the activation of a transcription factor cascade that starts with SKN-1, coupled with input from the Wnt/β-catenin asymmetry pathway through the nuclear effector POP-1. As development proceeds, transiently-expressed cell fate factors are succeeded by stable, tissue/organ-specific regulators. The pathway is complex and uses motifs found in all transcriptional networks. Here, the regulators that function in the C. elegans endomesoderm network are described. An examination of the motifs in the network suggests how they may have evolved from simpler gene interactions. Flexibility in the network is evident from the multitude of parallel functions that have been identified and from apparent changes in parts of the corresponding network in Caenorhabditis briggsae. Overall, the complexities of C. elegans endomesoderm specification build a picture of a network that is robust, complex, and still evolving.
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In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1–P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

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Methods in Cell Biology

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Bibliography of Books (19)

Posterior pattern formation in C. elegans involves position-specific expression of a gene containing a homeobox

Cell

Location of specific messenger RNAs in C. elegans by cytological hybridization

Dev. Biol.

Translational control of maternal glp-1 mRNA establishes an asymmetry in the C. elegans embryos

Cell

Ontogeny of maternal and newly transcribed mRNA analyzed by in situ hybridization during development of C. elegans

Dev. Biol.

Post-embryonic cell lineages of the hermaphrodite and male gonads in C. elegans

Dev. Biol.

Cell-specific transcriptional regulation of the major sperm protein in C. elegans

Dev. Biol.

Postembryonic cell lineages of the nematode Caenorhabditis elegans

Dev. Biol.

The embryonic cell lineage of the nematode Caenorhabditis elegans

Dev. Biol.

Postranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans

Cell

Cited by (69)

Versatile glycoside hydrolase family 18 chitinases for fungi ingestion and reproduction in the pinewood nematode Bursaphelenchus xylophilus

Stabilization of RNT-1 protein, runt-related transcription factor (RUNX) protein homolog of Caenorhabditis elegans, by oxidative stress through mitogen-activated protein kinase pathway

Immunofluorescence Microscopy

In situ Hybridization of Embryos with Antisense RNA Probes

Structure and evolution of the C. elegans embryonic endomesoderm network

Global Transcriptional Repression in C. elegans Germline Precursors by Regulated Sequestration of TAF-4