synMuv B proteins antagonize germline fate in the intestine and ensure C. elegans survival

Abstract

Previous studies demonstrated that a subset of synMuv B mutants ectopically misexpress germline-specific P-granule proteins in their somatic cells, suggesting a failure to properly orchestrate a soma/germline fate decision. Surprisingly, this fate confusion does not affect viability at low to ambient temperatures. Here, we show that, when grown at high temperature, a majority of synMuv B mutants irreversibly arrest at the L1 stage. High temperature arrest (HTA) is accompanied by upregulation of many genes characteristic of germ line, including genes encoding components of the synaptonemal complex and other meiosis proteins. HTA is suppressed by loss of global regulators of germline chromatin, including MES-4, MRG-1, ISW-1 and the MES-2/3/6 complex, revealing that arrest is caused by somatic cells possessing a germline-like chromatin state. Germline genes are preferentially misregulated in the intestine, and necessity and sufficiency tests demonstrate that the intestine is the tissue responsible for HTA. We propose that synMuv B mutants fail to erase or antagonize an inherited germline chromatin state in somatic cells during embryonic and early larval development. As a consequence, somatic cells gain a germline program of gene expression in addition to their somatic program, leading to a mixed fate. Somatic expression of germline genes is enhanced at elevated temperature, leading to developmentally compromised somatic cells and arrest of newly hatched larvae.

Fig. 1.

synMuv B mutants show temperature-sensitive expression of P-granule proteins in somatic cells. (A) PGL-1 (red) and PGL-3 (green) staining in wild-type (WT) and synMuv B mutant L1s at 26°C. Wild-type L1s display staining only in the two primordial germ cells (PGCs) Z2 and Z3 (arrowheads), whereas mutants show a range of ectopic expression. In some cells the levels of PGL-1 and PGL-3 differ (arrows). (B) PGL-3 staining in synMuv B mutants at 20°C and 26°C. PGCs (arrowheads). Scale bar: 10 μm.

Fig. 2.

Loss of germline chromatin modifiers suppresses high temperature arrest (HTA). L4 synMuv B mutants of the five genotypes shown were transferred to either empty vector or gene-specific RNAi bacteria at 26°C, and progeny were scored for HTA. n=41-146.

Fig. 3.

Germline and intestine genes are the predominant category of high temperature arrest (HTA) candidate genes. (A) The overlap of genes significantly upregulated in lin-35 versus wild type (WT) and those significantly downregulated in lin-35;mes-4(RNAi) versus lin-35 defines the HTA candidate genes. (B) Categories of HTA candidate genes based on published expression analysis (Reinke et al., 2004; Meissner et al., 2009; Wang et al., 2009) (see Materials and methods).

Fig. 4.

Meiosis genes are expressed in the soma of synMuv B mutants at 26°C. (A) REC-8 staining is bright in the primordial germ cells (PGCs; arrowheads) and undetectable or dim in the soma of wild type (WT), whereas synMuv B mutants show enhanced somatic staining. (B) HTP-3 staining is only detected in the PGCs (arrowheads) of wild type, whereas synMuv B mutants show somatic staining also, but only at 26°C. Scale bar: 10 μm.

Fig. 5.

Ectopic expression of HTP-3 begins in embryogenesis. HTP-3 (green) and ELT-2 (red) staining of wild-type (WT) and lin-9 mutant embryos at progressively later stages. HTP-3 staining is limited to the primordial germ cells (PGCs) in wild type after the E8 stage, but appears in additional somatic cells in lin-9 embryos. Scale bar: 10 μm.

Fig. 6.

Genes are more highly misexpressed in high temperature arrest (HTA)-prone genotypes. Quantitative RT-PCR of mRNA levels of eight HTA candidate genes in seven synMuv B mutant L1s relative to wild-type L1s at 20°C and 26°C. lin-9, lin-15B, lin 35 and lin-37 are HTA-prone mutants; lin-61 shows HTA at low frequency; lin-53 and lin-36 do not show HTA. Expression of the act-2 gene was used as an internal control. Black asterisks indicate a significant difference between the given genotype and wild type. Red asterisks indicate a significant difference between the given genotype and both wild type and lin-53. Black asterisks over a bracket indicate a significant difference between expression at 20°C and 26°C (P<0.05). Histograms are based on the mean of three biological replicates; error bars indicate s.e.m.

Fig. 7.

The intestine is responsible for high temperature arrest (HTA). (A) ELT-2 staining (green) highlights intestinal nuclei. In lin-15B and lin-35 mutant L1s the majority of somatic nuclei with HTP-3 staining (red) are intestinal nuclei. By contrast, in lin-52 mutant L1s the majority of somatic nuclei with HTP-3 signal are not intestinal. Red arrowheads indicate primordial germ cells (PGCs). Scale bar: 10 μm. (B) Gut granules, seen as increased birefringent material by differential interference contrast imaging, accumulate in the intestines of wild-type worms after 12 hours of feeding at either 26°C or 20°C. lin-15B and lin-35 mutant L1s are able to accumulate gut granules after feeding at 20°C but not after feeding at 26°C. Scale bar: 10 μm. See Fig. S4 in the supplementary material for autofluorescence images of gut granules. (C) Phenotypes of progeny of lin-35 mutant mothers, or after RNAi of lin-35 in wild type (WT) or RNAi-resistant rde-1 mutants or rde-1 mutants expressing RDE-1(+) in their intestine at 26°C. (D) A transgene array carrying lin-35(+) driven by the elt-2 promoter rescues HTA. From lin-35(n745); bnEx51[elt-2p::lin-35::GFP] mothers, the GFP− progeny that did not inherit the transgene arrested at high frequency at 26°C, whereas the GFP+ progeny that inherited the transgene arrested at low frequency.