C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis

Abstract

Maintenance of mitotically cycling germline stem cells (GSCs) is vital for continuous production of gametes. In worms and insects, signaling from surrounding somatic cells play an essential role in the maintenance of GSCs by preventing premature differentiation. In addition, germ cell proteins such as the Drosophila Pumilio and Caenorhabditis elegans FBF, both members of the PUF family translational regulators, contribute to GSC maintenance. FBF functions by suppressing GLD-1, which promotes meiotic entry. However, factors that directly promote GSC proliferation, rather than prevent differentiation, are not known. Here we show that PUF-8, another C. elegans member of the PUF family and MEX-3, a KH domain translational regulator, function redundantly to promote GSC mitosis. We find that PUF-8 protein is highly enriched in mitotic germ cells, which is similar to the expression pattern of MEX-3 described earlier. The puf-8(-) mex-3(-) double mutant gonads contain far fewer germ cells than both single mutants and wild-type. While these cells lack mitotic, meiotic and sperm markers, they retain the germ cell-specific P granules, and are capable of gametogenesis if GLP-1, which normally blocks meiotic entry, is removed. Significantly, we find that at least one of these two proteins is essential for germ cell proliferation even in meiotic entry-defective mutants, which otherwise produce germ cell tumors. We conclude PUF-8 and MEX-3 contribute to GSC maintenance by promoting mitotic proliferation rather than by blocking meiotic entry.

Fig. 1

PUF-8 is localized preferentially on the P granules of mitotic germ cells. Green fluorescence in panels L1–L4 shows the distribution pattern of PUF-8::GFP in the germline. L1–L3 — intact larva; L4 — dissected gonad. Yellowish fluorescence seen is due to the auto fluorescence of gut granules. All worms shown here are puf-8(−) mutant rescued with the PUF-8::GFP transgene. The lower panels are enlarged views of two germ cell nuclei from the above strain immunostained with P granule-specific antibodies; left — P granules, middle — PUF-8::GFP and the right — merged.

Fig. 2

PUF-8 and MEX-3 are essential for germ cell proliferation. (A) Dissected adult gonads of the indicated genotype stained with DAPI. Orientation of the gonad: Left — distal and Right — proximal (in this as well as in all other figures). (B) Germ cell proliferation during larval development visualized by the expression of GFP::PGL-1 transgene in live worms of the indicated genotype.

Fig. 3

PUF-8 and MEX-3 are essential for mitotic progression. Dissected adult gonads of the indicated genotypes immunostained with the mitotic markers, anti-REC-8 (left) or anti-PH3 (right) antibodies, and DAPI (red) are shown.

Fig. 4

PUF-8 and MEX-3 are not required for germ cell identity or entry into meiosis. (A) Dissected adult gonads of the indicated genotypes immunostained with antibodies against the germ cell marker GLH-1. (B) Similar gonads immunostained for the LIN-12/Notch receptor GLP-1, which inhibits entry into meiosis. (C) Intact worms stained with DAPI. The germ cells are outlined. Sperm are visible as small dots of DAPI staining only in glp-1(−) and puf-8(−) mex-3(-) glp-1(−) worms.

Fig. 5

puf-8(−) mex-3(−) double mutant germ cells do not initiate meiosis. Left panel: dissected adult gonads of the indicated genotypes immunostained with antibodies against the synaptonemal complex protein HIM-3. Right panel: similar gonads immunostained with antibodies against the sperm marker MO (red) and DAPI (green).