A germ cell determinant reveals parallel pathways for germ line development in Caenorhabditis elegans

Abstract

Despite the central importance of germ cells for transmission of genetic material, our understanding of the molecular programs that control primordial germ cell (PGC) specification and differentiation are limited. Here, we present findings that X chromosome NonDisjunction factor-1 (XND-1), known for its role in regulating meiotic crossover formation, is an early determinant of germ cell fates in Caenorhabditis elegans. xnd-1 mutant embryos display a novel 'one PGC' phenotype as a result of G2 cell cycle arrest of the P4 blastomere. Larvae and adults display smaller germ lines and reduced brood size consistent with a role for XND-1 in germ cell proliferation. Maternal XND-1 proteins are found in the P4 lineage and are exclusively localized to the nucleus in PGCs, Z2 and Z3. Zygotic XND-1 turns on shortly thereafter, at the ∼300-cell stage, making XND-1 the earliest zygotically expressed gene in worm PGCs. Strikingly, a subset of xnd-1 mutants lack germ cells, a phenotype shared with nos-2, a member of the conserved Nanos family of germline determinants. We generated a nos-2 null allele and show that nos-2; xnd-1 double mutants display synthetic sterility. Further removal of nos-1 leads to almost complete sterility, with the vast majority of animals without germ cells. Sterility in xnd-1 mutants is correlated with an increase in transcriptional activation-associated histone modification and aberrant expression of somatic transgenes. Together, these data strongly suggest that xnd-1 defines a new branch for PGC development that functions redundantly with nos-2 and nos-1 to promote germline fates by maintaining transcriptional quiescence and regulating germ cell proliferation.

Keywords: C. elegans; Germ line; Nanos; Primordial germ cells; Proliferation; XND-1.

Fig. 1.

XND-1 is expressed in primordial germ cells. (A-C) C. elegans embryos stained with anti-XND-1 (magenta), anti-PGL-1 (white) to mark the germ lineage, and DAPI (green) to show embryonic nuclei. All embryos are oriented with anterior to the left. (A-A‴) Wild-type embryos showed XND-1 expression in P₄ and its daughters, Z2 and Z3. (A) Inset shows that XND-1 was absent in the germline blastomere of 12-cell stage embryos, (A′-A‴) whereas its accumulation and expression was seen in the cytoplasm and the nucleus of the P₄ cell at the 40-cell stage. (B) xnd-1 M+Z− embryos showing maternal XND-1 expression similar to that in wild type. (C) Zygotic expression of XND-1 was assessed by staining embryos resulting from a cross of 3×FLAG::GFP::xnd-1 transgenic males with wild-type hermaphrodites. XND-1 was not detected until the 310-cell stage (C′), at which time its accumulation was seen in the cytoplasm of PGCs. Shortly thereafter, at the ∼320+cell stage, XND-1 localization became nuclear (C″,C‴). Scale bars: 5 µm.

Fig. 2.

xnd-1 is essential for germ cell proliferation. Photomicrograph of age-matched adult animals stained with DAPI to show the germ lines (dotted lines, distal to the left). (A) Zygotic loss of xnd-1 led to reduced germline size. Compare wild type (top) with the smaller xnd-1 M+Z− (bottom). (B) Comparison of germline size in xnd-1 M−Z+ (top) versus xnd-1 M−Z− (bottom) revealed XND-1 zygotic rescue of germ cell proliferation defects. Anterior is to the left. Scale bars: 20 µm. mit, mitotic zone; TZ, transition zone; P, pachytene; Dp, diplotene.

Fig. 3.

xnd-1 mutant embryos missegregate P granules and exhibit PGCs defects. (A) C. elegans embryos stained with anti-PGL-1 (magenta) and DAPI (green). Anterior is to the left. xnd-1 mutant embryos missegregate P granules into the D cell and its daughters. (B) Classification of 100+ cell stage xnd-1(−) embryos based upon the number of blastomeres having PGL-1 staining, shown in magenta in photomicrograph to the right of the graph. (1) One PGC: Embryos in this class have only one PGL-1-positive cell. (2) One PGC+somatic: Embryos have one blastomere with strong PGL-1 staining (PGC) and one or more additional blastomeres with weak PGL-1 staining (presumptive somatic cell). (3) Two PGCs: Embryos with two PGL-1-positive cells. (4) Two PGCs+somatic: Embryos in this class have two blastomeres with strong PGL-1 staining and one or more blastomeres with weak PGL-1 staining. Scale bars: 5 µm.

Fig. 4.

xnd-1 regulates the PGC cell cycle. (A) L1 larvae expressing PGL-1::GFP (green) and mCherry::H2B (red). L1 larvae with two (top) or one PGCs (bottom) can easily be identified. Insets show maximum projection z-stacks of the PGCs. Arrows point to the PGCs. (B) Snapshots from time-lapse movies of wild-type and xnd-1(–­) embryos expressing PGL-1::GFP (green) to mark germline blastomeres (yellow arrows) and mCherry::H2B (red) to mark nuclei. Time stamp is in h:m:s:ms format. (C,D) Wild-type and xnd-1(−) embryos of 550-cell stage stained with anti-PGL-1 to mark the PGCs (white), anti-CYB-1 to detect Cyclin B (red) and DAPI to mark nuclei (green). (E-N) Wild-type and xnd-1(−) embryos of indicated cell stages stained with anti-PGL-1 to mark the PGC (white), anti-pCDK-1 to mark cells arrested in G2 (red) and DAPI to mark nuclei (green). Wild-type or xnd-1(−) embryos (>100-cell stage) with two PGCs never showed accumulation of pCDK-1 in the PGC (G,I-L), whereas xnd-1(−) embryos (>100-cell stage) with one PGC showed strong PGC accumulation of pCDK-1 (H,M,N). Scale bars: 5 µm.

Fig. 5.

xnd-1(−) and nos-2(−) animals are sterile and lack germ lines. Graph of sterility and percentage of no germ cell animals of the indicated genotypes (See Table S2 for more details).

Fig. 6.

xnd-1(−) PGCs show hallmarks of aberrant transcriptional activation. (A-C) H3K4me2 persists and accumulates in Z2/Z3 of xnd-1 and nos-2 late embryos. (A) Photomicrographs show close-ups of PGCs in >100-cell embryos of the indicated genotypes. Embryos were stained with anti-PGL-1 to mark PGCs (blue), anti-H3K4me2 (red) and DAPI to mark nuclei (green). Scale bars: 2 µm. (B) The percentage of embryos with H3K4me2-positive PGCs for the indicated genotypes. Data are represented as mean±s.d. (C) The correlation between the percentage embryos with H3K4me2-positive PGCs with the percentage sterility for the indicated genotypes. (D) unc-119::GFP and unc-33::GFP expression in the germ lines of wild-type and xnd-1 M−Z− adults. Germ lines are outlined and arrowheads point to the distal tip cells. Both transgenes are aberrantly expressed in xnd-1 mutant animals from mid-pachytene through diplotene. Adult gonad images were obtained as multiple images and then merged to create a final image of an entire gonad. Scale bars: 20 µm.

Fig. 7.

Models for the control of germline fates by XND-1 and NOS-2. In Model 1, XND-1 and NOS-2 regulate common targets via different modes of regulation, transcriptional regulation by XND-1 and post-transcriptional regulation by NOS-2. In Model 2, XND-1 and NOS-2 regulate distinct sets of targets genes which are both required to confer fertility.