HOP-1 Presenilin Deficiency Causes a Late-Onset Notch Signaling Phenotype That Affects Adult Germline Function in Caenorhabditis elegans

Abstract

Functionally redundant genes present a puzzle as to their evolutionary preservation, and offer an interesting opportunity for molecular specialization. In Caenorhabditis elegans, either one of two presenilin genes (sel-12 or hop-1) facilitate Notch activation, providing the catalytic subunit for the γ secretase proteolytic enzyme complex. For all known Notch signaling events, sel-12 can mediate Notch activation, so the conservation of hop-1 remains a mystery. Here, we uncover a novel "late-onset" germline Notch phenotype in which HOP-1-deficient worms fail to maintain proliferating germline stem cells during adulthood. Either SEL-12 or HOP-1 presenilin can impart sufficient Notch signaling for the establishment and expansion of the germline, but maintenance of an adult stem cell pool relies exclusively on HOP-1-mediated Notch signaling. We also show that HOP-1 is necessary for maximum fecundity and reproductive span. The low-fecundity phenotype of hop-1 mutants can be phenocopied by switching off glp-1/Notch function during the last stage of larval development. We propose that at the end of larval development, dual presenilin usage switches exclusively to HOP-1, perhaps offering opportunities for differential regulation of the germline during adulthood. Additional defects in oocyte size and production rate in hop-1 and glp-1 mutants indicate that the process of oogenesis is compromised when germline Notch signaling is switched off. We calculate that in wild-type adults, as much as 86% of cells derived from the stem cell pool function to support oogenesis. This work suggests that an important role for Notch signaling in the adult germline is to furnish a large and continuous supply of nurse cells to support the efficiency of oogenesis.

Keywords: Notch signaling; gene redundancy; germline; oogenesis; presenilin; proliferation.

Figure 1

HOP-1 is required for maximum fecundity and duration of reproductive span. (A) Plot of self-brood size for hermaphrodites with reduced hop-1 function. hop-1(+) activity is reduced in the hermaphrodite being assayed (zygotic genotype) as well as her mother (maternal genotype), as indicated; “−” represents the hop-1(ar179) null allele and “+” represents the wild-type (WT) allele. Full genotypes are (left to right): N2, hop-1(ar179), hop-1(ar179) derived from hop-1(ar179)/dpy-5(e61) unc-13(e1091) mothers, hop-1(ar179)/+; unc-32(e189)/+; him-5(e1490)/+ from hop-1(ar179); unc-32(e189) mothers mated to him-5(e1490) males, and hop-1(ar179)/dpy-5(e61) from hop-1(ar179)/dpy-5(e61) mothers. Horizontal lines indicate mean; n values are (left to right): 10, 17, 11, 14, and 9. Difference between the last two groups and WT are not statistically significant (P > 0.3); ** P < 0.005 and *** P < 0.001. (B) Plot of brood size for mated hermaphrodites (herms) or females. For each experimental pair, the animal indicated above the plot was mated with either hop-1(ar179) (red) or control hop-1(+) (gray) worms. Pair 1: total cross progeny brood produced by mating to fog-2(q71) females to indicated him-8(e1489) males. Pair 2: total brood produced by mating him-8(e1489) males to indicated young adult (day 1) hermaphrodites. Pair 3: cross progeny brood produced by mating him-8(e1489) males to indicated adult day 4 hermaphrodites after exhaustion of self-progeny [data are derived only from animals that successfully produced progeny, which was 90% for N2 day 4 hermaphrodites (n = 30) and 30% for hop-1 day 4 hermaphrodites (n = 33)]. Pair 4: cross progeny brood produced by mating him-8(e1489) males to indicated fog-2(q71) females. The control data for pair 1 and pair 4 (WT female × WT male) are the same, replotted for ease of comparison. Horizontal lines indicate mean; n values are (left to right): 17, 11, 7, 10, 10, 10, 17, and19. Differences between the two genotypes assayed within each pair are all statistically significant (P < 1 × 10⁻⁶), except for pair 1 (P = 0.32). (C) Graph of percentage of unmated or mated hermaphrodites that produce progeny (≥ 1) each day over 10 days of adulthood. hop-1(ar179) hermaphrodites in red and N2 hermaphrodites in blue; n = 10 for unmated N2, n = 14 for unmated hop-1(ar179), n = 14 for mated N2, and n = 17 for mated hop-1(ar179).

Figure 2

HOP-1 is required to maintain the germline proliferative zone in adulthood. (A) Representative hermaphrodite gonads dissected from day 2 (72 hr posthatching) and day 3 adults (96 hr posthatching); only the distal portion of each gonad arm is shown. Open arrow indicates distal tip of the gonad and closed arrow indicates distal boundary of the transition zone at which overt meiotic differentiation is recognized by crescent-shaped leptotene/zygotene nuclei. Bar, 20 µm. (B) Mean size of proliferative zone (number of nuclei) for gonads dissected from wild-type (WT) N2 and hop-1(ar179) hermaphrodites at different time points through adulthood at 20°. Under these conditions, hermaphrodites [WT and hop-1(ar179)] reach the L4/adult molt by 48–50 hr posthatching, and begin adulthood by 55 hr posthatching (see Materials and Methods for developmental timing measurements). N ≥ 20 dissected gonads for each data point except hop-1 day 4 adult (Ad), for which n = 15. Error bars are SEM. Difference between hop-1 and N2 mean is statistically significant at each time point except 55 hr (P = 0.66 for 55 hr and P < 0.0001 for all other time points). The penetrance of the hop-1(ar179) defect at each time point is shown in distribution charts in Figure S1 in File S1. (C) Mean size of proliferative zone (number of nuclei) for gonads dissected from WT, hop-1(ar179), and sel-12(ok2078) males at different time points from L4 through adulthood at 20°. All strains carry him-8(e1489). N ≥ 20 dissected gonads for each data point except day 3 Ad (n ≥ 16) and sel-12 day 4 Ad (n = 9). Error bars are SEM. Difference between WT and sel-12 mean is not statistically significant at any time point (P = 0.076 for 55 hr and P > 0.78 at all other time points). Difference between WT and hop-1 mean is statistically significant at all time points (P = 0.018 for 45 hr and P < 0.0001 for all other time points). (D) Representative gonads of WT, hop-1(ar179), and sel-12(ok2078) males [all him-8(e1489)]. The gonads of all L4 males contain mitotically dividing germ cells in the most distal region (the distal gonad is reflexed against the remainder of the gonad; examples of cells in the brief mitotic metaphase are indicated with arrows). Germ cells in the bend region of the gonad have entered meiosis, and those more proximally have developed into spermatocytes (solid arrowhead) and mature spermatids (open arrowhead). The same organization persists in day 2 Ad gonads of WT and sel-12 males, but not in hop-1 gonads where all germ cells have switched to meiosis and developed into spermatids. All animals are positioned with their anterior to the left, and gonads are outlined with a dotted line. Bar, 50 µm.

Figure 3

hop-1(ar179) suppresses the late-onset tumor caused by glp-1(ar202). glp-1(ar202) and hop-1(ar179); glp-1(ar202) hermaphrodites were raised at the permissive temperature (15°) through day 1 of adulthood, and then shifted to the restrictive temperature (25°) to induce glp-1(ar202) hyperactivity on day 2 of adulthood. The penetrance of the late-onset tumor phenotype, characterized by excess accumulation of undifferentiated germ cells, is plotted for glp-1(ar202) and for hop-1(ar179); glp-1(ar202) gonad arms, as assayed either 24 or 48 hr after temperature shift to 25°. Gonad arms were characterized according to the following criteria: Low = proliferative zone is normal or slightly enlarged, and meiotic cells are present in the mid- and proximal gonad; Med = proliferative zone extends well past the gonad bend, but meiotic cells are present in the proximal gonad; and High = proliferative zone fills entire gonad, and no meiotic cells are visible in the gonad (“full tumor”). N = 70, 82, 78, and 86 gonad arms (left to right).

Figure 4

Correlation of GLP-1/Notch activity with hermaphrodite fecundity. Mean brood size is plotted for glp-1(bn18)^ts animals shifted from 15 to 25° at the indicated time. N = 10–20 animals for each time point; error bars are SEM. The last data point represents continuous maintenance at 15° without a shift to 25°. Relative developmental stage of animals is indicated below the plot, as determined by examining the progression of vulval and gonad morphogenesis of additional glp-1(bn18)^ts animals at the indicated time points (see Materials and Methods). Horizontal dashed lines represent the comparable hop-1 brood size expected under these conditions: mean brood of 115 progeny (thick line) and range of 88–140 (thin lines) (see Materials and Methods and Figure S2 in File S1). Vertical dashed lines indicate corresponding times at which a temperature shift of glp-1(bn18)^ts animals would be expected to yield the hop-1-like brood size.

Figure 5

Timing of hop-1 phenotype onset is modified in animals with altered sel-12 dosage. Penetrance of male gonad proliferation defect is plotted at four different ages for each of four different genotypes that vary presenilin dosage. Dissected gonads are categorized by severity of phenotype according to size of the proliferation zone (number of nuclei distal to the meiotic transition zone). The sel-12 gene is located on the X chromosome and is not dosage compensated (Jans et al. 2009), so males are hemizygous, except for tra-2 pseudomales, which have two X chromosomes. n ≥ 22 gonad arms for each genotype at each time point. Age (left to right) for each genotype: 45, 55, 72, and 96 hr posthatching (20°). Full genotypes (left to right): him-8(e1489); sel-12(+)/ø, hop-1(ar179); him-8(e1489); sel-12(+)/ø, hop-1(ar179); him-8(e1489); sel-12(ok2078)/ø [no zygotic sel-12(+), and half dose of maternal sel-12(+)] hop-1(ar179); tra-2(e1095); sel-12(+)/sel-12(+). D, day.

Figure 6

Expression dynamics of sel-12 and hop-1 through development. RNA-sequencing expression data from ModENCODE (Gerstein et al. 2010) was used to calculate expression values (FPKM) by WormBase (http://www.wormbase.org, release WS260, date Aug 2017). The FPKM values for each experiment in each library in each developmental stage were averaged, and the median average FPKM value for two (or four for EE) independent poly-A RNA-Seq libraries is plotted here, with error bars representing the range of average FPKM values for each stage. The “soma L4” strain has no germline due to the glp-1(q224) mutation, which prevents germline expansion during larval development. EE, early embryo; LE, late embryo; FPKM, fragments per kilobase of transcript per million mapped reads.

Figure 7

hop-1(ar179) hermaphrodites produce enlarged oocytes. (A) Representative DIC images of live day 1 adult animals of indicated genotype (only one gonad arm is shown). The length of the most mature oocyte (−1 oocyte, double-headed arrow) and the adjacent spermatheca (arrowhead) are indicated. For reference, a gonad from a temperature-shifted glp-1(bn18)^ts animal [according to Nadarajan et al. (2009)] displays the enlarged oocyte phenotype (Looc). Black Bar, 50 µm. (B) Penetrance of the Looc phenotype, using the criteria of Nadarajan et al., whereby a gonad is considered to have the Looc phenotype if the volume of the −1 oocyte is at least 20% greater than the corresponding average wild-type volume (Nadarajan et al. 2009).

Figure 8

hop-1(ar179) and glp-1(bn18)^ts hermaphrodites have a slower rate of embryo production. (A) Mean embryo-laying rate for N2 and hop-1(ar179) hermaphrodites at 20°, as measured in a 6-hr window once a day for the first 4 days of adulthood. Error bars are SD; n = 17 for hop-1 and n = 12 for N2. (B) Continuous monitoring of embryo-laying rate for N2, hop-1(ar179), and glp-1(bn18)^ts hermaphrodites at 15°, as measured in 6–18-hr successive windows throughout the laying period. Error bars are SEM; n = 11 for N2, n = 14 for hop-1(ar179), and n = 17 for glp-1(bn18)^ts. Dashed lines connect average values for each time point; solid lines represent moving average with a period of two data points. Embryo-laying rates at 15° are roughly half of those at 20°.