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

HOP-1 is necessary for maximum fecundity in hermaphrodites

hop-1 mutant hermaphrodites are self-fertile and produce viable progeny (Li and Greenwald 1997); however, the extent of germline function in these animals had not been investigated. We first probed germline output in HOP-1-deficient hermaphrodites by measuring the number of self-progeny produced by hermaphrodites that lacked zygotic and maternal hop-1(+) activity. For our analyses, we used the hop-1(ar179) allele, which contains an internal deletion that removes most of the coding sequence (see Materials and Methods). On average, hop-1(ar179) hermaphrodites were found to produce 56% the number of self-progeny produced by wild-type hermaphrodites in similar conditions (Figure 1A). The reduced self-brood size of hop-1(ar179) is a recessive phenotype, and either zygotically or maternally supplied hop-1(+) activity can contribute to increasing brood size, with the largest effect coming from the zygotic contribution (Figure 1A). Two independent missense alleles of hop-1 also cause a reduced fecundity phenotype: hop-1(gk862343) hermaphrodites have an average brood size of 172 ± 40 (n = 9) and hop-1(gk751127) hermaphrodites have an average brood size of 259 ± 40 (n = 14).

Decreased fecundity of hop-1 mutants can be attributed to decreased oocyte production and/or function

The self-brood of a hermaphrodite is normally limited by the number of sperm produced, so we compared sperm production in wild-type and HOP-1-deficient hermaphrodites. Sperm are stored in two spermathecas, one in each of two gonad arms. At the beginning of adulthood, hop-1(ar179) hermaphrodites contained an average of 152 ± 31 sperm per gonad arm (n = 34), which is not significantly different from that of wild-type hermaphrodites [161 ± 16 (n = 7); P = 0.278] and should be sufficient to produce a wild-type sized brood (2 × 152 progeny). Thus, the fully penetrant fecundity defect of hop-1(ar179) hermaphrodites cannot be explained by insufficient sperm production.

To test the functionality of sperm produced in the absence of hop-1(+) function, we turned to hop-1(ar179) males. Although this is not a direct test of hermaphrodite sperm function, sperm development proceeds similarly in hermaphrodites and males, and therefore we reasoned that a major role for HOP-1 in sperm development might be revealed by this assay. Young hop-1(ar179) males were found to sire broods to equivalent size as those sired by wild-type males under the same mating conditions, indicating that there was no significant defect in the functionality of the sperm produced by HOP-1-deficient males (Figure 1B, first pair).

To eliminate the role of sperm in brood size, exogenously supplied wild-type sperm were introduced into hop-1(ar179) hermaphrodites by mating. If the hop-1 brood defect were caused by defective hermaphrodite sperm, then hop-1(ar179) animals would be expected to yield wild-type broods when mated to wild-type males. We found the opposite result: the unlimited supply of sperm allowed wild-type hermaphrodites to achieve a mean brood size of 796 ± 98 (n = 7), while mated hop-1(ar179) hermaphrodites yielded an average brood size of only 242 ± 66 (n = 10) (Figure 1B, second pair).

To completely remove the contribution of hermaphrodite-produced sperm, brood production was analyzed for two types of mated hop-1 animals that lacked their own sperm: either hop-1(ar179) hermaphrodites that had exhausted their own sperm supply (day 4 adults) or female hop-1(ar179) animals that produce only oocytes due to the feminizing fog-2(q71) mutation. In both cases, the fecundity of hop-1(ar179) animals was dramatically reduced relative to otherwise wild-type animals (Figure 1B, third and fourth pairs), suggestive of a defect in oocyte production or function.

In wild-type hermaphrodites, progeny production continues until the sperm supply is exhausted. We reasoned that if oocyte production is compromised in hop-1(ar179) animals, then hop-1(ar179) hermaphrodites that had ceased to produce progeny might contain unutilized sperm. To this end, we analyzed DAPI-stained gonads from day 4 and day 6 adult hermaphrodites. At this age, almost all wild-type hermaphrodite gonads are devoid of spermatids (1.8% N2 gonads contain > 10 spermatids; n = 109). In contrast, 43% of hop-1(ar179) gonads contain > 10 spermatids (n = 106), and most of these have > 20 spermatids, which was never observed among the wild-type gonads. Although we cannot eliminate a possible role for HOP-1 in hermaphrodite sperm function, our results demonstrate an important role for HOP-1 in oocyte production and/or function, and defects in this role could fully account for the fecundity defect of hop-1(ar179) hermaphrodites.

HOP-1-deficient animals have reduced reproductive span

Optimal oocyte production/function could require HOP-1 function throughout the life of a hermaphrodite. However, an alternative model is suggested by the above mating experiments, in which the hermaphrodite reproductive span was extended by the presence of an unlimited sperm supply. Under these conditions, the need for HOP-1 function is more pronounced in older animals than in younger animals. For example, day 4 adult hop-1(ar179) hermaphrodites showed the most severe defect in fecundity: only 30% produced cross progeny upon mating, and most of these produced < 25 progeny (n = 33). In contrast, 90% of wild-type day 4 adult hermaphrodites produced cross progeny, and most produced > 300 progeny (n = 30) (Figure 1B). These results suggest that the role of HOP-1 in oocyte production/function might become increasingly important with age, and thus play a key role in allowing an extended reproductive span in the presence of unlimited sperm.

To directly assess reproductive span, we followed hop-1(ar179) hermaphrodites over the course of 10 days of adulthood in the presence of wild-type males, and measured the proportion of animals that produce progeny each day. Because males are present, sperm are no longer limiting, and the duration of the reproductive span can be measured. In these experiments, > 90% of control wild-type hermaphrodites continued producing progeny through 8 days of adulthood. In contrast, none of the hop-1(ar179) hermaphrodites continued to produce progeny after the sixth day, and most produced their last progeny on the fourth day of adulthood (Figure 1C). This analysis demonstrates that HOP-1 function allows a roughly twofold extension of the reproductive span of hermaphrodites.

HOP-1 is necessary for sustained germ cell proliferation in the adult

In hermaphrodites, sperm and oocytes are derived from a progenitor stem cell population that proliferates in the distal end of each of two gonad arms. Proliferation begins during early larval development and continues throughout adulthood, generating > 1000 germ cell progenitors per gonad arm (Kimble and White 1981). Maintenance of a proliferative germline stem cell pool is dependent on Notch signaling from the somatic distal tip cell (DTC), which surrounds the distal region of the gonad (Austin and Kimble 1987; Henderson et al. 1994). In response to Notch ligand, the underlying germ cell progenitor cells maintain an undifferentiated mitotic fate, but as they move proximally through the gonad, they evade the effects of Notch signaling from the DTC and transition to meiosis. The first 40 germ cells to enter meiosis do so during the L3–L4 stages, and ultimately differentiate into sperm. All germ cells that enter meiosis subsequently will take on an oogenic fate, which includes oocytes and nurse cells [for review see Hansen and Schedl (2013), Pasternak et al. (2004)]. The adult gonad contains an assembly line of germ cells from the most distal pool of proliferating stem cells to the most proximal mature oocyte. Morphological changes in early meiotic prophase nuclei can be used to estimate where the transition to meiosis occurs along the distal–proximal axis of the gonad. The region distal to this transition zone is referred to as the proliferative zone, and contains proliferating germ cell progenitors as well as germ cells in meiotic S phase in preparation for entry into meiotic prophase (Hansen et al. 2004; Crittenden et al. 2006; Maciejowski et al. 2006; Cinquin et al. 2010; Fox et al. 2011). Excessive Notch signaling increases the size of the proliferative zone, as meiotic entry is delayed to a more proximal position (Berry et al. 1997; Pepper et al. 2003). Conversely, decreased Notch signaling results in a smaller proliferative zone because germ cells enter meiosis prematurely, diminishing the size of the stem cell pool (Austin and Kimble 1987; Michaelson et al. 2010; Fox and Schedl 2015). In extreme cases, where no GLP-1/Notch receptor activity is present, germline proliferation fails to occur, and instead the first several larval germ cells enter meiosis and differentiate into sperm. In this case, no other germ cells—mitotic or meiotic—populate the adult gonad, resulting in a sterile gonad (Austin and Kimble 1987). The same phenotype is observed upon depletion of both HOP-1 and SEL-12 presenilins (Li and Greenwald 1997; Westlund et al. 1999; Francis et al. 2002), demonstrating the essential role of presenilin function in germline Notch signaling.

The fact that both sperm and oocytes are produced by HOP-1-deficient hermaphrodites indicates that sufficient Notch signaling occurs in the distal gonad to prevent meiotic entry and allow at least some germ cell proliferation. However, the reduced fecundity of hop-1(ar179) hermaphrodites led us to hypothesize that either the level, or the duration, of Notch signaling might be reduced in a HOP-1-deficient gonad, resulting in a smaller number of proliferating germ cells. We measured the size of the proliferative zone in gonads dissected from hop-1(ar179) hermaphrodites over the course of adulthood, and compared them to those of similarly aged wild-type hermaphrodites. Almost all hop-1(ar179) hermaphrodites began adulthood with a gonad proliferative zone that was comparable in size to that of wild-type young adults; however, all hop-1(ar179) gonads failed to maintain this proliferative zone through adulthood (Figure 2, A and B and Supplemental Material, Figure S1 in File S1). Within the first 5–10 hr of adulthood, the mean proliferative zone size of hop-1 gonads dropped to only 53% that of similarly aged wild-type gonads (60 hr posthatch). At this time point, only 52% of hop-1(ar179) young adult gonads had a proliferative zone that contained > 100 germ cell progenitors (compared to 100% for N2 gonads), and 29% lacked a proliferative zone all together [n = 21 for hop-1(ar179) and n = 24 for N2; see Figure S1 in File S1]. The penetrance of this phenotype increased as the hop-1(ar179) hermaphrodites aged, such that by day 3 of adulthood, no hop-1(ar179) gonads contained > 50 nuclei in the proliferative zone, and most gonads had morphologically distinguishable meiotic nuclei in the distal-most region of the gonad, indicating premature meiotic entry of germ cell progenitors (Figure 2A and Figure S1 in File S1). In comparison, wild-type day 3 or day 4 adult hermaphrodites all maintained a proliferative zone of ≥ 100 nuclei (Figure 2, A and B and Figure S1 in File S1). These results demonstrate that the key contribution of the HOP-1 presenilin is not in the establishment of a germ cell proliferative population, but rather in the maintenance of this proliferating pool through adulthood.

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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.

Analysis of male hop-1(ar179) gonads also revealed HOP-1 dependence for the maintenance of an adult stem cell population. hop-1(ar179) male gonads successfully established a proliferative zone during larval development, but the proliferative zone was quickly depleted during the end of larval development and early adulthood (Figure 2C). The gonads of almost all day 2 adult males contained only differentiated spermatocytes and spermatids, even in the distal-most region of the gonad, where Notch signaling would normally maintain the undifferentiated germ cell progenitor fate. In contrast, similarly aged wild-type adult males maintained a distal population of undifferentiated germ cells.

The absolute need for HOP-1 function to maintain the adult germline stem cell pool demonstrates that SEL-12 and HOP-1 are not functionally redundant for this role, but it is possible that they are both additively required, and that SEL-12-deficient gonads might also display a similar adult depletion of the gonad proliferative zone. We analyzed the gonads of young adult sel-12(ok2078) hermaphrodites and found that the proliferative zone was comparable to wild-type (average of 235 ± 36 nuclei for 55 hr and 240 ± 49 nuclei for 60 hr; n = 21 for each time point; P > 0.1 for N2 vs. sel-12 at both time points). Unfortunately, we were unable to accurately assess the persistence of this proliferative zone through adulthood because the sel-12 egg-laying defect caused accumulating embryos to impinge upon the gonad, ultimately leading to matricide. Instead, to directly compare SEL-12- and HOP-1-deficient gonads through adulthood, we turned to male gonads. Unlike male hop-1(ar179) gonads, sel-12(ok2078) male gonads were found to maintain a proliferative zone of wild-type size through at least day 4 of adulthood (Figure 2, C and D). These results demonstrate that the SEL-12 presenilin is entirely dispensable for maintaining germ cell proliferation in adulthood, and indicate that the HOP-1 presenilin is both necessary and sufficient for this role.

HOP-1 is necessary for GLP-1/Notch signal transduction in the adult gonad

A likely explanation for the loss of germ cell proliferation in adult hop-1(ar179) gonads is that the Notch receptor cannot be activated in adult HOP-1-deficient gonads. We tested this hypothesis by monitoring the ability of a hyperactive glp-1/Notch allele to induce late-onset “germline tumors” in adult HOP-1-deficient gonads. The glp-1(ar202) point mutation causes temperature-sensitive hyperactivity of the glp-1/Notch receptor that is still dependent on γ secretase activity (Pepper et al. 2003). Excessive Notch activation throughout the gonad leads to ectopic germ cell proliferation that, in extreme cases, is described as a germline tumor because the gonad becomes filled with proliferating germ cells at the expense of differentiating germ cells. To focus our inquiry on adult glp-1/Notch activation, we allowed hop-1(ar179); glp-1(ar202) hermaphrodites to complete larval development and day 1 of adulthood at the permissive temperature, and then shifted them to the restrictive temperature to induce the hyperactivity of the glp-1(ar202) allele in day 2 adults. The effect of adult glp-1(ar202) induction can be seen by comparing gonads after 24 and 48 hr at the restrictive temperature. In an otherwise wild-type background, the percentage of gonads exhibiting excessive proliferation at the expense of meiotic differentiation increased from 21% at 24 hr to 66% after 48 hr; similarly, the percentage of tumorous gonads increased from 5.7% at 24 hr to 24% at 48 hr (Figure 3). In contrast, in a HOP-1-deficient background, the penetrance of these phenotypes changed very little between 24 and 48 hr (Figure 3), indicating that in HOP-1-deficient adults, glp-1(ar202) cannot be activated. In these experiments, we cannot fully eliminate residual glp-1(ar202) hyperactivation that might have occurred during larval development at the permissive temperature. Nonetheless, the ability of the hop-1(ar179) mutation to suppress glp-1(ar202)-induced late-onset germline tumors supports the conclusion that Notch signal transduction in adult germ cells requires the HOP-1 presenilin.

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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).

The hop-1 mutant phenotype is a late-onset glp-1/Notch mutant phenotype

We propose that the hop-1(ar179) phenotype represents a novel late-onset glp-1/Notch phenotype, in which Notch signaling is functional in the early gonad but nonfunctional later in adulthood. This scenario would explain how hop-1 animals can successfully establish and expand a germline, but cannot maintain it in adulthood. In contrast, SEL-12-deficient gonads can both establish and maintain the germ cell stem cell fate, and gonads that lack both HOP-1 and SEL-12 function fail to establish a proliferative germline at all (Li and Greenwald 1997; Westlund et al. 1999). Given these results, we propose that the wild-type germline normally switches from a dual presenilin mode, in which either HOP-1 or SEL-12 executes Notch activation, to an exclusive HOP-1 presenilin mode, thus accounting for the late-onset Notch phenotype of HOP-1-deficient gonads.

If a late-onset Notch defect is responsible for the hop-1 fecundity defect, then we predicted that a hop-1-like brood could be phenocopied in a wild-type hermaphrodite by experimentally turning off Notch signaling at the time at which the gonad normally switches from dual presenilin to HOP-1 dependence. We used a loss-of-function temperature-sensitive glp-1(bn18)^ts allele (Kodoyianni et al. 1992; Qiao et al. 1995) to test this idea, and to estimate the developmental timing of the hypothesized switch. glp-1(bn18)^ts hermaphrodites were shifted from permissive (15°) to restrictive temperature (25°) at different times through development and adult life, and fecundity was then assessed by measuring self-broods. The results of this analysis reveal a clear linear relationship between the amount of time spent at permissive temperature (functional Notch signaling) and fecundity (Figure 4). This correlation is consistent with the well-established relationship between Notch signaling and the persistence of the stem cell pool (Austin and Kimble 1987), and establishes the minimum time required for Notch signaling to allow hermaphrodite self-fertility. Since all animals shifted during late L3 (60 hr posthatching at 15°, n = 19) generated ≥ 14 progeny, with an average brood size of 40 progeny, we conclude that Notch signaling through the L3 stage is sufficient to support self-fertilization in the hermaphrodite gonad (Figure 4).

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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.

To estimate the developmental time point at which animals normally switch from dual presenilin to HOP-1, we asked which of the temperature-shifted populations (represented in Figure 4) most closely recapitulated a hop-1-like brood. Additional analysis of glp-1(bn18)^ts animals maintained at the permissive temperature (see below and Figure S2 in File S1) revealed that the embryo output rate was slightly slower than that of glp-1(+) animals at the same temperature, and thus for our interpretation, we adjusted the expected hop-1-like brood size accordingly. By the time that wild-type hermaphrodites produce 190 progeny [the mean brood size of hop-1(ar179) animals at 15° is 190 ± 38, n = 8], glp-1(bn18)^ts animals have produced an average of 115 progeny (Figure S2 in File S1); thus, we used 115 as our benchmark for the size of brood that should be attainable by glp-1(bn18) animals before switching to HOP-1 dependence. By these criteria, the low-fecundity phenotype of hop-1 was best phenocopied by glp-1(bn18)^ts animals that were shifted to restrictive temperature during the mid-L4 stage (80 hr posthatch at 15°), pinpointing mid-L4 as the developmental time point for the switch between the dual presenilin and HOP-1 modes.

The large range of brood sizes and proliferative zone measurements observed for hop-1(ar179) hermaphrodites may reflect variation in the exact timing of the switch to HOP-1 dependency (compare wild-type and hop-1 distribution, and error bars in Figure 1A and Figure 2, B and C). Using a glp-1(bn18)^ts temperature-shift graph (Figure 4) and a glp-1(bn18)^ts /glp-1(+) conversion graph (Figure S2 in File S1), we predicted that loss of Notch signaling in early L4 would result in a brood of roughly 125 progeny, while loss of Notch signaling at the end of L4 would result in a brood of roughly 250 progeny. In our experiments, the range of observed hop-1(ar179) broods at 15° was 150–267 progeny (n = 8), consistent with a loss of Notch signaling occurring at slightly different times within the L4 stage.

Declining SEL-12 presenilin availability/activity in the gonad may cause the switch to HOP-1 dependency

A switch to HOP-1 dependence during the L4 stage might indicate that at this developmental time, the SEL-12 presenilin normally becomes unavailable or inactive in the germline. Several observations support this hypothesis. First, reducing the sel-12(+) dosage (maternal and zygotic) by half in hop-1(ar179) animals reduces the average brood size by 34% [126 ± 37, n = 18 for hop-1(ar179); sel-12(ok2078)/+ vs. 191 ± 35, n = 17 for hop-1(ar179)]. This diminished brood size is consistent with an earlier cessation of Notch signaling, as predicted by the above glp-1 temperature-shift experiment. Second, comparison of hop-1(ar179) males and hermaphrodites reveals an earlier loss of germ cell proliferation in males as compared to hermaphrodites (compare Figure 2, B and C). This difference could be caused by a variety of factors, one of which is the fact that males have only one genetic dose of sel-12, compared to the two doses of sel-12 in a hermaphrodite. sel-12 is one of the X chromosome genes that escapes dosage compensation, and is expressed twofold higher in hermaphrodites compared to males (Jans et al. 2009). To further explore this possibility, we measured the persistence of the gonad proliferative zone in hop-1(ar179) tra-2(e1095) pseudomales that have two copies of the X chromosome, and therefore double the genetic dosage of sel-12 compared to normal males. The proliferative zone was found to persist longer in hop-1(ar179) tra-2(e1095) XX pseudomales compared to hop-1(ar179) XO males (Figure 5). Reciprocally, hop-1(ar179) males that had reduced sel-12 function exhibited proliferative zone loss earlier in development than did hop-1(ar179) males with normal sel-12 activity (Figure 5). Although these experiments are not a direct demonstration of the influence of sel-12, they are consistent with the idea that the timing of the switch to HOP-1 dependence reflects a developmental time at which sel-12 function declines below a certain threshold.

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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.

Further support for this model and specification that sel-12 reduction might occur at the mRNA level comes from analysis of sel-12 and hop-1 expression data (Figure 6) from genome-wide RNA-sequencing (RNA-Seq) expression analysis conducted by ModENCODE (Gerstein et al. 2010). The partial genetic redundancy of sel-12 and hop-1 predicts that these genes have overlapping expression patterns; however, our results predict that in the L4 gonad, sel-12 activity becomes limiting while hop-1 is readily available in the gonad. Indeed, sel-12 mRNA measurements reveal that at the L4 stage, detectable sel-12 expression is almost entirely somatic rather than germline, and does not increase significantly upon transition to adulthood. In stark contrast, the hop-1 gene shows a steep increase in expression after the L2 stage, with the highest expression attained in adults. Furthermore, hop-1 expression appears to be almost entirely germline expression, as sterile L4 animals show very little hop-1 expression. Independently, Wang et al. identified 4699 genes expressed in dissected gonads from adult hermaphrodites, and hop-1, but not sel-12, is represented in the collection (Wang et al. 2009). Together, these expression data are consistent with a switch in the L4 gonad from dual presenilin mode to exclusive HOP-1 mode, and further suggest that this switch can be at least partially explained by presenilin mRNA levels.

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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.

HOP-1-deficient animals produce enlarged oocytes

The hop-1(ar179) phenotype presents a unique opportunity to analyze oogenesis independently of germline development, because a germline stem cell pool is established and sufficient sperm and oocytes are generated to yield self-fertility. Without HOP-1 presenilin, we have demonstrated that germ cell proliferation ceases and fecundity is reduced. We observed two additional defects in the hop-1(ar179) adults. First, as has been documented for other Notch signaling mutants (Nadarajan et al. 2009), the mature oocytes of hop-1(ar179) hermaphrodites are often abnormally large (Figure 7). In 41% of hop-1(ar179) gonads, the most mature oocyte was ≥ 50% larger in volume than the average size of the corresponding oocyte in a wild-type gonad; by comparison, only 3.4% of N2 gonads exhibited such large oocytes [n = 76 for hop-1(ar179) and n = 58 for N2]. In contrast, sel-12(ok2078) gonads rarely contained such large oocytes (3.6% n = 28). Although Notch signaling has been shown to be required to promote timely oocyte cellularization and appropriate oocyte size, the mechanism for this influence remains unknown (Nadarajan et al. 2009). Our results demonstrate that the HOP-1 presenilin, and not the SEL-12 presenilin, is essential for this role of Notch signaling in oogenesis.

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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).

Notch signaling is necessary in the adult gonad for high embryo output rate

As an indicator of oogenesis productivity, we analyzed the rate of embryo production by hop-1(ar179) animals over the course of the hermaphrodite egg-laying period. Oogenesis is the rate-limiting step to embryo production in wild-type adult hermaphrodites with sperm, and thus changes in oogenesis output should be reflected in the rate of embryo output (McCarter et al. 1999). We expected embryo productivity to cease prematurely in hop-1(ar179) hermaphrodites because of the diminishing pool of germ cell progenitors, but were surprised to find a reduced embryo output rate throughout adulthood (Figure 8A). Continuous monitoring at 15° revealed greater resolution in this comparison: young adult hop-1(ar179) animals begin producing embryos at a rate comparable to that of wild-type hermaphrodites, but while wild-type animals dramatically increase output rate through the first 2 days of laying, hop-1 animals fail to increase production rate beyond the rate attained in the first half-day of laying (Figure 8B).

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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°.

To determine whether the reduced embryo output rate of hop-1 hermaphrodites reflects a defect in Notch signaling, we also analyzed the embryo output rate of hermaphrodites with reduced glp-1/Notch function. A temperature of 15° is semipermissive for glp-1(bn18)^ts function, allowing enough Notch signaling in the distal gonad to generate and maintain a proliferative zone, but one that is only 70% the size of a wild-type proliferative zone at 15° (Michaelson et al. 2010; Fox et al. 2011; and C. Goutte, unpublished results). We found that throughout adulthood at 15°, glp-1(bn18)^ts hermaphrodites exhibited a distinct reduction in embryo output rate relative to wild-type (Figure 8B). Despite the reduced rate, embryo production increased gradually through the first 2 days of laying, closely paralleling the changes in wild-type embryo output over the course of adulthood. Surprisingly, glp-1(bn18)^ts animals continued to produce embryos for a longer time than wild-type, ultimately producing close-to-wild-type-sized broods (Table 1). This result has important general implications for the study of fecundity, because it demonstrates that a reduced rate of embryo production is not necessarily indicative of reduced fecundity. For the purposes of this study, the reduced embryo output rate of glp-1(bn18)^ts animals corroborates the suggestion that Notch signaling is necessary for efficient oogenesis.

Interestingly, the two different temporal profiles of hop-1 and glp-1 embryo production rate mimic the temporal profiles of their defects in germline proliferation: hop-1 gonads begin adulthood with a normal proliferative zone, which then becomes depleted over the course of the adult laying period, and the embryo production rate is initially comparable to wild-type but then becomes progressively more and more reduced relative to wild-type. In contrast, glp-1(bn18)^ts hermaphrodites produce embryos at a rate that is roughly 70% that of wild-type, regardless of the age of the animal, and the proliferative zone size is 70% that of wild-type animals. This correlation raises the possibility of a simple, yet unrecognized, link between the size of the proliferative zone and the rate of oocyte production.

The reduced embryo production rate of hop-1(ar179) cannot be explained simply by a reduced number of available oocytes generated by the proliferative zone, because such a model would predict a delay of at least 50 hr (the time required for a germ cell to progress from meiotic entry to mature oocyte; McCarter et al. 1999; Jaramillo-Lambert et al. 2007) between the onset of the proliferative zone phenotype and the embryo output phenotype. Instead, reduced embryo output is observed in hop-1 animals at the beginning of adulthood, nearly coincident with the diminishment of the proliferative zone (Figure 2B). As an alternative explanation, we considered the function of germ cells upon exiting the proliferative zone. In a wild-type hermaphrodite gonad, germ cells in early meiotic prophase are transcriptionally active and function as nurse cells to provide material that is taken up by older oocytes developing in the proximal gonad (Wolke et al. 2007). It is estimated that at least half of these cells are later culled from the gonad by apoptosis (Gumienny et al. 1999; Jaramillo-Lambert et al. 2007). We hypothesized that limited nurse cell function could provide an alternative explanation for the reduced hop-1 embryo output rate, and would better account for the close timing of this phenotype with the reduction in the proliferative zone.

To gauge the contribution of the stem cell pool to nurse cell production, we used our analysis of N2 and glp-1(bn18)^ts to estimate the percentage of germ cell progenitors that take on a nurse cell role rather than develop into an oocyte. We reasoned that nurse cell production in each gonad could be quantified by calculating the number of germ cells that enter meiosis but do not give rise to oocytes. To do this, we used proliferative zone output rates that have been calculated for N2 and glp-1(bn18)^ts at 15° by Fox and Schedl (2015) and compared them to the embryo output rates that we measured at 15°. Since each mature oocyte gives rise to an embryo, the embryo output rate per gonad (half the total embryo output rate) was used to represent the number of oocytes produced per gonad per hour, and was subtracted from the total number of germ cells that enter meiosis each hour (Table 1). These calculations indicate that, for roughly 15 germ cells that enter meiosis per hour at 15°, two will become oocytes and 13 will function only as nurse cells. Thus, at 15°, nurse cells represent 86% of the proliferative zone output in a wild-type hermaphrodite gonad, and the proliferative zone must supply a total of 7.4 germ cells to generate each oocyte. Repeating this analysis for glp-1(bn18)^ts animals indicates that roughly 1.5 oocytes and 11 nurse cells are contributed by the proliferative zone each hour at 15°. Despite the 31% reduction in proliferative zone output rate, glp-1 animals maintained roughly the same ratio of germ cells to oocyte as wild-type (Table 1). These calculations indicate that there is a high demand on the proliferative zone to generate roughly seven germ cells for each oocyte, and support a direct relationship between the size of the proliferative zone and the rate of oogenesis.