Description
In
C. elegans
, exogenous and endogenous double-stranded RNAs are recognized and bound by
RDE-4
, which directs them to Dicer for cleavage (Boyle et al., 2026; Parker et al., 2006; Tabara et al., 2002; Thivierge et al., 2011). Subsequent processing of these RNAs results in double-stranded primary small interfering RNAs (siRNAs) (Duchaine et al., 2006; Lee et al., 2006; Yigit et al., 2006), which bind to Argonaute proteins. The Argonautes
ALG-3
and
ALG-4
(
ALG-3
/4) are redundant and expressed in spermatocytes during spermatogenesis (Conine et al., 2010; Han et al., 2009; Vasale et al., 2010). Their disruption
causes defects in spermatocyte maturation and sperm function (Conine et al., 2010).
ALG-3
/4 can repress or promote the expression of their targets, with the latter process being enhanced at 25 °C (Conine et al., 2010, 2013). One gene with decreased levels of corresponding endo-siRNA, mRNA, and protein in
alg-3
/4
mutant males, i.e. a direct target of
ALG-3
/4, is
lea-1
(Conine et al., 2013).
The
lea-1
gene is robustly upregulated in long-lived mutants of the insulin/IGF-1 (IIS) pathway such as the insulin/IGF-1 receptor
daf-2
mutant (Chen et al., 2015) and PI3K
catalytic subunit
age-1
mutant
(Liontis et al., 2026). As is the case for the lifespan extension of IIS mutants (Kenyon et al., 1993; Klass, 1983; Murakami & Johnson, 1996), the upregulation of
lea-1
in
daf-2
mutants requires the downstream activation of the forkhead box O (FOXO) transcription factor
DAF-16
(Chen et al., 2015). However,
lea-1
does not appear to play a role in aging and is dispensable for the longevity of
daf-2
mutants (Hibshman & Goldstein, 2021; Zečić et al., 2022).
We previously found that
alg-3
/4
mutations cause the disruption of Eph receptor (EphR) perimembrane localization and the elevation of PTEN protein levels in the neighboring mature oocyte (Liontis et al., 2026). These effects caused by
alg-3
/4
mutations were associated with the enhancement of the lifespan and healthspan of
age-1
(
hx546
)
mutant hermaphrodites, but not males (Liontis et al., 2026). In addition, paternal sperm carrying the
alg-3
/4
mutations was not sufficient to elevate oocyte PTEN levels in progeny, and paternal sperm carrying wild-type
alg
-3/4 was also not sufficient to rescue the particularly low brood size of
age-1
;
alg-3
/4
mutants (Liontis et al., 2026). Taken together, our previous findings are consistent with spermatogenically-expressed
ALG-3
/4 regulating the oocyte in the same generation in a cell non-autonomous manner.
To further understand the spatial dynamics of genes regulated by
ALG-3
/4, we aimed here to measure the non-autonomous effects of
ALG-3
/4 on the expression of their target gene
LEA-1
. In addition, we were interested in assessing the interaction between the
ALG-3
/4 and IIS pathways in regulating a common gene target. Although
LEA-1
does not appear to play a role in aging, it uniquely satisfies the conditions of being expressed in various tissues, including spermatocytes and oocytes, being regulated by both
ALG-3
/4 and IIS, and having a readily available reporter strain,
LP858
, where its coding region was endogenously tagged with a fluorescent protein using CRISPR (mNeonGreen::
LEA-1
) (Hibshman & Goldstein, 2021).
We confirmed that
ALG-3
/4 promote
LEA-1
protein levels in males by observing a strong decrease in mNeonGreen::
LEA-1
levels in the spermatocytes of
alg-3
(
tm1155
);
alg-4
(
ok1041
)
mutant males
(
Figure 1A,
B)
. We then assessed
LEA-1
levels in hermaphrodites
(
Figure 1C
–G)
. Intriguingly, we found a strong downregulation of mNeonGreen::
LEA-1
in the pachytene stage germ cells and proximal (−1) oocyte of
alg-3
/4
mutant hermaphrodites
(
Figure 1C,
D, G)
. Given that
ALG-3
and
ALG-4
are specifically expressed in spermatocytes (Charlesworth et al., 2021; Conine et al., 2010), this finding is consistent with non-autonomous effects of
ALG-3
/4. Consistent with
lea-1
being the most statistically significantly upregulated gene in
age-1
(
hx546
)
mutants according to our RNA-sequencing data (Liontis et al., 2026), mNeonGreen::
LEA-1
levels were considerably increased throughout the germline of
age-1
mutant hermaphrodites
(
Figure 1C,
E, G)
. In these
age-1
mutants, the
alg-3
/4
mutations caused a statistically significant but markedly attenuated reduction in mNeonGreen::
LEA-1
levels
(
Figure 1C,
F, G)
. In other words, the
age-1
(
hx546
)
background largely (but not entirely) suppressed the effect of
alg-3
/4
mutations in reducing
LEA-1
levels in pachytene germ cells and the proximal oocyte.
Finally, we crossed wild-type (WT) and
alg-3
/4
mutant worms to observe the effects of maternal or paternal loss of
alg-3
/4
on the
LEA-1
levels of progeny. Cross-progeny of
alg-3
/4
mutant parents displayed significantly decreased mNeonGreen::
LEA-1
levels in their proximal oocytes, compared to cross-progeny of WT worms, as expected from these control conditions
(
Figure 1H
)
. Surprisingly, progeny of WT males crossed to
alg-3
/4
mutant hermaphrodites, like those of
alg-3
/4
mutant males crossed to WT hermaphrodites, also exhibited a similar decrease in mNeonGreen::
LEA-1
levels in their proximal oocyte
(
Figure 1H
)
. This suggests that the process resulting in the downregulation of
lea-1
in
alg-3
/4
mutants can be inherited both maternally and paternally. Given that these progeny animals are heterozygous for the
alg-3
/4
mutations, an alternative explanation is that
alg-3
/4
are haploinsufficient, at least with respect to their regulation of
lea-1
. This interpretation is unlikely because
alg-3
/4
heterozygotes do not phenocopy the sterility at 25 °C of
alg-3
/4
homozygous mutants (Conine et al., 2010, 2013). In addition, it was shown that male
alg-3
/4
heterozygotes do not display a decrease in
lea-1
pre-mRNA levels compared to WT, unless they descend directly from a homozygous
alg-3
/4
mutant parent (Conine et al., 2013). Overall, although it is unclear how
ALG-3
/4 promote
LEA-1
expression outside of spermatocytes,
ALG-3
/4 must be functional in both the father and hermaphrodite mother to sustain
LEA-1
expression in the oocyte of their progeny.
In this work, we found that
ALG-3
/4 not only promotes
LEA-1
levels in male spermatocytes but also in the oocyte and precursor germ cells of hermaphrodites
(
Figure 1I
)
. Given that
LEA-1
is repressed by IIS, this is a system where
ALG-3
/4 and
AGE-1
have opposite effects on gene expression. In contrast,
ALG-3
/4 and
AGE-1
both inhibit
DAF-16
activity in their regulation of lifespan (Liontis et al., 2026). The downregulation of
LEA-1
despite the increased activation of
DAF-16
in
alg-3
/4
mutants is paradoxical, suggesting that there is likely an additional signaling pathway regulated by
ALG-3
/4, separate from the MSP/EphR/PTEN system (Liontis et al., 2026), at play
(
Figure 1I
)
. This is further supported by the fact that the increase in
DAF-18
(PTEN) levels in the oocyte of
alg-3
/4
mutants could not be inherited (Liontis et al., 2026), in contrast to the decrease in
LEA-1
levels
(
Figure 1H
)
. In both cases, the dysregulation of these genes in
alg-3
/4
mutants cannot be simply reasoned as disruptions of
ALG-3
/4
in paternal sperm. Instead, our results suggest that impairments causing
LEA-1
downregulation in
alg-3
/4
mutant males and hermaphrodites are likely inherited throughout development and adulthood, whereas those causing
DAF-18
elevation are present only in the generation where both copies of
alg-3
/4
are mutated. It is therefore likely that
DAF-18
and
LEA-1
are regulated by separate pathways controlled by
ALG-3
/4. Overall, our studies of
ALG-3
/4 reveal multiple cell non-autonomous gene regulatory mechanisms.
Methods
C. elegans
strains and maintenance
Worms were cultured and assayed at 20 °C on solid nematode growth media (NGM) seeded with
E. coli
OP50
. The mNG::
LEA-1
strain
LP858
was outcrossed once to eliminate an unexpected dumpy (dpy)-like phenotype. The resulting strain was then crossed to the
age-1
and
alg-3
;
alg-4
mutant strains, which have already been outcrossed six times.
Fluorescence microscopy
Compound microscopy was performed using the Zeiss AxioImager Z1. Worms were mounted on 2% agarose pads, paralyzed with 10–20 mM levamisole in M9 buffer, placed under a thin glass coverslip, and immediately imaged. A constant exposure time was always used for images shown in the same figure panel. In addition, controls were always present on the exact same agarose pad as experimental animals (e.g., mutants). The ImageJ (Fiji) software was used for quantification of mNeonGreen::
LEA-1
fluorescence and this analysis was performed while blinded to the strains' genotype. Mean fluorescence was normalized to controls in each independent experimental replicate, and results were pooled to produce the graphs shown in the figure. Random counterbalancing of the order in which strains were imaged was done between each independent replicate. Regions of interest were delimited manually: spermatocytes for L4 males; pachytene-stage germ cells in the proximal Rachis region and the proximal −1 oocyte's cytoplasmic region for young Day 1 adult hermaphrodites. At this stage, oocytes were large and mature but no more than 1 embryo was present.
Statistical analyses and graphs
Statistics were analyzed as was done previously (Liontis et al., 2026). Briefly, biological replicates (n) represent the number of animals, whereas independent experimental replicates (N) represent the number of independent experiments started on a different day with independent populations of animals. When comparing means, the F-test for variance and Anderson-Darling, D'Agostino-Pearson, Shapiro-Wilk, or Kolmogorov-Smirnov tests for normality were computed first. The resulting appropriate unpaired Student's t-test (equal vs. unequal variance) or Mann-Whitney test (nonparametric) was then used to assess a two-tailed significant difference between two groups. Tests were computed and graphs were generated using GraphPad Prism 10.
Error bars represent the standard error of the mean.
Parental effects on
LEA-1
All strains were homozygous for mNeonGreen::
LEA-1
. Three males and one hermaphrodite at the L3 – L4 stage were transferred to each plate, with several plates for each cross. Hermaphrodites were considered to have mated when a significant number of progeny (F1) were males (up to 50%). F1 late L4 hermaphrodites that were identified as cross-progeny, i.e. not older than their sibling F1 males, were transferred to a new plate to prevent new crossing as a confounding factor. These F1 animals were imaged 7 hours after being transferred, at which point they reached young adulthood.