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Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 1 of 28
Mechanical force of uterine occupation
enables large vesicle extrusion from
proteostressed maternalneurons
Guoqiang Wang1†, Ryan J Guasp1†, Sangeena Salam1†, Edward Chuang1,
Andrés Morera1, Anna J Smart1, David Jimenez1, Sahana Shekhar1,
Emily Friedman1, Ilija Melentijevic1, Ken C Nguyen2, David H Hall2, Barth D Grant1,
Monica Driscoll1*
1Department of Molecular Biology and Biochemistry, Nelson Biological Laboratories,
Rutgers, The State University of New Jersey, Piscataway, United States; 2Department
of Neuroscience, Albert Einstein College of Medicine, Bronx, United States
Abstract Large vesicle extrusion from neurons may contribute to spreading pathogenic protein
aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenera-
tive disease. Factors that regulate the extrusion of large vesicles, such as exophers produced by
proteostressed C. elegans touch neurons, are poorly understood. Here, we document that mechan-
ical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher
production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C.
elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early
embryo production can strongly suppress exopher extrusion from the ALMR neurons during the
peak period. Conversely, restoring egg production at the late reproductive phase through mating
with males or inducing egg retention via genetic interventions that block egg- laying can strongly
increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher
production are associated with expanded uterus lengths and genetic interventions that suppress
ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of
fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead
eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized
eggs, constitute the exopher- inducing stimulus. We conclude that the mechanical force of uterine
occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our
observations draw attention to the potential importance of mechanical signaling in extracellular
vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention
to mechanobiology in neurodegenerative disease.
eLife assessment
This important study explores the potential influence of physiologically relevant mechanical forces
on the extrusion of vesicles from C. elegans neurons. The authors provide compelling evidence to
support the idea that uterine distension per se can induce vesicular extrusion from adjacent neurons.
Overall, this work will be of interest to neuroscientists and investigators in the extracellular vesicle
and proteostasis fields.
RESEARCH ARTICLE
*For correspondence:
driscoll@dls.rutgers.edu
†These authors contributed
equally to this work
Competing interest: The authors
declare that no competing
interests exist.
Funding: See page 24
Preprint posted
16 November 2023
Sent for Review
22 December 2023
Reviewed preprint posted
19 February 2024
Reviewed preprint revised
31 July 2024
Version of Record published
10 September 2024
Reviewing Editor: Patrick J Hu,
Vanderbilt University Medical
Center, United States
Copyright Wang, Guasp,
Salam etal. This article is
distributed under the terms
of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 2 of 28
Introduction
In neurodegenerative disease, prions and protein aggregates can transfer among cells of the nervous
system to promote pathology spread (Peng etal., 2020; Davis etal., 2018). Determination of the
factors that enhance or deter pathological transfer is, therefore, a central goal in the effort to clini-
cally address neurodegenerative disease. Study of aggregate transfer in the context of the mamma-
lian brain is a major experimental challenge as events are rare, sporadic, and transiently apparent,
and tissue is not easily accessible for in vivo observation. We model aggregate transfer by proteost-
ressed ALMR touch receptor neurons in the living C. elegans nervous system (Melentijevic etal.,
2017; Cooper et al., 2021; Arnold etal., 2023), an experimental system that enables molecular
and genetic manipulation and evaluation in a physiological context, directly through the transparent
cuticle (Corsi etal., 2015).
More specifically, C. elegans adult neurons can extrude large vesicles called exophers (~5µm;
100X larger than exosomes) that carry potentially deleterious proteins and organelles out of the
neuron (Melentijevic etal., 2017; Cooper etal., 2021; Arnold etal., 2023). Disrupting proteostasis
via diminished chaperone expression, autophagy, or proteasome activity, or over- expressing aggre-
gating proteins like human Alzheimer’s disease associated fragment Aβ1- 42, expanded polyglutamine
Q128 protein, or high concentration mCherry fluorophore, increases exopher production from the
affected neurons. Neurons that express proteotoxic transgenes maintain higher functionality if those
neurons produce exophers as compared to those that do not, suggesting that exopher- genesis can
be neuroprotective, at least in young adult neurons. Extruded exopher contents can be transferred
to neighboring glial- like hypodermal cells for content degradation in the lysosomal network (Wang
etal., 2023). Several mammalian models feature similar biology (Nicolás-Ávila etal., 2020; Lamp-
inen etal., 2022; Davis etal., 2014; Nicolás-Ávila etal., 2021; Nicolás-Ávila etal., 2022), and thus
eLife digest Neurons are specialized cells in the brain and nervous system that transmit signals
between the brain and the rest of the body, enabling humans and animals to react to internal and
external stimuli. For this communication system to function effectively, neurons must remain healthy.
Neurons maintain their function in a variety of ways, including by removing excess or damaged
cellular components (such as organelles and protein aggregates) that could compromise neuron func-
tion. One way to do this is by extruding organelles and aggregates. During ‘extrusion events’, the
material to be removed is gathered within a budding portion of the plasma membrane, which forms
a vesicle that ejects the material from the neuron. However, the factors driving the extrusion process
remained unknown.
To investigate, Wang, Guasp, Salam et al. conducted experiments in the roundworm Caenorhab-
ditis elegans, finding that the number of extrusion events in a certain type of neuron increases at the
peak of reproduction. More specifically, a greater number of extrusion events were associated with
the presence of fertilized eggs, which accumulate in the uterus before they are laid. Disrupting eggs,
sperm or the fertilization process suppressed the increase in extrusion events, suggesting the pres-
ence of fertilized eggs is responsible.
To determine how the eggs might trigger extrusion events, Wang et al. stretched the uterus using
dead eggs, unfertilized eggs or by injecting fluid, finding that each of these approaches increased the
number of extrusion events. Further analysis suggests that this mechanical stretching of the uterus
signals to the neurons that reproduction has started, encouraging the neurons to remove old compo-
nents and optimize their function. Wang et al. hypothesize that this stretch response could support
neuronal behaviors that aid in successful reproduction, such as sensing food and selecting where to
lay eggs.
The findings increase our understanding of the factors that trigger vesicle extrusion in living organ-
isms. These observations could have implications for human neurodegenerative diseases such as
Alzheimer’s disease, in which protein aggregates accumulate in neurons. It is possible that mechan-
ical signals generated by factors associated with Alzheimer’s disease, such as high blood pressure,
could influence neuronal extrusion and contribute to some of the mechanisms underlying aggregate
transfer in neurodegenerative diseases.
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we speculate that the basic transfer biology represents a conserved process that can be recruited for
animal- wide proteostasis balance.
Neuronal exophers are generated only in adult animals, with an unexpected pattern of a peak
at young adult days 2–3 and then later in age with more variable onset (Melentijevic etal., 2017).
While using chemical regent 5- fluoro- 2'-deoxyuridine (FUdR) to block progeny production for aging
studies, we found that blocking reproduction strongly limited the early adult peak of exopher produc-
tion. Here, we report data that support that early adult exopher production is sensitive to the load
of eggs in the reproductive tract. We document that uterine expansion, rather than chemical signals
emanating from fertilized eggs, correlates strongly with the level of exopher production and suggest
a model in which mechanical signaling, normally induced across generations from egg to parent via
uterine stretch, is a license for proteostressed neurons to release potentially toxic materials in large
extracellular vesicles.
Mechanical signaling exerts a profound impact on virtually all cell types, and has been implicated
in traumatic brain injury and neurodegenerative disease, yet remains poorly understood (Hall etal.,
2021). Our observations direct enhanced experimental attention to studies on how mechanical force
can influence extracellular vesicle formation and aggregate transfer in the living brain and in neuro-
degenerative disease.
Results
The six C. elegans gentle touch receptor neurons (AVM, ALML, and ALMR located in the anterior
body, and PVM, PLML and PLMR located in the posterior body) can be readily visualized in vivo by
expression of fluorescent proteins under the control of the touch neuron- specific mec- 4 promoter
(Figure1A). We commonly monitor exophers extruded by the ALMR neuron, which typically produces
more exophers than the other touch neurons (Melentijevic etal., 2017; Arnold etal., 2020), using
a strain in which fluorophore mCherry is highly expressed in touch neurons and is avidly eliminated
(strain ZB4065 bzIs166[Pmec- 4::mCherry], hereafter referred to as mCherryAg2 for simplicity). ALMR
exopher production occurs with a distinctive temporal profile, such that at the L4 larval stage ALMR
rarely, if ever, produces an exopher, but in early adult life, the frequency of exopher events increases,
typically reaching a peak of 5–20% of ALMR neurons scored at adult day 2 (Ad2); exopher detec-
tion then falls to a low baseline level after Ad3 (Melentijevic etal., 2017; Arnold etal., 2020), a
pattern that parallels adult reproduction (Figure1B). Late in life, exophers can reappear with variable
frequency, but here we focus on the young adult exopher generation.
Sterility-inducing drug FUdR suppresses ALMR exophergenesis
In experiments originally designed to study exopher events in aging animals, we sought to generate
age- synchronized populations by blocking progeny production from early adult stages using DNA
synthesis inhibitor FUdR. Unexpectedly, we found that 51μM FUdR strongly suppressed exopher
events as quantitated at Ad2 (Figure1C). FUdR is commonly used in C. elegans to inhibit the prolifer-
ation of germline stem cells and developing embryos, but has also been noted to impair RNA metab-
olism (Burnaevskiy etal., 2018) and improve adult proteostasis (Angeli etal., 2013). To probe which
FUdR outcome might confer exopher suppression, we first addressed whether disruption of progeny
production by alternative genetic means might suppress exophergenesis, which would implicate a
viable germline as a factor in exopher modulation.
Germline elimination and germline tumors suppress ALMR exopher
production
In the assembly line- like hermaphrodite C. elegans gonad, germline stem cells close to the signaling
distal tip cell (DTC) proliferate by mitosis (diagram of C. elegans gonad and germ cell development in
Figure1D). We disrupted germ cell production using the glp- 4(bn2ts) valyl- tRNA synthetase 1 mutant.
At the restrictive temperature (25°C), glp- 4(bn2ts) causes germ cell arrest during the initial mitotic
germ cell divisions, effectively eliminating the germline (Beanan and Strome, 1992). We constructed
an mCherryAg2; glp- 4(bn2ts) strain and quantitated ALMR exopher production in animals grown at
the restrictive temperature, scoring exophers during early adulthood (Figure1E). ALMR neurons in
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 4 of 28
Figure 1. Exophergenesis is dependent on the presence of the germline, ooyctes, and sperm. (A)Exophers produced by ALMR are readily visualized
in living C. elegans. Top, positions of the six C. elegans touch receptor neurons: AVM (Anterior Ventral Microtubule cell), ALMR (Anterior Lateral
Microtubule cell, Right), ALML (Anterior Lateral Microtubule cell, Left), PVM (Posterior Ventral Microtubule cell, Right), PLMR (Posterior Lateral
Microtubule cell, Right), and PLML (Posterior Lateral Microtubule cell, Left). Bottom panels are representative pictures (n > 100, scale bar = 10 μm) of an
ALMR neuron without (lower left) or with (lower right) exopher production from strain ZB4065 bzIs166[Pmec- 4::mCherry], which expresses elevated mCherry
in the touch receptor neurons. Over- expression of mCherry in bzIs166 is associated with enlargement of lysosomes and formation of large mCherry foci
that often correspond to LAMP::GFP- positive structures; ultrastructure studies reveal considerable organelle morphological change not seen in low
reporter- expression neurons; polyQ74, polyQ128, Aβ1- 42 over- expression all increase exophers (Melentijevic etal., 2017; Arnold etal., 2023). Most
genetic compromise of different proteostasis branches--heat shock chaperones, proteasome, and autophagy--enhance exophergenesis, supporting
exophergenesis as a response to proteostress. (B)Both exopher production and reproduction typically peak around Ad2 in the Pmec- 4::mCherry strain.
Left axis: the frequency of exopher events in the adult hermaphrodite C. elegans strain ZB4065 at 20°C; Mean ± SEM of nine independent trials
(~50 animals in each trial). Right axis: daily progeny count (Mean ± SEM) from 10 wild- type N2 hermaphrodites. (C)5- uoro- 2'-deoxyuridine (FUdR),
which inhibits progeny production, suppresses early adult exopher production. Data are the percentage of ALMR exopher events among>50 Ad2
hermaphrodites in each trial (total of 3 independent trials) for strain ZB4065 at 20°C in absence (control) or presence of 51μM FUDR. ***p<0.001 in
Cochran–Mantel–Haenszel test. (D)Illustration of the roles of germline development genes tested for impact on exophers. The C. elegans reproductive
system comprises a bilobed gonad in which germ cells (light blue, dark nuclei) develop into mature oocytes, which are fertilized in the spermatheca
(sperm indicated as dark dots) and held in the uterus until about the 30- cell gastrulation stage, at which point eggs (dark blue) are laid. Indicated are
the steps impaired by germline developmental mutations we tested. (E)Germline stem cells are required for efcient exopher production. Data show
the percentage of ALMR exopher events (Mean ± SEM) among 50 adult hermaphrodite C. elegans that express the wild- type GLP- 4 protein or the GLP-
4(ts) protein encoded by glp- 4(bn2) at 25°C. All animals express Pmec- 4mCherry, and the glp- 4(ts) mutants lack a germline when reared at the restrictive
temperature (25°C). Eggs collected from both wild- type and the glp- 4(ts) mutants were grown at 15°C for 24hr before being shifted to 25°C (at L1
stage) to enable development under restrictive conditions; three independent trials of 50 animals represented by each dot; ***p<0.001or *p<0.05 in
Cochran–Mantel–Haenszel test. Note that in wild- type (WT), temperature shift normally induces a modest elevation in exopher numbers (typically a
Figure 1 continued on next page
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the germline- less glp- 4 mutant produced significantly fewer exophers on Ad1- 4 as compared to age-
and temperature- matched controls.
Both oogenesis and spermatogenesis are critical for early adult
exopher production
Given that lack of functional germ cells impaired neuronal exopher production, we sought to test
whether oocytes or sperm might be specifically required for exophergenesis, taking advantage of C.
elegans genetic reagents available for the manipulation of gamete development.
Oogenesis is required for the peak of early adult exophergenesis
In the C. elegans hermaphrodite, the differentiation of germline stem cells begins during the L4
larval stage with spermatogenesis, after which sperm production is shut down and oogenesis begins
(Zanetti et al., 2012). Genetic mutants that produce only sperm or only oocytes have been well
characterized. To test a mutant that produces sperm but no oocytes, we employed fem- 3(q20ts), a
temperature- sensitive gain- of- function mutant that causes germ cells to exclusively differentiate into
sperm (Ellis and Schedl, 2007; Ahringer and Kimble, 1991). We constructed an mCherryAg2; fem-
3(q20ts) line, reared animals at non- permissive temperature 25°C, and scored ALMR exophers in
mutant and control animals during early adulthood. In the sperm- only, oocyte- deficient fem- 3(q20ts)
background, exophergenesis is mostly diminished over the first five days of adulthood (Figure1F). We
infer that oocytes must be present for early adult ALMR exopher production and conclude that sperm
alone are not sufficient to drive exopher elevation.
Spermatogenesis is required for the peak of early adult exophergenesis
The presence of functional sperm can stimulate ovulation to trigger oocyte maturation. To assess
neuronal exopher production in the reciprocal reproductive configuration in which animals have
oocytes but no sperm, we disrupted spermatogenesis using two approaches. First, we used a
temperature- sensitive fem- 1 mutation to produce animals with oocytes but no sperm (Doniach
and Hodgkin, 1984). We crossed fem- 1(fc17ts) into the mCherryAg2 strain and examined exopher
production from ALMR neurons in hermaphrodites at the restrictive temperature of 25°C. We found
that ALMR exopher production in fem- 1(fc17) mutants was suppressed at 25°C (Figure1G).
Second, we used the auxin- inducible degron system (AID) (Nishimura etal., 2009; Zhang etal.,
2015) to degrade SPE- 44, an essential protein required for spermatid differentiation (Kasimatis etal.,
2018; Kulkarni etal., 2012). In the AID system, the addition of auxin to the culture induces rapid
degradation of proteins genetically tagged with a specific auxin- dependent degron sequence. AID
targeting of SPE- 44- degron is highly effective in disrupting sperm maturation (Kasimatis etal., 2018).
few % increase, supplemental data in Cooper etal., 2021) and is thus not itself a factor in exopher production levels. (F)Oogenesis is required for
efcient exopher production. Spermatogenesis occurs but oogenesis is blocked when fem- 3(gf) mutant hermaphrodites are cultured at 25°C. Data show
the percentage of ALMR exopher events (Mean ± SEM) in hermaphrodite C. elegans that express the wild- type FEM- 3 protein or the temperature-
dependent gain- of- function (gf) FEM- 3 protein (25°C; three independent trials, n=50/trial), ***p<0.001; **p<0.01;or *p<0.05 in the Cochran–Mantel–
Haenszel test. (G)Spermatogenesis is required for efcient exopher production. There is no spermatogenesis in fem- 1(lf) at the restrictive temperature
of 25°C, while oogenesis is normal in the hermaphrodite. Shown is the percentage of ALMR exopher events (Mean ± SEM) in adult hermaphrodites
that express the wild- type FEM- 1 protein or the temperature- dependent loss of function (lf) FEM- 1 protein (25°C; three independent trials, 50 animals/
trial). ****p<0.0001or ***p<0.001 in Cochran–Mantel–Haenszel test. (H)Spermatogenesis is required for efcient exopher production. Data show the
percentage of ALMR exopher events (Mean ± SEM) in hermaphrodite C. elegans that express the SPE- 44::degron fusion. SPE- 44 is a critical transcription
factor for spermatogenesis (Kulkarni etal., 2012), and is tagged with a degron sequence that enables targeted degradation in the presence of auxin
in line ZB4749. In the auxin- inducible degron (AID) system, auxin is added to the plates in 0.25% ethanol, so ‘control’ is treated with 0.25% ethanol
and ‘no sperm’ is treated with 1mM auxin applied to plates in 0.25% ethanol from egg to adult day 1; 4–6 independent trials of 50 animals per trial.
****p<0.0001or ***p<0.001 in Cochran–Mantel–Haenszel test. Note that under no sperm or non- functional sperm production, oocytes still transit
through the spermatheca and enter the uterus (as shown by the DIC picture); unfertilized oocytes can be laid if the egg- laying apparatus is intact. (I)
Summary: Genetic interventions that block major steps of germ cell development strongly block ALMR exophergenesis in the adult hermaphrodite. The
dual requirement for sperm and oocytes suggests that fertilization and embryogenesis are required events for inducing ALMR exophergenesis.
The online version of this article includes the following source data for gure 1:
Source data 1. Daily progeny count for panel B, and exopher score for panels B, C, E, F, G, H.
Figure 1 continued
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We treated strain ZB4749 (fxIs1[Ppie- 1::TIR1::mRuby] zdIs5[Pmec- 4::GFP]; bzIs166[Pmec- 4::mCherry]; spe-
44(fx110[spe- 44::degron])) with auxin during larval developmental stages to block sperm maturation
and then transferred Ad1 animals to NGM plates without auxin, a condition that disrupted sperm
maturation but allowed oocyte generation. We find that blocking sperm maturation by targeting
SPE- 44 for degradation abolished ALMR exopher production (Figure1H) even though oogenesis is
not significantly impacted (Kasimatis etal., 2018). We infer that functional sperm must be present
for early adult ALMR exopher production and conclude that oogenesis alone is not sufficient to drive
exopher elevation in early adult life.
Fertilization and early embryonic divisions are required for the early
adult exophergenesis peak
The requirement of sperm and oocytes for neuronal exopher production raises the obvious question
as to whether fertilized eggs/embryos are required for ALMR exophergenesis (Figure1I). C. elegans
genes impacting fertilization and embryonic development have been studied in exquisite detail
(Greenstein, 2005; Stein and Golden, 2018; Rose and Gönczy, 2014; Schneider and Bowerman,
2003). We tested embryonic development genes known for roles at particular stages for impact on
neuronal exopher levels using RNAi knockdown approaches.
In C. elegans fertilization, as the mature oocyte encounters the sperm- filled spermatheca, a single
sperm enters the mature ovulated oocyte (fertilization time 0), triggering the rapid events of egg
activation, which include polyspermy block, eggshell formation, completion of meiotic divisions and
extrusion of polar bodies. The multi- step eggshell formation (Figure2A) is completed within 5min
of sperm entry, by which time the zygote has been passed into the uterus, where the maternal chro-
mosomes execute both meiotic divisions (meiosis accomplished by~20min after fertilization). After
the establishment of egg polarity cues, the first mitotic cell division occurs~40min after fertilization
(Stein and Golden, 2018; Schneider and Bowerman, 2003). Eggs are held in the mother’s uterus
until they are laid at the~30-cell stage (gastrulation). In wild- type, ~8 fertilized eggs occupy the
uterus when egg laying begins at~6hr of adult life (Medrano and Collins, 2023).
Our data revealed an unexpected theme: we found that disruption of tested early- acting genes
essential for eggshell assembly (chitin- binding domain protein cbd- 1 González et al., 2018;
Figure2B, chitin precursor synthesis gene gna- 2 Johnston etal., 2006; Figure2C and D, permea-
bility barrier- required CDP- ascarylose synthesis gene perm- 1 Olson etal., 2012; Figure2E), and/or
needed for the progression through the 1cell or 2cell stages of embryonic development (Severson
etal., 2002; Rappleye etal., 1999; Rappleye etal., 2003; Benenati etal., 2009) (profilin pfn- 1 and
coronin- related pod- 1 (Figure2F)), cause potent suppression of ALMR exophergenesis (Figure2G).
In contrast, RNAi knockdowns of genes playing important roles for later stage embryonic develop-
ment (4- cell to 8-cell stages mex- 3, mom- 2, gastrulation genes end- 1/–3 or gad- 1) (Figure2F) confer
negligible impact on ALMR exophergenesis (Figure2H). Our data support a fertilization requirement
for neuronal exopher stimulation and suggest that the exophergenesis signal/condition that promotes
the early adult wave of exophergenesis is associated with the very earliest stages of embryonic devel-
opment. Of note, disruption of eggshell biosynthesis has the immediate downstream consequence of
disruption of early polarity establishment and first divisions, such that genetic separation of eggshell
production from first division proficiency is not possible. For example, pod- 1 RNAi has been reported
to be associated with eggshell deficits (Rappleye et al., 1999), although no eggshell deficits are
oberseved for pfn- 1 RNAi (Sönnichsen etal., 2005). We thus conclude that either eggshell integrity
or biochemical events associated with the first embryonic cell divisions are required for neuronal
exophergenesis.
Restoring fertilized eggs later in life can extend ALMR exophergenesis
Having found that fertilized eggs are necessary for the early adult elevation in exopher production,
we asked whether the presence of eggs is sufficient for stimulation of exopher production by intro-
ducing fertilized eggs later in adult life when they are not normally present. In C. elegans, sperm are
made in the L4 stage and are stored in the spermatheca to fertilize oocytes that mature in the adult.
Sperm are limiting for hermaphrodite self- fertilization, with more oocytes made than can be fertilized
by self- derived sperm. Unmated hermaphrodites thus cease egg laying around adult day 4 because
they run out of self- supplied sperm. However, if males mate with hermaphrodites, increased progeny
Research article Neuroscience
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Figure 2. Events required for adult day 2 (Ad2) elevation of exopher production occur during the earliest stages consequent to fertilization and are
largely completed by the 4- cell stage. (A)Diagram of eggshell layers, post- fertilization timeline for layer formation, and indication of steps at which
RNAi disrupts eggshell biogenesis. The formation of the multilayered eggshell is accomplished via a hierarchical assembly process from outside to
inside, with outer layers required for later formation of inner layers. The outmost vitelline layer assembles in part prior to fertilization, dependent
on chitin- binding domain protein (CBD- 1) González etal., 2018. The next eggshell layer is made up of chitin, which confers eggshell stiffness and
requires the gna- 2- encoded enzyme glucosamine- 6- phosphate N- acetyltransferase (GNPNAT1) for precursor biosynthesis (Johnston etal., 2006). The
innermost lipid layer of eggshell is called the permeability layer, which is lipid- rich and is needed to maintain osmotic integrity of the embryo. PERM- 1
Figure 2 continued on next page
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numbers can be produced due to increased sperm availability. More germane to our experiment, if
males are mated to the reproductively senescing hermaphrodite to replenish sperm, hermaphrodites
can produce fertilized eggs for a few additional days (Figure3A).
To determine if the presence of eggs might be sufficient to drive exopher production after Ad3, we
mated males to Ad3 reproductively senescing hermaphrodites. We found that restored egg produc-
tion is associated with increased and extended exopher production, provided that hermaphrodites
were mated to fertilization- proficient males (Figure3B). We conclude that adult ALMR exophergen-
esis is driven by the presence of fertilized eggs and that the older age decrease in exopher produc-
tion (~Ad4) under standard culture conditions is more likely attributed to the lack of fertilized egg
accumulation at this life stage, rather than to the existence of a chronological limit on the biochemical
capacity to elevate exopher levels at older ages.
Genetically induced egg retention elevates ALMR exopher production
Fertilized eggs might release a signal or create a condition that stimulates exophergenesis. If such
an influence were limiting, increasing the egg concentration in the body might increase exopher
numbers. To address whether elevating the young adult egg load can increase exophers, we took
multiple distinctly- acting genetic approaches to limit egg laying and promote egg retention in the
body (Figure3C). We tested four genetic conditions that lower or block egg- laying: a null mutation
of prolyl hydroxylase egl- 9, for which disruption induces a mild egg- laying defect (Trent etal., 1983);
a null allele of proprotein convertase subtilisin/kexin type 2 egl- 3 (Salem etal., 2018) that perturbs
neuropeptide processing and confers a severe egg- laying defective phenotype (Trent etal., 1983);
a reduction- of- function mutation in SOX transcription factor SEM- 2 (sem- 2(n1343)) that eliminates
production of the sex myoblasts needed to generate egg laying muscle; and RNAi directed against
the lin- 39 homeobox transcription factor HOXA5 ortholog required for vulval cell fate specification
(Wagmaister etal., 2006; Sternberg, 2005).
(Olson etal., 2012) (among others, including FASN- 1 [Rappleye etal., 2003], POD- 1 [Rappleye etal., 1999], and EMB- 8 [Benenati etal., 2009])
is required for permeability barrier formation (Stein and Golden, 2018; Johnston and Dennis, 2012). Note that eggshell biogenesis is critical for
polyspermy barrier, spermathecal exit, meiotic chromosome segregation, polar body extrusion, AP polarization and internalization of membrane and
cytoplasmic proteins, and correct rst cell divisions (Johnston and Dennis, 2012), so genetic separation of eggshell malformation from the earliest
embryonic formation is not possible. (B) cbd- 1, a gene encoding an essential component of the eggshell vitelline layer, is critical for Ad2 exopher
elevation. Exopher scores in Ad2 animals (strain ZB4757: bzIs166[Pmec- 4::mCherry] II) that were treated with RNAi against cbd- 1, total of three trials
(50 hermaphrodites per trial), **p<0.01 in Cochran–Mantel–Haenszel test, as compared to the empty vector (EV) control. (C) gna- 2, a gene required
for chitin precursor biosynthesis and chitin layer formation, is critical for Ad2 exopher elevation. (C) Exopher scores in Ad2 animals treated from the
L1 stage with RNAi against gna- 2. The gna- 2 gene encodes enzyme glucosamine- 6- phosphate N- acetyltransferase (GNPNAT1) required for chitin
precursor biosynthesis. (D)Exopher scores in Ad2 animals harboring a null mutation in the essential gna- 2 gene. gna(∆) homozygous null worms are
GFP negative progeny of stain ZB4941: bzIs166[Pmec- 4::mCherry]; gna- 2(gk308) I/hT2 [bli- 4(e937) let-?(q782)] qIs48[Pmyo- 2::GFP; Ppes- 10::GFP; Pges- 1::GFP]
(I;III). Data represent a total of three trials (50 hermaphrodites per trial), **** p<0.0001 in Cochran–Mantel–Haenszel test, as compared to wild- type
animals. (E) perm- 1, a gene required for permeability barrier synthesis, is critical for Ad2 exopher elevation. Exopher scores in Ad2 animals treated
with RNAi against perm- 1, which encodes a sugar modication enzyme that acts in the synthesis of CDP- ascarylose. Data represent a total of 3 trials
(50 hermaphrodites per trial), *p<0.05 in Cochran–Mantel–Haenszel test, as compared to the EV control. Note that previously characterized strong
exopher suppressors pod- 1, emb- 8, and fasn- 1 (Melentijevic etal., 2017; Cooper etal., 2021) are also needed for egg shell permeability barrier layer
formation (Rappleye etal., 1999; Benenati etal., 2009). (F)Diagram of select genes required for specic stages of embryonic development. pfn- 1
RNAi (arrest at the one- cell stage Schonegg etal., 2014; pod- 1 RNAi (arrest at the two- cell stage Luke- Glaser etal., 2005; arrest stage phenotype
for other genes is not precisely documented, but these genes play signicant roles at the indicated stages; images annotated according to WormAtlas
(https://doi.org/10.390/wormatlas.4.1). (G)RNAi targeting of early acting embryonic development genes lowers exopher production. Exopher scores
from Ad2 animals that were treated with RNAi against genes characterized to be essential for 1- cell to 2- cell stage embryonic development. Total of
three trials (50 hermaphrodites per trial). *p<0.05or ****p<0.0001 in Cochran–Mantel–Haenszel test, as compared to the empty vector control. Strong
exopher suppressor pod- 1 has been previously reported (Melentijevic etal., 2017). We found RNAi directed against gene emb- 8 (as early as 2cell
arrest, but arrest at the 1- to 50cell stage reported [Schierenberg etal., 1980]) to be more variable in outcome (not shown). (H)RNAi targeting of
genes that disrupt 4cell stage and later embryonic development does not alter exopher production levels. Exopher scores in Ad2 animals that were
treated with RNAi against genes that are essential for 4cell to gastrulation stages of embryonic development. Total of three trials (50 hermaphrodites
per trial). ns, not signicant in Cochran–Mantel–Haenszel test, as compared to the empty vector control.
The online version of this article includes the following source data for gure 2:
Source data 1. Exopher score for panels B, C, D, E, G, H.
Figure 2 continued
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Figure 3. Anterior Lateral Microtubule cell (ALMR) exophergenesis levels are markedly inuenced by the number of fertilized eggs retained in the
uterus. (A)Later life mating extends the C. elegans reproductive period. Progeny count for each wild- type hermaphrodite in the presence (green) or
absence (black) of males (1 hermaphrodite+/-5males) from Ad1 to Ad6. Males are present from Ad4 to Ad6. Total of 12 wild- type hermaphrodites
scored for each condition. Data shown are mean ± SEM. ****p<0.0001 in two- way ANOVA with Šídák’s multiple comparisons test. (B)Introducing
fertilized eggs in late adulthood extends the period of elevated exophergenesis. Males carrying functional (green) or nonfunctional (red) sperm
(spe- 45(tm3715)) were added to plates housing hermaphrodites as endogenous stores of sperm are depleted at Ad3. Data shown are mean ±
SEM of percentage hermaphrodite ALMR neurons exhibiting an exopher event on days indicated. Total of three trials, and 50 hermaphrodites for
each treatment at a single time point. ****p<0.0001 in Cochran–Mantel–Haenszel test, as compared to the sterile male group or no male control.
(C)Hypothesis: Genetic interventions (mutations/RNAi) that increase egg retention increase ALMR exophergenesis. (D)Genetic interventions that
induce egg retention elevate exopher levels. egl- 9(sa307). ALMR exopher scores Ad2. Strain ZB4757: bzIs166[Pmec- 4::mCherry] II vs. strain ZB4772:
bzIs166[Pmec- 4::mCherry] II; egl- 9(sa307) V. Total of three trials (50 worms per trial, each trial one dot); **** p<0.0001 in Cochran–Mantel–Haenszel test,
as compared to the wild- type control. (E)Genetic interventions that induce egg retention elevate exopher levels. egl- 3(gk238). ALMR exopher scores
Ad1. Strain ZB4757: bzIs166[Pmec- 4::mCherry] II vs. strain ZB4904: bzIs166[Pmec- 4::mCherry] II; egl- 3(gk238) V. Total of three trials (50 worms per trial, each
trial one dot); ****p<0.0001 in Cochran–Mantel–Haenszel test, as compared to the wild- type control. (F)Genetic interventions that induce egg retention
elevate exopher levels. sem- 2(n1343). ALMR exopher scores Ad1. Strain ZB4757: bzIs166[Pmec- 4::mCherry] II vs. strain ZB4902: sem- 2(n1343) I; bzIs166[Pmec-
4::::mCherry] II. Exophers were scored on Ad1 because of the damaging excessive bagging that ensues in this background. Total of three trials (50 worms
per trial, each trial one dot); ****p<0.0001 in Cochran–Mantel–Haenszel test, as compared to the wild- type control. (G)Genetic interventions that induce
egg retention elevate exopher levels. lin- 39 RNAi on a strain expressing Pmec- 4::mCherry from a multi- copy transgene. ALMR exopher scores Ad2. Strain
ZB4757: bzIs166[Pmec- 4::mCherry] II treated with RNAi against lin- 39. Total of six trials (50 worms per trial, each trial one dot); ****p<0.0001 in Cochran–
Mantel–Haenszel test, as compared to the empty vector control. (H)Genetic interventions that induce egg retention elevate exopher levels. lin- 39 RNAi
on a strain expressing Pmec- 18::mKate from a single copy transgene. ALMR exopher scores Ad2. Strain OD2984: ltSi953 [Pmec- 18::vhhGFP4::zif- 1::operon-
linker::mKate::tbb- 2 ’'UTR+Cbr- unc- 119(+)] II; unc- 119(et3) III treated with RNAi against lin- 39. Total of three trials (50 worms per trial, each trial one dot);
****p<0.0001 in Cochran–Mantel–Haenszel test, as compared to the empty vector control. (I)Egg- laying modulating neurotransmitters octopamine (OA)
and serotonin (5- HT) inuence ALMR exophergenesis levels. Data show the mean ± SEM of percentage hermaphrodite ALMR neurons exhibiting an
Figure 3 continued on next page
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We found that the associated massive egg retention correlated with a dramatic elevation of exopher
numbers for each genetic impediment to egg laying (egl- 9 (Figure 3D); egl- 3 (Figure 3E); sem- 2
(Figure3F), lin- 39 RNAi (Figure3G)). For example, under the treatment of lin- 39 RNAi, ~60% of the
wild- type strain expressing mCherry in ALMR by a multi- copy transgene produced ALMR exophers on
Ad1, compared to only~7% of the same strain treated with empty vector (EV) control (Figure3G). We
conclude that regardless of the genetic strategy employed to trap eggs in the body, egg retention can
lead to high ALMR exopher production.
In complementary studies, we examined the impact of egg retention on ALMR exophergenesis
when we expressed fluorescent protein mKate from a single copy transgene (i.e. in the absence of
an over- expressed reporter). We found that the empty vector control treatment without induction
of egg retention is associated with no ALMR exophergenesis in the single copy mKate transgenic
strain (0% in all three trials). However, when we treated with lin- 39 RNAi to induce egg retention, we
measured~60% exophergenesis (Figure3H). Thus regardless of the expression levels of exogenous
proteins in the ALMR neuron, the egg retention condition can induce high exopher production.
Hyperactive egg-laying and consequent low egg retention are
associated with low exopher production
Neurotransmitters octopamine (OA) and serotonin (5- HT) have been well- documented to play
opposing roles in C. elegans egg- laying behavior (Chase and Koelle, 2007). Feeding C. elegans
octopamine strongly suppresses egg- laying to induce egg retention, while supplementing with 5- HT
causes hyperactive egg- laying (Horvitz etal., 1982). Consistent with outcomes in animals physically
blocked for egg- laying, treatment with egg retention- promoting OA enhanced ALMR exophergenesis
(Figure3I).
To test the outcome of 5- HT- induced enhanced egg laying, we measured the impact of 5- HT conse-
quent to 6- hr food withdrawal, a condition that we previously found markedly enhances ALMR exoph-
ergenesis (Cooper etal., 2021), and, therefore, is expected to increase the dynamic range for scoring
exopher suppression. We find that 5- HT treatment, which lowers egg retention, strongly suppresses
fasting- associated ALMR exophergenesis (Figure3I). Although perturbing neuronal signaling holds
complex consequences for whole- animal physiology, our findings are consistent with a model in which
high egg load increases neuronal exophergenesis, and low egg retention decreases exophergenesis.
The egg- laying circuit Is controlled in part by Goα inhibition—null allele goa- 1(n1134) removes this
inhibition such that eggs are often laid at very early developmental stages (2–4cell stage) rather than
being retained in the uterus until gastrulation (~30cell stage) (Waggoner etal., 2000). We intro-
duced goa- 1(n1134) into the mCherryAg2 background and scored ALMR exopher events at Ad2. We
confirmed that goa- 1(n1134) retains few eggs in the uterus and is associated with a significantly lower
number of ALMR exopher events as compared to the age- matched wild- type control (Figure3J).
In sum, manipulation of uterine egg occupancy is strongly correlated with the extent of ALMR
neuronal exophergenesis—high egg retention promotes high exophergenesis and low egg retention
is associated with low exophergenesis.
exopher event at Ad2 after 48hr of treatment with 20mM octopamine (OA) or 4mM serotonin (5- HT) on OP50 bacteria seeded NGM plates. Because
5- HT (which increases egg laying) was hypothesized to suppress exopher production, we assayed under conditions of 6 hr food limitation, which
markedly raises the exopher production baseline, enabling easier quantication of suppression effects (Cooper etal., 2021). Total of three trials and 50
hermaphrodites per trial for each condition. ***p<0.001or ****p<0.0001 in Cochran–Mantel–Haenszel test, as compared to the control group treated
with M9 buffer (solvent for OA or 5- HT). (J)Mutant goa- 1(n1134), with hyperactive egg- laying and low egg retention in the uterus (pictures on the left,
representative of 20, and scale bar = 50 μm), has low exopher scores at Ad2. Strain ZB4757: bzIs166[Pmec- 4::mCherry] II vs. strain ZB5352: goa- 1(n1134) I;
bzIs166[Pmec- 4::mCherry] II. Boxes highlight egg zone. **p<0.01 in Cochran–Mantel–Haenszel test.
The online version of this article includes the following source data for gure 3:
Source data 1. Daily progeny count for panel A, and exopher score for panels B, D, E, F, G, H, I, J.
Figure 3 continued
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ALMR neuron proximity to the egg zone correlates with
exophergenesis frequency
The ALML and ALMR anterior touch neurons share developmental, morphological, and functional
similarities (Chalfie and Sulston, 1981), yet paradoxically, ALMR consistently produces exophers at
higher levels than ALML (Melentijevic etal., 2017; also Figure4G). The ALM neurons are embedded
within the C. elegans hypodermis, but the ALMR soma is situated in the vicinity of the gonad and its
resident eggs, whereas the ALML soma, on the opposite side of the animal, is positioned closer to the
intestine (Figure4A and B; Goodman, 2006).
To ask if proximity to the gonad is correlated with exopher production, we randomly selected 128
Ad2 hermaphrodites that expressed mCherry in the touch receptor neurons, imaged in brightfield
to visualize the egg zone of each animal, and imaged again in the red channel to visualize the touch
neuron, recording the relative positions of ALMR and the egg zone (Figure4C). We also assessed
whether the ALMR neuron had produced an exopher or not.
We found that 37/128 ALMRs examined had produced an exopher (Exopher+), and that 36/37
(95%) of the Exopher+ ALMR neuronal somas that had produced exophers were positioned within
the visualized egg zone (Figure4D). For the Exopher- ALMRs that had not produced exophers, 63/91
(70%) had neuronal somas located in the egg zone. Thus, although ALMR soma positioning in the
egg zone does not guarantee exophergenesis in the mCherryAg2 strain, the neurons that did make
exophers were nearly always in the egg zone (p<0.01 in Chi- Square test, Figure4D).
To further test for the association of egg zone proximity to ALMR and exopher production, we
genetically shifted ALM position. During development, the ALM neurons migrate posteriorly to near
the mid- body (Sym etal., 1999), and most commonly, ALM somas are situated posterior to AVM.
ALM soma positions, however, can be influenced by migration and specification cues. In particular,
transgenic introduction of a mec- 3 promoter fragment bearing an internal deletion (fusion of the
–1 to –563 sequences to the –1898 to –2372 mec- 3 promoter fragment, plasmid pJC4) can induce
anterior ALM migration during development, sometimes resulting in final ALM positions anterior to
AVM (Toms etal., 2001; Figure4E). We took advantage of the partially deleted mec- 3 promotor
sequences in pJC4 to manipulate the ALM position. In these studies, we introduced pJC4 with the
co- transformation marker pRF4 (rol- 6(su1006)) that disrupts the cuticle to induce rolling of transgenic
animals into the Pmec- 4::mCherry background. Rol hermaphrodites have a strikingly high baseline of
ALMR exophergenesis (~40% exophers in rollers vs.~20% in the wild- type). Strikingly, we found that
when ALMs are situated anterior to AVM, ALMR exophergenesis drops to~5% (4/73) vs 71% for the
posterior position (55/78) (Figure 4F). Although we cannot exclude that physiological changes in
differently- positioned touch neurons underlie reduced exophergenesis, data are consistent with a
model in which proximity to the egg zone correlates with exophergenesis.
Another way to increase egg proximity to ALMR is to disrupt egg- laying capacity, which confers egg
retention and uterine expansion. We hypothesized that in the sem- 2(rf) mutant, which is associated
with considerable internal egg accumulation, additional touch neurons should experience increased
proximity to eggs in the blocked uterus. Indeed, we find that in the sem- 2(rf) background, every touch
neuron that is positioned in the general region of the expanded uterus (ALML, AVM, PVM) increases
exopher production, but the posterior PLM neurons, which cannot be approached by the gonad, do
not produce exophers (Figure4G). Thus, touch neurons can be stimulated to produce exophers if
the egg domain is artificially brought closer to them. Data are consistent with a model in which ALMR
normally makes most exophers because of its closest natural positioning near the egg- filled uterus.
Possibilities are that a diffusible signal may eminate from the filled uterus or that mechanical pressure
associated with a filled uterus might signal enhanced exophergenesis.
Uterine expansion associated with high egg load correlates with high
exophergenesis
How might the presence of eggs signal to the maternal neurons to induce exophers? We consider
two main possibilities: (1) eggs filling the uterus might exert physical pressure that activates essential
stretch- signaling for young adult neuronal exopher release. This mechanical stretch signal might act
directly (for example introducing chronic and/or dynamic pressure on the touch neurons), or indi-
rectly (possibly inducing the stretched uterus/somatic gonad to release chemical signals that promote
neuronal exopher formation); (2) early fertilized eggs might release a short- range chemical signal that
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Figure 4. Anterior Lateral Microtubule cell (ALMR) exophergenesis levels correlate with proximity to the egg zone. (A)ALMR is positioned close to the
uterus and Anterior Lateral Microtubule cell (ALML) is situated on the opposite side, close to the intestine. A diagram of ALMR and ALML positions
relative to major body organs in cross- section that is anterior to the ‘egg zone.’ Image drawing based on the EM pictures of adult hermaphrodite slice
#273 of WormAtlas. DNC: dorsal nerve cord; VNC: ventral nerve cord; Hyp7: hypodermal cell 7; CAN(L/R): canal- associated neurons (left/right); neurons
in red. (B)Electron microscopy cross- section image of the uterus region indicating ALMR soma and eggs within the adult uterus. Note ALMR is close
to the egg- lled uterus, ALML is on the opposite side closer to the intestine. The ALML soma is not evident in this cross section. Scale bar = 10μm.
(C)Illustration of the egg zone denition, distance between outermost eggs. The measure of this distance corresponds to uterine length. (D)ALMRs
Figure 4 continued on next page
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contributes to young adult proteostasis reorganization (Labbadia and Morimoto, 2014; Labbadia
and Morimoto, 2015) to promote exophergenesis.
To begin to dissect the role of egg pressure in promoting exophergenesis, we analyzed the phys-
ical relationships of neurons, eggs, and uterine shape. Eggs can readily be observed to distort tissue
structure in young adult C. elegans. For example, in a strain that expresses GFP to label the hypo-
dermis and expresses mCherry to label the touch neurons, the distortion of the hypodermis by eggs
can be easily visualized as dark non- fluorescent eggs project through the observation plane of the
hypodermis (Figure5A). Thus, the uterus can approach and pressure surrounding tissue, including
touch neurons.
We quantitated the absolute uterine length as an indicator for stretch in relation to exopher
production levels under representative conditions of high and low exophergenesis. We found that
egg retention mutants that exhibit high exophergenesis, egl- 3(Δ) (Figure5B), egl- 9(Δ) (Figure5C),
and sem- 2(rf) (Figure 5D), had significantly longer egg zones (i.e. uterus length) as compared to
wild- type. In contrast, cbd- 1 RNAi (Figure5E), sperm- less induction with SPE- 44 AID (Figure5F),
and hyperactive egg- laying mutant goa- 1(Δ) (Figure5G), which we find to be strong exophergenesis
suppressors, are all associated with short uterine egg zones. Knocking down either the 4cell stage
gene mex- 3 or the gastrulation gene gad- 1, which are associated with neither egg retention nor
exopher elevation, does not have an extended egg zone/uterine length (Figure5H), so not all devel-
opmental compromises are associated with uterine extension.
Even when eggshell production and early embryonic divisions are
disrupted, forced uterine expansion can elevate exopher levels
Our initial studies suggest extended uterine length is correlated with high exopher levels, but high
egg retention is also a feature of an extended uterus. To begin testing a requirement for fertilized
eggs per se in the exopher influence, we asked whether egg viability is essential for promoting early
adult exophergenesis. We manipulated egg integrity/uterine contents in egg retention mutants by
egg/embryo perturbation, testing for impact on exophergenesis.
Under conditions of cbd- 1 RNAi, eggshell development and embryonic development are blocked;
eggs that form are fragile and can be malformed consequent to passing through the spermatheca
into the uterus; embryonic development does not proceed and egg remnants tend to be sticky (John-
ston etal., 2010). As shown in Figure2B (and again in Figure6A), treating WT reproductive animals
(that have functional egg- laying capacity) with cbd- 1 RNAi to kill embryos exerts a potent block on
ALMR exophergenesis. We proceeded to test the consequence of cbd- 1 RNAi in mutants that cannot
positioned close to the lled gonad produce exophers more frequently than ALMRs that are positioned a bit more distally. We selected Ad2 mCherry
animals at random, then identied whether or not ALMR had produced an exopher, and subsequently determined whether ALMR was positioned within
the egg zone or outside the egg zone as indicated (neuronal soma positioning differences are a consequence of developmental variation). Neurons
with somas positioned further from the egg area produced fewer exophers than neurons within the egg zone indicated; total of 91 worms for the ‘No
Exopher’ group and 37 for the ‘Exopher +’ group; **p<0.01 in Chi- Square test. (E)The Aamodt group (Toms etal., 2001) previously reported that
high copy numbers of plasmid pJC4 containing the mec- 3 promoter region (−1 to –563, and –1898 to –2372 of the mec- 3 translational start) exhibited
increased abnormal positioning of ALM neurons anterior to AVM. We introduced plasmid pJC4 along with transformation reporter pRF4 rol- 6(su1006)
in the background of mCherryAg2 (note this revealed that rol- 6(su1006) is a strong exopher enhancer) and identied neurons that were positioned
posterior to AVM (normal, close to the uterus) and those that were positioned anterior to AVM (further away from the uterus). (F)ALMR neurons
genetically induced to adopt positions further away from the uterus generate fewer exophers then those close to the uterus. We counted numbers of
exophers produced in Ad2 Rol hermaphrodites for each position type. Strain ZB5046: Ex [(pJC4) Pmec- 3::GFP+pRF4]; bzIs166[Pmec- 4::mCherry] II. Total
of three trials (61(34a + 27p); 39(19a + 20p); 51(20a + 31p) animals per trial); ****p<0.0001 in Cochran–Mantel–Haenszel test. (G)When eggs cannot
be laid in the sem- 2(rf) mutant, the eggs that accumulate in the body are brought in closer proximity to ALML, AVM, and PVM touch neurons with a
resulting increase in their exopher production. Strain ZB4757: bzIs166[Pmec- 4::mCherry] II vs. strain ZB4902: sem- 2(n1343) I; bzIs166[Pmec- 4::mCherry] II. Left,
Exopher scoring (Mean ± SEM) of all six touch receptor neurons in Ad1 wild- type hermaphrodite; Right, Exopher scoring (Mean ± SEM) of all six touch
receptor neurons in Ad1 sem- 2(rf) hermaphrodite. Total of 4 trials (50 worms per trial) for each. Wild- type, egg laying procieint animals on Ad1 exhibit
low exopher levels, but when eggs accumulate early in the sem- 2 mutant, exophers markedly increase in ALMR and other touch neurons that are in the
vicinity of an expanded uterus. PLM neurons are situated posterior to the anus and are not subject to uterine squeezing effects.
The online version of this article includes the following source data for gure 4:
Source data 1. Contingency data for panel D, and exopher score for panels F, G.
Figure 4 continued
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extrude eggs or their remnants, and, therefore, would retain defective eggshell/dead embryos in the
uterus.
We subjected the sem- 2(rf) egg- laying defective mutant to cbd- 1 RNAi so that the uterus would
fill with defective eggshell/dead embryo remains. Strikingly, we found that cbd- 1 RNAi/dead embryo
retention in the sem- 2(rf) background is still associated with significantly elevated levels of ALMR
exophergenesis (~45%), while in egg- laying proficient wild- type, barely any ALMR exophergenesis is
observed under cbd- 1 RNAi conditions (<1%) (69/150 for sem- 2 egg- laying blocked cbd- 1 RNAi vs.
1/150 for egg- laying proficient cbd- 1 RNAi, Figure6A).
Figure 5. Uterine length measures for egg- laying defective and gastrulation defective mutants support the correlation of high exopher production and
uterine expansion. (A)Eggs can distort tissues in their vicinity. Shown (representative of 10, scale bar = 20 μm) is strain ZB4942: fxIs1[Ppie- 1::TIR1::mRuby]
I; bzIs166[Pmec- 4::mCherry] II; spe- 44(fx110[spe- 44::degron]) IV; pwSi93[Phyp7::oxGFP::lgg- 1], with touch neurons expressing mCherry (red); and the
hypodermis expressing GFP. Dark round areas (white arrows) are eggs that press into the hypodermis when viewed in this focal plane. On the right,
slit- like dark regions correspond to hypodermal seam cells. (B)Uterus length of WT vs. egl- 3(∆); strain ZB4757: bzIs166[Pmec- 4::mCherry] II vs. ZB4904:
bzIs166[Pmec- 4::mCherry] II; egl- 3(gk238) V. n = ~20 hermaphrodites from one trial. ***p<0.001 in two- tailed t- test. Note that we did not normalize uterine
length to body length in B- E. (C)Uterus length of wild- type (WT) vs. egl- 9(∆) ZB4757: bzIs166[Pmec- 4::mCherry] II vs. ZB4772: bzIs166[Pmec- 4::mCherry] II;
egl- 9(sa307) V. n = ~20 hermaphrodites from one trial. ****p<0.0001 in two- tailed t- test. (D)Uterus length of WT vs. sem- 2(rf); strain ZB4757: bzIs166[Pmec-
4::mCherry] II vs. ZB4902: sem- 2(n1343) I; bzIs166[Pmec- 4::mCherry] II. n = ~20 hermaphrodites from one trial. ****p<0.0001 in two- tailed t-test. (E)Uterine
length is short under cbd- 1 RNAi compared to WT + empty vector RNAi. Uterus length of strain ZB4757: bzIs166[Pmec- 4::mCherry] treated with RNAi
against cbd- 1 or control empty vector feeding RNAi. n = ~20 hermaphrodites from one trial. ****p<0.0001 in two- tailed t- test. (F)When sperm
maturation is blocked in egg- laying competent animals, leaving oocytes to occupy reproductive structures, the uterus length is short. Uterus length of
strain ZB4749: fxIs1[Ppie- 1::TIR1::mRuby] zdIs5[Pmec- 4::GFP] I; bzIs166[Pmec- 4::mCherry] II; spe- 44(fx110[spe- 44::degron]) IV. 1mM auxin treatment induces
the no sperm status. n = ~20 hermaphrodites from one trial. ****p<0.0001 in two- tailed t- test. (G)Uterine length is short in the hyperactive egg- laying
mutant which has low occupancy of eggs in the uterus. Uterus length of strain N2: wild- type vs. strain MT2426: goa- 1(n1134) I. n=20 hermaphrodites
from one trial. ****p<0.0001 in two- tailed t-test. (H)Knocking down the 4cell stage gene mex- 3 or the gastrulation gene gad- 1 has normal uterine
length. Uterus length of strain ZB4757: bzIs166[Pmec- 4::mCherry] treated with RNAi against the mex- 3 or gad- 1 gene. mex- 3 RNAi disrupts embryonic
deveopment at the 4cell stage, while gad- 1 RNAi disrupts gastrulation at the stage at which eggs are normally laid and perturbs later development but
not egg shell formation and egg laying. n = ~20 hermaphrodites from one trial. Not signicant (ns) in two- tailed t- test as compared to the empty vector
control.
The online version of this article includes the following source data for gure 5:
Source data 1. Uterus length data for panels B, C, D, E, F, G, H.
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Figure 6. Uterine expansion correleates strongly with Anterior Lateral Microtubule cell (ALMR) exophergenesis regardless of whether eggs, oocytes,
or debris are retained. (A)Despite cbd- 1 RNAi mediated disruption of eggshell formation and earliest embryonic cell divisions, exopher levels are high
in the sem- 2(rf) egg retention background. The percentage of ALMR exopher events among 50 Ad2 wild- type (left) or Ad1 sem- 2(rf) hermaphrodite C.
elegans that are treated with either empty vector control RNAi or RNAi targeting cbd- 1 in each trial (total of 3 independent trials). sem- 2 mutants bag
extensively at Ad2 and cannot be tested. Diagram indicates uterine lling status of test sem- 2(rf) + cbd- 1 RNAi. Strain ZB4757: bzIs166[Pmec- 4::mCherry] II
vs. strain ZB4902: sem- 2(n1343) I; bzIs166[Pmec- 4::mCherry] II. ****p<0.0001 in Cochran–Mantel–Haenszel test. (B)Uterine length remains long in the egg-
laying defective sem- 2(rf) + cbd-1 RNAi. Uterus length of strain ZB4757: bzIs166[Pmec- 4::mCherry] II vs. ZB4902: sem- 2(n1343) I; bzIs166[Pmec- 4::mCherry] II
treated with RNAi against cbd- 1. n = ~20 hermaphrodites from one trial, Ad2, ****p<0.0001 in two- tail unpaired t- test. (C)Blocking sperm maturation in
a sem- 2(rf) mutant, which lls the uterine space with oocytes, induces exophers in the absence of eggs. The percentage of ALMR exopher events among
50 sem- 2(rf) hermaphrodite C. elegans that express the SPE- 44 AID system (‘control’ is treated with 0.25% ethanol vehicle and ‘no sperm’ is treated with
1mM auxin in 0.25% ethanol from egg to adult day 2) in each trial (total of 3 independent trials). L4 stage is the last larval stage before adult. Diagram
indicates uterine oocyte lling status of test sem- 2(rf) + SPE- 44 AID. Strain ZB4953: sem- 2(n1343) fxIs1[Ppie- 1::TIR1::mRuby] I; bzIs166[Pmec- 4::mCherry] II;
spe- 44(fx110[spe- 44::degron]) IV. (D)When sperm maturation is disrupted in mutants blocked for egg laying, leaving oocytes to occupy reproductive
structures, the uterus expands as oocytes accumulate. Uterus length of strain ZB4749: fxIs1[Ppie- 1::TIR1::mRuby] zdIs5[Pmec- 4::GFP] I; bzIs166[Pmec-
4::mCherry] II; spe- 44(fx110[spe- 44::degron]) IV vs. ZB4953: sem- 2(n1343) fxIs1[Ppie- 1::TIR1::mRuby] I; bzIs166[Pmec- 4::mCherry] II; spe- 44(fx110[spe-
44::degron]) IV. 1mM auxin treatment induces the no sperm status to both strains. n = ~20 hermaphrodites from one trial, Ad2. ****p<0.0001 in
two- tail unpaired t- test. (E)Disrupting sperm maturation in lin- 39 RNAi animals blocked for egg- laying lls the uterine space with oocytes, and induces
Figure 6 continued on next page
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 16 of 28
Importantly, under conditions of defective eggshell/dead embryo retention associated with cbd- 1
RNAi; sem- 2(rf), the uterine egg zone is expanded (Figure6B), extending the correlation of exopher
production with uterine length. We conclude that intact eggshells and earliest embryonic divisions
are not required for the boost in exopher production observed when uterine contents are forced to
accumulate—uterine retention of dead eggs and egg remnants is sufficient for exopher elevation if
the egg laying apparatus is defective. Expansion of the uterine compartment, rather than eggshell/
embryo integrity, tracks with exopher elevation.
Forced uterine expansion via oocyte accumulation can elevate exopher
levels
Although uterine retention of malformed inviable embryos is sufficient to elevate neuronal exophers
when egg laying is blocked, the defective cbd- 1 embryos or debris might still release egg- associated
chemical signals. To test for a requirement of any fertilization- dependent egg signals in the egg- laying
compromised mutants, we asked whether uterine filling with only oocytes can suffice to promote
neuronal exopher elevation. Unfertilized oocytes cannot initiate embryonic development or egg- shell
biosynthesis; nor can oocytes elevate ALMR exophergenesis in hermaphrodites that are proficient at
egg- laying (Figure1G&H).
We tested two distinct uterine retention conditions—sem- 2(rf) and lin- 39 RNAi—in which we used the
auxin- inducible degron system to disrupt sperm maturation such that only oocytes filled the gonad. Note
that in the absence of sperm, oocytes do not mature but are ‘ovulated’ at a reduced rate compared to
in the presence of sperm; oogenesis continues such that~25 oocytes are typically found stacked in the
gonad (Figure6—figure supplement 1; McGovern etal., 2007). For both genetic retention strategies,
we found that build- up of retained oocytes in egg- laying blocked animals was both sufficient to elevate
exophers (Figure6C&E) and expand the uterus (Figure6D&F; Figure6—figure supplement 1). More-
over, the oocyte retention was similarly efficacious in elevating exopher production to egg retention,
increasing ALMR exophergenesis to approximately 80% in the sem- 2(rf) mutant (Figure6C). We conclude
that fertilization, egg shells, and egg remnants are not essential for the early adult exopher peak. Expan-
sion of the uterus with unfertilized oocytes can suffice to elevate neuronal exopher formation.
exophers in the absence of eggs. The percentage of ALMR exopher events among 50 adult day 1 or 2 SPE- 44 AID no sperm hermaphrodite C. elegans
treated with either control RNAi or lin- 39 RNAi in each trial (total of 3 independent trials). Diagram indicates uterine oocyte lling status of test lin- 39
RNAi + SPE- 44 AID. ZB4749: fxIs1[Ppie- 1::TIR1::mRuby] zdIs5[Pmec- 4::GFP] I; bzIs166[Pmec- 4::mCherry] II; spe- 44(fx110[spe- 44::degron]) IV. ****p<0.0001 in
Cochran–Mantel–Haenszel test. (F)When sperm maturation is blocked, leaving oocytes to occupy reproductive structures, the uterus length is short;
but if oocytes cannot be laid in the lin- 39 RNAi background, the uterus expands as oocytes accumulate. Uterus length of strain ZB4749: fxIs1[Ppie-
1::TIR1::mRuby] zdIs5[Pmec- 4::GFP] I; bzIs166[Pmec- 4::mCherry] II; spe- 44(fx110[spe- 44::degron]) IV +1mM auxin treatment to eliminate sperm maturation. n
= ~20 hermaphrodites. ****p<0.0001 in two- tail unpaired t- test. (G)Blocking oocyte production in the background of lin- 39 RNAi- mediated disruption
of the egg- laying apparatus eliminates early adult exophergenesis. We used SPE- 44 AID to block sperm maturation and fem- 3(q20) to prevent oocyte
production; lin- 39 RNAi to disrupt egg- laying capacity. Diagram indicates empty uterus status of test fem- 3(q20); spe- 44 AID; lin- 39(RNAi) strain.
Exopher scoring of Ad2 ZB4749: fxIs1[Ppie- 1::TIR1::mRuby] zdIs5[Pmec- 4::GFP] I; bzIs166[Pmec- 4::mCherry] II; spe- 44(fx110[spe- 44::degron]) IV +1mM auxin
or ZB5042: bzIs166[Pmec- 4::mCherry] II; fem- 3(q20)ts IV, treated with either control empty vector (EV) RNAi or lin- 39 RNAi at 25°C. Total of three trials (50
worms per trial). ****p<0.0001 in Cochran–Mantel–Haenszel test. (H)Blocking oocyte production in the background of sem- 2(rf)- mediated disruption of
the egg- laying apparatus eliminates early adult exophergenesis. We used cbd- 1 RNAi to disrupt eggshell and fem- 3(q20) to prevent oocyte production;
sem- 2(n1343) to disrupt egg- laying capacity. Exopher scoring of adult day 2 hermaphrodites, treated with either control empty vector (EV) RNAi or cbd- 1
RNAi at 25°C. Total of three trials (50 worms per trial). Diagram indicates empty uterus status of test fem- 3(q20); cbd- 1 RNAi; sem- 2(rf) strain. (I)The
uterus length is correlated with ALMR exophergenesis. Data shown are the mean of uterus length (X- aixs) and percentage ALMRs with exopher (Y- axis)
for different genotypes/treatments measured in this study. The correlation line is based on a linear t model and the Pearson r and p value is based on
the correlation assay. Uterus length from short to long: SPE- 44 AID; cbd- 1 RNAi; wild- type (adult day 1); mex- 3 RNAi; gad- 1 RNAi; wild- type (adult day 2);
egl- 3(∆); egl- 9(∆); sem- 2(rf); SPE- 44 AID+ lin- 39 RNAi; SPE- 44 AID+ sem- 2(rf).
The online version of this article includes the following source data and gure supplement(s) for gure 6:
Source data 1. Exopher score for panels A, C, E, G, H, I, and uterus length data for panels B, D, F, I.
Figure supplement 1. Representative pictures of oocytes retention (red rectangle) in the uterus of Adult day 2 hermaphrodite.
Figure 6 continued
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 17 of 28
Lack of a functional egg-laying apparatus does not induce exopher
elevation when the uterus is not filled
The above- described experiments left open the possibility that the lack of a functional egg- laying
apparatus itself might be causative in the elevation of exopher production. To address this possi-
bility, we compared disruption of sperm (permissive for oocyte accumulation) to disruption of oogen-
esis (effectively empties the uterus) when egg- laying capacity was compromised by lin- 39 RNAi
(Figure6G). lin- 39 RNAi+ oocyte retention promotes exopher formation, but eliminating oocytes
(fem- 3(gf)) eliminates exopher elevation even when egg- laying is blocked by lin- 39 RNAi. That is to
say, although oocyte accumulation with uterine expansion suffices to elevate exophers, removing
the oocytes and uterus occupancy eliminates the exopher boost. We observe the same outcome of
suppressed exopher formation when cbd- 1 RNAi- induced dead embryo retention in the sem- 2(rf)
egg- laying defective mutant (which is exopher- inducing) is prevented from oocyte production by fem-
3(gf) (Figure6H). Thus, disruption of egg laying on its own is not the driving factor in high exopher-
genesis; rather, uterine filling is required.
We revisited the relationship of uterine length and exopher level by adding data from the studies
with oocyte retention to reinforce the conclusion that ALMR exophergenesis is strongly correlated
with the level of uterus stretching caused by the accumulation of uterine contents (Figure6I).
Sustained physical distortion of the gonad by fluid injection can rapidly
elevate exopher production
To independently test for a role in physical stretch/filling of the uterus in exopher induction, we
distorted the gonad compartment by injecting dye- containing M9 buffer into a very young adult. The
animal subjects we tested were vulva- less (lin- 39 RNAi) and also subjected to SPE- 44 AID to block
sperm production. These vulva- less+ spermless hermaphrodites normally exhibit high ALMR exoph-
ergenesis at late Ad1 and Ad2 (Figure6C&D) due to oocyte accumulation. To avoid oocyte influence,
we conducted our physical expansion studies just as animals reached Ad1, a time when ALMR exoph-
ergenesis is typically not observed (Figure7A).
We picked the L4 stage vulva- less (lin- 39 RNAi)+ sperm less (SPE- 44 AID) hermaphrodites for
age synchronization (20°C, grown for 12hr after L4 selection) and then injected dye containing M9
buffer into the uteri of these very young adult days 1 hermaphrodites, scoring for exopher produc-
tion~10min after the injection (Figure7B).
Control animals (mock- injected animals that were stabbed without fluid delivery) exhibited
no ALMR exophers. By contrast, we found that when we held injection pressure continuously for
2 or more minutes, ~20% of the ALMs scored exhibited an exopher event shortly after injection
(Figure7C&D). Injection experiments using animals with functional vulvae (in which injected mate-
rial is rapidly extruded through the vulval opening) failed to induce ALMR exophers (Figure 7E),
supporting that ALMR exophergenesis caused by 2min injection of dye- containing M9 is due to the
physical distortion of gonad rather than the chemical impact of the dye- containing M9.
In sum, exopher induction by uterine accumulation of eggs, malformed eggs, dead embryos,
oocytes, or fluid- induced expansion supports a model in which early adult ALMR exophergenesis is
elevated by physical distortion of the uterus that occurs with reproduction (Figure7F).
Discussion
Exopher production by proteo- stressed C. elegans touch neurons occurs with a striking temporal
pattern that features an early adult peak of exophergenesis coincident with the period of maximal
egg production. Here, we report that the early exophergenesis peak is dependent on uterine occu-
pation, which normally is conferred by fertilized egg accumulation prior to the deposition of eggs.
Uterine expansion that is associated with the filling of a blocked uterus with unfertilized oocytes or
by fluid alone can also induce high neuronal exopher production, supporting a model in which the
physical distention associated with uterine occupancy, rather than chemical signals derived from fertil-
ized eggs per se, is a required component of the signaling relay between reproduction and young
adult neuronal debris elimination. Trans- tissue cross- talk to the maternal nervous system thus appears
accomplished via mechanical force transduction. Our findings hold implications for mechano- biology
in neuronal proteostasis management.
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 18 of 28
Figure 7. Anterior lateral microtubule cell (ALMR) exophergenesis can be induced by uterine compartment distortion that accompanies uid
injection. (A)Summary of timing of ALMR exophergenesis from age synchronized spermless+ vulvaless hermaphrodite. Total of three trials and 50
hermaphrodites in each trial. Strain ZB4749: fxIs1[Ppie- 1::TIR1::mRuby] zdIs5[Pmec- 4::GFP] I; bzIs166[Pmec- 4::mCherry] II; spe- 44(fx110[spe- 44::degron]) IV,
cultured on the 1mM auxin treated nematode growth media (NGM) agar plates seeded with HT115 E. coli expressing dsRNA against the C. elegans lin-
39 gene. Eventually, oocytes accumulate in this strain, but at worst very few are evident in the timeframe in which we performed injections. Data shown
are mean ± SEM at each time point. The data demonstrate that there is no ALMR exophergenesis in this background before the 18th hr after L4 sync.
Testing the impact of injection on ALMR exophergenesis before the 17th hr after L4 thus monitors injection consequences during a timeframe in which
no exophers are normally produced. (B)Illustrated experimental design for testing the ALMR exophergenesis response to physically expanding the
gonad via 2min continuous uid injection. We performed 2+ min duration injections of M9 buffer mixed with food color dye 1:10 or 1.5:10 dye/M9 ratio
(to verify successful injection; dye contains water, propylene glycol, FD&C reds 40 and 3, propylparaben) into the uteri of sperm- less only (EV control) or
sperm- less+ vulvaless (treated with lin- 39 RNAi) animals. Strain: ZB4749 as in panel A, treated with either empty vector (EV) or RNAi against lin- 39 in the
presence of 1mM auxin. lin- 39 RNAi disrupts vulval development such that injected uids are retained to expand the uterus. In injections with animals
that have functioning egg- laying apparatus, uids can exit the animal and do not expand the uterus. (C)2min sustained injection into egg- laying
blocked, reproduction blocked animals can induce rapid exophergenesis: representative picture of an ALMR exophergenesis event consequent to
injection. (D)2min sustained injection into egg- laying blocked, reproduction blocked animals can induce rapid exophergenesis: exopher scoring of the
control mock injected and 2min injected animals. Strain ZB4749: fxIs1[Ppie- 1::TIR1::mRuby] zdIs5[Pmec- 4::GFP] I; bzIs166[Pmec- 4::mCherry] II; spe- 44(fx110[spe-
44::degron]) IV treated with auxin and lin- 39 RNAi to induce the sperm- less and vulva- less status, respectively. Data represent a total of three trials
(6–10 worms in each trial). p<0.05 in Cochran–Mantel–Haenszel test, as compared to the no- injection uid control. (E)2min sustained injection induces
rapid ALMR exophergenesis only from vulvaless animals. Data showing the exopher scoring of the sperm- less EV control (with functional vulva) or
sperm- less+vulvaless (treated with lin- 39 RNAi) worms. Strain: ZB4749 (genotype in panel A legend) treated with either EV or RNAi against lin- 39 in the
presence of 1mM auxin to disrupt sperm maturation. Data represent a total of 6 trials (6–10 worms in each trial). 3 out of 6 trials showed ALMR exopher
induction by M9 injection to the vulvaless worms; while not a single trial produced ALMR exopher induction by M9 injection to animals with WT vulvae.
If the vulva is intact, injected uids are observed to leak out, consistent with the assumption that the gonads of egg- laying procient animals will not
sustain required expansion. (F)Summary. Eggs, dead egg accumulation, oocyte accumulation, or injection pressure all lead to ALMR exophergenesis.
These varied interventions have a similar impact on the uterus, which is the uterine distortion by mechanical forces. We propose that early adult ALMR
exophergenesis requires mechanical or stretch- associated force generated by the uterine cargos.
The online version of this article includes the following source data for gure 7:
Source data 1. Exopher score for panels A, D, E.
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 19 of 28
The mechanical landscape of reproduction that influences neuronal
exophergenesis
C. elegans reproduction features physical expansion and contraction of multiple tissue/cell types--the
gonad houses the expanding germline, and the spermatheca expands and retracts vigorously as each
mature oocyte enters and exits. The uterus also stretches to house eggs and can contract locally as
eggs transit or are expelled via the action of the vulval and uterine muscles. The filled reproductive
apparatus can thus clearly exert both constitutive and sporadic pressure on surrounding tissues as it
enacts its essential functions.
What is the source of the force that promotes exopher production? Elegant work on the egg- laying
circuit (comprising the somatic gonad, the HSN and VC neurons, and the vulval, and uterine muscle)
has provided evidence for mechanical signaling within the egg- laying circuit that regulates initiation,
promotion, and termination of egg- laying (Medrano and Collins, 2023; Ravi etal., 2018; Kopchock
etal., 2021; Ravi etal., 2021). Working backward, exopher- promoting force seems unlikely to derive
from the vulva or vulval muscle contractions, since when these cells are genetically disrupted in lin- 39
RNAi or in sem- 2 mutants, high levels of exophers still are generated. Changes in spermatheca volume,
which expands and contracts dramatically as mature oocytes enter via valve opening/closure (Tan
and Zaidel- Bar, 2015), might be sensed, but spermatheca contractions are reported to be normal in
hyperactive egg- laying goa- 1 (Govindan etal., 2006), which is an exopher- suppressing background.
Under no- sperm conditions, oocyte transit rates are lower than for fertilized eggs, and sperm- derived
signals influence spermathecal valve opening (McGovern etal., 2007; McCarter etal., 1999; Miller
etal., 2001), but if the egg- laying apparatus is genetically compromised and oocytes accumulate
in the absence of sperm, exopher levels are high, suggesting deficits in spermatheca operations or
sperm signals per se do not drive exophergenesis. Given that under normal reproductive conditions
of egg- laying proficiency, correctly shelled eggs are required for the early peak in exopher produc-
tion, a plausible hypothesis was that fertilized eggs might produce an essential diffusible factor that
stimulates neuronal exopher- genesis. However, exophers can be produced abundantly in the absence
of fertilized eggs when the vulva is unable to open and release uterine contents, resulting in uterine
distention due to debris filling. Thus, the simplest model we envision for the reproductive cues that
influence maternal neuronal exophergenesis is that a filled uterus (under normal conditions the conse-
quence of hard- shelled eggs that occupy it) is sensed and required for the early adult peak in exopher
production.
How might force be sensed and transduced?
Mechanotransduction is the sensing of a mechanical signal, such as pressure or stretch, and conver-
sion into a cellular response. Members of several ion channel families have been implicated in sensing
of touch, hearing, shear stress, and pressure, including Piezo, TRP, and DEG/ENaC families (Delmas
and Coste, 2013). These are rational candidates for mechanosensors in neurons, the uterus, or other
cells that might act in a relay between the uterus and neuron.
At the same time, classic mechanosensory channels have extremely rapid gating and might not be
the best- suited candidates for acting in the sustained and locally dynamic forces anticipated for the
reproductive uterine environment. Adhesion G- Protein Coupled Receptors, which have extracellular
adhesion motifs and seven transmembrane domains characteristic of the GPCR class (lat- 1, lat- 2,
cdh- 6 in C. elegans [Mee etal., 2004; Willson etal., 2004; Hutter etal., 2000]), or components of
the YAP/TAZ transcriptional program (Panciera etal., 2017) may integrate responses to forces trans-
mitted via the cytoskeleton and could be considered as potential players in the required signaling.
Determination of the identity of mechanotransducers and assignment of the site of action to the
neuron, the uterus, or an intermediate relay cell type remains for future studies. Modeling will also
need to incorporate the fact that fluid injections, which require 2min long sustained application of the
filling stimulus to induce exophers, could provoke exopher production on a rapid timeframe, typically
recorded only 10min after the injection period. Thus the proteostressed touch neurons appear poised
to eliminate contents upon mechanical stimulation.
Mechanical signaling in reproduction across species
Uterine stretch may be a more prevalent mechanism for inducing maternal nervous system response
than currently appreciated. Distention of the female fly reproductive tract by egg passage through the
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 20 of 28
tract (normal biology) or by artificial means (experimental fluid injection) can induce behavioral attrac-
tion of the mother to acetic acid, thought to signal a favorable food environment for offspring (Gou
etal., 2014). In this case, DEG/ENaC channel family member PPK1 expressed in a subset of mechano-
sensitve neurons that tile the reproductive tract and respond to its contraction/distention is required.
The pathway to the behavioral change remains to be determined. Uterine stretch in mammals has also
been to reported influence maternal behavior (Kristal, 2009).
Why link exophers to reproduction?
Turek et al. report that exophers produced by C. elegans muscle cells follow a similar time course of
highest production at adult day 2, and demonstrated a dependence of the temporal muscle exopher-
genesis pattern on eggs, and commonly with highest exopher production in muscles in the vicinity of
the uterus (Turek etal., 2021) (muscle exophers may be released to supply nutrients to developing
progeny). Together with our observations, data raise the possibility that the onset of reproduction
and the initial filling of the uterus triggers, or generates a ‘license’ for EV/exopher production across
tissues. In the case of stressed touch receptor neurons that have been our focus, evidence suggests
that deleterious protein aggregates and/or excess proteins and organelles are handed off to neigh-
boring glial- like hypodermal cells for degradation (Wang etal., 2023). Clearing the nervous system
(and other organs) for optimal function might confer a selective advantage for successful maternal
reproduction.
In this regard, it is fascinating that the peak exopher production period is coincident with a
proteostasis reconfiguration that has been well documented to accompany reproduction onset
in young adult C. elegans. In brief, during larval development, C. elegans exhibits high activity
of HSF- 1 and consequently, HSF- 1- dependent chaperone expression, but HSF- 1 activity is turned
down in adult life (Labbadia and Morimoto, 2014; Labbadia and Morimoto, 2015; Sala and
Morimoto, 2022; Sala etal., 2020). At the same time in early adult life, proteasome activity is rela-
tively enhanced (at least as measured in the hypodermis) (Liu etal., 2011). These measures may
reflect a general proteostasis reorganization (chaperone activity, proteosome activity) that occurs in
early adult life in response to reproduction (Labbadia and Morimoto, 2014; Sala and Morimoto,
2022). Our observations on neuronal exophers suggest that exopher- mediated content elimina-
tion may constitute another co- regulated branch of this proteostasis reconfiguration. Importantly,
the HSF- 1 turn- down in young adult life is blocked by cbd- 1 RNAi (and additional early eggshell/
development gene RNAi) (Sala etal., 2020). Thus the presence of eggs can signal across tissues
to turn- down hsf- 1 proteostasis- related activities in the mother. We speculate that this young adult
reconfiguration of proteostasis might reflect a mechanism to optimize successful reproduction,
possibly both fine- tuning nervous system function and shifting resources balance to favor progeny
as suggested by the disposable soma theory of aging proposed by Kirkwood (Kirkwood and Holl-
iday, 1979).
Of note, we do not observe exophers in larval stages (Melentijevic etal., 2017). We speculate
that young adult physiology might be temporally tweaked such that some tissues have optimized
capacity to manage/degrade large aggregates and organelles at an early adult developmental
‘clean up’ time, possibly analogous to how a town service for bulky oversized garbage pick- up
might be limited to particular days during the year. As exopher production appears generally
beneficial for neuronal function and survival (Melentijevic etal., 2017; Yang etal., 2022), the
early life extrusion phase appears a positive feature of reproductive life. More broadly, proteome
‘clean up’ phases may be programmed as key steps at specific transitions during development
and homeostasis, for example, as occurs in the temporal lysosome activation that clears aggre-
gate debris in C. elegans maturing oocytes (Bohnert and Kenyon, 2017) or in the maturation of
mouse adult neuronal stem cells via vimentin- dependent proteasome activity during quiescence
exit (Morrow etal., 2020).
Across species, the production of exopher- like vesicles may also be enhanced by mechanical
signals anchored outside of reproduction. For example, mice cardiomyocytes that are constantly
under mechanical stress due to contraction activities produce exopher- like vesicles (Nicolás-Ávila
etal., 2020). Mouse kidney proximal tubular epithelia cells (PTEC) under constant mechanical stress
due to both fluid shear stresses and absorption- associated osmotic pressure, also release exopher- like
vesicles (Huang etal., 2023).
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 21 of 28
Large vesicle extrusion, mechanobiology, and neurodegenerative
disease
The impact of mechanical force on in vivo production of extracellular vesicles has not been a major
focus of the EV field, although a range of studies have considered force consequences (such as fluid
shear responses, stretch) in cultured cells. Overall, however, EV biogenesis and uptake appear to be
markedly influenced by biomechanical force type, magnitude, and duration (Thompson and Papout-
sakis, 2023). At the same time, the neurodegeneration field has generated myriad studies linking
Alzheimer’s disease susceptibility and AD pathology signatures such as extracellular accumulation of
amyloid-β protein and/or intracellular accumulation of tau as outcomes of mechanical stress- based
stimuli such as traumatic brain injury, arterial hypertension, and normal pressure hydrocephalus
(Ramos- Cejudo etal., 2018; Malone etal., 2022). Mechanical stress may trigger or promote protein
misfolding, aggregation, and extrusion. Examples of recent implication of mechanical stimuli in
AD- related outcomes include that stretch in the brain vascular system can increase APP and B- secre-
tase expression to increase Aβ production (Gangoda etal., 2018) and that microglial mechano-
sensing via the Piezo1 mechanotransducing channel limits progression of Aβ pathology in mouse
models (Hu et al., 2023). Our study reveals a capacity of mechanical force to influence neuronal
release of large vesicles containing neurotoxic species, inviting more serious consideration of the roles
of mechanobiology in maintaining proteostasis and influencing aggregate transfer within the context
of a living nervous system.
Table 1. Strain list.
Strain Name Genotype Index
N2 wild- type wild- type
ZB4065 bzIs166[Pmec- 4::mCherry] II wild- type
ZB4757 bzIs166[Pmec- 4::mCherry] II (ZB4065 outcrossed to N2 six times) wild- type
ZB4768 glp- 4(bn2)ts I; bzIs166[Pmec- 4::mCherry] II glp- 4(ts)
ZB5042 bzIs166[Pmec- 4::mcherry] II; fem- 3(q20) IV fem- 3(gf)
ZB4915 bzIs166[Pmec- 4::mCherry] II; fem- 1(hc17) IV fem- 3(lf)
ZB4749 fxIs1[Ppie- 1::TIR1::mRuby] zdIs5[Pmec- 4::GFP] I; bzIs166[Pmec- 4::mCherry]
II; spe- 44(fx110[spe- 44::degron]) IV. SPE- 44
ZB4941 bzIs166[Pmec- 4::mCherry]; gna- 2(gk308) I/hT2 [bli- 4(e937) let-?(q782)]
qIs48[Pmyo- 2::GFP; Ppes- 10::GFP; Pges- 1::GFP] (I;III) gna- 2(∆)
AD295 spe- 45(tm3715); him- 5(e1490); asEx89 [spe- 45 ‘fosmid 1’
mixture+Pmyo::gfp] spe- 45(tm3715)
ZB4772 bzIs166[Pmec- 4::mCherry] II; egl- 9(sa307) V egl- 9(∆)
ZB4904 bzIs166[Pmec- 4::mCherry] II; egl- 3(gk238) V egl- 3(∆)
ZB4902 sem- 2(n1343) I; bzIs166[Pmec- 4::::mCherry] II sem- 2(rf)
ZB5352 goa- 1(n1134) I; bzIs166[Pmec- 4::mCherry] II goa- 1(∆)
ZB5046 Ex [(pJC4) Pmec- 3::gfp+pRF4]; bzIs166[Pmec- 4::mCherry] II pJC4 +rol- 6(su1006)
ZB4942 fxIs1[Ppie- 1::TIR1::mRuby] I; bzIs166[Pmec- 4::mCherry] II; spe-
44(fx110[spe- 44::degron]) IV; pwSi93[Phyp7::oxGFP::lgg- 1]Figure5A
ZB4953 sem- 2(n1343) fxIs1[Ppie- 1::TIR1::mRuby] I; bzIs166[Pmec- 4::mCherry] II;
spe- 44(fx110[spe- 44::degron]) IV sem- 2(rf)
ZB5709 sem- 2(n1343) I; bzIs166[Pmec- 4::mCherry] II; fem- 3(q20) IV. sem- 2(rf); fem- 3(q20)
OD2984 ltSi953 [Pmec- 18::vhhGFP4::zif- 1::operon- linker::mKate::tbb- 2
3'UTR+Cbr- unc- 119(+)] II; unc- 119(ed3) III Single- copy transgene
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 22 of 28
Materials and methods
Strains and maintenance
All strains used in this study carry the transgene bzIs166[Pmec- 4::mCherry] to mark the six touch receptor
neurons: ALMR, ALML, AVM, PVM, PLMR, and PLML. The genotype of C. elegans strains used in this
study are listed in Table1. We maintained all C. elegans strains on nematode growth media (NGM)
seeded with OP50- 1 Escherichia coli in a 20°C or 15°C incubator. We kept all animals on food for at
least 10 generations before using them in a test.
Age synchronization
For the majority of experiments, we used a bleaching protocol or an egg- laying protocol for age
synchronization, otherwise, we picked L4 animals for synchronization.
Temperature sensitive mutants
We maintained the age- synchronized temperature- sensitive mutations in a 15°C incubator. For either
fem- 1 or fem- 3 mutants, we directly placed the isolated eggs into a 25°C incubator in each exper-
imental test. Since the egg- hatching of glp- 4 mutant is out of sync at 25°C, we placed the isolated
eggs in a 15°C incubator for 24hr before transferring them into the 25°C incubator for experimental
tests.
Auxin inducible degradation
We dissolved auxin (indole- 3- acetic acid, 98+%, A10556, Alfa Aesar) in 95% ethanol to make a 400mM
stock, then we prepared a 40mM auxin solution by diluting the 400 mM stock solution in M9 medium
(which contains 1mM MgSO4) and applied 200ul of the 40mM solution on to the NMG- agar plate
(which is 60mm in diameter and contains~8–9g medium). We left the plate on an open bench at
room temperature for one or two days to dry out the auxin solution, then seeded the plate with 200ul
OP50- 1 E. coli and waited for another two or three days before storing the plates in a 4°C environ-
mental room. We only used the plates which had been stored in the 4°C environmental room for at
least a week, so the concentration of auxin can be equilibrated into 1mM. We prepared the ethanol
control plates under the same procedure, and the final concentration of ethanol in the control plates
should be~0.25%.
To proceed auxin inducible degradation of SPE- 44, we placed the isolated eggs on auxin- treated
plates or the ethanol control plates. After~72hr of culture in a 20°C incubator, the animal reached
the age of adult day 1 and the auxin- treated worms became sterile. Then, we transferred the worms
into a regular NGM- agar plate without auxin or ethanol.
Microscopy and image processing
We took the DIC or fluorescent pictures with a Zeiss compound microscope or spinning disc confocal
microscope driven by MetaMorph software, then processed the pictures with Fiji ImageJ software.
Exopher scoring
Exopher numbers vary in experiments between 5–30%, mostly 10–15%range, and typically reach
peak at adult day 2 (Melentijevic etal., 2017). Exphers can remain intact for approximately 2hr,
but the vesicle form of exophers is mostly identifiable in the following 24hr. Therefore, the exopher
count includes both the intact form and the fragmented degraded form, also known as ‘starry night’
(Wang etal., 2023). We scored the exopher count with the protocol published in JoVE (Arnold etal.,
2020). We age- synchronized the animal via egg- laying, bleaching, or L4 picking. Then, we examined
at least 50 animals for each genotype or treatment with the FBS10 Fluorescence Biological Micro-
scope (KRAMER scientific), repeated for at least three trials.
RNAi treatment
All RNAi clones used in this study come from the Ahringer RNAi library. The NGM- agar RNAi plate
contains 1mM IPTG and 25 μg/ml carbenicillin. The food on top of the medium is HT115 bacteria
expressing dsRNA against a targeted gene or carrying an empty vector (EV, L4440) as the control. The
treatment is from eggs to the last day of each test.
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 23 of 28
FUdR treatment
The concentration of FUdR on NGM- agar plate is~51mM. The treatment started from adult day 1.
Male mating experiment
In each trial, we prepared~2000 age synchronized adult day 1 hermaphrodites and did exopher
counting for 50 worms from adult day 1 to day 3. In adult day 3, we divided the worms into three
groups (~400 worms in each group, and~100 worms per plate). Group 1 is the control worms without
males. We added~100 sterile males into group 2 and~100 normal males into group 3 for each plate.
Electron microscopy
We prepared mCherry animals for TEM analysis by high pressure freezing and freeze substitution
(HPF/FS), and followed a standard to preserve ultrastructure. After HPF in a Baltec HPM- 010, we
exposed animals to 1% osmium tetroxide, 0.1% uranyl acetate in acetone with 2% water added,
held at −90°C for 4days before slowly warming back to −60°C, −30°C, and 0°C, over a 2 day
period. We rinsed the samples several times in cold acetone and embedded the samples into a plastic
resin before curing them at high temperatures for 1–2days. We collected serial thin sections on
plastic- coated slot grids and post- stained them with 2% uranyl acetate and then with 1:10 Reynold’s
lead citrate, and examined with a JEOL JEM- 1400 Plus electron microscope. By observing transverse
sections for landmarks such as the 2nd bulb of the pharynx, it was possible to reach the vicinity of the
ALM soma before collecting about 1500 serial thin transverse sections.
The microinjection experiment
We mounted animals on coverslips with dried 2% agarose pads covered in halocarbon 700 oil. We
then placed coverslips onto an Axiovert S100 TV Inverted Microscope (Carl Zeiss) and injected animals
with capillary needles filled with M9+ 10% red dye under 40psi of pressure. Needles were pulled
from borosil capillary tubing (1.0mm OD, 0.5mm ID; FHC) using a P- 97 Micropipette Puller (Sutter
Instrument). Puller parameters were as follows: Heat 474 (Ramp- 25); Pull 90; Vel 100; Time 180. For
the control mock injection, we transiently poked the animal in the uterine region with a needle and
then transferred the animal onto an NGM- agar plate. For the injected group, we applied injection
pressure for 2min with an estimated flow rate of approximately 25 fL/s and then transferred the
animal onto an NGM- agar plate. After each injection or mock injection, we immediately examined the
exopher status of the ALMR neuron in less than 10min.
Statistics
The exopher phenotype (+ or -) for each animal is a nominal variable, so we analyzed the exopher data
by Cochran–Mantel–Haenszel test. There is no power analysis for each experiment, but each exper-
iment has at least three independent trials. For analyzing progeny count, we used two- way ANOVA
with Šídák’s multiple comparisons test. For analyzing the uterus length, we used an unpaired two- tail
t- test.
Acknowledgements
We thank Dr. Andrew Singson, Dr. Amber Krauchunas, and Dr. Xue Mei for sharing reagents and
providing comments in experimental design. We also thank the Caenorhabditis Genetics Center
(CGC, founded by the National Institutes of Health - Office of Research Infrastructure Programs
(P40 OD010440)) for providing some strains. Funding sources: NIH R24 OD010943 to DHH; NIH
K12GM093854 to EC; NIH 5R01GM135326 to BDG; NIH R37AG56510 to MD; and NIH R01AG047101
to MD and BDG.
Research article Neuroscience
Wang, Guasp, Salam etal. eLife 2024;13:RP95443. DOI: https://doi.org/10.7554/eLife.95443 24 of 28
Additional information
Funding
Funder Grant reference number Author
NIH Ofce of the Director R24OD010943 David H Hall
National Institute of
General Medical Sciences 5R01GM135326 Barth D Grant
National Institute on Aging R37AG56510 Monica Driscoll
National Institute on Aging R01AG047101 Barth D Grant
Monica Driscoll
National Institute of
General Medical Sciences K12GM093854 Edward Chuang
The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.
Author contributions
Guoqiang Wang, Ryan J Guasp, Sangeena Salam, Conceptualization, Data curation, Formal anal-
ysis, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing
– review and editing; Edward Chuang, Conceptualization, Data curation, Validation, Investigation,
Methodology, Writing – original draft; Andrés Morera, Anna J Smart, Conceptualization, Data
curation, Formal analysis, Validation, Investigation, Methodology, Writing – original draft, Writing
– review and editing; David Jimenez, Sahana Shekhar, Emily Friedman, Investigation; Ilija Melenti-
jevic, Resources, Investigation; Ken C Nguyen, Validation, Investigation, Methodology; David H Hall,
Resources, Data curation, Funding acquisition, Validation, Methodology, Writing – original draft; Barth
D Grant, Conceptualization, Resources, Funding acquisition, Methodology; Monica Driscoll, Concep-
tualization, Resources, Supervision, Funding acquisition, Validation, Methodology, Writing – original
draft, Project administration, Writing – review and editing
Author ORCIDs
Guoqiang Wang
https://orcid.org/0000-0002-3694-7103
Barth D Grant
https://orcid.org/0000-0002-5943-8336
Monica Driscoll
https://orcid.org/0000-0002-8751-7429
Peer review material
Reviewer #1 (Public Review): https://doi.org/10.7554/eLife.95443.3.sa1
Reviewer #2 (Public Review): https://doi.org/10.7554/eLife.95443.3.sa2
Reviewer #3 (Public Review): https://doi.org/10.7554/eLife.95443.3.sa3
Author response https://doi.org/10.7554/eLife.95443.3.sa4
Additional files
Supplementary files
• MDAR checklist
Data availability
Figure 1- source data, Figure 2- source data, Figure 3- source data, Figure 4- source data, Figure 5- source
data, Figure 6- source data, and Figure 7- source data contain the numerical data used to generate the
figures.
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