Developmental Biology 475 (2021) 265–276

Contents lists available at ScienceDirect

Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology

Developmental plasticity and the response to nutrient stress in

Caenorhabditis elegans
Sabih Rashid 1, Christopher Wong 1, Richard Roy *
Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montreal, QC, H3A 1B1, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: Developmental plasticity refers the ability of an organism to adapt to various environmental stressors, one of
Caenorhabditis elegans which is nutritional stress. Caenorhabditis elegans require various nutrients to successfully progress through all the
Developmental plasticity larval stages to become a reproductive adult. If nutritional criteria are not satisfied, development can slow or
Starvation
completely arrest. In poor growth conditions, the animal can enter various diapause stages, depending on its
AMPK
Diapause
developmental progress. In C. elegans, there are three well-characterized diapauses: the L1 arrest, the dauer
Dauer larva diapause, and adult reproductive diapause, each associated with drastic changes in metabolism and germline
Germ line development. At the centre of these changes is AMP-activated protein kinase (AMPK). AMPK is a metabolic
Quiescence regulator that maintains energy homeostasis, particularly during times of nutrient stress. Without AMPK, meta-
bolism is disrupted during dauer, leading to the rapid consumption of lipid stores as well as misregulation of
metabolic enzymes, leading to reduced survival. During the L1 arrest and dauer diapause, AMPK is responsible for
ensuring germline quiescence by modifying the germline chromatin landscape to maintain germ cell integrity
until conditions improve. Similar to classic hormonal signalling, small RNAs also play a critical role in regulating
development and behaviour in a cell non-autonomous fashion. Thus, during the challenges associated with
developmental plasticity, AMPK summons an army of signalling pathways to work collectively to preserve
reproductive fitness during these periods of unprecedented uncertainty.

1. Caenorhabditis elegans as a model for developmental plasticity
The development of an organism is heavily influenced by environ-
mental factors. These include temperature, weather, and even the pres-
ence of other species, all of which can impact development at various
levels (R

egniere et al., 2012; Prudic et al., 2011; O’Leary et al., 2017).
However, while different species display diverse regulation of develop-
ment through environmental factors, growth in all organisms is influ-
enced by nutrient availability. Lack of essential nutrients can slow or
even arrest growth outright, and in some cases trigger organisms to un-
dergo alternative developmental programs (Lafuente and Beldade,
2019). This developmental plasticity serves as an adaptive means for
organisms to survive acute or chronic environmental stress, allowing
them to adjust their physiology and growth until environmental condi-
tions improve (Braendle and F
elix, 2008; Sommer, 2020). This form of
organismal adaptation requires that key cellular sensors must respond to
such cues and transduce this information globally through a robust,
well-established network of signalling pathways.
Caenorhabditis elegans is an ideal model to study various aspects of
developmental plasticity. In the wild, they are free-living nematodes that
feed primarily on microorganisms present in decaying organic material
(Fr
ezal and F
elix, 2015). Because of their simple anatomy, quick devel-
opment time, and adaptation to laboratory environments, a vast array of
molecular and genetic tools have been developed to interrogate almost
every aspect of their cellular and organismal function.
Development in C. elegans is regulated by the activity and cross talk
between a number of conserved signalling pathways (Greenwald et al.,
1983; Friedman et al., 1988; Kimura et al., 1997; Long et al., 2002; Jia
et al., 2004; Lapierre and Hansen, 2012; Blackwell et al., 2019).
Numerous temporally oscillating proteins and small RNAs, which regu-
late the timing of development, have also been identified (Lee et al.,
1993; Johnstone and Barry, 1996). The effects of these pathways are
particularly robust, resulting in an essentially invariant pattern of cell
division and development, provided that environmental fluctuation is
minimal. However, during periods of environmental challenge, C. elegans
can also modulate its developmental rate and even undergo alternative

* Corresponding author.
E-mail address: richard.roy@mcgill.ca (R. Roy).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ydbio.2021.01.015
Received 13 October 2020; Received in revised form 24 December 2020; Accepted 29 January 2021
Available online 4 February 2021
0012-1606/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Rashid et al.
developmental trajectories to survive bouts of environmental stress to
ultimately enhance fitness.
Nutrition is one of the major determinants of C. elegans growth and
development. In addition to the absence of food, which can slow or arrest
development, C. elegans also requires the presence of various micro-
nutrients for proper growth. These include vitamins, amino acids, heme,
and cholesterol (Zeci
c et al., 2019). These and other nutrients are
required throughout development or at specific life stages, often
reflecting a distinct developmental requirement for a given metabolite.
Heme deficiency, for instance, will only cause larval arrest at the L4
stage, prior to which C. elegans can develop without the nutrient and not
suffer any apparent consequences (Rao et al., 2005).
Based on the numerous nutritional needs of the organism, a chemi-
cally defined minimal medium has been developed, which provides the
essential nutrients required to support C. elegans development (Szewczyk
et al., 2003). The medium can serve as a powerful tool to test the effects
of various metabolite supplements on growth. Given the natural habitat
of C. elegans, animals can subsist on a wide variety of diets, each of which
modulates their growth in different ways. In some cases, the specific
dietary metabolites within their food that regulate growth have been
identified (MacNeil et al., 2013). Recent efforts have attempted to cate-
gorize the various natural diets of C. elegans in order to better understand
how the organism’s developmental and metabolic processes have
evolved alongside its environment (Schulenburg and F
elix, 2017).
One notable feature of C. elegans development is their ability to
reversibly arrest their development in response to environmental stress.
The two most well-studied of these “diapauses” occur following
embryogenesis at the L1 diapause, or later in larval development during
the dauer diapause (Fig. 1). The L1 diapause occurs during the first (L1)
larval stage and is characterized by a global developmental quiescence
that is triggered in emergent larvae that hatch in the absence of nutrients
(Hong et al., 1998; Baugh and Sternberg 2006). While wild type animals
can survive for up to ten days without food, their capacity to initiate
post-embryonic development decreases with the duration of the
diapause, suggesting that this developmental plasticity comes at a cost
(Zhang et al., 2011). Curiously, as animals remain in the diapause, they
begin to exhibit features typical of aged adult animals including accu-
mulation of proteotoxic aggregates, though many of these are completely
Fig. 1. C. elegans can undergo developmental arrest at various stages in
response to starvation. Upon encountering energetic or environmental
stresses, the animal can enter a diapause stage depending on its current devel-
opmental stage. In all of the described diapause stages, the germ cells undergo
G2 stage cell cycle arrest.
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Developmental Biology 475 (2021) 265–276
reversed upon refeeding and execution of the larval developmental
program (Roux et al., 2016).
In addition to the L1 arrest, C. elegans can arrest their development in
later larval stages, following a ‘nutritional checkpoint’ that exists
following some of the later larval moults (Schindler et al., 2014).
C. elegans may also enter an adult reproductive diapause (ARD) upon
starvation in the L4 stage, which is characterized by a shrinking of the
germ line and an increase in longevity (Angelo and Gilst, 2009).
While temporary developmental arrests may potentially provide a
short-term survival advantage, animals can adjust their developmental
progression for longer diapauses by entering into an alternate L3 stage
called the dauer diapause. Dauer larvae are characterized by quiescent
behaviour, altered metabolism, a hardened protective cuticle, and sig-
nificant changes in gene regulation (Cassada and Russell, 1975; Wang
and Kim, 2003). In this alternate stage, wild-type animals can survive for
up to four months, until more favourable environmental conditions arise.
In their natural habitat, wild-type animals display a behaviour called
nictation, where the animal stands on its tail and waves its head, pre-
sumably as an attempt to attach onto a passing host (F
elix and Braendle,
2010). This likely increases the chance of larval dispersal followed by a
subsequent change in environment. Because this can lead to dramatic
changes in nutrient status in a very short period of time, dauer larvae are
able to quickly readjust their metabolism and resume their growth,
allowing them to return to a reproductive mode of development.
Nevertheless, animals that have transited through the dauer diapause
have an altered life history that is recorded in their transcriptome (Hall
et al., 2010), suggesting that developmental plasticity of such a drastic
nature can have long-term consequences for the organism.
2. Metabolic responses to nutrient stress
2.1. The dauer detour
When faced with overcrowding, high temperatures, or a lack of food,
late stage L1 larvae can alter their developmental trajectory and delay
reproduction by executing a preparatory second larval stage (L2d) fol-
lowed by a quiescent third larval stage (dauer) to enhance survival until
environmental conditions become more favourable and capable of sup-
porting their progeny (Cassada and Russell, 1975). This dauer diapause is
notable as it results in significant changes in animal morphology and
behaviour, as opposed to simply slowing or arresting growth, and thus
serves as a striking example of developmental plasticity. (Riddle et al.,
1981; Hu, 2007). In addition, this diapause is highly conserved among
numerous nematode species, including various parasites, where it aids in
host infection (Hotez et al., 1993; Hawdon and Schad, 1991). This has led
to some speculation that the evolution of some nematode species from
soil-dwelling roundworms to parasites that live within hosts may have
been aided by the dauer adaptation (Crook, 2014), which facilitated the
infection of other organisms by allowing parasites to adapt to the often
harsh internal environments typical of their hosts (Harvey et al., 2000).
Dauer formation is mediated by a dauer pheromone which is secreted
continuously by the animals (Golden and Riddle, 1982; 1984; Fielenbach
and Antebi, 2008). When levels of the pheromone in a population surpass
a threshold, animals will activate the dauer program. The ensuing
response is relayed to amphid neurons in the head region of the larva
(Bargmann, 1991), and the subsequent morphological and metabolic
adaptations are regulated by a number of signalling pathways, including
the insulin/IGF-1, TGFβ, and cyclic GMP signalling pathways (Kimura
et al., 1997; Ren et al., 1996; Riddle and Albert, 1997; Birnby et al.,
2000). This complex regulation is expected, given the substantial
changes in gene expression that distinguish the dauer larva from L3
larvae which undergo continuous reproductive development (Jeong
et al., 2009).
Animals that have passed through dauer exhibit significantly altered
gene expression profiles compared to animals that develop in replete
conditions (Hall et al., 2010). This transcriptional memory may be
S. Rashid et al.
preserved epigenetically through changes in histone modifications, as
post-dauer animals generally exhibit decreased levels of H3K4me3 and
H4panAc. Post-dauer animals also exhibit a detectable extension in
lifespan and larger brood sizes, potentially increasing reproductive
fitness. It is so far unclear whether there are negative consequences
associated with the altered post-dauer chromatin signature. Hall et al.
speculate that this phenotypic diversity that arises due to this alternate
life history may provide an evolutionary advantage, in addition to the
survival advantage conferred by the stress-resistant dauer stage.
2.2. Redox state and the regulation of dauer formation
Dauer formation is triggered by increased levels of dauer pheromone
in a growing population of animals. This affects a number of downstream
steps that converge on DAF-12, a nuclear hormone receptor-like tran-
scription factor that can bind several steroidal ligands known as
dafachronic acids (DAs) (Motola et al., 2006) (Fig. 2). Reproductive
growth is maintained when DAF-12 is bound by DAs, but when DAF-12 is
in its unliganded state it binds to DIN-1S, forming a transcriptional
complex that blocks gene expression required for reproductive develop-
ment, while activating dauer-specific genes (Antebi et al., 2000; Ludewig
et al., 2004).
DA formation is itself regulated by levels of the electron donor
NADPH. Penkov et al., discovered that a tug of war between DA synthesis
and the biosynthesis of the saccharide trehalose helps determine dauer
formation. Synthesis of trehalose from glucose-6-phosphate (G6P) pre-
vents G6P entry into the pentose phosphate pathway (Wamelink et al.,
2008), thus reducing levels of cellular NADPH which is required for DA
synthesis by DAF-9 (Motola et al., 2006). Without DAs to bind it, DAF-12
is activated and dauer formation is promoted. Notably, trehalose levels
are increased during starvation, promoting stress resistance and
longevity (Hibshman et al., 2017). Dauer formation thus invokes com-
mon stress response pathways, highlighting the coordination of devel-
opmental progression and metabolic adaptation. Other methods of
reducing cellular NADPH, such as disrupting the activity of isocitrate
dehydrogenase, also induces dauer formation, suggesting internal
metabolite concentrations, as well as redox state, are significant con-
tributors to dauer formation. DAF-12 itself appears to induce the
biosynthesis of trehalose, reducing levels of NADPH in response to dauer
pheromone, thus creating a feedback loop to promote dauer formation.
NADþ, meanwhile, is sufficient to trigger dauer exit in C. elegans
(Mylenko et al., 2016). NADþ, and potentially other bacterial compo-
nents, can thus serve as signals that indicate the presence of food,
Fig. 2. Dauer formation in C. elegans is tightly regulated and invokes numerous metabolic changes. Environmental stressors such as lack of food or high
population density can trigger entry into the dauer stage. In this stage, a metabolic shift occurs that promotes the catabolism of existing energy stores through
anaerobic reactions in order to sustain development. Animals may exit dauer when environmental conditions, such as food availability, improve.
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Developmental Biology 475 (2021) 265–276
promoting exit and resumption of reproductive development.
In addition to the effects of NADPH in regulating dauer formation,
previous work suggests that a redox catalyzing enzyme called thio-
redoxin trx-1 also regulates dauer formation through interactions with
the cGMP pathway (Sanzo-Machuca et al., 2019). This study reveals a
redox-dependent role of trx-1 in maintaining the longevity of long-lived
mutants, including the daf-2 (insulin-like receptor) mutant. Interestingly,
the loss of the catalytic or regulatory subunits of AMPK also severely
suppressed the lifespan extension conferred by daf-2, while also
dramatically decreasing survival of the dauer larva (Narbonne and Roy,
2006). AMPK plays a role in maintaining redox balance during periods of
metabolic stress, such as glucose starvation (Ren and Shen, 2019). AMPK
is also a target of Trx1 in mice (Shao et al., 2014), suggesting that the
dauer survival defect seen in AMPK mutants could be compounded by a
misregulation of redox balance, although it is unclear whether the defect
is a result of damage due to elevated ROS levels, or reductive stress
resulting from the progressive accumulation of toxic redox/metabolic
by-products (Tilton et al., 1991).
2.3. Metabolic shifts in the dauer stage
Given that dauer larvae often must survive for extended durations
until their surrounding environmental conditions improve, their global
metabolism must adjust to accommodate a potentially lengthy quies-
cence. Oxidative metabolism is greatly reduced in dauer larvae,
compared to animals that bypassed this diapause (Wang and Kim, 2003).
Instead, dauer larvae shift towards glyoxylate and gluconeogenic path-
ways to generate energy. The shift towards gluconeogenesis, in partic-
ular, results in the accumulation of glycogen and trehalose. SAGE and
microarray analysis of dauer gene expression also indicates increases in
genes involved in β-oxidation of fatty acids (Jones et al., 2001).
Many of these changes in metabolic gene expression represent a shift
towards a regulated consumption of existing energy stores, as dauer
larvae do not feed, and thus must subsist entirely on stored nutrients. The
shift towards gluconeogenesis is regulated primarily by AAK-2, one of the
two catalytic components of AMPK, while the switch to the glyoxylate
cycle was mediated by AAK-2 and DAF-12 (Penkov et al., 2020). This
shift occurs as a result of stoichiometric ratios of enzymes within dauer
larvae. Specifically, ICL-1, a glyoxylate cycle enzyme, is upregulated in
dauer larvae, whereas this ratio is reduced in aak-2 mutants. In the
absence of an appropriate metabolic adjustment, the survival of the dauer
larva may be compromised. Loss of the insulin signalling effector daf-16,
for example, leads to a misregulation of catabolism, causing many sugars,
S. Rashid et al.
lipids, and amino acids to be rapidly degraded in daf-16 mutants, ulti-
mately resulting in premature expiration compared to wild-type dauer
larvae (Penkov et al., 2020). A similar loss of energy stores is observed in
dauer animals lacking AMPK, further suggesting daf-16 and AMPK may
work in concert to regulate long-term survival in dauer through meta-
bolic regulation (Narbonne and Roy, 2009). Recently, it has been shown
that the addition of ethanol can increase the duration of dauer survival by
allowing the animals to metabolize it as an energy source (Kaptan et al.,
2020). This supplementation induces a metabolic shift to improve sur-
vival, and specifically prevents the deterioration of mitochondria
commonly seen in dauer animals. The mitochondria may thus serve as
important metabolic regulators of survival during the dauer stage.
Analysis of gene expression in dauer larvae indicate that various
fermentation pathways are upregulated, suggesting that dauer larvae are
able to utilize carbon sources for energy in the absence of oxygen, which
may be important for animals that do not feed and thus have reduced
internal oxygen (Wang and Kim, 2003; Holt and Riddle, 2003). Some
nematode species are able to undergo anaerobic mitochondrial fermen-
tation, converting phosphoenolpyruvate (PEP), a glycolytic intermediate,
into oxaloacetate (Komuniecki and Harris, 1995). Malate de-
hydrogenases, or malic enzymes, can reversibly convert oxaloacetate to
malate, which were upregulated in dauer larvae (McElwee et al., 2006).
Malate dismutation in anaerobic mitochondria may be one possible
method of producing energy and maintaining redox balance in dauer
larvae (McElwee et al., 2006). Indeed, there is evidence that C. elegans
may utilize alternate, anaerobic metabolic pathways such as fumarate
respiration, as rhodoquinone, a mitochondrial component required for
anaerobic respiration is found in all stages of the C. elegans life cycle
(Takamiya et al., 1999).
2.4. Essential nutrients
C. elegans have a specific set of essential nutrients required
throughout their lifespan, without which they will stop developing
entirely, or face numerous deleterious effects (Braeckman et al., 2008;
Zeci
c et al., 2019) (Fig. 3). In their natural environment their diet consists
primarily of different bacterial species, although fungal, plant, and ani-
mal matter has also been identified in their intestine (Schulenberg,
2017). This finding may indicate that a diet solely of bacteria is not
sufficient for the animal’s dietary essentials. Indeed, media used in lab-
oratories must also be supplemented with cholesterol in order to facili-
tate proper growth, indicating that a diet that consists solely of E. coli is
insufficient. The regulation of developmental rate by various metabolites
is likely mediated through insulin/IGF-1 and TOR signalling, as both of
these signalling pathways are responsible for sensing and responding to
nutrients, and have defined roles in developmental regulation (Iser and
Wolkow, 2007; Jia et al., 2004). However, there are instances where
metabolites can mediate C. elegans development independent of major
signalling pathways (MacNeil et al., 2013). Nucleotides, for example, are
metabolites that are required for proper development, and germ cell
proliferation is inhibited when nucleotide uptake is low by compro-
mising biosynthetic or scavenging pathways (Chi et al., 2016; Jia et al.,
2020).
The minimal medium used for axenic growth of C. elegans contains
precise amounts of every metabolite that animals require for develop-
ment (Szewczyk et al., 2003). However, animals generally grow slowly in
this medium compared to traditional bacterial diets. The ‘typical’
C. elegans life cycle has been defined primarily by its growth on labora-
tory strains of E. coli, such as OP50 or HT115. In the natural world,
C. elegans development is highly plastic, with growth rates varying
dramatically based on its diets (Samuel et al., 2016). This highlights the
possibility that C. elegans can respond to natural metabolites in remark-
ably different ways. Vitamin B12 found in diets of Comamonas aquaticas,
for example, can alter gene expression and cause accelerated develop-
mental rates and altered timing of egg-laying (Watson et al., 2014).
Conversely, bacteria lacking certain essential nutrients may induce
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Developmental Biology 475 (2021) 265–276
developmental arrest in C. elegans. This arrest may allow animals to
temporarily slow or halt developmental processes until foraging allows
them to identify food sources that satisfy their needs, which is likely not
difficult given the diversity of their natural environment (Schulenberg,
2017).
2.5. Amino acids
C. elegans require several amino acids in their diet, including the same
nine amino acids essential in humans (histidine, lysine, tryptophan,
phenylalanine, methionine, threonine, leucine, isoleucine and valine), as
well as arginine (Braeckman et al., 2009). The remaining amino acids can
be synthesized de novo. Amino acids serve as the basic building blocks for
proteins, but certain amino acids can be used for the synthesis of me-
tabolites which serve as energy sources in low-nutrient conditions (Kühnl
et al., 2005). Supplementation with amino acids can also extend the
lifespan of animals in a daf-16-dependent manner (Edwards et al., 2015).
Conversely, lack of essential amino acids can have severe conse-
quences on C. elegans development. The branched amino acids, leucine,
isoleucine, and valine, may be required for progression past the L2 larval
stage (Jia et al., 2016). Mutations in the branched-chain α-ketoacid de-
hydrogenase, part of the branched chain amino acid (BCAA) catabolic
pathway, can cause developmental arrest as well as embryonic lethality
(Jia et al., 2016). In addition to serving as protein building blocks, these
amino acids are also required for the synthesis of certain lipids, and have
been implicated in metabolic disease in humans, suggesting that they
have physiological roles beyond protein synthesis (Newgard, 2012).
In order for animals to regulate developmental plasticity in response
to nutrient signals, they must integrate nutrient sensing with downstream
regulation of metabolism and growth. In C. elegans, the amphid neurons
play a role in amino acid sensing. Specifically, MGL-1 and MGL-2, both of
which are metabotrophic glutamate receptors that are expressed in the
amphids, are involved in food sensing, as loss of mgl-1 results in animals
that do not delay reproduction even in the absence of food (Jeong and
Paik, 2017). Additionally, leucine-mediated rescue of starvation pheno-
types, such as stress resistance and increased longevity, is abolished
when mgl-1 and mgl-2 are disrupted, suggesting that they play a role
specifically in amino acid sensing (Kang and Avery, 2009). Signals from
these neural receptors likely get transduced to downstream signalling
pathways, which further regulate development in response to nutrient
signals. The TOR pathway is a primary candidate for this regulation, as it
has a known role in mediating amino acid signals in other organisms
(Jewell et al., 2013). In particular, Rag GTPases are likely involved, as
they activate the TOR pathway in the presence of amino acids (Sancak
et al., 2008). Interestingly, a cocktail of amino acids can trigger devel-
opment in quiescent animals undergoing L1 arrest, and this also occurs
following hypodermal activation of Rag-TORC1, suggesting amino acid
signalling activates TOR to promote development (Fukuyama 2015).
Thus, developmental plasticity requires the integration of external
environmental cues with an exquisitely controlled network of metabolic
modulators of developmental progression.
2.6. Vitamin deficiencies
In C. elegans, as in most organisms, vitamins act as cofactors and
donor molecules to catalyze a wide spectrum of metabolic reactions (Bito
et al., 2013). Many vitamins are essential for C. elegans development,
including riboflavin, thiamin, and vitamin B12 (Dougherty et al., 1959).
Like amino acids, a deficiency of these essential vitamins can slow or
arrest development. However, the mechanisms by which development is
regulated by the presence or absence of vitamins remain largely un-
known. Deficiencies of vitamin B2 result in a decrease of TOR activity in
animals, as well as a reduction of protease secretion in the intestine, a
process by which animals sense and avoid low quality food (Qi et al.,
2017). Vitamin B12, as well as vitamin B9, or folate, act in the methio-
nine/SAM cycle (Yilmaz and Walhout, 2014) and the reduction of folate
S. Rashid et al.
intake in C. elegans results in reproductive defects, as well as a shorter
lifespan (Austin et al., 2010).
Conversely, supplementation with vitamins can have significant ef-
fects on the physiology of C. elegans. Addition of low doses of folic acid
can increase lifespan and delay aging through inhibition of both TOR and
insulin signalling, suggesting that it may affect C. elegans developmental
rate as well (Rathor et al., 2015). Vitamin D can also affect longevity by
activating stress response genes including skn-1/Nrf, which regulates
oxidative stress responses, and ire-1/ERN1, which is required for the
unfolded protein response (Mark et al., 2016). Dietary variation in
Comamonas aquaticas lead to an increased developmental rate through
the regulation of gene expression that is independent of TOR and insulin
signalling (MacNeil et al., 2013). Specifically, this effect is induced by
Vitamin B12 via its role as a cofactor in the methionine/SAM cycle
(Watson et al., 2014). This suggests that the supply of certain vitamins
may limit the growth rate of C. elegans, and faster rates of development
may be more prevalent than that of the standard wild-type Bristol strain
fed a laboratory-derived E. coli diet. It is, however, unclear whether a
faster developmental rate would confer a fitness advantage to C. elegans
in a natural context. Given the number of development adjustments
C. elegans likely undergoes in nature, most of which primarily involve the
slowing or ceasing of development in order to improve survival (Fr
ezal
and F
elix, 2015), it is certainly advantageous to tune developmental rate
to the quality of food that is consumed, as a hard-wired rapid rate of
development would oblige animals to increase nutrient intake to a level
that cannot be systematically accommodated by their natural
environments.
2.7. L1 arrest
The L1 arrest shares similarities with the dauer diapause, particularly
Fig. 3. C. elegans have diverse nutritional needs. Various nutrients, such as vitamins, heme, and amino acids, are required for optimal growth of C. elegans.
Conversely, deficiencies in these nutrients can induce a variety of defects, from shortened lifespans, reduced reproductive capabilities, to developmental arrest.
Beneficial effects from nutrients are depicted in green, deleterious effects from a lack of those nutrients are in red.
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Developmental Biology 475 (2021) 265–276
in terms of its major regulators. Both daf-16 and AMPK appear to play
vital roles in both contexts, as loss of these factors lead to an inability to
arrest, thus leading to decreased survival in starved L1 animals (Mu~ noz
and Riddle, 2003; Baugh and Sternberg, 2006; Narbonne and Roy, 2009;
Baugh, 2013). L1 diapause is also regulated by a number of insulin-like
peptides, which are ligands that can serve as both agonists and antago-
nists for the insulin receptor DAF-2 (Pierce et al., 2001). Specifically,
ins-6, ins-4, and daf-28 appear to regulate L1 arrest in concert, as over-
expression of these ILPs leads to reduced survival during L1 starvation, as
animals continue to grow despite the lack of nutrients (Chen and Baugh,
2014). Conversely, the loss of these ILPs leads to increased survival in the
L1 diapause. Interestingly, these ILPs appear to play a role in regulating
development by facilitating communication between neurons and the
intestine (Hung et al., 2014), suggesting that intestinal cells are crucial
for relaying information in order to appropriately tune developmental
progression to nutrient status.
Notably, while the L1 diapause promotes survival in the short term
when faced with starvation, it also comes with several drawbacks,
including damage to the gonad, as well as a delay in development
following recovery from the arrest (Lee et al., 2012). Indeed, while ani-
mals can survive for up to 21 days in this arrest, they display numerous
characteristics typical of aging, including mitochondrial fragmentation
and granulated tissues (Roux and Kenyon, 2016). However, many of
these phenotypes are reversed upon feeding of L1 worms, though protein
aggregation does persist even into adulthood.
Neuronal signals also impinge on survival during the L1 diapause, as
survival was seen to increase in mutants lacking unc-31 function, which
plays a role in neuroendocrine signalling through ligands such as ILPs
(Lee and Ashrafi, 2008). Indeed, the mere sensing of food, without
ingestion, was sufficient to trigger gene expression changes in
L1-arrested larvae, including upregulation of the IIS pathway (Kaplan
S. Rashid et al.
et al., 2018). In particular, expression of the ILP daf-28, a DAF-2 agonist,
was increased in the amphid neurons. This suggests external nutritional
cues are transduced by neurons, leading to downstream effects that
prepare larvae for development in response to food availability.
Coupling nutrient sensing with the maintenance of quiescence is
likely crucial for prolonged survival of L1 arrested larvae, although the
precise molecular mechanisms behind this have not been elucidated.
Regulation by small RNAs, such as microRNAs, may also contribute to
survival of the starved L1 larvae. AIN-1 and AIN-2, components of the
miRNA-induced silencing complexes (miRISC), are required for L1 sur-
vival during the arrest (Zhang and Han, 2011). Indeed, miRNAs such as
mir-71 promote survival during the L1 arrest survival through the inhi-
bition of insulin receptor/PI3K pathway genes (Zhang et al., 2011), while
the miRNA mir-235, the ortholog of mammalian mir-92, (Kasuga et al.,
2013), is required for the quiescence of blast cells. Specifically, mir-235
targets nhr-91, a homolog of mammalian germ cell nuclear factor
(GCNF), to suppress its expression during the L1 arrest and thus inhibit
blast cell progression. Notably, mir-235 is negatively regulated by the IIS
pathway, suggesting its activity is also nutritionally controlled (Kasuga
et al., 2013).
2.8. AMPK, a major regulator of the starvation response
AMPK is a highly conserved enzyme that has numerous roles in
regulating metabolism. AMPK’s function during periods of energy stress
has been well characterized, particularly in C. elegans. Loss of AMPK
further reduces the viability of animals that remain in the L1 diapause for
extended periods, suggesting that the kinase has an important role in
managing the metabolic stress caused by acute starvation throughout the
L1 diapause (Lee et al., 2012; Demoinet et al., 2017).
Conversely, animals lacking AMPK display numerous defects during
and after the dauer diapause (Narbonne and Roy, 2009; Kadekar and
Roy, 2019). AMPK mutant animals have greatly reduced survival in the
dauer stage (Narbonne and Roy, 2009). Given the role of AMPK in
regulating enzyme levels (Penkov et al., 2020), the reduced dauer sur-
vival may be a result of metabolic misregulation that causes animals to be
unable to respond to the nutrient stresses of dauer. Loss of AMPK also
interferes with the ability of dauer animals to effectively utilize energy
stores, a process that is crucial for extended dauer survival. Specifically,
triglyceride stores are rapidly metabolized in AMPK dauer larvae, sug-
gesting these animals have a much smaller reservoir of energy on which
to subsist (Narbonne and Roy, 2009). Those AMPK mutant larvae that do
survive through dauer display numerous somatic defects, such as pro-
truding or burst vulva, as well as dauer germline hyperplasia and
post-dauer sterility (Kadekar and Roy, 2019).
3. Protecting the germ line during acute nutrient stress
3.1. Germline quiescence during the dauer stage
The capacity of C. elegans to exercise its inherent developmental
plasticity often manifests in various contexts where animals are obliged
to adapt to starvation, such as by altering their cell cycle and/or delaying
aspects of tissue development or differentiation (Budirahardja and
G€onczy, 2009). A crucial process in this adaptation is the preservation of
totipotent germ cells during extended periods of nutrient deprivation in
order to ensure that animals are able to reproduce once they resume
reproductive development (Fukuyama, 2018).
When the L1 larva hatches from the egg, it contains four primordial
gonadal cells, two of which make up the somatic primordium, while the
other two correspond to the primordial germ cells (PGCs) that will pro-
liferate to generate the entire germ line (Hirsh et al., 1976). During
C. elegans development, germ cell proliferation is dependent on activa-
tion of the Notch receptor GLP-1, which is present on the germ cells, by
its Delta-like ligand LAG-2 and APX-1, which are expressed on the distal
tip cells (Austin and Kimble, 1987; Nadarajan et al., 2009). This germline
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stem cell population expands throughout and beyond the late L3 larval
stage (Michaelson et al., 2010; Schindler et al., 2014), after which the
proximal-most germ cells become displaced outside the signalling range
of the DTCs, resulting in a switch from mitotic divisions to the onset of
meiosis (Austin and Kimble, 1987; Henderson et al., 1994; Nadarajan
et al., 2009).
In both the L1 diapause and the dauer stage, the germline stem cells
undergo a G2/M phase cell cycle arrest (Narbonne and Roy, 2006;
Ouellet et al., 2008; Seidel and Kimble, 2015) (Fig. 1) Interestingly, no
germ cell proliferation occurs throughout the dauer diapause despite
active Notch signalling. The C. elegans ortholog of the PTEN tumour
suppressor, DAF-18 is required, not only for the formation of the dauer
larva, but also the extended survival conferred by this stage (Solari et al.,
2004). In addition, the C. elegans orthologues of various components of
the LKB1/AMPK signalling pathway were found to be critical to establish
and/or maintain cell cycle quiescence in the dauer stage (Narbonne &
Roy, 2006, 2009). The loss of AMPK leads to supernumerary germ cells in
the dauer germ line, although the defect occurs during the execution of
dauer, when animals are still feeding and nutrients are present, and not
during the diapause per se.
The integrity of the germ cells is compromised in AMPK mutant dauer
larvae, and they therefore do not produce functional sperm or oocytes,
rendering the post-dauer mutants sterile (Kadekar and Roy, 2019). While
wild type animals that transit through dauer display characteristic
chromatin modifications and corresponding gene expression, animals
that lack AMPK signalling misregulate both endogenous small RNA
pathways and their chromatin landscape in the germ line. These alter-
ations impact gene expression in the dauer and post-dauer animals, and
are likely the basis of the observed post-dauer sterility (Kadekar and Roy,
2019). For example, the expression levels of certain transcripts (ppk-2,
pmk-1, spe-26, pro-2, mek-2) are altered in wild-type animals that transit
through the dauer stage compared to those animals that did not (Ow
et al., 2018). In animals that lack both catalytic subunits and hence all
AMPK signalling (aak(0) mutants), the expression of these five genes was
dramatically misregulated compared to wild-type animals (Kadekar and
Roy, 2019). Since these incorrectly deposited chromatin marks persist
into the post-dauer animals, the abnormal expression of these genes also
persists, likely disrupting a multitude of reproductive processes. Thus,
the changes that must take place in the germ line to adapt gene expres-
sion to the energetic stress of the dauer stage are under the control of
AMPK. Through its activation during the dauer diapause, it remodels the
chromatin landscape to regulate germline quiescence and preserve germ
cell integrity for the duration of the arrest, whether it will be 24 h or 24
days.
3.2. Germline quiescence during the L1 arrest
During the L1 arrest, larvae adjust their germline development,
instructing the PGCs to undergo a reversible arrest until a nutrient con-
tingency is met. A fusion protein GFP:histone-H2A, which marks all
chromosomes in the early embryonic cells, revealed that the embryonic
germline precursor cells Z2 and Z3 undergo G2 cell cycle arrest
(Fukuyama et al., 2006) (Fig. 1). These precursor cells condense their
chromosomes before embryogenesis and remain condensed for the
remainder of the L1 arrest. Conversely, somatic cells only condense their
chromosomes briefly during mitosis (Fukuyama et al., 2006). Moreover,
immunofluorescence analysis of γ-tubulin revealed that the Z2 and Z3
precursor cells contain two centrosomes in the starved L1 larvae rather
than a single centrosome in the continuously fed counterparts consistent
with a G2 and not a G1 arrest. When starved animals were recovered with
a DNA synthesis inhibitor hydroxyurea, 98% of larvae contained more
than three PGCs, indicating that these cells were able to divide without
any requirement for S phase (Fukuyama et al., 2006).
The germ cell arrest that occurs during the L1 arrest stage is depen-
dent on the activity of daf-18/PTEN, a negative regulator of the insulin-
like signalling pathway (Fukuyama et al., 2006; Ogg and Ruvkun, 1998).
S. Rashid et al.
Genetic analysis reveals that DAF-18 functions downstream of age-1/-
PI3K and therefore partially bypasses the requirement for insulin-like
signalling in order to respond to nutritional changes. In blast cells,
cki-1, a cyclin-dependent kinase inhibitor is required for quiescence
during the L1 arrest (Fukuyama et al., 2003; Hong et al., 1998; Kosti
c
et al., 2003). In contrast to somatic cells, cki-1 is not required for germ
cell arrest. Instead, germ cell arrest is dependent on the mitotic spindle
checkpoint protein mdf-1/Mad1p, a downstream effector of DAF-18 and
AKT-1/Akt/PKB signalling (Watanabe et al., 2008).
The role of AMPK in blocking somatic cell divisions during the L1
diapause was recently expanded to include the post-embryonic Q neu-
roblasts. These cells divide and also migrate in aak(0) mutants (Zheng
et al., 2018). Therefore, the loss of AMPK allows a subset of somatic cells
to execute aspects of their post-embryonic developmental program
without satisfying a food signal contingency that normally ensures global
developmental quiescence.
AMPK is required for germ cell arrest during the L1 diapause
(Demoinet et al., 2017). It is also critical to block the aberrant chromatin
modifications that occur in response to starvation. Animals that lack all
AMPK signalling (aak(0)) die prematurely in the L1 diapause, but if they
are recovered before they expire, most of these animals will become
sterile adults (~70% of the population), while those individuals
(remaining 30%) that are fertile produce few progeny (Demoinet et al.,
2017). In L1 arrested aak(0) mutants, the activating chromatin mark
H3K4me3 accumulates abnormally in the PGCs and persists. In fact, the
marks continue to remain elevated in subsequent generations that were
never starved. Instead they accumulate and presumably cause atypical
germline gene expression, consequently disrupting germ cell integrity.
The loss of AMPK in this context results in reproductive defects, not only
in starved animals, but also their non-starved progeny, revealing a
transgenerational effect likely conferred through the accumulation of the
ectopic histone modifications. This transgenerational epigenetic conse-
quence of developmental plasticity that is revealed by the loss of AMPK
may represent a more common evolutionary phenomenon whereby or-
ganisms prime not only themselves, but also future generations, for
potentially limiting nutrient conditions (Heijmans et al., 2008).
The compromise of components of the COMPASS-like complex during
the L1 diapause is sufficient to partially suppress both the sterility and the
reduced brood size in aak(0) mutants (Demoinet et al., 2017). In
C. elegans, the COMPASS-like complex catalyzes the trimethylation of
H3K4. RBR-2 is a histone demethylase that catalyzes the demethylation
of H3K4 (Mariani et al., 2016). Based on similarities in somatic defects
and reduced brood sizes between aak(0) and rbr-2 mutants, the mis-
regulation of the levels of H3K4me3 could be a cause of sterility in aak(0)
mutants (Demoinet et al., 2017). The compromise of any one of the
COMPASS-like complex components during the L1 diapause was suffi-
cient to restore the fertility and correct the reduced brood size. Inter-
estingly, most of the somatic defects of starved aak(0) mutants were not
suppressed when treated with set-2 and set-16 RNAi, suggesting that these
defects could be due to an H3K4me3-independent pathway. Bio-
informatic analysis also indicates that all members of the COMPASS-like
complex components with the exception of DPY-30, a histone methyl-
transferase complex regulatory subunit, possess consensus AMPK phos-
phorylation motifs. In the PGCs of starved L1 diapause mutants, the levels
of SET-2 were higher compared to starved wild-type PGCs, indicating
that AMPK could be involved in regulating the levels of SET-2 and
COMPASS-like complex elements in the PGC nuclei. The absence of
AMPK activity therefore results in an increase in H3K4me3 abundance
and inappropriate gene expression in the starved aak(0) mutant germ
cells.
H3K4me3 is most often associated with transcriptional start sites and
enhanced elongation (Lin et al., 2011). Interestingly, the aberrant tran-
scriptional elongation that occurs as a result of the aberrant chromatin
H3K4me3 levels may also contribute to the sterility in aak(0) animals,
since treatment with a well-characterized transcription elongation in-
hibitor partially restored fertility in the post-L1 diapause aak(0) mutant
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Developmental Biology 475 (2021) 265–276
animals (Demoinet et al., 2017). Therefore, in response to acute starva-
tion, L1 diapause larvae tune their metabolic state through
AMPK-mediated adjustment, while the immortal germ line lineage is
protected by at least three AMPK-dependent pathways: modulation of
TOR activity to block premature PGC divisions; regulation of the chro-
matin modifying COMPASS-like complex; and regulation of the 14-3-3
protein PAR-5, to block the deposition of inappropriate epigenetic
instructions.
3.3. Adult reproductive diapause
While the dauer and L1 diapauses have been well characterized, the
existence of ARD, a hypometabolic stage, has been debated since it was
first introduced approximately 10 years ago (Angelo and Van Gilst, 2009;
Carranza-García & Navarro, 2020). Early and late L4 animals that
encounter starvation can enter the ARD by altering their somatic and
germline tissue. When L4 larval stage animals commit towards the
reproductive adult stage and encounter starvation, they can execute the
ARD. Animals can spend up to 30 days in this state without compro-
mising their adult lifespan (Angelo and Van Gilst, 2009). During this
period, the gonad undergoes shrinkage. While the majority of somatic
cells continue to undergo cell division and endoreduplication without
disruption, vulval development is arrested (Angelo and Van Gilst, 2009;
Seidel and Kimble, 2011). Therefore, because of this defect, entry into the
ARD stage is optimal at the early L4 larval stage, before any embryos
begin to develop. Should the animals enter ARD at the late L4 larval
stage, animals will produce embryos that are retained within the uterus,
resulting in internal hatching and death of the mother (Seidel and Kim-
ble, 2011).
The germ line undergoes drastic changes during ARD. After three
days of starvation in wild-type animals, the number of the germ cells
decreases from 150 to 30 germ cells per arm, causing the gonad to shrink
(Angelo and Van Gilst, 2009; Seidel and Kimble, 2011). In wild-type
animals, starvation affects the number of oocytes produced. In replete
conditions, each gonad arm can possess six to ten oocytes with ovulation
occurring once every 23 min. In ARD animals, each gonad arm only holds
one oocyte and the rate of ovulation decreases to once every 8 h (Seidel
and Kimble, 2011). By the tenth day, embryo production ceases.
Starvation also alters the cell cycle of the germ cells. In the absence of
food, the mitotic germ cell cycle becomes quiescent (Angelo and Van
Gilst, 2009). The cell cycle slows and eventually most germ cells arrest at
the G2 phase. In young adults, the germ cells arrest their cell cycle after
30 min of starvation, while L4 larval stage animals do not halt their cell
cycle immediately. Instead, they will moult into adulthood then slow
down and arrest their mitotic germ cell cycle (Seidel and Kimble, 2015).
Not surprisingly, starvation also affects the rate of meiosis in the germ
cells. The expression of GLD-1, a meiotic marker, revealed that the rate of
meiosis in the gonad of the starved animals is lower than continuously
fed animals, suggesting that nutrient availability affects the development
of the germ cells (Seidel and Kimble, 2015). However, most of these
phenotypes are reversible upon the resumption of feeding.
After spending five days in ARD, the mitotic index of germ cells,
defined as the ratio of cells undergoing mitosis to the total number of
proliferative cells, is very low (Roy et al., 2016). Yet after only 1.5 h
following the resumption of feeding, the germ cells enter M phase and the
mitotic index recovers substantially. Consequently, the DNA content
matches the changes in the mitotic index. The DNA content is high in the
first 24 h post-ARD, but by 48 h, there is a significant reduction in DNA
content that persists for the following four days, which correlates with
the decline in sperm number. Refed or recovered animals return to their
original body size following ARD and regenerate a normal-sized gonad,
although the recovered animals do not have as many germ cells and
therefore produce approximately 70% of the brood size of their contin-
uously fed counterparts (Angelo and Van Gilst, 2009; Carranza-García
and Navarro, 2019). Although germ cell proliferation and gonad size
largely recover after ARD, this period of nutrient deprivation in ARD
S. Rashid et al.
dramatically affects overall germ cell numbers.
There is debate regarding the molecular mechanisms that underlie
successful entry and exit from ARD. One group reported that the tran-
scription factor nhr-49/HNF4 is responsible for the gonad size reduction
during starvation and fertility recovery upon re-feeding (Angelo and Van
Gilst, 2009; Carranza-García and Navarro, 2019). ced-3 mutants which
are deficient in apoptosis could not undergo gonad shrinkage and did not
recover from ARD, suggesting that germline apoptosis was critical for
surviving this brief period of starvation (Angelo and Van Gilst, 2009).
Later, another group reported that apoptosis is not required for recovery
from ARD, suggesting that the gonad shrinkage is not dependent on
germline apoptosis (Carranza-García and Navarro, 2019). Studies also
show that LIN-35/Rb, the C. elegans homolog of the retinoblastoma
protein which regulates starvation-induced germ cell apoptosis, is not
required for gonad shrinking or recovery from the ARD stage
(L
ascarez-Lagunas et al., 2014). While ARD does not use any of the ca-
nonical longevity pathways, autophagy, and fat metabolism for other
forms of diapause, ARD requires the HLH-30/TFEB transcription factor
that also plays an important role in the L1 arrest through its ability to
regulate efficient entry and exit of this quiescent stage (Gerisch et al.,
2020).
Proper RNA metabolism is required for ARD recovery. While germ
cells undergo G2 cell cycle arrest, somatic cell divisions or endor-
eduplication are not postponed during prolonged starvation (Burnaev-
skiy et al., 2018) (Fig. 1). cye-1 mutants, which are cyclin E-deficient, are
able to pass through ARD normally, suggesting that cell cycle reactivation
in somatic tissue was not required for recovery. Three-week starved ARD
animals that were treated with a thymidylate synthase inhibitor 5-fluo-
ro-20 -deoxyuridine (FUDR) were unable to restore their body and gonad
size, normal pumping rate, or thrashing after ARD, suggesting that
nucleic acid metabolism is critical for ARD recovery. To examine if
mitochondrial DNA synthesis was the underlying cause for the defects
seen in FUDR-treated recovering ARD animals, polg-1 mutants, which are
deficient for mitochondrial DNA polymerase, were subjected to ARD and
allowed to recover. These mutants recovered normally and had an un-
altered mitochondrial DNA content, suggesting that the recovery from
ARD does not require mitochondrial DNA synthesis in the soma. To assess
if RNA metabolism is a requirement for ARD recovery, animals were
treated with FUDR and specific nucleotides were supplemented into the
growth media. Only supplementation of uracil, uridine, and deoxyur-
idine were able to reverse the defects associated with FUDR treatment.
The type of RNA molecules that are important for morphological and
functional restoration during the recovery from ARD still remain
uncharacterized.
4. Coordinating developmental and physiological adaptation
during situations of developmental plasticity
Hormones enable communication between tissues to regulate physi-
ology and behaviour in many organisms. They play a key role in regu-
lating development in response to nutritional stress to ensure the survival
of the organism. As previously mentioned, dauer formation is triggered
by reduced levels of a bile acid-like hormone Dafachronic acid, resulting
in a number of somatic and germline changes that include global cell
cycle and developmental arrest that are mediated by the nuclear receptor
called DAF-12 (Antebi et al., 2000).
In addition to regulating dauer entry and co-regulating the dauer
metabolic transition, DAF-12 also functions as a ligand-regulated miRNA
switch in response to nutrient availability (Shen et al., 2012). Some
daf-12 mutants display a heterochronic delay of the third larval stage
program and reiterate the L2 seam cell division programs (Antebi et al.,
2000; Hammell et al., 2009). In the daf-12 mutant, the expression levels
of the let-7-related miRNAs mir-48, mir-84, and mir-241 are reduced,
suggesting that active DAF-12 functions as a positive regulator of these
miRNAs. Consistent with these findings, mutants with a triple knockout
of these let-7-related miRNAs repeat the L2 seam cell division, resembling
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Developmental Biology 475 (2021) 265–276
a daf-12 mutant. Liganded DAF-12 promotes the transcription of the
let-7-related miRNA family, which in turn downregulates the expression
of hbl-1/hunchback during the L2/L3 transition, allowing the animal to
execute the L3 larval stage gene expression program. Interestingly, these
let-7-related miRNAs negatively feed back on DAF-12. This relationship
illustrates how nuclear receptors and small RNAs couple physiological
and environmental events.
Similar to traditional peptide hormones and their signalling path-
ways, small RNAs are emerging as beacons of developmental and phys-
iological adaptation, tuning gene expression to match environmental
change. Perhaps the most notable among these is the pioneer microRNA
lin-4, which is required for post-embryonic developmental timing (Lee
et al., 1993). When lin-4 is expressed in the late L1 larval stage, it binds to
the 30 UTR of lin-14 and lin-28 mRNA to suppress their expression. In lin-4
mutants, the L1 program is reiterated, resulting in seam cell defects,
absence of egg-laying structures, long body shape, and uncoordinated
movements in the adult animals. Thus, lin-4 is a small RNA that is crucial
for timely progression through the early larval stages. Although this
example demonstrates the importance of small RNAs in coordinating
cellular/developmental decisions that ultimately determine the entire
developmental trajectory of the organism, a hormone can be generally
defined as a molecule that is produced in one tissue and can exert its
effects in either the same (autocrine) or another tissue(s) (paracrine),
often invoking some degree of cell non-autonomy.
As in the case with many peptide hormones, neurons have recently
been placed at the centre of small RNA-mediated effects in C. elegans. It
has been demonstrated that double-stranded RNA (dsRNA) expressed in
the neurons can travel to other somatic tissues as well as the germ line to
silence its target(s) (Devanapally et al., 2015). Fluorescently labelled
dsRNA that is injected into the body cavity accumulates within the oo-
cytes likely through the non-specific import of material from the body
cavity. These cell non-autonomous acting dsRNAs are known as mobile
RNAs, and the silencing that they trigger in the germ line relies on sec-
ondary RNAs produced by RDE-1 processing, the secondary nuclear
Argonuate HRDE-1, and the RNase D homolog MUT-7. Thus, extracel-
lular dsRNA produced in the neurons could be taken up and processed
within the parental germ line where they can elicit robust gene silencing
and transgenerational inheritance.
Endogenous siRNAs of neuronal origin may also produce a heritable
response to regulate behaviour in subsequent generations through the
modification of gene expression in the germ line in a manner that is
dependent on the activity of the nuclear Argonaute proteins HRDE-1 and
CSR-1 (Posner et al., 2019). The transmission of these endogenous siR-
NAs is dependent on RDE-4, a neuron-enriched small RNA binding pro-
tein and was previously shown to play a role in neuronal migration,
learning, and memory (Juang et al., 2013; Sims et al., 2016; Tonkin and
Bass, 2003). Although it was not specifically shown, findings from this
group imply that neuronal-derived small RNAs travel to the germ line.
Small RNAs produced in animals expressing RDE-4 exclusively in the
neurons can affect gene expression cell non-autonomously in affected
progeny for up to three generations, underscoring an adaptive means of
communicating information that is sensed by the neurons and trans-
mitted, quite far from the source of their biosynthesis, to the germ line.
However, despite the excitement generated by this possible link between
the neurons and the germ line, it is important to note that the authors did
not show that RDE-4-dependent neuronal small RNAs have homology to
the germline genes that were differentially expressed in the presence of
neuronal RDE-4.
Perhaps even more intriguing, small RNAs have recently been shown
to reinforce learning avoidance behaviours in a transgenerational
manner. C. elegans consume a variety of bacterial food sources in the
environment. C. elegans that have consumed Pseudomonas aeruginosa, a
pathogenic strain known to cause disease in plants and animals, develop
an avoidance behaviour within 4 h of consuming the bacteria (Moore
et al., 2019). This learned behaviour is inherited up to four generations
through the germ line, despite the previous generations never
S. Rashid et al.
Fig. 4. Neuron-derived small RNAs play various roles in C. elegans. Small
RNAs synthesized in the neurons can influence behaviour and somatic devel-
opment. Small RNAs can be inherited transgenerationally to influence
P. aeruginosa avoidance, chemotaxis behaviour, and proper L2/L3 larval stage
transition. Small RNAs could potentially play a role in preserving germline
quiescence and germ cell integrity during the dauer stage in AMPK animals.
encountering P. aeruginosa. The progeny of the exposed mother express
daf-7/TGFβ at significantly high levels in the ASI neurons, while daf-7
mutants do not inherit this avoidance behaviour. Mutations that disrupt
the nuclear RNAi pathway also perturb the normal aversion to
P. aeruginosa, suggesting that small RNA-mediated epigenetic adaptation
might be involved in learning and transmitting this avoidance behaviour.
RNA-sequencing revealed that microRNAs and piRNAs are significantly
altered upon exposure to P. aeruginosa. Mutants that lack prg-1, the
C. elegans ortholog of the PIWI Argonaute, display a naïve choice pref-
erence and normal pathogenic avoidance after exposure, however, this
avoidance behaviour is not inherited. Moreover, exposure to the purified
small RNAs isolated from P. aeruginosa is sufficient to induce an avoid-
ance behaviour (Kaletskey et al., 2020). Further analysis shows that a
single non-coding RNA, P11, that targets the C. elegans maco-1 is
required. In addition to the requirement of intact RNAi and piRNA
pathways, the ASI neuron and the germ line are required for transmitting
this transgenerational inheritance behaviour. These data are consistent
with a regulatory role of neuronally-derived small RNAs that are trans-
mitted to future generations through the germ line to affect learned
pathogenic avoidance behaviour in order to identify nutritious food
sources, although how these small RNAs are packaged and expedited to
their final destinations remains undetermined.
Like hormones, small RNAs coordinate development among different
tissues according to both developmental and environmental cues (Fig. 4).
Although many have unique functions in development, like many hor-
mones, their effects depend greatly on their downstream effectors and/or
the response of their target tissues. However, although hormones elicit
changes within the organism to adjust to immediate environmental sig-
nals, the effects of small RNAs can be both immediate or they can be long-
term, even transgenerational, adjusting gene expression to enhance the
survival of progeny over multiple generations.
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Developmental Biology 475 (2021) 265–276
5. Conclusion
The natural environment of any organism can pose numerous, often
unpredictable challenges to health and survival. Developmental plas-
ticity is thus crucial for organisms to be able to adapt and maximize their
fitness. This is particularly true for C. elegans, and we have highlighted
the potential of this animal to adapt to nutrient stress at various points of
their life cycle. This adaptation may be as simple as slowing growth when
faced with a dearth of essential nutrients, as seen in animals grown on
axenic ‘minimal media’ (Szewczyk et al., 2003), or even accelerating
development upon encountering excess quantities of specific metabo-
lites, as is the case with animals fed Vitamin B12 (MacNeil et al., 2013).
Alternatively, developmental plasticity can involve more drastic
shifts, such as a developmental arrest in larvae that hatch in the absence
of food, or the thoroughly studied dauer diapause, where animals couple
nutrient signals with dramatic changes in gene expression, ultimately
resulting in altered metabolism, morphology, and even behaviour. The
dauer stage in particular highlights the extent to which developmental
plasticity can benefit survival. By entering the dauer diapause, animals
can survive environmental stresses for extended periods, then resume
reproductive development only when conditions improve. This adapta-
tion not only benefits survival of individuals, but may also have led to
evolutionary advantages, such as allowing nematodes to evolve from soil-
dwelling bacterivores to infectious parasites (Crook, 2014).
We also attempted to highlight the extensive metabolic challenges
faced by even simple organisms such as C. elegans when faced with acute
starvation. Lack of adequate food resources will trigger arrest at several
points during C. elegans development, highlighted by phenomena such as
the L1 diapause or the ARD. Even after development has ceased, drastic
shifts in metabolic pathways must occur in order for animals to survive
the period of nutrient stress seen prominently in dauer larvae, which
prioritize fatty acid oxidation, gluconeogenesis, and fermentation path-
ways to provide them with the energy they require for survival. Aside
from severe caloric deprivation, lack of several key metabolites, such as
amino acids or vitamins, can limit the development of C. elegans through
distinct mechanisms, highlighting the diverse and even “process-specific”
nutritional needs that organisms require.
Developmental plasticity in response to nutrient stress often includes
cell cycle quiescence in animals, including the germ cells. Maintaining
the integrity of the germ line is crucial, as the totipotent nature of the
germ cells must be preserved so that following the arrest, fertilization
will trigger appropriate embryonic and post-embryonic developmental
progression. AMPK is a notable mediator of quiescence in the germ line,
and is required for the survival and post-arrest fertility in both the L1 and
dauer diapauses through interactions with a number of different signal-
ling pathways and mechanisms, highlighting the complexity of gene
regulation during these developmental arrests (Narbonne and Roy, 2006,
2009; Demoinet et al., 2017). Of particular interest is the idea that AMPK
signalling may be mediated partly through small RNAs (Kadekar and
Roy, 2019). Indeed, emerging data indicate that small RNAs play a
crucial role as hormone-like signals throughout development. Small
RNAs, particularly those produced in the neurons, may also act trans-
generationally to regulate behaviour, and likely many other aspects of
physiology. How these small RNAs toggle between mounting an imme-
diate versus a long-term transgenerational adaptive response should help
us to understand how developmental plasticity may at times confer an
advantage to the individual, while at other times confer a benefit to the
population as a whole.
Acknowledgements
We thank Sider Penkov for valuable insights and discussion con-
cerning many of these ideas. RR is supported by a project grant from the
Canadian Institutes for Health Research (CIHR).
S. Rashid et al. Developmental Biology 475 (2021) 265–276

References Developmental Strategies. Diversity and Commonality in Animals. Springer, Tokyo.

https://doi.org/10.1007/978-4-431-56609-0_5.
Fukuyama, M., Gendreau, S.B., Derry, W.B., Rothman, J.H., 2003. Essential embryonic
Angelo, G., Van Gilst, M.R., 2009. Starvation protects germline stem cells and extends
roles of the CKI-1 cyclin-dependent kinase inhibitor in cell-cycle exit and
reproductive longevity in C. elegans. Science 326 (5955), 954–958. https://doi.org/
morphogenesis in C. elegans. Dev. Biol. 260 (1), 273–286. https://doi.org/10.1016/
10.1126/science.1178343.
s0012-1606(03)00239-2.
Antebi, A., Yeh, W.H., Tait, D., Hedgecock, E.M., Riddle, D.L., 2000. daf-12 encodes a
Fukuyama, M., Rougvie, A.E., Rothman, J.H., 2006. C. elegans DAF-18/PTEN mediates
nuclear receptor that regulates the dauer diapause and developmental age in
nutrient-dependent arrest of cell cycle and growth in the germline. Curr. Biol. 16 (8),
C. elegans. Genes Dev. 14 (12), 1512–1527.
773–779. https://doi.org/10.1016/j.cub.2006.02.073.
Austin, J., Kimble, J., 1987. glp-1 is required in the germ line for regulation of the
Fukuyama, M., Kontani, K., Katada, T., Rougvie, A.E., 2015. The C. elegans hypodermis
decision between mitosis and meiosis in C. elegans. Cell 51 (4), 589–599. https://
couples progenitor cell quiescence to the dietary state. Curr. Biol. 25 (9), 1241–1248.
doi.org/10.1016/0092-8674(87)90128-0.
https://doi.org/10.1016/j.cub.2015.03.016.
Austin, M.U., Liau, W.-S., Balamurugan, K., Ashokkumar, B., Said, H.M., LaMunyon, C.W.,
Gerisch, B., Tharyan, R.G., Mak, J., Denzel, S.I., Popkes-van Oepen, T., Henn, N.,
2010. Knockout of the folate transporter folt-1 causes germline and somatic defects in
Antebi, A., 2020. HLH-30/TFEB is a master regulator of reproductive quiescence.
C. elegans. BMC Dev. Biol. 10 (1), 46. https://doi.org/10.1186/1471-213x-10-46.
Dev. Cell 53 (3), 316–329. https://doi.org/10.1016/j.devcel.2020.03.014 e5.
Baugh, L.R., 2013. To grow or not to grow: nutritional control of development during
Golden, J.W., Riddle, D.L., 1982. A pheromone influences larval development in the
Caenorhabditis elegans L1 arrest. Genetics 194 (3), 539–555. https://doi.org/
nematode Caenorhabditis elegans. Science 218 (4572), 578–580.
10.1534/genetics.113.150847.
Golden, J.W., Riddle, D.L., 1984. A pheromone-induced developmental switch in
Baugh, L.R., Sternberg, P.W., 2006. DAF-16/FOXO regulates transcription of cki-1/Cip/
Caenorhabditis elegans: temperature-sensitive mutants reveal a wild-type temperature-
Kip and repression of lin-4 during C. elegans L1 arrest. Curr. Biol. 16 (8), 780–785.
dependent process. Proc. Natl. Acad. Sci. U.S.A. 81 (3), 819–823.
https://doi.org/10.1016/j.cub.2006.03.021.
Greenwald, I.S., Sternberg, P.W., Horvitz, H.R., 1983. The lin-12 locus specifies cell fates
Birnby, D.A., Link, E.M., Vowels, J.J., Tian, H., Colacurcio, P.L., Thomas, J.H., 2000.
in Caenorhabditis elegans. Cell 34 (2), 435–444. https://doi.org/10.1016/0092-
A transmembrane guanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate a
8674(83)90377-x.
common set of chemosensory behaviors in Caenorhabditis elegans. Genetics 155 (1),
Hall, S.E., Beverly, M., Russ, C., Nusbaum, C., Sengupta, P., 2010. A cellular memory of
85–104.
developmental history generates phenotypic diversity in C. elegans. Curr. Biol. : CB 20
Bito, T., Matsunaga, Y., Yabuta, Y., Kawano, T., Watanabe, F., 2013. Vitamin B12
(2), 149–155. https://doi.org/10.1016/j.cub.2009.11.035.
deficiency in Caenorhabditis elegans results in loss of fertility, extended life cycle, and
Hammell, C.M., Karp, X., Ambros, V., 2009. A feedback circuit involving let-7-family
reduced lifespan. FEBS open bio 3, 112–117. https://doi.org/10.1016/
miRNAs and DAF-12 integrates environmental signals and developmental timing in
j.fob.2013.01.008.
Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 106 (44), 18668–18673. https://
Blackwell, T.K., Sewell, A.K., Wu, Z., Han, M., 2019. TOR signalling in Caenorhabditis
doi.org/10.1073/pnas.0908131106.
elegans. development, metabolism, and aging. Genetics 213 (2), 329–360. https://
Harvey, S.C., Gemmill, A.W., Read, A.F., Viney, M.E., 2000. The control of morph
doi.org/10.1534/genetics.119.302504.
development in the parasitic nematode Strongyloides ratti. Proceedings: Biol. Sci. 267
Braeckman, B.P., 2009. Intermediary metabolism. In: WormBook, pp. 1–24. https://
(1457), 2057–2063. https://doi.org/10.1098/rspb.2000.1249.
doi.org/10.1895/wormbook.1.146.1.
Hawdon, J.M., Schad, G.A., 1991. Albumin and a dialyzable serum factor stimulate
Braendle, C., F
elix, M.A., 2008. Plasticity and errors of a robust developmental system in
feeding in vitro by third-stage larvae of the canine hookworm Ancylostoma caninum.
different environments. Dev. Cell 15 (5), 714–724. https://doi.org/10.1016/
J. Parasitol. 77 (4), 587–591.
j.devcel.2008.09.011.
Heijmans, B.T., Tobi, E.W., Stein, A.D., Putter, H., Blauw, G.J., Susser, E.S.,
Budirahardja, Y., G€ onczy, P., 2009. Coupling the cell cycle to development.
Slagboom, P.E., Lumey, L.H., 2008. Persistent epigenetic differences associated with
J. Development 136 (17), 2861–2872. https://doi.org/10.1242/dev.021931.
prenatal exposure to famine in humans. Proc. Natl. Acad. Sci. U.S.A. 105 (44),
Burnaevskiy, N., Chen, S., Mailig, M., Reynolds, A., Karanth, S., Mendenhall, A.,
17046–17049. https://doi.org/10.1073/pnas.0806560105.
Kaeberlein, M., 2018. Reactivation of RNA metabolism underlies somatic restoration
Henderson, S.T., Gao, D., Lambie, E.J., Kimble, J., 1994. lag-2 may encode a signaling
after adult reproductive diapause in C. elegans. Elife 7, e36194. https://doi.org/
ligand for the GLP-1 and LIN-12 receptors of C. elegans. Development 120 (10),
10.7554/eLife.36194.
2913–2924.
Carranza-García, E., Navarro, R.E., 2019. Apoptosis contributes to protect germ cells from
Hibshman, J.D., Doan, A.E., Moore, B.T., Kaplan, R.E., Hung, A., Webster, A.K.,
the oogenic germline starvation response but is not essential for the gonad shrinking
Bhatt, D.P., Chitrakar, R., Hirschey, M.D., Baugh, L.R., 2017. daf-16/FoxO promotes
or recovery observed during adult reproductive diapause in C. elegans. PloS One 14
gluconeogenesis and trehalose synthesis during starvation to support survival. eLife
(6), e0218265. https://doi.org/10.1371/journal.pone.0218265.
6, e30057. https://doi.org/10.7554/eLife.30057.
Carranza-García, E., Navarro, R.E., 2020. Insights into the hypometabolic stage caused by
Hirsh, D., Oppenheim, D., Klass, M., 1976. Development of the reproductive system of
prolonged starvation in L4-adult Caenorhabditis elegans hermaphrodites. Front Cell
Caenorhabditis elegans. Dev. Biol. 49 (1), 200–219.
Dev. Biol. 8, 124. https://doi.org/10.3389/fcell.2020.00124.
Holt, S.J., Riddle, D.L., 2003. SAGE surveys C. elegans carbohydrate metabolism: evidence
Cassada, R.C., Russell, R.L., 1975. The dauerlarva, a post-embryonic developmental
for an anaerobic shift in the long-lived dauer larva. Mech. Ageing Develop. 124 (7),
variant of the nematode Caenorhabditis elegans. Dev. Biol. 46 (2), 326–342. https://
779–800. https://doi.org/10.1016/s0047-6374(03)00132-5.
doi.org/10.1016/0012-1606(75)90109-8.
Hong, Y., Roy, R., Ambros, V., 1998. Developmental regulation of a cyclin-dependent
Chen, Y., Baugh, L.R., 2014. ins-4 and daf-28 function redundantly to regulate C. elegans
kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis
L1 arrest. Dev. Biol. 394 (2), 314–326. https://doi.org/10.1016/
elegans. Development 125 (18), 3585–3597.
j.ydbio.2014.08.002.
Hotez, P., Hawdon, J., Schad, G.A., 1993. Hookworm larval infectivity, arrest and
Chi, C., Ronai, D., Than, M.T., Walker, C.J., Sewell, A.K., Han, M., 2016. Nucleotide levels
amphiparatenesis: the Caenorhabditis elegans Daf-c paradigm. Parasitol. today 9 (1),
regulate germline proliferation through modulating GLP-1/Notch signaling in
23–26. https://doi.org/10.1016/0169-4758(93)90159-d.
C. elegans. Genes Dev. 30 (3), 307–320. https://doi.org/10.1101/gad.275107.115.
Hu, Patrick J., 2007. Dauer. In: WormBook, pp. 1–19. https://doi.org/10.1895/
Crook, M., 2014. The dauer hypothesis and the evolution of parasitism: 20 years on and
wormbook.1.144.1.
still going strong. Int. J. Parasitol. 44 (1), 1–8. https://doi.org/10.1016/
Hung, W.L., Wang, Y., Chitturi, J., Zhen, M., 2014. A Caenorhabditis elegans
j.ijpara.2013.08.004.
developmental decision requires insulin signaling-mediated neuron-intestine
Demoinet, E., Li, S., Roy, R., 2017. AMPK blocks starvation-inducible transgenerational
communication. Development 141 (8), 1767–1779. https://doi.org/10.1242/
defects in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 114 (13),
dev.103846.
E2689–E2698. https://doi.org/10.1073/pnas.1616171114.
Iser, W.B., Wolkow, C.A., 2007. DAF-2/Insulin-Like signalling in C. elegans modifies
Devanapally, S., Ravikumar, S., Jose, A.M., 2015. Double-stranded RNA made in
effects of dietary restriction and nutrient stress on aging, stress and growth. PloS One
C. elegans neurons can enter the germline and cause transgenerational gene silencing.
2 (11), e1240. https://doi.org/10.1371/journal.pone.0001240.
Proc. Natl/ Acad. Sci. U. S. A. 112 (7), 2133–2138. https://doi.org/10.1073/
Jeong, H., Paik, Y.-K., 2017. MGL-1 on AIY neurons translates starvation to reproductive
pnas.1423333112.
plasticity via neuropeptide signalling in Caenorhabditis elegans. Dev. Biol. 430 (1),
Dougherty, E.C., 1959. Introduction to axenic culture of invertebrate metazoan: a goal.
80–89.
Ann. N. Y. Acad. Sci. 77, 27–54. https://doi.org/10.1111/j.1749-
Jeong, P.Y., Kwon, M.S., Joo, H.J., Paik, Y.K., 2009. Molecular time-course and the
6632.1959.tb36891.x.
metabolic basis of entry into dauer in Caenorhabditis elegans. PloS One 4 (1), e4162.
Edwards, C., Canfield, J., Copes, N., Brito, A., Rehan, M., Lipps, D., Brunquell, J.,
https://doi.org/10.1371/journal.pone.0004162.
Westerheide, S.D., Bradshaw, P.C., 2015. Mechanisms of amino acid-mediated
Jewell, J.L., Russell, R.C., Guan, K.L., 2013. Amino acid signalling upstream of mTOR.
lifespan extension in Caenorhabditis elegans. BMC Genet. 16 (1), 8. https://doi.org/
Nat. Rev. Mol. Cell Biol. 14 (3), 133–139. https://doi.org/10.1038/nrm3522.
10.1186/s12863-015-0167-2.
Jia, K., Chen, D., Riddle, D.L., 2004. The TOR pathway interacts with the insulin
F

elix, M.-A., Braendle, C., 2010. The natural history of Caenorhabditis elegans. Curr. Biol.
signalling pathway to regulate C. elegans larval development, metabolism and life
20 (2), R965–R969. https://doi.org/10.1016/j.cub.2010.09.050.
span. Development (Cambridge, England) 131 (16), 3897–3906. https://doi.org/
Fielenbach, N., Antebi, A., 2008. C. elegans dauer formation and the molecular basis of
10.1242/dev.01255.
plasticity. Genes Dev. 22 (16), 2149–2165. https://doi.org/10.1101/gad.1701508.
Jia, F., Cui, M., Than, M.T., Han, M., 2016. Developmental defects of Caenorhabditis
Fr
ezal, L., F

elix, M.A., 2015. C. elegans outside the Petri dish. eLife 4, e05849. https://
elegans lacking branched-chain α-ketoacid dehydrogenase are mainly caused by
doi.org/10.7554/eLife.05849.
monomethyl branched-chain fatty acid deficiency. J. Biol. Chem. 291 (6),
Friedman, D.B., Johnson, T.E., 1988. A mutation in the age-1 gene in Caenorhabditis
2967–2973. https://doi.org/10.1074/jbc.M115.676650.
elegans lengthens life and reduces hermaphrodite fertility. Genetics 118 (1), 75–86.
Jia, F., Chi, C., Han, M., 2020. Regulation of nucleotide metabolism and germline
Fukuyama, M., 2018. Nutritional control of the germline development in Caenorhabditis
proliferation in response to nucleotide imbalance and genotoxic stresses by EndoU
elegans. In: Kobayashi, K., Kitano, T., Iwao, Y., Kondo, M. (Eds.), Reproductive and

274
S. Rashid et al.
nuclease. Cell Rep. 30 (6), 1848–1861. https://doi.org/10.1016/
j.celrep.2020.01.050 e5.
Johnstone, I.L., Barry, J.D., 1996. Temporal reiteration of a precise gene expression
pattern during nematode development. EMBO J. 15 (14), 3633–3639.
Jones, S.J., Riddle, D.L., Pouzyrev, A.T., Velculescu, V.E., Hillier, L., Eddy, S.R.,
Stricklin, S.L., Baillie, D.L., Waterston, R., Marra, M.A., 2001. Changes in gene
expression associated with developmental arrest and longevity in Caenorhabditis
elegans. Genome Res. 11 (8), 1346–1352.
Juang, B.T., Gu, C., Starnes, L., Palladino, F., Goga, A., Kennedy, S., L’Etoile, N.D., 2013.
Endogenous nuclear RNAi mediates behavioral adaptation to odor. Cell 154 (5),
1010–1022. https://doi.org/10.1016/j.cell.2013.08.006.
Kadekar, P., Roy, R., 2019. AMPK regulates germline stem cell quiescence and integrity
through an endogenous small RNA pathway. PLoS Biol. 17 (6), e3000309 https://
doi.org/10.1371/journal.pbio.3000309.
Kaletsky, R., Moore, R.S., Vrla, G.D., Parsons, L.R., Gitai, Z., Murphy, C.T., 2020.
C. elegans interprets bacterial non-coding RNAs to learn pathogenic avoidance.
Nature 586 (7829), 445–451. https://doi.org/10.1038/s41586-020-2699-5.
Kang, C., Avery, L., 2009. Systemic regulation of starvation response in Caenorhabditis
elegans. Genes Dev. 23 (1), 12–17. https://doi.org/10.1101/gad.1723409.
Kaplan, R., Webster, A.K., Chitrakar, R., Dent, J.A., Baugh, L.R., 2018. Food perception
without ingestion leads to metabolic changes and irreversible developmental arrest in
C. elegans. BMC Biol. 16 (1), 112. https://doi.org/10.1186/s12915-018-0579-3.
Kaptan, D., et al., 2020. Exogenous ethanol induces a metabolic switch that prolongs the
survival of Caenorhabditis elegans dauer larva and enhances its resistance to
desiccation. Aging cell 19 (10), e13214. https://doi.org/10.1111/acel.13214.
Kasuga, H., Fukuyama, M., Kitazawa, A., Kontani, K., Katada, T., 2013. The microRNA
miR-235 couples blast-cell quiescence to the nutritional state. Nature 497 (7450),
503–506. https://doi.org/10.1038/nature12117.
Kimura, K.D., Tissenbaum, H.A., Liu, Y., Ruvkun, G., 1997. daf-2, an insulin receptor-like
gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277
(5328), 942–946.
Komuniecki, R., Harris, B.G., 1995. Carbohydrate and energy metabolism in helminths.
In: Marr, J.J., Muller, M. (Eds.), Biochemistry and Molecular Biology of Parasites.
Academic Press, London, pp. 49–66.
Kosti
c, I., Li, S., Roy, R., 2003. cki-1 links cell division and cell fate acquisition in the
C. eleganssomatic gonad. Dev. Biol. 263 (2), 242–252. https://doi.org/10.1016/
j.ydbio.2003.07.001.
Kühnl, J., Bobik, T., Procter, J.B., Burmeister, C., H€ oppner, J., Wilde, I., Lüersen, K.,
Torda, A.E., Walter, R.D., Liebau, E., 2005. Functional analysis of the methylmalonyl-
CoA epimerase from Caenorhabditis elegans. FEBS J. 272 (6), 1465–1477. https://
doi.org/10.1111/j.1742-4658.2005.04579.x.
Lafuente, E., Beldade, P., 2019. Genomics of developmental plasticity in animals. Front.
Genet. 10, 720.
Lapierre, L.R., Hansen, M., 2012. Lessons from C. elegans: signalling pathways for
longevity. Trends Endocrinol. Metabol.: TEM (Trends Endocrinol. Metab.) 23 (12),
637–644. https://doi.org/10.1016/j.tem.2012.07.007.
L

ascarez-Lagunas, L.I., Silva-García, C.G., Dinkova, T.D., Navarro, R.E., 2014. LIN-35/Rb
causes starvation-induced germ cell apoptosis via CED-9/Bcl2 downregulation in
Caenorhabditis elegans. Mol. Cell Biol. 34 (13), 2499–2516. https://doi.org/10.1128/
mcb.01532-13.
Lee, B.H., Ashrafi, K., 2008. A TRPV channel modulates C. elegans neurosecretion, larval
starvation survival, and adult lifespan. PLoS Genet. 4 (10), e1000213 https://
doi.org/10.1371/journal.pgen.1000213.
Lee, R.C., Feinbaum, R.L., Ambros, V., 1993. The C. elegans heterochronic gene lin-4
encodes small RNAs with antisense complementarity to lin-14. Cell 75 (5), 843–854.
https://doi.org/10.1016/0092-8674(93)90529-y.
Lee, I., Hendrix, A., Kim, J., Yoshimoto, J., You, Y.J., 2012. Metabolic rate regulates L1
longevity in C. elegans. PloS One 7 (9), e44720. https://doi.org/10.1371/
journal.pone.0044720.
Lin, C., Garrett, A.S., De Kumar, B., Smith, E.R., Gogol, M., Seidel, C., Krumlauf, R.,
Shilatifard, A., 2011. Dynamic transcriptional events in embryonic stem cells
mediated by the super elongation complex (SEC). Genes Dev. 25 (14), 1486–1498.
https://doi.org/10.1101/gad.2059211.
Long, X., Spycher, C., Han, Z.S., Rose, A.M., Müller, F., Avruch, J., 2002. TOR deficiency
in C. elegans causes developmental arrest and intestinal atrophy by inhibition of
mRNA translation. Curr. Biol. 12 (17), 1448–1461. https://doi.org/10.1016/s0960-
9822(02)01091-6.
Ludewig, A.H., Kober-Eisermann, C., Weitzel, C., Bethke, A., Neubert, K., Gerisch, B.,
Hutter, H., Antebi, A., 2004. A novel nuclear receptor/coregulator complex controls
C. elegans lipid metabolism, larval development, and aging. Genes Dev. 18 (17),
2120–2133. https://doi.org/10.1101/gad.312604.
MacNeil, L.T., Watson, E., Arda, H.E., Zhu, L.J., Walhout, A.J., 2013. Diet-induced
developmental acceleration independent of TOR and insulin in C. elegans. Cell 153
(1), 240–252. https://doi.org/10.1016/j.cell.2013.02.049.
Mariani, L., Lussi, Y.C., Vandamme, J., Riveiro, A., Salcini, A.E., 2016. The H3K4me3/2
histone demethylase RBR-2 controls axon guidance by repressing the actin-
remodeling gene wsp-1. Development 143 (5), 851–863. https://doi.org/10.1242/
dev.132985%JDevelopment.
Mark, K.A., Dumas, K.J., Bhaumik, D., Schilling, B., Davis, S., Oron, T.R., Sorensen, D.J.,
Lucanic, M., Brem, R.B., Melov, S., Ramanathan, A., Gibson, B.W., Lithgow, G.J.,
2016. Vitamin D promotes protein homeostasis and longevity via the stress response
pathway genes skn-1, ire-1, and xbp-1. Cell Rep. 17 (5), 1227–1237.
McElwee, J.J., Schuster, E., Blanc, E., Thornton, J., Gems, D., 2006. Diapause-associated
metabolic traits reiterated in long-lived daf-2 mutants in the nematode Caenorhabditis
elegans. Mech. Ageing Develop. 127 (5), 458–472. https://doi.org/10.1016/
j.mad.2006.01.006.
275
Developmental Biology 475 (2021) 265–276
Michaelson, D., Korta, D.Z., Capua, Y., Hubbard, E.J., 2010. Insulin signalling promotes
germline proliferation in C. elegans. Development 137 (4), 671–680. https://doi.org/
10.1242/dev.042523.
Moore, R.S., Kaletsky, R., Murphy, C.T., 2019. Piwi/PRG-1 Argonaute TGF-β mediate
transgenerational learned pathogenic avoidance. Cell 177 (7), 1827–1841. https://
doi.org/10.1016/j.cell.2019.05.024 e1812.
Motola, D.L., Cummins, C.L., Rottiers, V., Sharma, K.K., Li, T., Li, Y., Suino-Powell, K.,
Xu, H.E., Auchus, R.J., Antebi, A., Mangelsdorf, D.J., 2006. Identification of ligands
for DAF-12 that govern dauer formation and reproduction in C. elegans. Cell 124 (6),
1209–1223. https://doi.org/10.1016/j.cell.2006.01.037.
Mu~ noz, M.J., Riddle, D.L., 2003. Positive selection of Caenorhabditis elegans mutants with
increased stress resistance and longevity. Genetics 163 (1), 171–180.
Mylenko, M., Boland, S., Penkov, S., Sampaio, J.L., Lombardot, B., Vorkel, D.,
Verbavatz, J.M., Kurzchalia, T.V., 2016. NADþ is a food component that promotes
exit from dauer diapause in Caenorhabditis elegans. PloS One 11 (12), e0167208.
https://doi.org/10.1371/journal.pone.0167208.
Nadarajan, S., Govindan, J.A., McGovern, M., Hubbard, E.J., Greenstein, D., 2009. MSP
and GLP-1/Notch signaling coordinately regulate actomyosin-dependent cytoplasmic
streaming and oocyte growth in C. elegans. Development 136 (13), 2223–2234.
https://doi.org/10.1242/dev.034603.
Narbonne, P., Roy, R., 2006. Inhibition of germline proliferation during C. elegans dauer
development requires PTEN, LKB1 and AMPK signalling. Development 133 (4),
611–619. https://doi.org/10.1242/dev.02232%JDevelopment.
Narbonne, P., Roy, R., 2009. Caenorhabditis elegans dauers need LKB1/AMPK to ration
lipid reserves and ensure long-term survival. Nature 457 (7226), 210–214. https://
doi.org/10.1038/nature07536.
Newgard, C.B., 2012. Interplay between lipids and branched-chain amino acids in
development of insulin resistance. Cell Metabol. 15 (5), 606–614. https://doi.org/
10.1016/j.cmet.2012.01.024.
O’Leary, J.K., Barry, J.P., Gabrielson, P.W., Rogers-Bennett, L., Potts, D.C., Palumbi, S.R.,
Micheli, F., 2017. Calcifying algae maintain settlement cues to larval abalone
following algal exposure to extreme ocean acidification. Sci. Rep. 7 (1), 5774.
https://doi.org/10.1038/s41598-017-05502-x.
Ogg, S., Ruvkun, G., 1998. The C. elegansPTEN homolog, DAF-18, acts in the insulin
receptor-like metabolic signalling pathway. Mol. Cell. 2 (6), 887–893. https://
doi.org/10.1016/s1097-2765(00)80303-2.
Ouellet, J., Li, S., Roy, R., 2008. Notch signalling is required for both dauer maintenance
and recovery in C. elegans. Development 135 (15), 2583–2592. https://doi.org/
10.1242/dev.012435%JDevelopment.
Ow, M.C., Borziak, K., Nichitean, A.M., Dorus, S., Hall, S.E., 2018. Early experiences
mediate distinct adult gene expression and reproductive programs in Caenorhabditis
elegans. PLoS Genet. 14 (2), e1007219 https://doi.org/10.1371/
journal.pgen.1007219.
Penkov, S., Raghuraman, B.K., Erkut, C., Oertel, J., Galli, R., Ackerman, E., Vorkel, D.,
Verbavatz, J.M., Koch, E., Fahmy, K., Shevchenko, A., Kurzchalia, T.V., 2020.
A metabolic switch regulates the transition between growth and diapause in
C. elegans. BMC Biol. 18 (1), 31. https://doi.org/10.1186/s12915-020-0760-3.
Pierce, S.B., Costa, M., Wisotzkey, R., Devadhar, S., Homburger, S.A., Buchman, A.R.,
Ferguson, K.C., Heller, J., Platt, D.M., Pasquinelli, A.A., Liu, L.X., Doberstein, S.K.,
Ruvkun, G., 2001. Regulation of DAF-2 receptor signalling by human insulin and ins-
1, a member of the unusually large and diverse C. elegans insulin gene family. Genes
Dev. 15 (6), 672–686. https://doi.org/10.1101/gad.867301.
Posner, R., Toker, I.A., Antonova, O., Star, E., Anava, S., Azmon, E., Rechavi, O., 2019.
Neuronal small RNAs control behaviour transgenerationally. Cell 177 (7),
1814–1826. https://doi.org/10.1016/j.cell.2019.04.029 e1815.
Prudic, K.L., Jeon, C., Cao, H., Monteiro, A., 2011. Developmental plasticity in sexual
roles of butterfly species drives mutual sexual ornamentation. Science 331 (6013),
73–75. https://doi.org/10.1126/science.1197114.
Qi, B., Kniazeva, M., Han, M., 2017. A vitamin-B2-sensing mechanism that regulates gut
protease activity to impact animal’s food behaviour and growth. eLife 6, e26243.
https://doi.org/10.7554/elife.26243.
Rao, A.U., Carta, L.K., Lesuisse, E., Hamza, I., 2005. Lack of heme synthesis in a free-living
eukaryote. Proc. Natl. Acad. Sci. U.S.A. 102 (12), 4270–4275. https://doi.org/
10.1073/pnas.0500877102.
Rathor, L., Akhoon, B.A., Pandey, S., Srivastava, S., Pandey, R., 2015. Folic acid
supplementation at lower doses increases oxidative stress resistance and longevity in
Caenorhabditis elegans. Age 37 (6), 113. https://doi.org/10.1007/s11357-015-9850-
5.
R
egniere, J., Powell, J., Bentz, B., Nealis, V., 2012. Effects of temperature on
development, survival and reproduction of insects: experimental design, data analysis
and modeling. J. Insect Physiol. 58 (5), 634–647. https://doi.org/10.1016/
j.jinsphys.2012.01.010.
Ren, Y., Shen, H.M., 2019. Critical role of AMPK in redox regulation under glucose
starvation. Redox Biol. 25, 101154. https://doi.org/10.1016/j.redox.2019.101154.
Ren, P., Lim, C.S., Johnsen, R., Albert, P.S., Pilgrim, D., Riddle, D.L., 1996. Control of
C. elegans larval development by neuronal expression of a TGF-beta homolog. Science
274 (5291), 1389–1391.
Riddle, D.L., Albert, P.S., 1997. Genetic and environmental regulation of dauer larva
development. In: Riddle, D.L., Blumenthal, T., Meyer, B.J., et al. (Eds.), C. elegans II,
second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY) (Chapter
26).
Riddle, D.L., Swanson, M.M., Albert, P.S., 1981. Interacting genes in nematode dauer
larva formation. Nature 290 (5808), 668–671. https://doi.org/10.1038/290668a0.
Roux, A.E., Langhans, K., Huynh, W., Kenyon, C., 2016. Reversible age-related
phenotypes induced during larval quiescence in C. elegans. Cell Metabol. 23 (6),
1113–1126. https://doi.org/10.1016/j.cmet.2016.05.024.
S. Rashid et al.
Roy, D., Michaelson, D., Hochman, T., Santella, A., Bao, Z., Goldberg, J.D.,
Hubbard, E.J.A., 2016. Cell cycle features of C. elegans germline stem/progenitor cells
vary temporally and spatially. Dev. Biol. 409 (1), 261–271. https://doi.org/10.1016/
j.ydbio.2015.10.031.
Samuel, B.S., Rowedder, H., Braendle, C., F
elix, M.-A., Ruvkun, G., 2016. Caenorhabditis
elegans responses to bacteria from its natural habitats. Proc. Natl. Acad. Sci. U.S.A.
113 (27), E3941–E3949.
Sancak, Y., Peterson, T.R., Shaul, Y.D., Lindquist, R.A., Thoreen, C.C., Bar-Peled, L.,
Sabatini, D.M., 2008. The Rag GTPases bind raptor and mediate amino acid signalling
to mTORC1. Science 320 (5882), 1496–1501. https://doi.org/10.1126/
science.1157535.
Sanzo-Machuca, A.,
Monje Moreno, J.M., Casado-Navarro, R., Karakuzu, O., Guerrero-
G
omez, D., Fierro-Gonz
alez, J.C., Swoboda, P., Mu~ noz, M.J., Garsin, D.A.,
Pedrajas, J.R., Barrios, A., Miranda-Vizuete, A., 2019. Redox-dependent and redox-
independent functions of Caenorhabditis elegans thioredoxin 1. Redox Biol. 24,
101178. https://doi.org/10.1016/j.redox.2019.101178.
Schindler, A.J., Baugh, L.R., Sherwood, D.R., 2014. Identification of late larval stage
developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and
steroid hormone signalling pathways. PLoS Genet. 10 (6), e1004426 https://doi.org/
10.1371/journal.pgen.1004426.
Schulenburg, H., F
elix, M.A., 2017. The natural biotic environment of Caenorhabditis
elegans. Genetics 206 (1), 55–86. https://doi.org/10.1534/genetics.116.195511.
Seidel, H.S., Kimble, J., 2011. The oogenic germline starvation response in C. elegans. PloS
One 6 (12), e28074. https://doi.org/10.1371/journal.pone.0028074.
Seidel, H.S., Kimble, J., 2015. Cell-cycle quiescence maintains Caenorhabditis elegans
germline stem cells independent of GLP-1/Notch. eLife 4, e10832. https://doi.org/
10.7554/eLife.10832.
Shao, D., Oka, S., Liu, T., Zhai, P., Ago, T., Sciarretta, S., Li, H., Sadoshima, J., 2014.
A redox-dependent mechanism for regulation of AMPK activation by Thioredoxin1
during energy starvation. Cell Metabol. 19 (2), 232–245. https://doi.org/10.1016/
j.cmet.2013.12.013.
Shen, Y., Wollam, J., Magner, D., Karalay, O., Antebi, A., 2012. A steroid receptor-
microRNA switch regulates life span in response to signals from the gonad. Science
338 (6113), 1472–1476. https://doi.org/10.1126/science.1228967.
Sims, J.R., Ow, M.C., Nishiguchi, M.A., Kim, K., Sengupta, P., Hall, S.E., 2016.
Developmental programming modulates olfactory behaviour in C. elegans via
endogenous RNAi pathways. eLife 5, ee1642. https://doi.org/10.7554/eLife.11642.
Solari, F., Bourbon-Piffaut, A., Masse, I., Payrastre, B., Chan, A.M.L., Billaud, M., 2004.
The human tumour suppressor PTEN regulates longevity and dauer formation in
Caenorhabditis elegans. Oncogene 24 (1), 20–27. https://doi.org/10.1038/
sj.onc.1207978.
276
Developmental Biology 475 (2021) 265–276
Sommer, R.J., 2020. Phenotypic plasticity: from theory and genetics to current and future
challenges. Genetics 215 (1), 1–13. https://doi.org/10.1534/genetics.120.303163.
Szewczyk, N.J., Kozak, E., Conley, C.A., 2003. Chemically defined medium and
Caenorhabditis elegans. BMC Biotechnol. 3, 19. https://doi.org/10.1186/1472-6750-
3-19.
Takamiya, S., Matsui, T., Taka, H., Murayama, K., Matsuda, M., Aoki, T., 1999. Free-living
nematodes Caenorhabditis elegans possess in their mitochondria an additional
rhodoquinone, an essential component of the eukaryotic fumarate reductase system.
Arch. Biochem. Biophys. 371 (2), 284–289. https://doi.org/10.1006/
abbi.1999.1465.
Tilton, W.M., Seaman, C., Carriero, D., Piomelli, S., 1991. Regulation of glycolysis in the
erythrocyte: role of the lactate/pyruvate and NAD/NADH ratios. J. Lab. Clin. Med.
118 (2), 146–152.
Tonkin, L.A., Bass, B.L., 2003. Mutations in RNAi rescue aberrant chemotaxis of ADAR
mutants. Science 302 (5651), 1725. https://doi.org/10.1126/science.1091340.
Wamelink, M.M.C., Struys, E.A., Jakobs, C., 2008. The biochemistry, metabolism and
inherited defects of the pentose phosphate pathway: a review. J. Inherit. Metab. Dis.
31 (6), 703–717.
Wang, J., Kim, S.K., 2003. Global analysis of dauer gene expression in Caenorhabditis
elegans. Development 130 (8), 1621–1634.
Watanabe, S., Yamamoto, T.G., Kitagawa, R., 2008. Spindle assembly checkpoint gene
mdf-1 regulates germ cell proliferation in response to nutrition signals in C. elegans.
EMBO J. 27 (7), 1085–1096. https://doi.org/10.1038/emboj.2008.32.
Watson, E., MacNeil, L.T., Ritter, A.D., Yilmaz, L.S., Rosebrock, A.P., Caudy, A.A.,
Walhout, A.J., 2014. Interspecies systems biology uncovers metabolites affecting
C. elegans gene expression and life history traits. Cell 156 (4), 759–770. https://
doi.org/10.1016/j.cell.2014.01.047.
Yilmaz, L.S., Walhout, A.J., 2014. Worms, bacteria, and micronutrients: an elegant model
of our diet. Trends Genet. 30 (11), 496–503. https://doi.org/10.1016/
j.tig.2014.07.010.
Zeci
c, A., Dhondt, I., Braeckman, B.P., 2019. The nutritional requirements of
Caenorhabditis elegans. Genes & nutrition 14, 15. https://doi.org/10.1186/s12263-
019-0637-7.
Zhang, X., Zabinsky, R., Teng, Y., Cui, M., Han, M., 2011. microRNAs play critical roles in
the survival and recovery of Caenorhabditis elegans from starvation-induced L1
diapause. Proc. Natl. Acad. Sci. U.S.A. 108 (44), 17997–18002.
Zheng, S., Qu, Z., Zanetti, M., Lam, B., Chin-Sang, I., 2018. C. elegans PTEN and AMPK
block neuroblast divisions by inhibiting a BMP-insulin-PP2A-MAPK pathway.
Development 145 (23), dev166876. https://doi.org/10.1242/dev.166876.