1. We are interested in the mechanisms that regulate the sensitivity of embryos
to nutritional status. Proper nutrition (composition and levels) is critical not
only for embryonic viability, but for a normal healthspan. We have previously
identified several mutants that are sensitive to elevated levels of dietary
glucose, including agl-1 (glycogen debranching enzyme), aak-2 (AMPK alpha
subunit), and unmapped mutants identified in a conditional glucose-sensitized
screen, gin-1(cv10) and gin-2(cv11). agl-1 sensitivity to elevated glucose is
dependent on insulin signaling, as mutations in daf-2 (insulin-like receptor)
suppress the agl-1 glucose-dependent embryonic lethality. The role of insulin
signaling pathway genes in embryonic development is also supported by the
observations that certain daf-2 alleles alone or in daf-16 double mutants
display embryonic defects [1]. Finally, over-expression of a daf-16::gfp reporter
increases agl-1 embryonic lethality. To assess the role of insulin signaling in
early embryonic development, we used CRISPR/Cas9 to introduce a GFP tag
into the daf-2 locus, creating a functional C-terminal tagged GFP fusion protein
[2]. DAF-2::GFP is expressed in the early embryo, and expression appears to be
influenced by nutritional status, as previously observed [3] . Specifically,
conditions of both elevated glucose or starvation (and dietary restriction)
appear to reduce DAF-2::GFP levels in the nervous system as well and in
embryos; certain diets appear to result in an increase in cytoplasmic versus
membranous DAF-2::GFP. The effects of defined media (diet) on DAF-2
expression and localization in the embryo will be presented. Surprisingly, we
discovered that DAF-2::GFP is also expressed in sperm, suggesting a paternal
contribution to early daf-2 embryonic function. This possible role is being
investigated.
Nutritional Regulation of Insulin Signaling During Embryogenesis
Caroline Granger, Yi Dong, Jeff Simske, Rammelkamp Center, MetroHealth Hospital, Cleveland, OH
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agl-1(jc11) agl-1; daf-2 agl-1(jc11) agl-1; daf-2
daf-2 is a strong suppressor of
agl-1 embryonic lethality
Live OP50 Glucose 0.2%
DAF-16::GFP localizes to the
nucleus in agl-1 animals
DAF-16::GFP over-expression
enhances agl-1 embryonic lethality
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agl-1(tm4809) agl-1(tm4809);
daf-16::GFP
daf-16::GFP
ABSTRACT
% Embryonic
Lethal
References
1. Gems, D., et al., Two pleiotropic classes of daf-2 mutation affect larval arrest,
adult behavior, reproduction and longevity in Caenorhabditis elegans. Genetics,
1998. 150(1): p. 129-55.
2. Dickinson, D.J., et al., Engineering the Caenorhabditis elegans genome using Cas9-
triggered homologous recombination. Nat Methods, 2013. 10(10): p. 1028-34.
3. Kimura, K.D., D.L. Riddle, and G. Ruvkun, The C. elegans DAF-2 insulin-like receptor
is abundantly expressed in the nervous system and regulated by nutritional status.
Cold Spring Harb Symp Quant Biol, 2011. 76: p. 113-20
DAF-2::GFP construction using CRISPR/Cas9
DAF-2::GFP Expression
Regulation of DAF-2::GFP Expression by Nutritional Status
daf-2 and agl-1 Interactions
agl-1 suppresses daf-2 longevity
daf-2; daf-16 cytokinesis and polarity phenotypes
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Days
agl-1(jc11);daf-2
wild-type
Ex::agl-1+;agl-1
agl-1(jc11)
agl-1(jc11)
agl-1::GFP; +/+
daf-2
Repair construct
CRISPR target – 3’ UTR
1. daf-2::gfp construct
2. N2 wild-type DNA
3. daf-2::gfp CRISPR allele
1 2 3
PCR
Verification
Earliest daf-16; daf-2 phenotypes suggest centrosomal/polarity defects
Does DAF-2 act upstream of other centrosomal components?
for: expressed in sperm
against: phenotype not as severe.
No DAF-2 in the one cell?
Embryonic expression in 4 cell embryos and after most likely zygotic
Embryonic Nutritional phenotypes ‘masked’ by earlier polarity and
cytokinesis defects
Polarity and Nutritional roles completely separated?
Expression in Centrosomes appears to be affected by
nutrition
Summary
Transmission of Paternal Nutritional Status
Males from food source 1 crossed to Hermaphrodites of food source 2
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Glu x Glu LOP50 x Glu LOP50 x LOP50 Glu x LOP50
% Viable
Embryos
Nuclear
Mostly
Nuclear
Mostly
Cytoplasmic Cytoplasmic
DAF-16::GFP 6 6 19 4
DAF-16::GFP;
agl-1
23 21 19 1
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wt agl-1
Cytoplasmic
Nuclear
p=.0014
wild type daf-2; daf-16 daf-2; daf-16
t=0
t=7’30”
daf-16; daf-2 double mutants show polarity defects (equal first blastomere division,
red arrow) and defects in either NMBD (nuclear membrane breakdown) or polar body
extrusion, resulting in apparent extra nuceolus (blue arrows). Finally, nucleoli often
fail to separate before cell division (white arrows).
Starved Fed sibs Re-fed, 4hrs
(left) Hatching into starvation results in expression only in the nerve ring and weak expression in
the basement membrane of the pharynx in DAF-2::GFP animals. (middle) Fed siblings show
expression in pharyngeal muscle and neurons. (right) In DAF-2::GFP animals re-fed for four hours,
expression returns to the XXX cell (arrow) and region posterior to the nerve ring.
Embryonic Expression
Sperm
Expression
8+cell embryosCentrosome
Nerve ring
Expression
(left) DAF-2::GFP expression is increased during glucose feeding, specifically along the lumenal
membrane of the pharynx. (middle) 3D reconstruction through pharynxes of re-fed UV animals
show increase neuronal expression (arrows) and possibly arborization of neurons from the nerve
ring (arrow heads). (right) Single plane from 3D reconstruction showing lumenal DAF-2::GFP
expression in the pharynx.
(above) DAF-2::GFP is expressed in the two-cell centrosome (stronger in AB) and in the
cytokinetic furrow between AB and P cells. (lower) Maternal expression is observed in the
sperm and (right) in the nerve ring, XXX cell, and unidentified neurons (arrow, arrowhead and
bar, respectively), matching expression described in reference 3.
DAF-2::GFP sperm expression suggests nutritional information is transmitted via
sperm. To test this possibility, agl-1(tm4809) males were grown on permissive, live
OP50 (LOP50) and restrictive glucose (Glu) media and then crossed with
hermaphrodites grown under both conditions. Over 1000 progeny from each cross
were scored. Presence of male cross progeny confirmed cross success.
% Embryonic
Lethal
#
Animals
%alive