Supplemental
Appendix
for
Sensory integration of food
and population density during the diapause exit
decision involves insulin
-
like signaling in
Caenorhabditis elegans
Mark G Zhang
a
, Maedeh Seyedolmohadesin
b
, Soraya Hawk
Mercado
a
, Arnaud
Tauffenberger
c,d
, Heenam Park
a
, Nerissa Finnen
a
, Frank Schroeder
c,d
, Vivek
Venkatachalam
b
, and Paul W Sternberg
a
a
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, CA,
9
1125
b
Physics Department, Northeastern University, Boston, MA,
02115
c
Boyce Thompson Institute
, Cornell University, Ithaca, NY,
14853
d
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY,
14853
Supplemental Appendix: Table of Contents
Supplemental Figures
................................
................................
................................
...
3
Figure S1. Supplemental Figure for Figure 1.
................................
..........................
3
Figure S2. Supplementary figure for Figure 2.
................................
........................
4
No Figure S3 (Figure 3 does not have a supplemental figure associated)
...........
6
Figure S4. Supplementary figure for Figure 4.
................................
........................
7
Figure S5. Supplemental figure for Figure 5.
................................
...........................
8
Figure S6. Supplemental figure for Figure 6.
................................
.........................
10
Supplemental Tables
................................
................................
................................
...
12
Table S1: List of strains used in this study
................................
...........................
12
Table S2: List of probes used in
ins
-
6
mRNA FISH
................................
..............
20
Supplemental Materials and Methods
................................
................................
........
22
C. elegans strains and maintenance
................................
................................
......
22
Dauer entry induction
................................
................................
..............................
22
Dauer exit assay
................................
................................
................................
.......
22
Molecular cloning and transgenesis
................................
................................
......
23
CRISPR Cas9 genome editing
................................
................................
.................
24
Imaging transgenic
ins
-
6
reporter strains throughout development
..................
24
Microscopy and image analysis
................................
................................
.............
24
mRNA fluorescence in
-
situ hybridization (FISH)
................................
..................
25
Calcium
imaging
................................
................................
................................
.......
26
Perturbation assays
................................
................................
................................
.
26
Ascaroside metabolomics
................................
................................
.......................
26
References
................................
................................
................................
...................
29
Supplemental Figures
Figure S1. Supplemental Figure for Figure 1.
(A) Dauer exit rates of mutants defective for single
neuropeptide genes. Refer to Table
S1 for list of strains. Bars indicate medians. Each dot is the dauer exit % from an assay
plate containing 50
-
100 animals each. ns, not significant, ***, p<0.001, **, p<0.01,
compared to “WT” by Welch ANOVA with Dunnett’s
T3 multiple comparison correction.
(B) Dauer exit rates of the double mutant
ins
-
6; daf
-
28
compared to single mutants
defective for each neuropeptide gene alone. Compared to experiments performed for
Fig. 1D, this assay was performed using a lower pheromone concentration that induces
almost wild
-
type dauers to exit. Bars indicate medians. Each dot is the dauer exit %
from an assay plate containing 50
-
100 animals each.
Figure S2. Supplementary figure for Figure 2.
(A
-
C) Quantification of
ins
-
6p::dYFP
transcriptional reporter signal (measured in
arbitrary units (a.u.)) in ASJ (A) or ASI (B) and
ins
-
6::mCherry
translational reporter
signal in the coelomocytes (C) during reproductive growth.
(D) An
ins
-
6
secretion reporter was built by driving
ins
-
6::mCherry
expression using the
ASJ
-
specific promoter
trx
-
1p
and the broadly expressing
let
-
858
3’ untranslated region
(UTR). Shown are the mCherry fluorescence intensities measured in the coelomocytes
for two independent extrachromosomal array lines bearing the
ins
-
6
secretion reporter
in dauers before transfer to favorable conditions (0 h.a.t.) and 6 hours after transfer to
favorable conditions (6 h.a.t.). While the T2A signal was included to simultaneously
meas
ure transcriptional activity driven the
trx
-
1p
promoter, we did not observe any
noticeable GFP signal.
(E) Design of
daf
-
28
transcriptional and translational reporters, which were constructed
similarly to those for
ins
-
6
. (F
-
H) Quantification of
daf
-
28p::dYFP
transcriptional reporter
signal (measured in arbitrary units (a.u.)) in ASJ (F) or ASI (G) and
daf
-
28::mCherry
translational reporter signal in the coelomocytes (H) during favorable growth (non
-
dauer
-
inducing) or dauer
-
inducing growth, as well as dauers after transfer to favorable
conditions (indicated by curved arrow) for the indicated lengths of time.
YA, young adult.
A, adult. h.a.t., hours after transfer to favorable conditions. See SI Appendix Materials
ins-6
secretionreporter
trx-1p
mCherry
let-8583'
T2A
gfp
ins-6
A
B
C
D
E
F
daf-28
transcriptionalreporter
daf-28
5'
dYFP
daf-283'
daf-28
translationalreporter
daf-285'
mCherry
daf-283'
daf-28
22 hpl (late L1)
32 hpl (late L2)
46 hpl (L4)
56 hpl (YA)
0
200
400
600
800
1000
ins-6p::dYFP
Fluorescence intensity (a.u.)
e268_ins-6_DR_reproductive_growth_asi
ASI
322.2
(10)
17.4
(10)
0.70
(10)
0.61
(10)
L1
L2
L4
Yo u n g
adult
22 hpl (late L1)
32 hpl (late L2)
46 hpl (L4)
56 hpl (YA)
0
2000
4000
6000
8000
ins-6::mCherry
Fluorescence intensity (a.u.)
e268_ins-6_DR_reproductive_growth_coel
2360
(10)
1637
(9)
1320
(10)
1109
(10)
Coelomocytes
L1
L2
L4
Yo u n g
adult
22 hpl (late L1)
32 hpl (late L2)
46 hpl (L4)
56 hpl (YA)
0
200
400
600
800
1000
ins-6p::dYFP
Fluorescence intensity (a.u.)
e268_ins-6_DR_reproductive_growth_asj
ASJ
L1
L2
L4
Yo u n g
adult
1.41
(10)
1.05
(10)
0.29
(10)
0.77
(10)
mzEx120G (line 1) 0 hpt
mzEx120G (line 1) 6 hpt
mzEx120L (line 2) 0 hpt
mzEx120L (line 2) 6 hpt
0
2000
4000
5000
10000
15000
ins-6::mCherry
Fluorescence intensity (a.u.)
E314 trx-1p::ins-6::mCherry::T2A::gfp signal during dauer exit R2
1299
(10)
2785
(11)
253.7
(12)
1227
(11)
Line 1
Line 2
✱✱
✱✱
hours after transfer
(h.a.t.)
0
6
0
6
L1
L4
YA
A
L1
L2d
Dauer
1 h.a.t.
2 h.a.t
3 h.a.t.
6 h.a.t.
24 h.a.t.
0
5000
10000
15000
20000
ins-6p::dYFP
Fluorescence intensity (a.u.)
e236_daf-28_DR_exit_dYFP_ASI
ASI
5,249
(8)
14,594
(8)
13,862
(8)
14,067
(8)
6,719
(8)
5,138
(8)
40.9
(10)
52.2
(10)
107
(10)
126
(12)
2,187
(10)
5,457
(10)
Favorable
Growth
Dauer-inducing
Growth
⤸
Transfer
L1
L4
YA
A
L1
L2d
Dauer
1 h.a.t.
2 h.a.t
3 h.a.t.
6 h.a.t.
24 h.a.t.
0
5000
10000
15000
20000
daf-28p::dYFP
Fluorescence intensity (a.u.)
e236_daf-28_DR_exit_dYFP_ASJ
ASJ
667
(8)
2829
(8)
3297
(8)
4711
(8)
267
(8)
130
(8)
5.9
(10)
17.6
(10)
13.1
(10)
115.0
(12)
34.5
(10)
9214
(10)
Favorable
Growth
Dauer-inducing
Growth
⤸
Transfer
L1
L4
YA
A
L1
L2d
Dauer
1 h.a.t.
2 h.a.t
3 h.a.t.
6 h.a.t.
24 h.a.t.
0
1000
2000
3000
4000
5000
daf-28::mCherry
Fluorescence intensity (a.u.)
e236_daf-28_DR_exit_mCherry
1238
(8)
2570
(8)
2883
(8)
1710
(7)
1112
(8)
1414
(8)
1577
(9)
1623
(10)
1473
(10)
1650
(12)
1720
(10)
2196
(10)
Coelomocytes
Favorable
Growth
Dauer-inducing
Growth
⤸
Transfer
G
H
and Methods for precise time points taken during animal development. Dotted lines
indicate different sets of animals used for imaging experiments. The same animals were
used between “Favorable growth” and “Dauer
-
inducing Growth”.
No Figure S3 (Figure 3 does not have a supplemental figure associated)
Figure S4. Supplementary figure for Figure 4.
(A) Dauers were transferred onto plates containing an intermediate
-
pheromone
concentration (0.05% w/v) with varying amounts of yeast extract and scored for exit 24
hours later. Bars indicate medians. Each dot is the dauer exit % from an assay plate
contain
ing 50
-
100 animals each.
(B)
ins
-
6p::dYFP
transcriptional reporter activity was measured in dauers that were
transferred to plates with an intermediate
-
pheromone concentration along with various
concentrations of yeast extract or a small volume of S
-
Basal washed live OP50 spotted
onto the plate. Three hours later, animals were imaged for YFP signal in ASJ. ns, not
significant, **, p<0.01 by Welch ANOVA with Dunnett’s T3 multiple comparisons
correc
tion when compared to “5x10
-
3
% w/v yeast extract”. Each dot represents one
animal. Medians are depicted by the orange bar. Medians and sample sizes are written.
0
5x10
-5
5x10
-4
5x10
-3
5x10
-2
5x10
-1
0
25
50
75
100
[Yeast Extract]
% w/v
Dauer Exit (%)
e209_yeast_extract_dauer_exit
A
B
5x10
-3
5x10
-2
5x10
-1
OP50
0
5000
10000
15000
20000
ins-6p::dYFP
Fluorescence intensity (a.u.)
e242_ins-6_DR_1
μ
Lph_YE_dYFP all data
5324
(10)
8107
(10)
9113
(10)
ns
✱✱
ns
7362
(10)
[Yeast extract]
% w/v
Figure S5. Supplemental figure for Figure 5.
(A,B) Individual calcium traces of ASJ in wild
-
type (A) or
unc
-
31
loss
-
of
-
function (B)
dauers in response to pheromone, food (bacterial supernatant), or a mixture of both.
Shown are traces from 10
-
12 ASJ neurons across six individual animals. Aggregate
traces are shown in Figure 5A, B.
A
B
C
0
15
30
-50
0
50
100
150
seconds
Δ
F/F0 (%)
unc-31
(ASJ): food
+stimulus
0
15
30
-100
-50
0
50
100
150
seconds
Δ
F/F0 (%)
unc-31
(ASJ): pheromone
+stimulus
0
15
30
-100
-50
0
50
100
150
seconds
Δ
F/F0 (%)
unc-31
(ASJ): food + pheromone
+stimulus
D
Dauers
Lo F: Lo P
0
5000
10000
15000
20000
ins-6p::dYFP
Fluorescence intensity (a.u.)
e228*_daf-2;ins-6_reporters_food:pher_dYFP_condensed
0
208.9
(12)
2360
(10)
daf-2
3
h.a.t.
✱
0
15
30
-50
0
50
100
150
seconds
Δ
F/F0 (%)
Wild type (ASJ): food
+stimulus
0
15
30
-100
-50
0
50
100
150
seconds
Δ
F/F0 (%)
Wild type (ASJ): pheromone
+stimulus
0
15
30
-100
-50
0
50
100
150
seconds
Δ
F/F0 (%)
Wild type (ASJ): food + pheromone
+stimulus
(C) Calcium traces of ASJ neurons (L and R) in wild
-
type L4 larvae in response to
control (buffer), supernatant, and LB. Shown are mean +/
-
SEM of 7 animals. Each plot
includes 15 seconds before stimulus delivery, 15 s of stimulus delivery, and 15 s period
after the delivery; the dotted vertical lines indicate the start and end of the stimulus
delivery. F0 was defined as average density of the 5 s period before the stimulus
delivery.
(D)
ins
-
6p::dYFP
transcriptional reporter activity in ASJ was measured in
daf
-
2(e1370)
loss
-
of
-
function mutant dauers that were transferred to no
-
pheromone conditions. Note
how the magnitude of
ins
-
6
transcriptional reporter activity is smaller compared to those
of comparable experiments such as in Fig. 2C and 4B. *, p<0.05 by Mann Whitney U
-
Test. Each dot represents one animal. Medians are depicted by the orange bar.
Medians and sample sizes are writt
en.