Germ-Granule Components Prevent Somatic Development in

Current Biology, Volume 24
Supplemental Information
Germ-Granule Components Prevent Somatic
Development in the C. elegans Germline
Dustin L. Updike, Andrew Kekūpa'a Knutson, Thea A. Egelhofer, Anne C. Campbell,
and Susan Strome
Supplemental Information
Table S1. Strains used in this study
Strain
Name
Genotype*
N2
OH9279
edIs6[unc-119p::UNC-119(first
101aa) ::GFP + ROL-6] IV
edIs6[unc-119p::UNC-119(first
101aa) ::GFP + ROL-6] IV
edIs6[unc-119p::UNC-119(first
101aa) ::GFP + ROL-6] IV
edIs6[unc-119p::UNC-119(first
101aa) ::GFP + ROL-6] IV
edIs6[unc-119p::UNC-119(first
101aa) ::GFP + ROL-6] IV
otIs118[unc-33p::GFP + UNC-4]
ntIs1[gcy-5p::GFP + LIN-15] V
otIs264[ceh-36p::tagRFP] III;
ntIs1 V; otIs305
otIs302[LSY-6::GFP +
F25B3.3::dsRed2]
otIs266[cat-1p::mCherry]
VH905
hdIs30[GLR-1::dsRed2]
DP132
DP132 L4
Stage
DP132 1st
Day Adults
DP132 2nd
Day Adults
DP132 4th
Day Adults
OH439
OH3192
OH10997
OH9729
No. of
F1s
examined
after
P-granule
RNAi
100
219
%
Sterile
F1s
No. of
sterile
F1s
examined
% Sterile
F1s with
germline
expression
of
transgene
40
76
Source
CGC
145
9
219
0
247
2.4
200
10
213
17
CGC
124
194
88
68
103
194
34
0
CGC
CGC
178
67
120
0
CGC
292
36
91
0
CGC
231
80
150
0
186
73
186
0
CGC
Don
Moerman
201
53
110
0
CGC
90
154
67
82
90
154
0
0
CGC
CGC
Oliver
Hobert
Ian Hope
Ian Hope
Bob
Horvitz
EG1285
OH1098
zuIs178 V; stIs10024;
stIs10060[cnd-1p::mCherry +
UNC-119]
oxIs12[unc-47p::GFP + LIN-15] X
otIs133[pttx-3p::RFP + UNC-4] II
OH122
mgIs25[UNC-97::GFP]
411
51
200
0
UL2484
UL2157
leEx[dhhc-14p::GFP + UNC-119]
leEx[nhr-52p::GFP + UNC-119]
212
222
86
74
125
140
0
0
MT11439
nEx1047[UNC-93::GFP + LIN-15]
285
86
150
0
OH11054
ntIs1 V; otIs305; otEx4445[snb1p::NLS-TagRFP]
82
74
48
0
CGC
OH10596
otEx4720[YFP::HLH-2 + ROL-6]
330
47
48
0
Oliver
Hobert
192
70
108
0
CGC
240
45
89
0
342
27
84
0
291
33
92
0
CGC
Strome
Lab
Strome
Lab
258
41
107
0
RW10060
RW10062
BC12881
SS974
SS947
RW10097
zuIs178 V; stIs10050[pha4p::HIS-24::mCherry + UNC-119]
sEx12881[grl-11p::GFP + DPY-5]
bnEx46[elt-2p::mCherry + myo2p::GFP]
bnEx35[GLH-1::GFP + myo3p::dsRed2]
zuIs178 V; stIs10088[hlh-1p::HIS24::mCherry]
CGC
trIs30[him-4p::MB::YFP + HMRPeter
1b::dsRed2 + UNC236
28
236
0
Roy
129nsp::dsRed2]
zuIs178 V; stIs10567[cehRW10724
21p::HIS-24::mCherry + UNC265
89
120
0
CGC
119]
wgIs177[EGLOP177
27::TY1::GFP::3xFLAG + UNC308
28
82
0
CGC
119]
BC10577
sEx10577[vab-3p::GFP + DPY-5]
220
53
105
0
CGC
ctIs39[hbl-1p::HBL-1(first
VT1142
185
68
114
0
CGC
133aa)::NLS::GFP + ROL-6] IV
zuIs178 V: stIs10064[end-3p::H1RW10064
mCherry::let-858 3’UTR + UNC171
73
103
0
CGC
119]
otIs305[hsp-16.2p::CHEOH9846
1::3xHA::BLRP + ROL-6] V;
CGC
ntIs1[gcy-5p::GFP + LIN-15] V
otIs306[hsp-16.2p::CHE-1::3xHA
+ ROL-6]; edIs6[unc-119p::UNCDUP3
this study
119(first 101aa)::GFP + ROL-6]
IV
glp-1(ar202gf) III; edIs6[uncSS1113
119p::UNC-119(first
this study
101aa)::GFP + ROL-6] IV
*F1s were scored on the 2nd day of adulthood unless otherwise noted. Fluorescent reporters examined are in
bold. Genep (e.g. unc-119p) indicates the promoter used to drive each transgene. Lowercase (DNA).
Uppercase (protein-coding region).
RP247
Supplemental Experimental Procedures
Microscopy: A Volocity acquisition and spinning disk confocal system (PerkinElmer) fitted on an inverted
microscope (Eclipse TE2000-E; Nikon) with an EM-CCD camera (Hamamatsu Photonics) was used to acquire
images in Figure 2A,C,D and Figure 3A,C,D (Nikon 60X oil objective) and Figure 3B (Nikon 40X air objective).
Leica AF6000 acquisition software on an inverted microscope (Leica DMI6000B) with a cooled CCD camera
(Leica DFC365FX) and a Leica 60X oil objective was used to acquire and deconvolve images in Figure 1 and
Figure 2B,E.
Strain construction: C. elegans strains were maintained as described previously [S1]. Strains used in this
study are listed in Table S1.
Immunocytochemistry: Whole worms and dissected germlines were fixed using methanol/acetone [S2].
Antibody dilutions were 1:30,000 rabbit anti-PGL-1 [S3], 1:1 mouse monoclonal SP56 (sperm) [S4], 1:1000
rabbit anti-DEPS-1 [S5], 1:10 mouse monoclonal anti-UNC-10 [S6], 1:10 chicken anti-HLH-1 [S7], 1:2000
mouse monoclonal mAb5.6 (MYO-3) [S8], 1:10,000 mouse monoclonal anti-H3S10ph (clone 140-3-5F5 from
Hiroshi Kimura) [S9], 1:1000 rabbit anti-GFP (Novus), 1:1000 rabbit anti-REC-8 (SDIX), and 1:500 Alexa Fluor
secondary antibodies (Life Technologies).
P-granule RNAi: 350 base pair fragments of glh-1 (cDNA base pairs 675-1024), glh-4 (749-1098), pgl-1 (13171666), and pgl-3 (265-613) were joined by PCR and cloned into the L4440 RNAi feeding vector to create
pDU49, which was transformed into HT115 bacteria. Feeding RNAi [S10] was performed at 24°C. Feeding
RNAi was commenced in the parental generation (P0) at the first larval stage (L1); progeny (F1) were
examined on the 2nd day of adulthood for GFP expression in the germline. For glp-1(ar202gf); unc-119::GFP
worms, feeding RNAi was initiated in the P0 generation at 15°C; P0 adults were shifted to the restrictive
temperature of 24°C, and their adult F1 progeny were examined for GFP expression in the germline. Control
RNAi feeding was performed with HT115 bacteria carrying the L4440 plasmid. Two-tailed p-values were
calculated using Fisher’s Exact Test.
mex-3; gld-1 RNAi: The first 400 base pairs of mex-3 cDNA and gld-1 cDNA were joined by PCR and cloned
into the L4440 RNAi feeding vector to create pDU50, which was transformed into HT115 bacteria. Feeding
RNAi was started at the 4th larval stage (L4) at 24°C, and adult F1 progeny were examined.
Single molecule FISH: Stellaris FISH probes (Biosearch Technologies) were designed for unc-119 with CAL
Fluor Red 610. Germlines from control and P-granule RNAi fed unc-119::GFP expressing worms were
dissected in egg buffer (25mM HEPES, 120mM NaCl, 2mM MgCl2, 2mM CaCl2, and 50mM KCl) on a GCP
(0.2% w/v gelatin, 0.02% w/v chrome alum, 0.05% w/v poly-L-lysine) coated slide. Slides were fixed in 3.7%
formaldehyde in PBS for 45 minutes, washed 2X with PBS, and incubated in 70% EtOH overnight at 4°C.
Slides were then washed with wash buffer (2XSSC, 10% formamide) and incubated with FISH probes in
hybridization buffer (2XSSC, 10% formamide, 10% w/v dextran sulfate) in the dark at 37°C for 4 hours. Slides
were rinsed in wash buffer, and then incubated in wash buffer for 30 minutes in the dark at 37°C. During the
last 30-minute 37°C incubation, 5ng/ml of DAPI was included in the wash buffer. 15ul of 2XSSC and a
coverslip were placed on each slide for imaging.
Expression profiling: Control or P-granule RNAi was performed on unc-119::GFP worms as described above.
100 germlines were dissected from F1 worms on the first day of adulthood. Undissected worms were scored
for sterility (84%, n=100) and germline GFP expression (9%, n=100) the following day. Total RNA was
prepared from dissected germlines using Trizol reagent (Life Technologies). cDNA was prepared using a
QuantiTect Reverse Transcription Kit (Qiagen), and qPCR was performed in quadruplicate on 384-panel
StellARray qPCR arrays (Bar Harbor Biotechnology) in a Roche LightCycler 480 system.
Induction of CHE-1 by heat shock: Control and P-granule RNAi was performed on hs::CHE-1; gcy-5::GFP and
hs::CHE-1; unc-119::GFP worms as described above. F1 progeny at the L4 stage were heat-shocked at 37°C
for 30 minutes and were examined for GFP expression in the germline the following day.
Supplemental References
S1.
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71–94.
S2.
Strome, S., and Wood, W. B. (1983). Generation of asymmetry and segregation of germ-line granules in
early C. elegans embryos. Cell 35, 15–25.
S3.
Kawasaki, I., Shim, Y. H., Kirchner, J., Kaminker, J., Wood, W. B., and Strome, S. (1998). PGL-1, a
predicted RNA-binding component of germ granules, is essential for fertility in C. elegans. Cell 94, 635–
645.
S4.
Ward, S., Roberts, T. M., Strome, S., Pavalko, F. M., and Hogan, E. (1986). Monoclonal antibodies that
recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans spermspecific proteins. J. Cell Biol. 102, 1778–86.
S5.
Spike, C. A., Bader, J., Reinke, V., and Strome, S. (2008). DEPS-1 promotes P-granule assembly and
RNA interference in C. elegans germ cells. Development 135, 983–993.
S6.
Hadwiger, G., Dour, S., Arur, S., Fox, P., and Nonet, M. L. (2010). A monoclonal antibody toolkit for C.
elegans. PLoS One 5, e10161.
S7.
Krause, M., Fire, a, Harrison, S. W., Priess, J., and Weintraub, H. (1990). CeMyoD accumulation defines
the body wall muscle cell fate during C. elegans embryogenesis. Cell 63, 907–19.
S8.
Priess, J. R., and Thomson, J. N. (1987). Cellular interactions in early C. elegans embryos. Cell 48,
241–50.
S9.
Egelhofer, T. A., Minoda, A., Klugman, S., Lee, K., Kolasinska-Zwierz, P., Alekseyenko, A. A., Cheung,
M.-S., Day, D. S., Gadel, S., Gorchakov, A. A., et al. (2011). An assessment of histone-modification
antibody quality. Nat. Struct. Mol. Biol. 18, 91–3.
S10. Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N.,
Moreno, S., Sohrmann, M., et al. (2003). Systematic functional analysis of the Caenorhabditis elegans
genome using RNAi. Nature 421, 231–237.

Download Report