Suppl. Material and Methods
Strains and genetics
Strains were maintained according to the standard protocol (BRENNER 1974). The following
mutant alleles and strains were used : LGI, lin-53(n833); LGII, lin-38(n751);
LGIII, hpl-2(tm1489), unc-49(e407), lin-36(n766); LGV sid-1(qt2); LGX, hpl-1(tm1624), lin15(n767), lin-15(n765). Extrachromosomal arrays: zhEx88[lin-31::lin-3hp + unc-119(+) +
myo-3::gfp] (DUTT et al. 2004), deIs4[lin-39TX::gfp + dpy-20(+) + ajm-1::gf] (WAGMAISTER
et al. 2006). The Bristol strain N2 was used as wild-type.
Constructs and Transgenic Lines
dpy-7 promoter constructs were generated by cloning hpl-1 and hpl-2a cDNAs into the
BamHI-KpnI and SmaI-KpnI sites of dpy-7p::nls::YFP (kind gift of Iva Greenwald)
respectively. lin-31p promoter constructs were generated by cloning hpl-1 and hpl-2a cDNAs
into the NotI site and SalI-NotI sites of pB253 (TAN et al. 1998) respectively. Constructs were
injected at a concentration of 10-20 μg/ml using pTG96 [sur-5::gfp] as co-transformation
marker (GU et al. 1998). For dpy-7 derivatives, pBluescript DNA was added as carrier
(70μg/ml). To generate low-copy arrays, constructs were injected at concentration of 0.10.5μg/ml with pBluescript (100μg/ml). The recipient strains were hpl-1(tm1624); hpl2(tm1489) unc-49(e407), lin-15(n767); hpl-2(tm1489) unc-49(e407), lin-38(n751); hpl2(tm1489) unc-49(e407).
For tissue specific RNAi, the plasmid dpy-7::lin-3hp (CUI et al. 2006) was injected at
50μg/ml with pTG96 as a co-transformation marker into lin-15(n767); hpl-2(tm1489) unc49(e407) animals. To obtain hpl-1(tm1624); hpl-2(tm1489) unc-49(e407) transgenic lines, lin15(n767);
hpl-2(tm1489) unc-49(e407) hermaphrodites
carrying
the
dpy-7::lin-3hp
extrachromosomal array were mated to hpl-1(tm1624) males and F2 Unc progeny were tested
by PCR for the presence of hpl-1(tm1624) and hpl-2(tm1489) and absence of lin-15(n767)
deletion alleles. Transgenic lines carrying the [lin-31::lin-3hp] extrachromosal array were
created by crossing hpl-1(tm1624); hpl-2(tm1489) unc-49(e407) and lin-15(n767); hpl2(tm1489) unc-49(e407) males to unc-119(e2498)); zhEx88[lin-31::lin-3hp+ unc-119(+) +
myo-::gfp] hermaphrodites (DUTT et al. 2004).
RNA interference
(ds) RNA was applied to worms by feeding (TIMMONS and FIRE 1998). Bacteria were seeded
on NGM agar plates containing IPTG and ampicillin. For lin-3 RNAi on lin-39::GFP
transgenic animals, starved L1 larvae were added onto the plates on the following day and
cultured at 25°C. Control animals were fed with bacteria carrying an empty L4440 vector.
GFP fluorescence in VPCs was scored in late L2 animals at the time of vulval induction on a
Zeiss Axioplan2 microscope coupled with a CoolSnap HQ Camera. For lin-3(RNAi) on
SynMuv mutants, animals were transferred to RNAi plates at the L4 stage and the Muv
phenotype scored in the F1 progeny.
Quantitive RT-PCR Analysis of lin-3 Transcripts
Synchronized wild-type and mutant L1 worms were grown on NGM plates with OP50
bacteria as food at 20°C, 24°C or 25°C. Larvae were harvested at early L3 stage. Total RNAs
were isolated by using Trizol reagent (Invitrogen). First-strand cDNA was prepared from 500
ng total RNA by using The RevertAid First Strand cDNA Synthesis Kit (Fermentas). The lin3 mRNA levels were determined using the Light Cycler FastStart DNA Master SYBR Green
1 RT-PCR kit (Roche) on LightCycler 1.5. rpl-26 expression was used for internal reference.
BRENNER, S., 1974 The Genetics of Caenorhabditis elegans. Genetics 77: 71-94.
CUI, M., J. CHEN, T. R. MYERS, B. J. HWANG, P. W. STERNBERG et al., 2006 SynMuv genes
redundantly inhibit lin-3/EGF expression to prevent inappropriate vulval induction in C.
elegans. Dev Cell 10: 667-672.
DUTT, A., S. CANEVASCINI, E. FROEHLI-HOIER and A. HAJNAL, 2004 EGF signal propagation during
C. elegans vulval development mediated by ROM-1 rhomboid. PLoS Biol 2: e334.
GU, T., S. ORITA and M. HAN, 1998 Caenorhabditis elegans SUR-5, a novel but conserved protein,
negatively regulates LET-60 Ras activity during vulval induction. Mol Cell Biol 18: 45564564.
TAN, P. B., M. R. LACKNER and S. K. KIM, 1998 MAP kinase signaling specificity mediated by the
LIN-1 Ets/LIN-31 WH transcription factor complex during C. elegans vulval induction. Cell
93: 569-580.
TIMMONS, L., and A. FIRE, 1998 Specific interference by ingested dsRNA. Nature 395: 854.
WAGMAISTER, J. A., J. E. GLEASON and D. M. EISENMANN, 2006 Transcriptional upregulation of the
C. elegans Hox gene lin-39 during vulval cell fate specification. Mech Dev 123: 135-150.