Download Supplementary Methods

Supplementary methods
Strains and germline transformation. All nematode strains were grown at 20-23°C
under standard conditions1. The strains used in this work include: N2; CX2345 adp1(ky20) II; CX2983 egl-4(ky95) IV; GR1321 tph-1(mg280) II; CB1111 cat-1(e1111) X;
CB1141 cat -4(e1141) V; CB1112 cat-2(e1112) II; MT9668 mod-1(ok103) V. Germ line
transformation was carried out as described2. The clone ofm-1::GFP, which is expressed
in coelomocytes, was used as a co-injection marker3. The injected strains were GR1321
tph-1(mg280) II and MT9668 mod-1(ok103) V. Tested DNA and the co-injection marker
were injected at 25 ng/µl. At least three transgenic lines were tested for each DNA clone.
Dissociation control for pathogen-induced learning. Wild type animals were grown
on OP50 until young adults, and then equally divided into groups to be used for training.
1. Animals were transferred onto pathogenic Pseudomonas PA14 for four hours, then
transferred onto non-pathogenic S. marcescens Db1140 for four hours. 2. Animals were
transferred onto non-pathogenic Pseudomonas 50E12 for four hours, then transferred
onto pathogenic Serraria ATCC 13880 for four hours. 3. Animals were transferred onto
Serratia Db1140 for four hours, then transferred onto PA14 for four hours. 4. Animals
were transferred onto Serratia ATCC 13880 for four hours, then transferred onto
Pseudomonas 50E12 for four hours. All four groups of animals were tested for their
preference between PA14 and S. marcescens ATCC 13880 in a binary choice assay. The
index for PA14 avoidance was calculated as [Number of worms on Serratia – Number of
worms on PA14] / Total number of animals on the plate. The experiment was repeated
six times. Results from groups 1 and 3 were combined and results from groups 2 and 4
were combined for Figure 1f (a total of 12 assays for each data point). Statistical
significance was determined by ANOVA.
Molecular biology. A tph-1 cDNA was isolated from the clone yk1176 and a mod-1
cDNA was isolated from the clone yk1233, both generously provided by Yoji Kohara.
cDNA sequences were confirmed by sequencing. The promoters used for tph-1
constructs were srh-142 (ADF chemosensory neurons)4 and ceh-2 (the pharyngeal
neurons NSM, I3, M3, M4)5. The promoters for mod-1 constructs were mod-16
(numerous neurons in the head, ventral nerve cord, and tail), ttx-37 (AIY interneurons)
and odr-2(2b)8(AIB and AIZ interneurons, ASG and IL2 sensory neurons, PVP, AVG
and RIF interneurons, SIA motor neurons). GFP was fused to the C-terminus of tph-1
cDNA and mod-1 cDNA to confirm the expression patterns of the transgenes.
Immunohistochemistry. Animals were stained for serotonin using a primary polyclonal
rabbit anti-serotonin antibody (ImmunoStar Inc.), and secondary Texas Red-labeled goat
anti-rabbit antisera (Jackson ImmunoResearch Lab.)9.
Statistical analysis. All results in the paper were robust to different methods of
statistical analysis. For each figure, the method that was most appropriate and
conservative is described below.
To test statistical significance in Figure 1b, the choice index of OP50-fed wild-type
animals was compared to the choice index of wild-type animals that were fed on test
bacterium and OP50, and P values were generated by ANOVA. Multiple comparisons
were corrected by Bonferroni t-test (*** P <0.001, n≥4 assays).
For Figure 1d, the learning index for wild type animals exposed to PA14 for 1 hour, 2
hours, or 4 hours was compared to the learning index of wild type animals cultivated on
PA14 and OP50 throughout life. P values were generated by ANOVA using the Dunnett
Test for multiple comparisons to one control (*P <0.05, n≥10 assays).
For Figure 1e, the choice index of wild type animals that were starved for 4 hours was
compared to the choice index of OP50-fed wild type animals, and to the choice index of
animals exposed to PA14 for four hours. P values were generated by ANOVA using the
Dunnett Test (*P <0.05, n≥14 assays).
For Figure 1f and 4a, statistical significance was determined by ANOVA (*** P<0.001,
** P<0.01, n≥12 assays).
For Figure 2b, in the four-choice maze assays, the percentage of OP50-fed animals that
accumulated on OP50 was compared to the percentage of PA14-exposed animals that
accumulated on OP50; the percentage of OP50-fed animals that accumulated on PA14
was compared to the percentage of PA14-exposed animals that accumulated on PA14.
Statistical significance was generated by ANOVA (*** P<0.001, n≥ 37 assays). An
alternative statistical analysis of the maze data in which the fraction of animals on OP50
and PA14 were compared to fraction on the “novel” bacteria P. fluorescens and S.
marcescens also showed that both attractive and aversive learning were highly significant
(P<0.001). Statistical significance in Figure 2d was determined in the same way (***
P<0.001, ** P<0.01, n≥23 assays).
For Figure 3a, learning indices of tph-1(mg280), cat-1(e1111), cat-4(e1114), and cat2(e1112) were compared to the learning index of wild type animals. P values were
determined by ANOVA with the Dunnett Test (*P <0.05, n≥16 assays). Figure 3c,
learning indices of tph-1 animals carrying an ADF::tph-1 transgene and tph-1 animals
carrying an NSM::tph-1 transgene were compared to the learning index of tph-1(mg280).
P values were determined by ANOVA with the Dunnett Test (n≥10 assays).
For Figure 3b and 3d, four-choice learning assays for animals trained with OP50 and
PA14, attractive learning indices of wild type animals, tph-1 animals carrying the
ADF::tph-1 transgene and tph-1 animals carrying both NSM::tph-1 and ADF::tph-1
transgenes were compared to the attractive learning index of tph-1(mg280) animals. P
values were generated by ANOVA with the Dunnett Test († P<0.05, n≥6 assays).
Statistical analysis of aversive learning was done in the same way (*P<0.05, n≥6 assays).
Attractive learning to P. fluorecesces and aversive learning to S. marcescens were
analyzed by ANOVA (P<0.05, n≥7 assays).
For Figure 4b and 4c, four-choice learning assays for animals trained with OP50 and
PA14, aversive learning indices of wild-type animals, mod-1 animals carrying a mod1::mod-1 transgene, mod-1 animals carrying a ttx-3::mod-1 transgene and mod-1 animals
carrying a odr-2(2b)::mod-1 transgene were compared to the aversive learning index of
mod-1(ok103) animals. P values were generated by ANOVA and the Dunnett Test for
multiple comparisons to one control (* P<0.05, n≥5 assays). Aversive learning on S.
marcescens was analysed by ANOVA (*P<0.05, n≥7 assays).
For Figure 5l, at each time point the learning index of animals treated with 2 mM
serotonin was compared to the learning index of animals without exogenous serotonin.
Statistical significance was determined by ANOVA and Bonferroni’s t-test for multiple
comparison (*P<0.05, n≥8 assays).
For Supplementary Figure 2, learning indices of adp-1(ky20), egl-4(ky95) and tol1(nr2033) were compared to the learning index of wild type animals. Statistical
significance was determined by ANOVA and the Dunnett Test (n≥8 assays). For
Supplementary Figure 3, statistical significance for wild type and different mutants was
determined by ANOVA (*P<0.05, n≥4 assays). For transgenic animals, the choice index
of PA14-exposed animals was compared to the choice index of OP50-fed animals
assayed on the same days, and statistical significance was determined by Student’s t-test
(*P<0.05, n≥10 assays).
For Supplementary Figure 4 and 5, statistical significance was determined by ANOVA
with the Dunnett Test (*P<0.05, n≥3 assays). For Supplementary Figure 7, statistical
significance was determined by ANOVA and Bonferroni’s t-test (**P<0.01, n≥19
animals each).
For Supplementary Figure 9a, 9b, 10a and 10b, statistical significance was determined by
ANOVA (***P<0.001, n≥7 assays). For Supplementary Figure 9c-e, 10c-h, the
percentages of OP50-fed animals accumulating on OP50 or PA14 were compared to the
percentages of PA14-exposed animals accumulating on OP50 or PA14 assayed on the
same days. Statistical significance was determined by Student’s t-test (**P<0.01,
*P<0.05, n≥5 assays).
1.
2.
3.
4.
5.
6.
7.
8.
9.
Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71-94 (1974).
Mello, C. C., Kramer, J. M., Stinchcomb, D. & Ambros, V. Efficient gene transfer
in C. elegans: extrachromosomal maintenance and integration of transforming
sequences. Embo J 10, 3959-70 (1991).
Miyabayashi, T., Palfreyman, M. T., Sluder, A. E., Slack, F. & Sengupta, P.
Expression and function of members of a divergent nuclear receptor family in
Caenorhabditis elegans. Dev Biol 215, 314-31 (1999).
Sagasti, A., Hobert, O., Troemel, E. R., Ruvkun, G. & Bargmann, C. I.
Alternative olfactory neuron fates are specified by the LIM homeobox gene lim-4.
Genes Dev 13, 1794-806 (1999).
Aspock, G., Ruvkun, G. & Burglin, T. R. The Caenorhabditis elegans ems class
homeobox gene ceh-2 is required for M3 pharynx motoneuron function.
Development 130, 3369-78 (2003).
Wenick, A. S. & Hobert, O. Genomic cis-regulatory architecture and trans-acting
regulators of a single interneuron-specific gene battery in C. elegans. Dev Cell 6,
757-70 (2004).
Altun-Gultekin, Z. et al. A regulatory cascade of three homeobox genes, ceh-10,
ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in
C. elegans. Development 128, 1951-69 (2001).
Chou, J. H., Bargmann, C. I. & Sengupta, P. The Caenorhabditis elegans odr-2
gene encodes a novel Ly-6-related protein required for olfaction. Genetics 157,
211-24 (2001).
Desai, C., Garriga, G., McIntire, S. L. & Horvitz, H. R. A genetic pathway for the
development of the Caenorhabditis elegans HSN motor neurons. Nature 336,
638-46 (1988).