SUPPLEMENTAL MATERIAL
SUPPLEMENTAL METHODS
C. elegans strains: The following is a summary of strains used in each figure. A
complete description of the vsIs integrated transgenes generated in this work can be found in
Supplemental Table S2.
Figure 1: LX960 lin-15(n765ts) X; vsIs97, LX959 vsIs13 IV; lin-15(n765ts) X, LX963
lin-15(n765ts) X; vsIs100, LX975 vsIs13 IV; lin-15(n765ts) X; vsIs97; vsIs100
Figure 2: MT2426 goa-1(n1134) I, LX940 goa-1(n1134) I; vsIs49 V; lin-15(n765ts) X,
LX941 goa-1(n1134) I; lin-15(n765ts) X; vsIs68, LX942 goa-1(n1134) I; lin-15(n765ts) X;
vsIs89, LX849 vsIs49 V; lin-15(n765ts) X, LX896 lin-15(n765ts) X, vsIs68, LX927 lin15(n765ts) X; vsIs89, LX850 lin-15(n765ts) vsIs50 X
Figure 3: MT1434 egl-30(n686sd) I, LX895 egl-30(n686sd) I; lin-15(n765ts) vsIs56 X,
LX894 egl-30(n686sd) I; lin-15(n765ts) X; vsIs66, LX969 egl-30(n686sd) I; lin-15(n765ts) X;
vsIs72, LX884 lin-15(n765ts) vsIs56 X, LX1034 lin-15(n765ts) X; vsIs110, LX968 lin15(n765ts) X; vsIs72
Figure 4: LX967 lin-15(n765ts) vsIs103 X, LX998 goa-1(n1134) I; lin-15(n765ts)
vsIs103 X
Figure 5: LX993 vsIs108 III; lin-15(n765ts) X, LX995 goa-1(n1134) I; vsIs108 III; lin15(n765ts) X, LX996 egl-30(n686sd) I; vsIs108 III; lin-15(n765ts) X, CX5974 kyIs262, LX1093
goa-1(n1134) I; kyIs262, LX1094 egl-30(n686sd) I; kyIs262, LX1020 vsIs108 III; lin-15(n765ts)
vsIs50 X, LX1018 goa-1(n1134) I; vsIs108 III; vsIs49 V; lin-15(n765ts) X, LX1090 egl30(n686sd) I; vsIs108 III; lin-15(n765ts) vsIs56 X
Figure 6: JT734 goa-1(sa734) I, MT1434 egl-30(n686sd) I, MT2426 goa-1(n1134) I,
LX940 goa-1(n1134) I; vsIs49 V; lin-15(n765ts) X, LX895 egl-30(n686sd) I; lin-15(n765ts)
vsIs56 X
Figure 7: LX1086 unc-4(e120) tph-1(mg280) II, LX1087 unc-4(e120) II, LX1088 tph1(mg280) II, LX1089 unc-4(e120) II; arDp2, LX1303 tph-1(mg280) II; vsEx549, LX1304 tph1(mg280) II; vsEx550, LX1305 tph-1(mg280) II; vsEx551, LX1306 tph-1(mg280) II; vsEx552,
LX850 lin-15(n765ts) vsIs50 X, LX884 lin-15(n765ts) vsIs56 X, LX1131 tph-1(mg280) II; lin15(n765ts) vsIs50 X, LX1132 tph-1(mg280) II; lin-15(n765ts) vsIs56 X
Supplemental Figure S2: LX967 lin-15(n765ts) vsIs103 X, LX993 vsIs108 III; lin15(n765ts) X
Supplemental Figure S3: LX993 vsIs108 III; lin-15(n765ts) X, LX995 goa-1(n1134) I;
vsIs108 III; lin-15(n765ts) X
Supplemental Figure S4: LX993 vsIs108 III; lin-15(n765ts) X, LX1091 ser-4(ok512)
vsIs108 III; lin-15(n765ts) X, LX1092 tph-1(mg280) II; vsIs108 III; lin-15(n765ts) X
Cell-Specific Transgenes: The ELM expression vector, pBH34.21, contains an ELM
promoter consisting of a multimer of the NdeI-box enhancer upstream of a minimal pes-10
promoter (HARFE et al., 1998). GFP expressed from this vector was seen in the sixteen egglaying muscles as well as the anal depressor muscles. The VC expression vector, pDM4, contains
a VC promoter consisting of a 500 bp VC enhancer element from lin-11 upstream of a minimal
pes-10 promoter (BANY et al., 2003). GFP expressed from this vector was observed in the six
VC neurons, a few additional ventral cord neurons in adults, and in the posterior intestine. Both
the ELM and VC expression vectors are derivatives of pPD97.78 (a gift from A. Fire, Stanford
University). All cDNAs expressed from the ELM and VC promoters were inserted between the
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AgeI and EcoRI sites. The HSN expression vector pJM60A contains an HSN promoter
consisting of a 3.1 kb fragment of the tph-1 promoter (MORESCO and KOELLE, 2004). GFP
expressed from this vector was seen in the HSNs and NSMs. As a control to distinguish whether
rescue effects were due to HSN or NSM expression, we used another vector, pJM53A, that
drives expression just in the NSMs. pJM53A is identical to pJM60A except that it contains a
smaller, 377 bp fragment of the tph-1 promoter (MORESCO and KOELLE, 2004). GFP expressed
from this vector was always observed in the NSMs, and very rarely in other serotonergic cells.
Both pJM60A and pJM53A are derivatives of pPD49.26 (MELLO and FIRE, 1995). All cDNAs
expressed from the HSN and NSM promoters were inserted between the NheI and KpnI sites. A
complete list of plasmids used in this paper can be found in Supplemental Table 1.
For the rescue experiments in Supplemental Figure S1, plasmids used for driving
expression of a G protein or PTX in specific cells were coinjected with plasmids driving GFP
expression in the same cells (to allow us to visualize and analyze the morphology of the cells)
and with the lin-15-rescuing plasmid pL15EK (to allow us to screen for animals that had taken
up the injected DNA) into lin-15(n765ts) animals. We initially analyzed the relative strength of
the three cell-specific promoters by quantitating the relative fluorescence intensity of GFP driven
by each promoter, and this data was used to determine the concentrations at which the constructs
were microinjected. We adjusted the concentrations of the G protein and PTX constructs in the
injection mixes to give comparable expression levels in each cell type, while still injecting the
same total amount of DNA. For the extra-chromosomal lines in Supplemental Figure S1, the G
protein and PTX expression constructs were injected with GFP expression constructs at the
following concentrations:
HSN-promoter::cDNA (100 ng/l) with HSN-promoter::GFP (70
ng/l); NSM-promoter::cDNA (122 ng/l) with NSM-promoter::GFP (48 ng/l); VC-
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promoter::cDNA (90 ng/l) with VC-promoter::GFP (80 ng/l); ELM-promoter::cDNA (10
ng/l) with ELM-promoter::GFP (10 ng/l). For control experiments, constructs consisting of
the cell-specific expression vectors with no insert were injected instead of the constructs
containing cDNA inserts.
For the rescue experiments in Figure 7D, plasmids used for driving expression of the tph1 cDNA were coinjected with plasmids used for driving GFP expression in the same cells (to
allow us to pick animals in which the extra-chromosomal transgenes were expressed in the cell
of interest) and with the myo-2 marker plasmid pJKL449.1 into tph-1(mg280) animals. The
constructs incorporated in the extra-chromosomal transgenes used in Figure 7D were injected at
the following concentrations: tph-1 cDNA construct or empty vector construct (100 ng/ul), GFP
expression construct (50 ng/ul), and pJKL449.1 (10 ng/ul).
Analysis of fluorescent presynaptic markers in the HSN neurons: All transgenes used
to express fluorescently-tagged SNB-1 or UNC-13S were generated to produce the lowest
expression levels of the fluorescent proteins that allowed adequate imaging using the most
sensitive settings on a Zeiss LSM 510 confocal microscope. Thus, the transgenes were produced
by adjusting the concentration of the DNA constructs injected down to the lowest level possible.
Further, we demonstrated that none of the transgenes had detectable effects in egg-laying assays.
Together, these strategies maximized our confidence that the fluorescent proteins used were not
being overexpressed to the point of interfering with synaptic function or causing inappropriate
subcellular localization. The presynaptic HSNL varicosities we visualized are of similar
dimension to the ventral cord presynaptic "puncta" of the NURRISH et al. (1999) study, but our
HSNL images were taken at considerably higher magnification and resolution in order to discern
changes in synaptic volume (by analyzing SNB-1::GFP) and UNC-13S::GFP localization within
4
the synaptic termini. For imaging of SNB-1::GFP and UNC-13S::GFP in the HSNL, animals
staged 40 hours post L4 were immobilized using 3 mM levamisole on a 3.5% agar pad per slide,
and a Z-stack image of the HSNL from one animal per slide was acquired.
To identify the synaptic varicosities in the SNB-1::GFP images, all voxels with green
fluorescence greater than a threshold significantly above background were selected using the
classifier in the Volocity software. Each region of interest (ROI) greater than 1 m3 in volume
within 10 m of the slit of the vulva was counted as a synaptic varicosity. The volume of each
synaptic varicosity was measured using the Volocity measurements tool, and the total synaptic
volume per neuron was determined by adding the volumes of all the varicosities from a single
neuron. The width of the HSNL process, the greatest length across the largest synaptic varicosity
in each animal, the number of anterior vs. posterior varicosities and the presence or absence of a
branch were also quantitated using the Volocity measurements tool to examine HSNL
morphology in wild-type and goa-1 mutant animals.
All of the images of UNC-13S::GFP plus DsRed2 in the HSNs of wild-type and goa-1
mutant animals were assigned a number and analysis was performed blind using Volocity.
Following three-dimensional reconstruction of the UNC-13S::GFP plus DsRed2 images, specific
regions of the HSN were classified as either part of the non-synaptic process (less than 1 m in
diameter) or as a synaptic varicosity. We determined the average intensity of UNC-13S::GFP in
both the non-synaptic process and synaptic varicosity regions, and then normalized those values
to the average DsRed2 intensity in the exact same regions. The DsRed2 fluorescence, which
filled out the HSN, was used to determine the regions of interest (ROIs) where the average
intensity of both the DsRed2 and UNC-13S::GFP was measured. Using the DsRed2
fluorescence, a 4 m long region of the HSN process with a diameter that never exceeded 1 m
5
was identified. This region was classified as the non-synaptic process. To determine the average
intensity of the DsRed2 in the process, all voxels greater than 200 arbitrary intensity units (a
threshold significantly above background) were selected as the ROI, and the mean intensity was
determined. The average UNC-13S::GFP intensity was measured in the exact same process ROI
that had been used to measure the DsRed2 intensity. Synaptic varicosity ROIs were identified as
areas where the diameter of the process exceeded 1 m and all DsRed2 voxels were greater than
200 arbitrary intensity units. The average intensity of DsRed2 and UNC-13S::GFP voxels in the
exact same synaptic varicosity ROIs were determined. For each HSNL, one process region and
up to two varicosity regions were analyzed. To normalize the UNC-13S::GFP to the DsRed2
levels, the average UNC-13S::GFP intensity from a specific ROI was divided by the average
DsRed2 intensity from the exact same ROI. The varicosity to process ratio was determined by
dividing the normalized varicosity value by that of the normalized process value for each
individual neuron.
Indirect immunofluorescence histochemistry: Anti-serotonin immunofluorescence
staining was carried out based on procedures by GARRIGA et al. (1993) and DUERR et al. (2001).
Synchronous populations of animals were isolated by placing staged L1 animals (produced by
hatching eggs in the absence of food) on plates with food and then growing these animals at 20º
C. Thirty hours after reaching the fourth larval stage, the animals were washed off plates with
M9 buffer and fixed with 4% paraformaldehyde in PBS for 18 hours. After the animals were
washed at least five times with PBS, the worms were rocked gently overnight at 37º C in a
solution consisting of 5% B-mercaptoethanol, 1% Triton X-100 in 0.12 M Tris-Cl, pH 7.5. After
the animals were washed five times in PBST (PBS with 0.5% Triton X-100) and transferred to a
clear 0.5 ml tube, 0.1 ml of collagenase solution (100 mM Tris-HCl pH 7.4, 1mM CaCl2, 1,000
6
units/ml Sigma collagenase type IV) was added and the worms were shaken at 37ºC, 230 rpm for
30 minutes. The animals were then washed five times with PBST, transferred to a new 0.5 ml
tube, and blocked with 10% normal goat serum in antibody buffer (0.5% Triton X-100, 1 mM
EDTA, 1% BSA, 5 mM sodium azide in PBS) for 1 hour at room temperature. Incubation with
0.1 ml of rabbit anti-serotonin antibody (Sigma, product number S5545, lot number 075K4779)
diluted 1:100 in antibody buffer was carried out overnight at room temperature without shaking.
After being rinsed with antibody wash buffer (0.5% Triton X-100, 1 mM EDTA, 0.1% BSA, 5
mM sodium azide in PBS) three times for fifteen minutes rocking, the animals were incubated in
0.1 ml of 1:250 Texas red goat anti-rabbit antiserum (JacksonImmuno, code number 111-075003, lot number 64146) in antibody buffer for 4 hours at room temperature. The animals were
washed three times, fifteen minutes rocking per each wash, in antibody wash buffer and then
resuspended in PBS. 5 l of stained animals were placed on a slide with 5 l of an anti-bleaching
solution (0.23 g DABCO in 10% PBS, 90% glycerol), covered with a cover slip and sealed with
nail polish.
7
SUPPLEMENTAL FIGURES
SUPPLEMENTAL FIGURE S1.- Effects of expressing proteins to rescue or inactivate G
protein signaling in the individual cells of the egg-laying system. (A) Average number of eggs
retained by transgenic animals in which GFP, GOA-1 plus GFP, or GOA-1Q205L plus GFP were
expressed in the individual cell types of the egg-laying system. This experiment is similar to that
shown in Figure 2, except that here extrachromosomal transgenes rather than integrated
transgenes were used. Although extrachromosomal transgenes cause mosaic expression, this
experiment allowed us to average data from multiple independent transgenes to ensure that the
results are not dependent on a particular transgene integrant. All results shown here are
consistent with those obtained with the integrated transgenes. Expression of GOA-1Q205L in the
HSNs, but not in any other cell type, rescued the hyperactive egg-laying phenotype of the goa-1
mutant. We performed additional experiments with the extrachromosomal transgenes that were
not carried out with integrated transgenes. Since the HSN promoter also causes expression in the
NSMs, we used an NSM-specific promoter and did not see rescue. This suggests that the rescue
effects observed with the HSN promoter are the result of GOA-1Q205L expression in the HSNs,
not the NSMs. Expression of GOA-1 without the activating Q205L mutation in the individual
cell types was not sufficient to rescue the goa-1 mutant egg-laying defect. Discussion of this lack
of rescue with wild-type GOA-1 can be found at the end of this figure legend. n = 50 animals per
genotype (10 animals from five independent transgenic lines). (B) Percent of early stage eggs
laid by transgenic animals in which GFP or the S1 subunit of PTX plus GFP was expressed in
the individual cell types of the egg-laying system. As in (A) these experiments were carried out
with extrachromosomal transgenes. Wild-type animals lay very few eggs at an early stage (8
cells or fewer), while goa-1 mutants lay most of their eggs at an early stage because they lay
8
eggs too often. Expression of PTX in the HSNs of wild-type animals, but not in any other cell
type caused the transgenic animals to lay a high percentage of early-stage eggs. Since expression
of PTX from the NSM promoter did not cause the transgenic animals to lay a high percentage of
early stage eggs, PTX expression in the HSNs and not the NSMs caused the hyperactive egg
laying. n = 100 eggs per genotype (20 eggs from five independent transgenic lines). The results
from both (A) and (B) suggest that GOA-1 functions in the HSNs to inhibit egg-laying behavior.
(C) Average number of eggs retained by transgenic animals in which GFP, EGL-30 plus GFP, or
EGL-30Q205L plus GFP were extra-chromosomally expressed in the individual cell types of the
egg-laying system. This experiment is similar to that shown in Figure 3, except that here
extrachromosomal transgenes rather than integrated transgenes were used. Expression of either
EGL-30 or EGL-30Q205L in the HSNs or ELMs, but not in any other cell type, resulted in partial
rescue of the egl-30 mutant egg-laying defective phenotype. Since rescue was not achieved with
the NSM promoter, this suggests that the rescue effects observed with the HSN promoter are the
result of EGL-30 (or EGL-30Q205L) expression in the HSNs, not the NSMs. n = 50 animals per
genotype (10 animals from five independent transgenic lines). All transgenic strains in this figure
carry the lin-15(n765) multivulva mutation, and were generated by microinjection of the
indicated expression constructs with the lin-15 rescuing marker plasmid pL15EK. Only nonmultivulva animals that were also expressing GFP in the specific cells of interest in the egglaying system were analyzed. This ensured that only animals that had inherited the transgene and
that further had not lost the transgene from the cells of interest due to mosaicism were analyzed.
For (A-C) error bars show the 95% confidence intervals and asterisks indicate significant
differences from the GFP alone controls, p <0.0001.
9
Despite our inability to rescue the goa-1 mutant with cell-specific expression of
wild-type GOA-1, our results are still consistent with the HSNs being the primary site of action
for GOA-1. Wild-type GOA-1 is necessary in the HSNs, since expression of PTX to inactivate
GOA-1 in the HSNs, but not in any other cell type, phenocopied the goa-1 mutant egg-laying
defect (Figure 2). The extremely high abundance of GOA-1 protein likely explains why
expression of GOA-1(Q205L), but not wild-type GOA-1, in the HSNs was sufficient to rescue
the hyperactive egg-laying of the goa-1 mutant. GOA-1 is a highly abundant protein: its
mammalian ortholog Go makes up 1-2% of brain membrane protein in mammals (STERNWEIS
AND
ROBISHAW, 1984), and GOA-1 constitutes a similar percentage of membrane protein in C.
elegans (A. Jose and M.R.K, unpublished data). The semi-dominance associated with goa-1
mutations (VAN SWINDEREN et al., 2001) indicates that this large amount of GOA-1 protein is
necessary for its function. It is likely that the relatively weak HSN promoter used in this work
did not express an adequate level of GOA-1 to rescue the goa-1 mutant. An alternative
explanation is that wild-type GOA-1, despite being necessary only the HSN (as shown by the
PTX experiments), must be provided in more than one cell of the egg-laying system to be
sufficient for rescue. This is unlikely, however, since expression of GOA-1 in the HSNs, ELMs
and VCs simultaneously was not sufficient to rescue the goa-1 mutant egg-laying defect (data
not shown).
SUPPLEMENTAL FIGURE S2.- Expression of fluorescent fusion proteins in the HSNs does
not affect egg-laying behavior. Average number of eggs retained by wild-type animals or
transgenic animals in which either SNB-1::GFP plus DsRed2 or UNC-13S::GFP plus DsRed2
were expressed in the HSN neurons. Expression of these fluorescent fusion proteins in the HSNs
10
did not disrupt egg-laying behavior, as the number of unlaid eggs retained by the transgenic
animals was not significantly different from the number retained by wild-type animals.
SUPPLEMENTAL FIGURE S3.- Localization of UNC-13S::GFP in the HSNs of wild-type
and goa-1 mutant animals. (A) Fluorescence of DsRed2 (B) UNC-13S::GFP or (C) merge in a
wild-type HSNL. In the merge, a process region is indicated with a bracket, and a representative
varicosity is surrounded by the box. (D) Fluorescence of DsRed2 (E) UNC-13S::GFP or (F)
merge, expressed in the HSNL of a goa-1(n1134) animal. For (A-F), images of the HSNL
process correspond with the region encompassed by the box in Figure 1B; brightness was
uniformly increased using Volocity (Improvision) to enable visualization; bar = 5 m. The UNC13S::GFP transgene was expressed at the lowest level possible while still allowing imaging to be
conducted so as to not cause abberant subcellular localization due to overexpression. Since this
transgene does not affect egg-laying behavior (Supplemental Figure S2), the UNC-13S::GFP
does not interfere with synaptic function and is not likely highly overexpressed. (G) The ratio of
UNC-13S::GFP mean fluorescence intensity in the HSNL varicosities to its intensity in the
HSNL non-synaptic process region, normalized to the DsRed2 control. A significant increase in
UNC-13S::GFP was observed in the synaptic varicosities compared to the HSNL process in
wild-type animals, and no significant difference in this ratio was observed in the goa-1(n1134)
mutant (p = 0.40). Error bars indicate standard error; data from 8 wild-type and 12 goa-1(n1134)
animals were averaged, using 1-2 varicosities per animal. We also used false-coloring to
qualitatively identify voxels of higher intensity within the varicosities to see if there was any
sub-varicosity localization of UNC-13S::GFP, and we did not detect a reproducible
11
concentration of UNC-13S::GFP in any specific region of the varicosities in goa-1 or wild-type
animals.
SUPPLEMENTAL FIGURE S4.- Mutations in tph-1 and ser-4 do not have a significant effect
on tph-1 reporter expression. Average intensity of DsRed2 driven by the tph-1 promoter in the
HSNL. Compared to the wild-type control, there was no change in the intensity of the DsRed2
expressed from the tph-1 promoter in the tph-1 mutant, which lacks serotonin, or in a deletion
mutant for ser-4, which encodes a G protein-coupled serotonin receptor. n = 30 for each
genotype and error bars show 95% confidence intervals.
12
SUPPLEMENTAL LITERATURE CITED
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GARRIGA, G., C. DESAI, and H. R. HORVITZ, 1993 Cell interactions control the direction of
outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans.
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MELLO, C., and A. FIRE, 1995 DNA transformation. Methods Cell Biol. 48: 451-482.
VAN SWINDEREN B., L. B. METZ, L. D. SHEBESTER, J. E. MENDEL, P. W. STERNBERG, and C. M.
CROWDER, 2001 Go regulates volatile anesthetic action in Caenorhabditis elegans.
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