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Supplemental Data
fkh-2 may act in the AWB lineage to regulate AWB neuron morphology
To determine whether FKH-2 acts cell-autonomously or cell non-autonomously,
we first attempted to rescue the AWB morphology defects by expressing a fkh-2 cDNA
under the str-1, lim-4 and odr-1 promoters which drive expression in the AWB neurons
(Troemel et al., 1997; Sagasti et al., 1999; L'Etoile and Bargmann, 2000). However, no
rescue was obtained upon expression of str-1p::fkh-2 or odr-1p::fkh-2, whereas
expression of lim-4p::fkh-2 resulted in increased dye-filling defects and loss of str1p::gfp expression, possibly indicating alteration of cell fate.
We reasoned that if FKH-2 acts cell-autonomously, then the ability or failure of
the AWB neurons to dye-fill should correlate more highly with the presence or absence
of fkh-2 rescuing sequences in the AWB, than in other cell types. We note, however, that
since the dye-filling defect of fkh-2(ok683) mutants is partially penetrant, a subset of
AWB neurons that dye-fill are expected to lack fkh-2 expression. Rescuing fkh-2 genomic
sequences were injected together with either an sru-38p::gfp (Colosimo et al., 2004) or
an odr-4::gfp fusion construct (Dwyer et al., 1998) into fkh-2(ok683) animals. The sru-38
promoter drives expression in the amphid AWB and ASH chemosensory neurons,
whereas the odr-4 promoter drives expression in a subset of chemosensory neurons
including the AWB, ASH and ASJ chemosensory neurons. The AWB and ASJ neurons
are closely related by lineage, while the ASH neurons are distant lineal relatives of the
AWB neurons.
Transgenic animals carrying rescuing fkh-2 sequences together with the sru38::gfp marker were dye-filled, and presence or absence of the rescuing sequences as
defined by sru-38p::gfp expression in the AWB or ASH neurons was correlated with the
ability or failure of the AWB neurons to dye-fill. We found that 74% and 58% of sides
with dye-filled AWB neurons exhibited gfp expression in the AWB and ASH neurons,
respectively (n=822 sides). Conversely, 2% and 28% of sides in which the AWB neurons
failed to dye-fill expressed gfp in the AWB or ASH neurons, respectively (n=46
neurons). These ratios are significantly different at p < 0.001 using a chi-square test. We
carried out a similar experiment using animals transgenic for fkh-2 genomic sequences
and the odr-4::gfp marker. We identify the AWB neurons in adult animals on the basis of
cell-specific marker expression (as in the case of sru-38::gfp expression), or by dyefilling. In adult transgenic animals expressing odr-4::gfp in multiple amphid neurons, we
are unable to definitively identify the AWB neurons in the absence of dye-filling. We
instead examined presence or absence of the array in the close and distant lineal relatives,
the ASJ and ASH neurons, respectively, that can be identified by dye-filling, and are not
developmentally affected by mutations in fkh-2. We found that while 76% and 81% of
ASJ and ASH neurons expressed odr-4::gfp on the same side as dye-filled AWB neurons
(n=89 sides), 14% and 60% of ASJ and ASH neurons expressed odr-4::gfp on the same
side when AWB was not dye-filled (n=43 sides). Taken together, these results strongly
suggest that FKH-2 acts in the AWB lineage to regulate neuronal morphology.
Supplemental Figure and Movie Legends
Figure S1. Movement of GFP-tagged DAF-10 and OSM-5. For each panel, shown are
still images (left) and kymographs (right) from representative movies. Velocity
histograms are shown below in each panel. In each still image, the genetic background is
indicated in the top left corner, the imaged cilia in the top right corner, and the fusion
protein examined in the bottom left corner. Dashed lines on the still images indicate the
continuous lines used to generate the kymographs. Velocities in the middle (M) and distal
(D) segments are indicated by black and gray bars, respectively in each histogram. Scale
bars in still images – 5 m. Scale bars in kymographs: horizontal bar – 5 m; vertical bar
- 5s. M – middle segment; D - distal segment.
Supplemental Movies
Anterior is at left in all movies.
Movie S1. Movement of GFP-tagged KAP-1 in wild-type AWB cilia. The display rate is
5 frames sec-1 with a total elapsed time of 38 sec.
Movie S2. Movement of GFP-tagged KAP-1 in AWB cilia of osm-3(p802) mutants. The
display rate is 5 frames sec-1 with a total elapsed time of 20 sec.
Movie S3. Movement of GFP-tagged OSM-3 in AWB cilia of wild-type animals. The
display rate is 5 frames sec-1 with a total elapsed time of 40 sec.
Movie S4. Movement of GFP-tagged OSM-3 in AWB cilia of kap-1(ok676) animals. The
display rate is 5 frames sec-1 with a total elapsed time of 40 sec.
Movie S5. Transport of GFP-tagged OSM-6 in wild-type ASH/ASI cilia. The display rate
is 5 frames sec-1 with a total elapsed time of 40 sec.
Movie S6. Transport of GFP-tagged OSM-6 in ASH/ASI cilia of osm-3(p802) mutants.
The display rate is 5 frames sec-1 with a total elapsed time of 40 sec.
Movie S7. Transport of GFP-tagged OSM-6 in wild-type AWB cilia. The display rate is
5 frames sec-1 with a total elapsed time of 28 sec.
Movie S8. Transport of GFP-tagged OSM-6 in AWB cilia of osm-3(p802) mutants. The
display rate is 5 frames sec-1 with a total elapsed time of 40 sec.
Movie S9. Transport of GFP-tagged OSM-6 in AWB cilia of kap-1(ok676) mutants. The
display rate is 5 frames sec-1 with a total elapsed time of 40 sec.
Movie S10. Transport of GFP-tagged OSM-6 in AWB cilia of fkh-2(ok683) mutants. The
display rate is 5 frames sec-1 with a total elapsed time of 32 sec.
Table S1. GFP fusion proteins are functional in the AWB neurons.
Strain
% wild-type AWB cilia
Wild-type
osm-6(p811)
osm-6(p811); Ex[str-1p::osm-6::gfp]
100
2
81
daf-10(e1387)
daf-10(e1387); Ex[str-1p::daf-10::gfp]
0
86
kap-1(ok676); osm-3(p802)
1
kap-1(ok676); osm-3(p802); Ex[str-1p::kap-1::gfp]
94
kap-1(ok676); osm-3(p802); Ex[str-1p::osm-3::gfp]
88
The cilia of adult animals grown at 25C were examined at 400X magnification. AWB
cilia were visualized by expression of str-1p::gfp.
n >55 each.
K1/M/osm-3
K1/M/WT
O3/D/ klp-11
O3/M/klp-11
O3/D/kap-1
O3/M/kap-1
O3/D/WT
O3/M/WT
D10/D/WT
D10/M/WT
O6/D/klp-11
O6/M/klp-11
O6/D/kap-1
O6/M/kap-1
O6/M/osm-3
O6/D/WT
O6/M/WT
Supplemental Table 2. Statistical analyses of pairwise comparisons of IFT particle component and kinesin motor
velocities in the ASH/ASI cilia.
O6/M/WT
__ S
S
S
S
S
S
S
S
S
S
S
S
S
O6/D/WT
S
__ S
S
S
S
S
S
S
O6/M/osm-3 S
S
__ S
S
S
S
S
S
S
S
S
S
S
S
S
O6/M/kap-1 S
S
__
S
S
S
S
S
S
S
O6/D/kap-1 S
S
__
S
S
S
S
S
S
O6/M/klp-11 S
S
__
S
S
S
S
S
S
O6/D/klp-11 S
S
__ S
S
S
S
S
S
S
D10/M/WT
S
S
S
S
S
S
__ S
S
S
S
S
S
S
D10/D/WT
S
S
S
__ S
S
S
S
S
O3/M/WT
S
S
S
S
S
S
S
__ S
S
S
S
S
S
O3/D/WT
S
S
S
S
S
__ S
S
S
O3/M/kap-1 S
S
S
S
S
S
S
S
__ S
S
S
S
O3/D/kap-1 S
S
S
S
S
S
S
S
S
__ S
S
S
O3/M/klp-11 S
S
S
S
S
__ S
S
S
O3/D/ klp-11 S
S
S
S
S
S
S
S
S
S
S
S
__ S
S
K1/M/WT
S
S
S
S
S
S
S
S
S
S
S
S
__ S
K1/M/osm-3 S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
__
O6 – OSM-6::GFP; D10 – DAF-10::GFP; O3 – OSM-3::GFP; K1 – KAP-1::GFP.
M – Middle segment; D – distal segment.
Thus, O6/D/kap-1 indicates the velocity of OSM-6::GFP in the ASH/ASI distal segments in kap-1 mutants.
S – indicates statistical significance at P < 0.01 using one-way ANOVA and Scheffe’s posthoc test for multiple paired comparisons.
S
S
S
S
__
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
__
S
S
S
__
S
S
S
S
S
S
S
S
S
S
__
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
__
S
S
S
S
S
S
S
S
S
S
S
S
S
__
S
S
S
S
S
S
S
S
S
__
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
__
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
__
S
S
S
K1/M/osm-3
S
S
S
S
K1/M/WT
O3/M/WT
D10/M/kap-1
D10/M/osm-3
D10/M/WT
S
S
S
S
S
S
__
S
S
S
S
O5/M/kap-1
S
S
S
S
S
__
S
S
S
S
S
-
O3/M/fkh-2
S
S
O3/M/klp-11
S
__
S
S
S
O3/M/kap-1
S
S
__
S
O5/M/WT
O6/D/klp-11
O6/D/fkh-2
O6/M/klp-11
S
O6/M/fkh-2
O6/D/kap-1
S
O6/M/osm-3
S
__
S
O6/D/WT
O6/M/WT
__ S
S
O6/D/WT
S
__ S
O6/M/osm-3
S
__ S
O6/M/kap-1
S
S
__
O6/D/kap-1
S
__ S
O6/M/klp-11 S
S
O6/D/klp-11 S
S
O6/M/fkh-2
S
S
O6/D/fkh-2
S
S
O5/M/WT
S
S
O5/M/kap-1
S
S
D10/M/WT
S
S
D10/M/osm-3
S
S
D10/M/kap-1 S
S
S
__
O3/M/WT
S
S
O3/M/kap-1
S
S
S
S
O3/M/klp-11 S
S
S
O3/M/fkh-2
S
S
K1/M/WT
S
K1/M/osm-3
S
S
Legends as in Supplemental Table 2.
O6/M/WT
O6/M/kap-1
Supplemental Table 3. Statistical analyses of pairwise comparisons of IFT particle component and kinesin motor velocities in
the AWB cilia.
S
S
__
S
S
S
S
S
S
S
S
S
S
__
S
S
S
S
S
__
REFERENCES
Colosimo ME, Brown A, Mukhopadhyay S, Gabel C, Lanjuin AE, Samuel AD, Sengupta
P (2004) Identification of thermosensory and olfactory neuron-specific genes via
expression profiling of single neuron types. Curr Biol, 14, 2245-2251.
Dwyer ND, Troemel ER, Sengupta P, Bargmann CI (1998) Odorant receptor localization
to olfactory cilia is mediated by ODR-4, a novel membrane-associated protein.
Cell, 93, 455-466.
L'Etoile ND, Bargmann CI (2000) Olfaction and odor discrimination are mediated by the
C. elegans guanylyl cyclase ODR-1. Neuron, 25, 575-586.
Sagasti A, Hobert O, Troemel ER, Ruvkun G, Bargmann CI (1999) Alternative olfactory
neuron fates are specified by the LIM homeobox gene lim-4. Genes Dev, 13,
1794-1806.
Troemel ER, Kimmel BE, Bargmann CI (1997) Reprogramming chemotaxis responses:
sensory neurons define olfactory preferences in C. elegans. Cell, 91, 161-169.