SUPPLEMENTARY DATA
Supplementary tables
Table S1. Ectopic expression of icd-1 does not suppress programmed cell death
during development
A. The expression of icd-1 under the control of a heat-inducible promoter does not block
the programmed death of cells during the development of the anterior pharynx
Strain
JR2028
JR2028
Heat shock
+
Average number of extra cells in
anterior pharynx ± STDEV
0.0 ± 0.2
0.1 ± 0.4
n
25
48
Range
0-1
0-1
JR2028 animals carry an integrated array of the transgene PHSicd-1, which allows the
heat-inducible expression of icd-11. Heat shock was applied during embryogenesis as
described by Bloss et al1. The number of extra cells in the anterior pharynx was
determined in animals at the fourth larval stage of development using DIC.
B. The expression of icd-1 in a ced-1(e1735) background under the control of a heatinducible promoter does not result in fewer cell corpses at the comma stage of
embryogenesis
Strain
ced-1(e1735)
ced-1(e1735); JR2028
Average number of corpses ± STDEV (n)
no heat shock
heat shock
24.2 ± 2.6 (10)
25.0 ± 2.9 (10)
23.0 ± 1.9 (20)
24.6 ± 3.7 (18)
The integrated PHSicd-1 array contained in JR20281 was transferred into a ced-1(e1735)
background2. Heat shock was applied during embryogenesis as described by Bloss et al1.
The number of cell corpses was determined in animals at the comma stage of
embryogenesis using DIC.
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Table S2. Ectopic expression of drp-1(wt) under the control of a heat-inducible promoter
results in the loss of Pceh-2dsRed-expressing cells
Genotype
bcIs51; PHSdrp-1(wt)
no heat shock
bcIs51; PHSdrp-1(wt)
heat shock
Average number of
Pceh-2dsRed-positive
living cells
Average number of
Pceh-2dsRed-positive
cell corpses
n
Range – sum of
Pceh-2dsRed-positive
cells and corpses
5.0
0
44
5
4.0
0.4
37
2-5
bcIs51 is an integrated array of the reporter Pceh-2dsRed, which is expressed in five neurons in the
anterior bulb of the pharynx during the second half of embryogenesis (starting at the 2-fold stage): the
two NSMs, the two M3s, and the I33. PHSdrp-1(wt) is an extrachromosomal array of the transgene
PHSdrp-1(wt), which drives the heat-inducible expression of drp-1(wt). Heat shock was applied to
bcIs51; PHSdrp-1(wt) embryos as described in Methods, except that adult animals were allowed to lay
eggs for 90’ and that heat shock was applied to embryos 4hr after the adults had been removed from
the plates. Embryos at the 3½- to 4-fold stage of embryogenesis were analyzed for the presence of Pceh2dsRed- positive cells and corpses using DIC and epi-fluoresence. The complete genotype of the
animals was unc-76(e911); bcIs51; PHS mitogfp+PHS drp-1(wt).
2
Table S3. Ectopic expression of drp-1(wt) under the control of a heat-inducible
promoter causes embryonic lethality, which is at least partially suppressed by ced9(n1950gf)
no heat shock
% transgenic % embryonic
Line
animals (n) lethality (n)
control - 1
58 (50)
0 (50)
control - 2
47 (92)
0 (92)
PHS drp-1(wt) - 2
69 (109)
1 (110)
PHS drp-1(wt) - 3
69 (156)
0 (156)
PHS drp-1(wt) - 5
76 (98)
0 (98)
ced-9(n1950gf); PHS drp-1(wt) - 9 58 (66)
0 (66)
+/+; PHS drp-1(wt) - 9*
57 (77)
0 (77)
ced-9(n1950gf); PHS drp-1(wt) - 10 52 (105)
2 (107)
+/+; PHS drp-1(wt) - 10*
59 (58)
3 (60)
heat shock
% transgenic % embryonic
animals (n)
lethality (n)
53 (232)
3 (240)
38 (88)
3 (91)
41 (89)
33 (133)
54 (95)
35 (147)
52 (42)
38 (68)
51 (111)
8 (121)
34 (109)
32 (161)
46 (137)
9 (150)
31 (136)
18 (165)
Animals carrying extrachromosomal arrays of the PHS drp-1(wt) transgene or control
extrachromosomal arrays were subjected to heat shock during embryogenesis as
described in Methods. The percent transgenic animals was determined by counting the
total number of adults and the number of non-Unc transgenic adults that developed from
the treated embryos. The percent embryonic lethality was determined by counting the
total number of embryos treated and the number of embryos that failed to hatch within
24hr of the treatment. The complete genotype of the animals were (from top to bottom):
unc-76(e911); PHS mitogfp lines 1, 2, unc-76(e911); PHS mitogfp+PHS drp-1(wt) lines 2, 3,
5, ced-9(n1950gf); unc-76(e911); PHS mitogfp+PHS drp-1(wt) line 9, unc-76(e911); PHS
mitogfp+PHS drp-1(wt) line 9, ced-9(n1950gf); unc-76(e911); PHS mitogfp+PHS drp-1(wt)
line 10, unc-76(e911); PHS mitogfp+PHS drp-1(wt) line 10. Lines marked with * were
crossed out of the ced-9(n1950gf) background to confirm that the transgenes were active
in a wild-type background.
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Table S4. Animals overexpressing drp-1(wt) contain larger numbers of cell corpses
than animals severely defective in engulfment
Genotype
+/+
ced-7(n1892); ced-5(n1812)
PHS egl-1
PHS drp-1(wt)
Heat shock
+
+
Average number of
corpses ± STDEV
7.3 ± 1.7
43.4 ± 4.9
49.3 ± 13.4
55.4 ± 16.1
n
16
15
10
10
Range
4-10
38-51
25-76
31-74
The strain ced-7(n1892); ced-5(n1812) carries a mutation in each of the two parallel,
partially redundant C. elegans engulfment pathways and is therefore severely defective in
engulfment4-6. PHS egl-17 and PHS drp-1(wt) animals carry extrachromosomal arrays of a
PHS egl-1 or PHS drp-1(wt) transgene, respectively, which allow the heat-inducible
expression of egl-1 or drp-1(wt). PHS egl-1 and PHS drp-1(wt) animals were subjected to
heat shock as described in Methods except that transgenic embryos were analyzed 3-4hr
after the heat shock rather than 2hr after the heat shock. The number of cell corpses was
determined in embryos at the 1½-fold stage of embryogenesis using DIC. The complete
genotypes of the animals were (from top to bottom): wild-type (N2), ced-7(n1892); ced5(n1812), unc-76(e911); PHS mito-gfp+PHS egl-1; unc-76(e911); PHS mito-gfp+PHS drp1(wt).
4
Supplementary figure legends
Figure S1. egl-1(n1084 n3082), ced-9(n1950gf), ced-9(n2812lf), ced-4(n1162), and
ced-3(n717) do not affect mitochondrial morphology in general. Representative
confocal images of rhodamine (bottom) and DIC (top) of wild-type, egl-1(n1084 n3082),
ced-9(n1950gf), ced-9(n2812lf); ced-3(n717), ced-4(n1162), and ced-3(n717) animals at
the comma to 1 ½-fold stage of embryonic development. Mitochondria were stained with
rhodamine B hexyl ester as described in Methods. Images of rhodamine-stained embryos
represent single confocal planes.
Figure S2. Reducing icd-1 function induces the appearance of small refractile
structures, which are not suppressed by mutations in egl-1, ced-9, ced-4, or ced-3.
icd-1 function1,8 was reduced by RNA-mediated interference (RNAi) using feeding as the
method for delivering dsRNA8. icd-1 RNAi resulted in embryonic lethality and the
appearance of small refractile structures in 50-75% (n=20 or more) of arrested embryos
in a wild-type background as well as in an egl-1(n1084 n3082), ced-9(n1950gf), ced4(n1162), or ced-3(n717) background. Representative DIC images of affected embryos.
Figure S3. Most refractile structures induced by icd-1 RNAi differ in their
appearance from cell corpses in wild-type animals and from ectopic cell corpses
induced by overexpression of egl-1 or drp-1(wt). Refractile structures in wild-type
embryos, in wild-type embryos ectopically expressing egl-1 or drp-1(wt) from a heatinducible transgene (PHSegl-17 and PHSdrp-1(wt)), or in wild-type embryos, in which icd-
5
1 function1 was reduced by RNAi (icd-1(RNAi)). Arrows point to refractile structures.
Representative DIC images of embryos at the comma to 1½-fold stage of embryonic
development. The complete genotypes of the animals analyzed were: N2 (‘wild-type’),
unc-76(e911); PHS mito-gfp+PHS egl-1 (‘egl-1 expression’); unc-76(e911); PHS mitogfp+PHS drp-1(wt) (‘drp-1(wt) expression’); icd-1(RNAi) (‘icd-1(RNAi)’).
6
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4.
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6.
7.
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