Wild type measurement
Raw cell cycle
Scaled cell cycle
Generation synchrony
Division timing
Expression
A-P position
L-R position
D-V position
A-P displacement
L-R displacement
D-V displacement
Total displacement
AB4-16 AB32
Large measurement
AB64
Small measurement
AB128
MS-32
E-8 C-16 D-4
Fig. S1. P-value chart for a pal-1(RNAi) embryo. Cells are across the x-axis and measurements are on the y-axis. Red represents
statistically significantly small measurements; blue denotes statistically significant large measurements; white shows measurements
that are within the wild-type range.
Fig. S2. Dev-scape graphical user interface. This graphical user interface assists users in entering information to perform the
analysis on their data. Detailed instructions for installing and using the software as well as interpreting its results are provided in
supplementary material Appendix S3.
x104
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
AB4-32 AB64 AB128
C
AB256
CND-1 expression
ABal
D
HND-1 expression
PHA-4 expression
ABa
MS1-32 MS64 E C
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
AB
ABar
ABpl
Expressing
Not Expressing
x103
8
6
4
2
0
-2
0
5
10
15
20
Minutes of cell cycle
NHR-25 expression
CND-1 and NHR-25 expression
P0
ABp
B
Expression level (a.u.)
Average expression level
A
ABpr
PHA-4 and ELT-2 expression
MS
EMS
P1
E C
P2
P3
Fig. S3. Details of expression classification and consistency. (A) Single-cell average expression levels are colored by the weight
assigned to them in a robust line fitting that identifies the background expression level. Cells with a positive expression level and
a weight below 0.3 are classified as expressing. Cells with a weight above 0.7 are classified as not expressing. Those with weights
between 0.3 and 0.7 are tested for increasing expression levels over time. (B) The expression trajectories of two cells with intermediate
weights, where the cell represented by the blue line passes the Wilcoxon rank-sum test and the cell represented by the red line fails the
test. (C) A complete lineage tree of cells that were classified as always expressing in wild-type embryos.
B
200
10
WT hyp
5
WT musc
0
pal-1 hyp
150
-5
pal-1 musc
50
pal-1 (RNAi)
Minutes
Wild type
cdt-1 (RNAi)
50
100
150
200
Wild type average division time
D
ABa
ABal
-10
C
100
ABar
1
20
0.8
40
0.6
0.4
60
0.2
80
AB
MS E C D
P0
AB
ABp
ABpl
Minutes
Current embryo division time
A
P1
EMS
ABpr
MS
E
C
0
P2
P3
Fig. S4. Proliferation consistency and disruptions. (A) Calculating the global pace of embryogenesis by comparing division timing
in three embryos with the wild-type average division timing. The wild-type and pal-1(RNAi) embryo clocks pass the test for linear
relationship to the wild-type average as quantified by their RMSD levels of 1.79 and 1.66. The embryo treated with cdt-1(RNAi) does
not pass the linearity test due to its large RMSD value of 20.28. (B) Generation asynchrony within a lineage group as shown by the
difference between the average division time of the lineage group and an individual cell’s division time. The wild-type embryo shows
asynchrony whereas a pal-1(RNAi) embryo shows synchrony. (C) Each cell’s history is represented by a vector where each element
corresponds to a time point and is assigned a value representing the proportion of the cell cycle that the corresponding time point is
away from a division. (D) The complete lineage of wild-type average cell cycle lengths with developmental time on the y-axis and
blue bars showing plus and minus one standard deviation.
A
Dorsal view
Eal
B
Ear
Wild type
Epl
Epr
correlated
anti-correlated
ABala
ABalp
ABara
ABarp
ABpla
ABplp
ABpra
MSa
MSp
E
C
D
P
ABprp
Fig. S5. Cell positions and movement. (A) (Left) Each color represents one E4 cell identity. Each dot represents that cell’s position in
one of 53 wild-type embryos. (Right) The covariance ellipsoids that summarize these distributions with the ellipsoid radii each being
one standard deviation in a principal component of the distribution. (B) Correlation matrix of the AB64 stage for a wild-type embryo.
Mean path (microns)
Single embryo paths (microns)
A
Anterior-Posterior axis
10
Left-Right axis
C
10
5
5
0
0
0
-5
-5
-5
0
10
20
30
Minutes of cell cycle length
D
F
5
5
5
0
0
0
-5
-5
-5
G4
0
10
20
30
Minutes of cell cycle length
0
10
20
30
Minutes of cell cycle length
H
I
4
4
2
2
2
0
0
0
-2
-2
-2
Noise vs noise change
(microns)
-4
0
10
20
30
Minutes of cell cycle length
-4
K
2
0
10
20
30
Minutes of cell cycle length
-4
L
2
1
1
0
0
0
-1
-1
-1
-2
-2
-2
-5
0
5
Current noise component
0
10
20
30
Minutes of cell cycle length
2
1
-5
0
5
Current noise component
Dorsal-Ventral axis
-10
0
10
20
30
Minutes of cell cycle length
-10
0
10
20
30
Minutes of cell cycle length
E
0
10
20
30
Minutes of cell cycle length
Single minus mean (microns)
10
5
-10
J
B
-5
0
5
Current noise component
Fig. S6. Signal to noise quantification for ABarppp. Cell movement along (A,D,G,J) the anterior-posterior axis, (B,E,H,K) the leftright axis and (C,F,I,L) the dorsal-ventral axis. (A-C) Cell migration paths in their respective components, aligned at their mean. The
migration paths are shown for 20 embryos with each embryo having a unique combination of color and line style. (D-F) The average
migration path in each component, or the average over all 20 lines in A-C. (G-I) The average path subtracted from each individual
path. We denote these values noise. (J-L) The magnitude of the noise at each time point plotted versus the change in noise level to the
following time point. The lack of bias in these distributions suggests the two axes are independent.
C cells
Shift embryo
Periphery cells
Other
Non-shift embryo
7 minutes after C2 to 4 division - There is no significant difference between embryos.
14 minutes after C2 to 4 division - C cells change direction in shift embryo.
25 minutes after C2 to 4 division - Periphery cells migrate clockwise in shift embryo while
there is minimal migration in non-shift embryo.
67 minutes after C2 to 4 division - Counterclockwise motion appears only in shift embryo.
Fig. S7. Circumferential shift in two wild-type embryos. The cell displacement patterns in 15-minute windows of one wild-type
embryo that does undergo an angular shift and one wild-type embryo that does not undergo an angular shift.
Movie 1. Covariance ellipsoids by time point. Each frame shows the variation in cell positions across 53 wild-type embryos at one
time point of development. The left side shows dorsal views and the right side shows ventral views. The ellipsoids are color coded by
their sublineages as in Fig. 4.
Movie 2. Average AB64 and AB128 displacement. The average cell positions and displacements of each cell at the AB64 and AB128
stages are shown. The view begins as dorsal with anterior to left and rotates about the anterior-posterior axis. The rods are color coded
by their sublineages as in Fig. 4.
Table S1. List of output files
Download Table S1
Table S2. C. elegans strains used
Download Table S2
Appendix S1. Wild-type statistics
Download Appendix S1
Appendix S2. pal-1(RNAi) example
Download Appendix S2
Appendix S3. Source code and instructions
Download Appendix S3