Mouse: Pregnant mice were intraperitoneally
injected with 0.6 mg IdU and after 1.5 hours with
1 mg BrdU. Embryos were harvested after 30 min.
Chick live imaging
Membrane-anchored GFP transgenic chicken
eggs were obtained from the Roslin Institute,
Edinburgh. Embryos were electroporated with
pCI-H2B-EGFP 8-15h before imaging.
For live imaging, we established a sagittal slice
culture protocol, based on a “clot” method (52, 53).
The brachial and anterior thoracic region was dissected in L15 medium, then transferred to a glass-bottom dish (Mat Tek) in a small drop of culture
medium [DMEM-F12 1:1, supplemented with N2,
B27 (Life Technologies)] containing 10 mg/ml fibrinogen (Calbiochem). Thrombin (0.5U/ml, Amersham)
was added, and a fibrin gel was allowed to form for
a few minutes. The slice was covered with ~0.5 ml
culture medium, and the dish was humidified. Z
stacks were collected for 3 hours on an inverted
Leica-SP5 confocal at 37°C.
Photoconversion in chick embryos
Stage HH16 chick embryos electroporated with
pPS-CFP2-N were cultured using the EC (early
chick) culture method (54), dorsal side up, and
immediately photoconverted along the entire
dorsoventral length using Leica MP-SP5 microscope, 10X/0.7NADry lens, 30% laser (405 nm)
power, ~25 s scan time. Either 15 hours or 0 hours
after photoconversion, the neural tube was flat-mounted in phosphate-buffered saline and immediately imaged.
DV boundary positions and pSmad
and Gli activity profiles
Image analysis was performed in Fiji (http://fiji.
sc/Fiji) and data analysis in MATLAB (
Math-works, MA, USA).
The mean fluorescence intensity in immunostained sections was quantified across a 10-mm
region adjacent to the apical lumen. The data
was background-subtracted and smoothed with
a 5-mm moving average. Boundaries were defined
as the positions where the intensity increased
above 10% of maximum.
pSmad and GBS-GFP intensity profiles were
normalized to the mean profile for each time
point, similarly to a described procedure (55), by
normalized intensity = a × (raw intensity), where
a is a fit parameter. Profiles that correlated poorly
with the mean (R2 < 0.5) were discarded. These
were usually sections damaged during dissection
and represented <5% of the data.
Seven independent time courses of pSmad and
GBS-GFP were collected. Sections in each time
course were stained and imaged together to minimize technical variability. The seven data sets
were pooled by normalizing to the median value
of F for each data set, where F is the 90th percentile of the fluorescence intensity of each profile.
Progenitor and neuron numbers
and differentiation rate
The number of progenitors per section was in-
ferred from the Sox2+ domain area (Fig. 1B) and
the average density of Sox2+ nuclei for each stage
(fig. S1, A and B) determined from a subset of
sections. The number of neurons was determined
by counting Dapi+,Sox2– nuclei. Islet1+ and inter-
spersed Islet1– nuclei [which are HB9/MNR2+
(not shown)] in the ventral horn were considered
MNs. The Pax3 boundary was used to separate
ventral interneurons V2 - V0 and dorsal interneurons.
Neurons (N) are produced from progenitors (P)
with a rate of differentiation g, i.e., dN =dt ¼ gP.
We obtained an estimate of g using g ¼ DN PiDt, where
Dt was the time interval between two time points,
DN was the difference in neuron number between
the end and the beginning of the time interval,
and Pi was the progenitor number in the middle of the interval. The error of g was calculated
by propagation of the standard deviations of
all participating variables, assuming that the errors are independent and uncorrelated. The 95%
CIs, calculated from the mean, SD, sample size,
and Student’s t value, were linearly interpolated
between time points.
Proliferation rate by
Three cell populations were distinguished by
anti-IdU/BrdU, anti-BrdU, and anti-Sox2 immunostaining: (i) Sox2+, IdU+, and BrdU– (denoted
IduL) are progenitors labeled by IdU during the
1.5-hour pulse, which have exited S phase before
BrdU addition; (ii) Sox2+, IdU+, and BrdU+; (iii)
Sox2+, Idu–, and BrdU–.
The IdU labeling index IduLI is defined as
the ratio of IduL over the total number of Sox2+
progenitors. It is related to the pulse duration
qðIduLÞ ¼1:5 hours and the total cell cycle
IduLI ¼ IduL
all Sox2þ cells ¼ qðIduLÞ qðtotÞ ln 2 ð3Þ
In turn, the proliferation rate l is related to
qðtotÞand IduLI as:
l ¼ ln2 qðtotÞ ¼ IduLI qðIduLÞ ð4Þ
Two assumptions are made: (i) cells divide
asynchronously and (ii) the length of the pulse
is shorter than the G2 phase. This is supported
by the random appearance of mitotic figures in
flat mounts and by the observation that no IduL
cells were found in telophase, indicating that they
are not completing mitosis within the experiment.
The relationship between the duration of mitosis,
the total cell cycle length, and the fraction of cells
in mitosis depends on the exact growth characteristics of the tissue (56). The MI for an exponentially growing tissue is defined as
MI ¼ pH3þ progenitors
all Sox2þ progenitors ¼ qðpH3Þ qðtotÞ ln 2 ð5Þ
where qðpH3Þ is the duration of the pH3-positive
phase of mitosis and qðtot Þ is the cell cycle length.
The proliferation rate l is related to qðtotÞ and
l ¼ ln 2 qðtotÞ ¼ MI qðpH3Þ ð6Þ
Thus, to calculate l, it is enough to know MI
and the duration of mitosis. MI was directly measured from sections. To determine the duration of
mitosis, we used the fact that l is related to both
MI (Eq. 6) and IduLI (Eq. 4). Hence,
qðpH3Þ ¼ qðIduLÞ MI
The average values of MI and IduLI from all
stages that were identical between the two types
of experiments were used for calculating qðpH3Þ.
The resulting mitosis duration is 24.6 T 7.0 min
for chick and 27.7 T 11.3 min for mouse. These are
similar to published values (24, 25).
MI in flat mounts (fig. S2, C and F) was estimated from the number of pH3+ cells per apical
area, normalized to the average apicobasal length
for the respective stage and domain measured in
sections (Fig. 1F and fig. S1C).
Cell cycle phase distribution
Mitotic cells were identified using Dapi S+G2 cells
using the Fucci-S/G2/M-Green fluorescence intensity. The fluorescence intensities of Olig2, Nkx2.2,
and Fucci-S/G2/M-Green were measured in individual nuclei. The Olig2 and Nkx2.2 levels were
background-subtracted and normalized to the
average intensity in the respective domain. Cells
with Olig2/Nkx2.2 ratio between 0.1 and 10 were
defined as coexpressing.
Coexpressing cells are assumed to undergo a
transition from an Olig2 to an Nkx2.2 stable state
or, with a smaller probability, vice versa (11). If
the transition occurs preferentially in G1 or S+G2
phase, the cell cycle phase distribution in cells
that are at the onset of the transition could appear different than in noncoexpressing populations. However, the distribution of both G1 and
S+G2 phases across the Olig2-Nkx2.2 level difference was normal (fig. S3C), suggesting that the
transition may occur at any time of the cell cycle.
Olig2 lineage tracing
Olig2KICreER mice (27) were crossed to CAG-CAT-EGFP (45). Cre expression was induced in Olig2+
progenitors by injecting 3 mg of tamoxifen. As a
result of the recombination, EGFP was induced
with a delay of 12 hours (fig. S10), allowing the
tracking of cells after they lose Olig2 expression.
Embryos were harvested 48 hours after injection;
hence, the effective time interval of EGFP labeling
was ~36 hours.
The change in the number of labeled progenitors P, regardless of their type, during the time
course of the experiment Dt ¼ t2 − t1 (t1, time
at the beginning; t2, end time) is
Pðt2Þ − Pðt1Þ
Dt ¼ lPðt1Þ −DN Dt ð8Þ
Here, Pðt2Þ are all labeled progenitors at t2 and
DN are all labeled neurons. Dt = 36 hours, and