significant change in the mitotic index (Fig. 6, A
and C, and fig. S9E). In the case of Ngn2 over-
expression, the differentiation rate of nonelectropo-
rated cells (fig. S9, F and G) was also affected and
became spatially uniform, suggesting that non-
autonomous mechanisms—e.g., feedback from
postmitotic cells—are involved in differentiation
control (32). Importantly, in all conditions the
relative size of the pMN domain became larger
on the electroporated compared to the contra-
lateral side (Fig. 6, B and D) and was similar to
the larger pMN relative size at the onset of the
experiment (~30 hours) (Fig. 2G). Because these
perturbations cause opposite effects on the over-
all tissue size, this result cannot be explained by
changes in the relative range of the signaling
gradients and respecification.
Discussion
Here, we show that progenitor domain proportions
in the neural tube continuously change through a
Fig. 5. Validation of the two-phase model. (A and B) Olig2KICreER-induced
EGFP expression (green) from the indicated times. Olig2 immunostaining,
red. (C) Quantification of rate of loss of Olig2 identity. Period of Olig2KICreER
activity, horizontal gray bars. (D) Distribution of cell cycle phases (based
on S/G2/M-Fucci expression) in progenitors expressing Olig2, Nkx2.2, or
both (Fig. 3E). (E to J) Mouse embryo culture at the indicated stages
without [(E) to (G)] or with [(H) to (J)] 5 mM cyclopamine. Sections in (H)
and (I) are from the same embryos as (E) and (F), respectively. (K)
Quantification of the experimental conditions in (E) to (J). The difference in
mean boundary position between control and cyclopamine treatment was
significant (Student’s t test, P < 0.05) for all boundaries at E8.5 and 3/6 at
E9.5-brachial. The different boundaries were measured in independent
experiments. Cyclopamine concentration for Nkx6.1 boundary in E8.5 is 3 mM.
(L and M) pSmad and GBS-GFP mean fluorescence intensity versus relative
distance from the ventral midline at different stages. (N) Profiles in (L) and
(M) were normalized to the maximum intensity in each time series and
summed to give combined pSmad and GBS-GFP activity. Error bars, mean T
SEM. For sample sizes, see table S2.
