conserved sequence that is independent of animal
size. Pattern is first established by morphogen-driven cell fate specification and then elaborated by
cell-type–specific regulation of differentiation rates.
How the spatiotemporal changes in the differentiation rate are regulated is a key question. The
length of G1, Notch signaling, and proneural genes
(e.g., Ascl1 and Ngn1/2), which are activated downstream of domain identity regulators such as Olig2,
could play a role (28, 33–38). The comparison of
mouse to chick and Minute embryos suggests
that the temporal changes in the differentiation
rate are conserved and independent of embryo size,
thus ensuring that domain proportions are comparable between individuals. This constrains the
possible molecular mechanisms controlling the
differentiation rate and implies that feedback
regulation might ensure its robustness (32). The
nonautonomous effect after Ngn2 electroporation
suggests a possible role for postmitotic neurons in
regulating the differentiation rate of progenitors,
although other mechanisms cannot be excluded.
The transition from specification to differen-
tiation phase correlates with the dynamics of
Shh and BMP signaling. These dynamics depend
on transduction cascades but are also likely to be
constrained by the effective ligand diffusion, deg-
radation, and the size of the morphogen source
(39). Despite the decrease in Shh and BMP signal-
ing, progenitor pattern is maintained during the
differentiation phase. This could be explained by
bistability produced by the transcriptional net-
work (11). In the floorplate, a transcriptional net-
work downstream of Fgf signaling contributes to
these cells becoming refractory to Shh and BMP
before E9.5 (40, 41). Our data suggests that neural
progenitors in the other domains tolerate a de-
crease in Gli and pSmad activity, although some
level of Shh signaling is clearly required during
the differentiation phase (23, 42).
During the specification phase, chick, mouse,
and Minute embryos are of similar initial size. This
might result from the early period of regulative
growth apparent from surgically manipulated
embryos (43, 44). Thus, the timing of the transition from specification to differentiation could be
connected to an optimal tissue size for the formation and activity of morphogen gradients. Further
quantitative analysis will be needed to understand
both morphogen-dependent and -independent
scaling mechanisms in other tissues and species.
Materials and methods
To generate Sox1CreER, the Sox1 reading frame was
replaced with CreERT2 (fig. S4A). The following
strains were previously described: Tg(GBS-GFP)
(Gli binding sites–green fluorescent protein) (11),
Olig2KICreER (27), CAG-CAT-EGFP (45), Fucci-S/
G2/M #504 (46), and Rpl24(Bst) (15, 47). Strains
were maintained on a Parkes background to maximize litter size.
Embryos were staged according to the number
of somites, where one somite is generated every
2 hours in mouse and 1.5 hours in chick (48).
Hamburger and Hamilton staging criteria were
used for late stages of chick development (49).
The chick data set was registered to mouse, so that
the brachial-level somite at which the measure-
ments were made was generated at equivalent
corresponding time (table S1).
For GBS-GFP and pSmad measurements, embryos were initially staged by somite number. The
dorsoventral lengths of the neural tubes were
fitted to DVlength ¼ aebt, where a and b are fit
parameters, t is time (fig. S8F), and then restaged
based on the fit.
Immunohistochemistry and imaging
Transverse sections were processed as described
(11). Idu/BrdU antigens were exposed by 40-min
deoxyribonuclease I treatment at 37°C, except
mouse E8.5, where 2N HCl was used.
Flat-mount preparation: the neural tube was cut
at the roofplate and then fixed in 4% paraform-
aldehyde and subsequently methanol. Antibody in-
cubations and washes were 24 hours each. The left/
right neural-tube halves were split, then mounted
with grease spacers between slide and coverslip.
Sections were imaged with 40x/1.25NAOil ob-
jective, flat mounts with 20x/0.7NADry on a Leica
TCS-SP5-MP. Single optical sections were taken,
except for GBS-GFP and pSmad analysis, where
a maximum projection of 3 z slices 1 mm apart
were used. For flat mounts, the entire apicobasal
depth of the progenitor layer was imaged with
z slices 1.5 mm apart.
Antibodies used were goat anti-Sox2 (R&D
systems, 1:100), rat anti-pH3 (Novus Biologicals,
1:2000), rabbit anti-Olig2 (Millipore, 1:1000), rabbit anti-Arx (50) (from J. Chelly, 1:1000), mouse
anti-Pax3(c) (Developmental Studies Hybridoma
Bank, 1:100), rabbit anti-Islet1 (51) (from T. Jessell,
1:3000), sheep anti-GFP (Biogenesis, 1:1000), mouse
anti-Nkx2.2 (DSHB, 1:25), rabbit anti-mAzami Green
(MBL, 1:100), rabbit anti-cleaved Caspase3 (Cell
Signaling, 1:500); rabbit anti-pSmad1/5/8 (from
E. Laufer), mouse anti-BrdU/IdU (1:80, BD clone
B44), rat anti-BrdU (1:80, Abcam, clone BU1/75).
Chick electroporation was performed in ovo at
HH14 and analyzed 48 hours later. Plasmids used:
pMIW-YAP (from Xinwei Cao), pCAGGS-NICD (from
Olivier Pourquie), pCAGGS-p21 (from Cheryll Tickle),
pCAGGS-Ngn2-IRES-GFP (from Francois Guillemot), pPS-CFP2-N (Evrogen), and pCI-H2B-EGFP
(from Tatjana Sauka-Spengler). The first three
were coelectroporated with pCAGGS-NLS-GFP to
mark transfected cells. Final concentrations were
0.5 mg/ml for pMIW-YAP and pPS-CFP2-N, and
1 mg/ml for all other plasmids.
Mouse embryo culture
Mouse embryo culture was performed as previously described (11).
Chick: 0.5 mg/ml IdU or 3.3 mg/ml BrdU (Sigma)
in PB, 10% sucrose, 0.5% Fast Green, were injected into the neural tube, and 50 ml was added
on top of the embryo. Embryos were treated with
IdU for 1.5 hours, then BrdU for 50 min.
1254927-8 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
Fig. 6. Perturbing differentiation changes the relative pMN size. (A) Chick electroporation at HH14
of Ngn2+GFP assessed after 48 hours. (B) Number of Sox2+ progenitors per hemisection on the control
versus Ngn2 electroporated side for 13 sections (five embryos) as described in (A). (C) Electroporation
of constitutive YAP+GFP, as in (A). Progenitors expand both dorsoventrally and apicobasally on the
electroporated side. (D) Quantification of (C) for 12 sections (six embryos). Error bars, mean T SEM.