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Fig. 6. XHes2 can regulate proneural gene transcription in both neural and
non-neural ectoderm. (A-I,K-V) Whole-mount in situ hybridization analysis
of neuro-specific gene expression in stage 14 (A-D,F-I) or 20 (E) or 24 (K-V)
Xenopus embryos upon injection of XHes2 variants or morpholinos.
Black arrowheads, posterior expression domains; black arrows, anterior
expression domains; white arrowheads, ectopic expression. (J) Real-time RT-PCR
analysis of neuro-specific gene expression in animal cap assays following
injection with XHes2-
C-VP16. (A,B)
XHes2 represses neurogenesis, as assessed by the downregulation of
XNgnr1 (89%, n=9) (A) and N-tubulin (88%,
n=16) (B). (C-E) Conversely, inhibition of endogenous
XHes2 with XHes2-Mo2 extends XNgnr1 expression in the
presumptive otic placodal region (D, injection into one of eight cells, 45%,
n=42; one of two cells, 62%, n=13). XHes2-Mo1 shows the same
phenotype (not shown, one of eight cells, 56%, n=63; one of two
cells, 100%, n=14) in contrast to the control Mo (C, one of eight
cells, 5%, n=60; one of two cells, 0%, n=37). (E) At some
later neurula stage embryos, ectopic patches of XNgnr1 (white
arrowhead) appear close to the otic placode region (arrow). Arrows in C,D
point to the presumptive otic placodal region. (F-I) Effects of XHes2
variants on proneural gene expression at stage 14. (F,H) XHes2-W does
not significantly alter XNgnr1 expression (slightly reduced in 39%,
n=13) (F), but still inhibits N-tubulin expression (91%,
n=11) (H). (G,I) In stage 14 embryos, XHes2-W-VP16 leads to
ectopic XNgnr1 expression (G1, 100%, n=19; G2 and G3 show
the non-injected and the injected lateral sides, respectively) but fails to
activate N-tubulin expression (I, 100%, n=15). Moreover,
transcription of N-tubulin is repressed in its endogenous domains
(I). (J) XHes2-C-VP16 induces neuronal differentiation of animal
caps. In early caps (stage 13), XHes2-C-VP16 induces the expression of
the proneural genes XNgnr1 and NeuroD, while expression of
late neuronal markers is low (Xath3) or even undetectable
(N-tubulin). From stage 22 onwards, Xath3 and
N-tubulin are significantly upregulated. Relative values are given as
mean±s.e.m of two independent experiments. (K-P) Effects of
XHes2 variants on proneural gene expression at stage 24. In
XHes2-W-VP16 overexpressing embryos, an ectopic activation of
XNgnr1 (L, 29%, n=14), NeuroD (N, 100%,
n=25) and N-tubulin (P, 85%, n=13) is observed.
Conversely, expression of XNgnr1 (K, 93%, n=14),
NeuroD (M, 100%, n=22) and N-tubulin (O, 85%,
n=13) is strongly reduced at this stage following XHes2 injection.
(Q-V) XHes2-W-VP16 induces ectopic sensory neurons. The sensory
neuron marker Xhox11L2 is ectopically activated by
XHes2-W-VP16 (R, 100%, n=13) while being repressed by XHes2
(Q, 100%, n=13). By contrast, transcription of the interneuron and
motoneuron markers Pax2 (S,T) and Xlim3 (U,V) is
downregulated by both XHes2 (S, 85%, n=26; U, 92%, n=12) and
XHes2-W-VP16 (T, 92%, n=24; V, 64%, n=14). Embryo
orientation: (A-I) dorsal views, anterior downwards; (K-V) anterior views,
dorsal upwards. is, injected side; nis, non-injected side.
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