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Fig. 6. An E-box contained within the ANPE element of the ngn1 gene is the
major Her5 response element. (A) ngn1 transgenic reporter lines (left
panel) (Blader et al., 2003)
and reporter constructs used in transient assays (right panel) used to locate
the response elements to Her5 within the ngn1 enhancer. (B-H')
Expression of gfp (revealed by in situ hybridisation, blue staining)
and pax2.1 (red staining, used to located the IZ) in the following
transgenic lines: –8.4ngn1:gfp (B,C),
–3.4ngn1:gfp (D,E), –3.1ngn1:gfp (F-H')
upon injection of MOher5 (C,E,G-H') or in non-injected
siblings (B,D,F). All panels are flat-mounted embryos, anterior towards the
top, at the three-somite (B-E) and eight-somite (F-H') stages; red
brackets indicate the IZ. Two different embryos are shown for injection in the
–3.1 line (G,H); H' is a highly magnified view of the
area boxed in H. Note that gfp expression is strongly induced across
the IZ upon block of Her5 activity in the –8.4 and
–3.4 lines, in a manner similar to endogenous ngn1
expression, but that the response of the –3.1 transgene is
minor and restricted to a few cells at the ventral midline. (I-K) Expression
of gfp (blue) and pax2.1 (red) in founder embryos injected
with –3.3ngn1:gfp (I) and
–3.3
Eboxngn1:gfp (J,K, two different embryos are
shown). Both constructs carry SceI sites at their extremities and
were co-injected with the meganuclease enzyme to trigger early integration
(Thermes et al., 2002). Note
the large number of ectopic gfp-positive cells in the entire medial
IZ domain in embryos expressing the mutated construct without blocking Her5
activity, demonstrating that the E-box located within the ANPE is the major
element mediating ngn1 repression at the IZ in vivo. (L-P) Expression
of gfp (blue) and pax2.1 (red) in transgenic embryos (lines
indicated bottom left). Uninjected embryos (L,N); embryos injected with
her5VP16 capped RNA (M,O,P). Embryos are observed at the eight-somite
(L,M) and one-somite (N-P) stage. Note that gfp expression in
–3.1ngn1:gfp embryos is unperturbed by Her5VP16 (M), while
ectopic expression is evident in –3.4ngn1:gfp embryos (two
examples shown in O,P, blue arrows indicate ectopic gfp-positive
cells, red arrows indicate pax2.1 expression in O).
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