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Fig. 5. Otx2 rescues the ZLI in OtxH embryos. Gene expression analysis of
embryos at prim-10 (28 hpf); the markers are indicated on each panel.
(A) Embryos were injected with 60 pg of the heat-shock-inducible
otx2-GFP DNA construct into the one-cell-stage blastomere. Embryos
were kept at 28°C until reaching the four-cell stage. Then, the double
morpholino (MO) mix was injected to block transcription of endogenous
otx1l and otx2. At the one-somite stage, the embryos were
heat-shocked for 1 hour at 37°C and were fixed at the prim-12 stage. To
reliably localise the Otx2-GFP-positive clone, an FITC-coupled antibody
staining against GFP and a Fast Red single in situ hybridisation for
shh were performed. Injection of otx2-GFP DNA and heat-shock
activation led to a random induction of GFP-positive clones in the OtxH
embryos. Confocal microscopy analysis of the embryos showed that ectopic
induction of shh expression is generally not observed in
otx2-GFP clones (B,B'). However, when these
clones were located within the MDT, shh expression was induced in
these cells (C,C' and cross-section in C'',
13/16-4/6 detected via 
-GFP staining and 9/10 via GFP-ISH).
(D,D') To mark the prethalamic-thalamic boundary, a double
in situ hybridisation of otx2 and shh was performed.
(E,E') To mark the prethalamus, a double staining with
dlx2a and shh was performed. (F-G') In control
(con) embryos, shh expression is absent at the ZLI. In addition, the
otx2 expression domain is smaller (F,F') and the dlx2a
domain is absent (G,G'). By contrast, in embryos in which
otx2-GFP is heat-activated, shh expression can be rescued in
the presumptive MDT (H,H'), and a cross-section reveals that the
induction is independent of the basal shh expression domain
(H'). The rescue of shh in small clones is sufficient to induce
the expression of dlx2a (5/10; I); for example, its expression
was induced uni-laterally in the neural tube (I'). T-shaped
brackets mark the plane of the section in the following picture. The dotted
line marks the border between the alar and basal plate. (J-K'')
otx2 mRNA (150 pg; + red-lineage tracer) was injected into one of 64
cells of OtxH shh-GFP embryos. At 32 hpf, embryos were fixed and
stained for GFP (green). The provision of an ectopic otx2 message is
able to rescue Shh-GFP expression within the ZLI in OtxH embryos (J-J'',
yellow arrowhead), as shown by a co-localisation of the red-lineage tracer of
the Otx2+ cells with the green GFP expression. The irx1b
expression profile was assessed in the same embryo (K-K''). When
Otx2+ cells are located within the presumptive thalamic territory,
they express irx1b (white arrowhead), shown by co-localisation of the
red lineage tracer with irx1b detection (blue). (K'') Merged
picture of Shh-GFP expression, the red lineage tracer and irx1b
expression. prim-10, primordium stage 10; PTh, prethalamus;
PTh*, rescued prethalamus; Th, thalamus;
ZLI, zona limitans intrathalamica; ZLI*, rescued
zona limitans intrathalamica.
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